<%BANNER%>

Directed Evolution of DNA Polymerases

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101209_AAAAAY INGEST_TIME 2010-12-09T08:55:11Z PACKAGE UFE0017563_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 1053954 DFID F20101209_AAAQFA ORIGIN DEPOSITOR PATH havemann_s_Page_075.tif GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
ef926e2cefa3aa00e4a81e3d06b3e0e2
SHA-1
31a06f5ef74dd15d999d04c826598408dc04c1ab
F20101209_AAAQEL havemann_s_Page_059.tif
d09d9ea55e81b296d83e23124c99adfc
82a28b1bc7b528fe74effe33dd08aaf0aa259277
111742 F20101209_AAAPZF havemann_s_Page_074.jp2
e840885562bbd9a8d6e7f9386150a996
34ecd4949d806cff803fb91af9b4d7840d239490
F20101209_AAAQDX havemann_s_Page_043.tif
3d02288c447d6d0aa804bba16875862d
c5ba58261d6273396b6c89a4de69055e0ceb9d4b
110052 F20101209_AAAPYR havemann_s_Page_058.jp2
d24f7ad10fc25703a99692e5c6358570
a87252cc0cf5f753fcd88aeae158420fcf4a7920
F20101209_AAAQFB havemann_s_Page_076.tif
c9cd67b1cd6b69b292631d2ce91e86f0
9cf04f8b57927b1f1697e2540dcb4045ab62ccd0
F20101209_AAAQEM havemann_s_Page_060.tif
d335ae14f6c1d1f3a05cc92b1de32ea7
73f885cbc683795ba2dfdd3e2d846e14cc90e5a3
118191 F20101209_AAAPZG havemann_s_Page_075.jp2
2e2865a2ac2f0df10c6bcb7d30f3b470
2118addfe83f17036beb2d74eb3fe88ca10c52b8
8423998 F20101209_AAAQDY havemann_s_Page_044.tif
a2495bab292988f1f895e6f737955d7e
09fb8f77dc03e5afdb8464b11b435755d29be9af
120844 F20101209_AAAPYS havemann_s_Page_059.jp2
5346560a7dee26ae942b951cccbd24db
c1d1caf81876f28c81d8933b5bd1a82e9acf27d4
F20101209_AAAQFC havemann_s_Page_077.tif
16d62d1304b19cf4ada0cd987b358d91
984847a3d75ba831556058e4dadd5bb8b9bc9017
F20101209_AAAQEN havemann_s_Page_061.tif
f9957843be48fdd262f710d461e30cbf
a5ceda2dcdac35c6d61ed90faa0d728ecda7606a
113333 F20101209_AAAPZH havemann_s_Page_076.jp2
3afe96c29f616dd6f51f3e70a4848dbe
8f00930b1dafe99399892e82dad11b31fa2a90df
1054428 F20101209_AAAQDZ havemann_s_Page_046.tif
6cc2f24052add562f3738d6a4c0bc73b
1c1f1b6f10f01f2e55d06c369ca4bee2d2013620
118181 F20101209_AAAPYT havemann_s_Page_060.jp2
e7c57d2cd2716eabf243c658955388ff
c61a8e66bc2cf497cc224c55c464f09d91ebaea8
25271604 F20101209_AAAQEO havemann_s_Page_062.tif
1b5f4a554db7ddeca7fa85cd10683372
786bbc19a32a88545880217d269783c3d764b57c
114343 F20101209_AAAPZI havemann_s_Page_077.jp2
3899947b44952053c875576a4510538b
a9e330b8e17def535378a37598662c47d1d41ae5
28196 F20101209_AAAPYU havemann_s_Page_061.jp2
2d266fe267fff69fa2069cd434a1a9fd
212b32928fbe23f9b9aeb5857233d849dc06c233
F20101209_AAAQFD havemann_s_Page_078.tif
25730bf3d55db84fe57df792d1b1946a
0bbcc3f1bba07e9d96a9c022ad474c6b92b7d906
F20101209_AAAQEP havemann_s_Page_063.tif
0441ddc6c7f6d6c098c07adb88ab1c9e
c5e8c87453d6599f86150f8f10a772930bee3553
114149 F20101209_AAAPZJ havemann_s_Page_078.jp2
4efbc41053f7b7f7b34a7170cbf3bfcd
999b6de8d5e95c8b478b702d6bd7177b196ed938
310599 F20101209_AAAPYV havemann_s_Page_062.jp2
1aedc4bc1e70791f8f3cc3937495e883
e51d626ac26ccbb04798bcd72321d57c4fe849b7
F20101209_AAAQFE havemann_s_Page_079.tif
f79b4d4e5eb8db0f55208dd67f7948fe
e9450dca39d6ab34f7c0098957b4f323b1984934
F20101209_AAAQEQ havemann_s_Page_064.tif
895e42ce06291ded191872b93070136b
ab5c101777bbd1d340b8e5863fb4eb85478e1202
110406 F20101209_AAAPZK havemann_s_Page_079.jp2
79264a352771999f4a4b54556a2249cd
d7ac7d482ec6acc7d69aed6fdf1952d210d8e4db
443144 F20101209_AAAPYW havemann_s_Page_063.jp2
2b2154c1f55835a3d344d20e18e487e0
bfb65e484b3fa4d4076fd6630934be75da101718
F20101209_AAAQFF havemann_s_Page_080.tif
d47acba288ad3d9245266b27eda5194b
ac60371592087b935cca7e7822ab4c88555b1b44
F20101209_AAAQER havemann_s_Page_065.tif
149a44b192cea7658d38e253520ea6d2
ce2e30dd7c68e2a368efc7de9adce0849bce14a0
107329 F20101209_AAAPZL havemann_s_Page_080.jp2
0dcc573eec5128afc08b99728a78ef41
4558d4769d988fa1c947c7966772a42f464a541c
F20101209_AAAQFG havemann_s_Page_081.tif
f2b746b4156cf3d40a2816289dab0317
32cc7cac1c03350d1535d93b843d62a59711c9a3
F20101209_AAAQES havemann_s_Page_066.tif
b19ca9362d2048a45799dec635324cc6
eab4df3c3299949f25d5f96e97a22110f3781bf5
111223 F20101209_AAAPZM havemann_s_Page_081.jp2
f72c8d3fd5e803397a391c831b437084
b34dfb38fd0d9e4ad866acd2e4e3fa10ed151ce1
1042918 F20101209_AAAPYX havemann_s_Page_065.jp2
efc131d526f2eb4209801a353df189eb
37b2d979cae5f8528336baecfd5023972285b68a
F20101209_AAAQFH havemann_s_Page_082.tif
b1b6b39ea4e7bfc4189c57e409a53136
8d03071e226113c5eeae8271f48823ba45b1d3fd
F20101209_AAAQET havemann_s_Page_067.tif
30771affe4ba0e0625ca08495f73dbb0
de30d4975869e369e83b0169b98dc8bc44e26fd2
106924 F20101209_AAAPZN havemann_s_Page_082.jp2
21be73f46695d22c8cc5eac821f9c445
63ec4fb59e934b78aa43ffb99f07b9db5f669278
524266 F20101209_AAAPYY havemann_s_Page_066.jp2
d9fe95f2c4e46707a8aeb263767d0f4a
4f34624016fe702fdcf077513e1149e89880e3cb
F20101209_AAAQFI havemann_s_Page_083.tif
759c0849ab092d954e61dfc8fd13f538
33f8b0c222af3c384286c734af7a93253bb8121e
F20101209_AAAQEU havemann_s_Page_068.tif
65c1398787b05409bcd286aa7cc8c35a
da3d531c705c1c045c2c091fd6ac56de9b2aed43
110103 F20101209_AAAPZO havemann_s_Page_083.jp2
d8e65a64d4b8329dbbdcacef131ac2f7
10fdc582e8d2f051870f184150773b1ed6ea404b
1051975 F20101209_AAAPYZ havemann_s_Page_068.jp2
06a886040d5d9a1dfa3787f5823356ba
2e49e30cce8e74bb8f5ba37346fdeb6323df6041
F20101209_AAAQFJ havemann_s_Page_084.tif
08081267db5034557e61679813156a1e
c2252225e1800908e54ce480c839728b9a4b8d42
F20101209_AAAQEV havemann_s_Page_069.tif
a60457d04bebfe866c2db51af68d0610
e374e2d4ca8b50dc446aff70ca8aebf93d4fc563
115257 F20101209_AAAPZP havemann_s_Page_084.jp2
8d0c5677a45d3b03580e93238cf2b507
9e8c697f2f213caa755279b74fad68f6ece6664a
F20101209_AAAQEW havemann_s_Page_070.tif
23f215e883bb1b8cb5325472fc17c2a8
89462585b22ce1d0f491b797d8709b13740a9efd
112291 F20101209_AAAPZQ havemann_s_Page_085.jp2
90ab5a4192bfb4934f4df9b8b3c9303a
765a1812e202c3bfb47f7638bfa3563aa7af0588
F20101209_AAAQFK havemann_s_Page_085.tif
e950dd1ffdfafa0856146514e31a495a
a413fcaefd547dfc2be147d7a7133d2342d1dd6b
F20101209_AAAQEX havemann_s_Page_071.tif
6a09d5ab6186cc3f15ae372081781720
f7e6d6b5b8d8c449cd6c59c6c15d89ab17f79a19
112516 F20101209_AAAPZR havemann_s_Page_086.jp2
ae69f033b06b00e07f0a1f96568fa810
c7eb615dcc29076a1f0956631525ea75fcc608aa
F20101209_AAAQGA havemann_s_Page_101.tif
fd26aa0f00761f9046fd89940abe6553
694708b8501a98e9b5424233937edfc70da53e17
F20101209_AAAQFL havemann_s_Page_086.tif
dd0ec88a0efcef24ec493507f3919769
0baf247e079fa3f55f9a81b10bdff094cb12f4e3
F20101209_AAAQEY havemann_s_Page_073.tif
3945cb65ff08a17a3684b19f8ed1a650
79383b7deb8413b40eec159a1232393d6fac4055
114523 F20101209_AAAPZS havemann_s_Page_087.jp2
164c02f3ed8a9d39bdfdc1cfc91a3e43
4cc06372a4ee66ffd494d858f81339828a82ce5c
F20101209_AAAQGB havemann_s_Page_102.tif
9a806a8416c59d4d60f132b425308f32
56fd116f24b136a6bd027f88d09d10e259b8fcca
F20101209_AAAQFM havemann_s_Page_087.tif
9659f5d4e9712398e7db153216675a1f
e69767781ae1d8f5d934ffd3079624745c5f71ea
F20101209_AAAQEZ havemann_s_Page_074.tif
00f5dbf9e80011028f79706d75a6d786
4eb2fcf64a02086b1189e82d4bad21e6edffb67d
38708 F20101209_AAAPZT havemann_s_Page_089.jp2
749057585e677656f77122fdce2c55e8
518d8e34ec4d1ebdea43f08969a37620031328e4
F20101209_AAAQGC havemann_s_Page_103.tif
e9ce7218163b9ccc6c1c47865eef9034
3f9d478bb98404340165bdf7e7bca9f0f07805a1
F20101209_AAAQFN havemann_s_Page_088.tif
1ada3ac1fddce938c15c51104b0469a4
b518fb928cee3cca849a43f47457c56be1e6c976
1051907 F20101209_AAAPZU havemann_s_Page_090.jp2
38c95b5f557f68e4b1398a5408d74edf
d7ecbdd80ec26e14f9424e80fd7bc70b45b07c35
F20101209_AAAQGD havemann_s_Page_104.tif
4fefa82e0d003771e9c9b6e88a223915
952372dd402c94c8ad168b4d4ad311b7de2f2b16
F20101209_AAAQFO havemann_s_Page_089.tif
d936577ef3e057fea409fdb67e8ac3d8
ef33353727f93fceb54f78ea028ca452eab6b05b
31336 F20101209_AAAPZV havemann_s_Page_091.jp2
339d98b196c297d2b121cc4f9e0cd816
37b89d48c54e25a3071e8343d7c6903d0456a2a5
F20101209_AAAQFP havemann_s_Page_090.tif
bbee518bfefaae851d138068d59d6b5d
62b439545292354c5a018f38bcc109ece83b6094
571168 F20101209_AAAPZW havemann_s_Page_092.jp2
0e72149dc8568269479cea46f2ebe2e1
9714bdfbf32be3fd7db71adbbfe9ba0075abf490
F20101209_AAAQGE havemann_s_Page_106.tif
1cf01b1789c572020e0a781661e1813c
0aca7a7621d2e40ba03ba41f6340ddca39a0d74c
F20101209_AAAQFQ havemann_s_Page_091.tif
54583fd0d7231abc4805625b4904ea31
cfcf64ab1a638dab2abf7f7419328b5c48b5ab2f
566699 F20101209_AAAPZX havemann_s_Page_093.jp2
21288aefb544d573201dc69df20c4b74
e7be97b285cc4974cc25f61fe232f68f26182ddc
F20101209_AAAQGF havemann_s_Page_107.tif
cb8a42eac1e8fb62b703f437395fda29
1b916a713f6528d55b2c7e459c3e7f94969ffa3c
F20101209_AAAQFR havemann_s_Page_092.tif
9b6b4232e20512248ca42ee698fbe190
2e11c85b99e687a76da332b2c5ad3a28683bfcf7
F20101209_AAAQGG havemann_s_Page_108.tif
a27770f04db38ef13403210b7ed402c8
e4289ba26e89f30f0ad37414455244052cca05de
F20101209_AAAQFS havemann_s_Page_093.tif
acc3b337db237298d9a41beaf6e68061
975523475b29ec9ebfc3bd5f375e06df366168c6
561230 F20101209_AAAPZY havemann_s_Page_094.jp2
0cbf0063630811d561f09cc45c402629
7b534dde28e67c7e7cc3058a0bbf752aef5cf015
F20101209_AAAQGH havemann_s_Page_109.tif
35b25b1f9328dcda3ab3f60828cdd004
d5bd877a495a43f36b1acbda185dc3babf0c08f1
F20101209_AAAQFT havemann_s_Page_094.tif
53f278ccbcda1ee20099176a0638573d
37bae0e5a6185c4896295ea4e926c06ddbfc4cfa
1051968 F20101209_AAAPZZ havemann_s_Page_095.jp2
792a7e1feba90c6063920bb60ebb43ba
78f84ceceab984ccb51d049693830a70d15e631c
F20101209_AAAQGI havemann_s_Page_110.tif
5fd3e286e3a29dd15c1f3a37b77c1826
c0d9b6ca660ed7ec8ccfd7f3454d1c847e23824f
F20101209_AAAQFU havemann_s_Page_095.tif
1c208027421f4c471ea242828cd50224
9dbd256c5faf3da459305ec2e2ff2c06887012e9
F20101209_AAAQGJ havemann_s_Page_111.tif
ca2cd7b8a40f45cf7f7d544fb9c79acf
e395de796125f4e776dc2ade67f26198f01bae6c
F20101209_AAAQFV havemann_s_Page_096.tif
656e02aa84ebcf6c56c45b4e859d74a5
59b6b9b77a13c301082ec48263976bc50642232c
F20101209_AAAQGK havemann_s_Page_112.tif
301c01c87d80f39bc14c89b66509fbf0
d2272dea7858dc68cb69a3c449e9ac33767b6124
F20101209_AAAQFW havemann_s_Page_097.tif
f39cc6dbfea2dd80211fa2e456e40e7f
785a7fe5b8ba2938b3134a1359b59a9397e878dd
F20101209_AAAQHA havemann_s_Page_130.tif
4d7348133d8d2e0f3d1eae543f22ab5a
7094a2910b489ecd7011930e679092926a695544
F20101209_AAAQGL havemann_s_Page_113.tif
2f1376084f794ec42c4a815421578863
a8075f394579509bc9f096ebc5b29af50ff53b8e
F20101209_AAAQFX havemann_s_Page_098.tif
29f7ad0d11d80ace98e835b4e1927340
b1cf66ec59eb1b7f6ff0c3a3ad9bba406686800e
F20101209_AAAQHB havemann_s_Page_131.tif
38c9fc9e18ae4da14e976ad0ab10f69a
641a065682c023cf7643e26802b79adaeb3310b9
F20101209_AAAQGM havemann_s_Page_114.tif
5bcc878ef592c989ea3b9f31b9759f33
c4c77d23c83477ce3ea913812354b5266b7ddda4
F20101209_AAAQFY havemann_s_Page_099.tif
fd22760d76c21d29ddb40bce9b9bb2a1
758a66dc8933dfa4450afe1a8aea84d91ef2527e
F20101209_AAAQHC havemann_s_Page_132.tif
d56d6a590fe14e03743efcb070b38343
1bc7e74dd77d8a802b0ae8f128db50856cf33ed4
F20101209_AAAQGN havemann_s_Page_115.tif
0f91f1ae9e623d7ddaebff703f0338f9
2f3b551c774f0d0729cafeed45d3c804e6b6fa3e
25265604 F20101209_AAAQFZ havemann_s_Page_100.tif
243455fc5e92140a185e02cd29f1eff7
e609fb64a2e9e3a100295b6f24c6866fd3c58704
F20101209_AAAQHD havemann_s_Page_133.tif
d55e7c315a20b1543481cca53b9e0bcf
39d5ddcc2254eae930fd76454284921af6f60e24
F20101209_AAAQGO havemann_s_Page_116.tif
0af479232ecfc81ff5f0712d0e80d303
50cfb738a1018cc5a44fdbdf314d4951204890f9
F20101209_AAAQHE havemann_s_Page_134.tif
d35017297bf05a3fcf27b28774ac6b78
41c454f007587f48b66845aab1f344befbcdd35b
F20101209_AAAQGP havemann_s_Page_117.tif
f2406806cdc8f65f569ff9122db17ede
b0afe8b37be1c2fb0058a27af422f962e209c468
F20101209_AAAQGQ havemann_s_Page_119.tif
fb0f1acfcf1682660c8764eba64c3943
ecdc691a37caef84d1cdf695d4ae3657dbba91ff
F20101209_AAAQHF havemann_s_Page_135.tif
099cbd1c4332e0744c1eaf9654081b70
5d7736e45fdad6168412a185232c9b3d44d6a894
F20101209_AAAQGR havemann_s_Page_120.tif
71a5e45affc5e88530220a7b5bafea83
067f6914199bf877c0c4e4c73ed1103630e96428
F20101209_AAAQHG havemann_s_Page_136.tif
1ff96f1acc266b397bbe5b4d231ac563
0ca48f97d5d5d709bb924fbd8a32fe6c626a7f77
F20101209_AAAQGS havemann_s_Page_121.tif
cd33954e756aef617fa573d5c8a8c4f6
a6413914c166f07cd127e4230272ad4bf884fd5a
F20101209_AAAQHH havemann_s_Page_137.tif
9f13efc6475de176a9f8f57a55ca326d
d7143eacd357220830e98bf57b3371a196c5e485
F20101209_AAAQGT havemann_s_Page_122.tif
333fe6db4fc934dcafc74e4c3eedb2c7
26b173d47cabc691e5c9ffcc30b7e2621479613c
F20101209_AAAQHI havemann_s_Page_138.tif
8dc161306c82ff9e6398bfd6230fd296
65405f32f74210fc8f61fe271a73f75fd00835ae
F20101209_AAAQGU havemann_s_Page_123.tif
0e8f2e80273499533896d5c35e44b273
44623efbc43caa37140c290c4b6d43514b45a195
F20101209_AAAQHJ havemann_s_Page_139.tif
4424ec5262ee69543f1f858f3bcb0865
5d2a277cda4b399ca204f1ae23987c215f46d509
F20101209_AAAQGV havemann_s_Page_124.tif
11f2bc02de4b9277643439dc4de2787c
8c83989128edcc5c8d30cdb526259dcb66a4a266
F20101209_AAAQHK havemann_s_Page_140.tif
72c88ed3f060a49313c2f5c6692e5f6f
c2960e43849dd27849248d38306455b8151d01fa
F20101209_AAAQGW havemann_s_Page_125.tif
700e1537e0a68a9bdb99629482f0ec3c
b789723a6328e13c58289786a28eb8e882f7090d
F20101209_AAAQHL havemann_s_Page_141.tif
76383531249b0d0035390ac8ccaa4adb
ea16498a22cd58ab40046b21815035c5288e6107
F20101209_AAAQGX havemann_s_Page_126.tif
01e7482455df2ba9818db788d8b63316
913bc7e22019dda3942d7d6b29d8aa29f6771fec
F20101209_AAAQIA havemann_s_Page_156.tif
5900453861232a7cc96f1540fbfb88b1
4618679987110e0fda564b225445fb4bb5122c45
F20101209_AAAQHM havemann_s_Page_142.tif
bffb29d78c0cc174ceb45eab835e8d99
3738b771ec2583c2b2b8ff1784125f77f783c2cf
F20101209_AAAQGY havemann_s_Page_128.tif
813519b50af1de6f08806ae86e6c307a
63cda873f7b4fc1a3394ffb3181ffdb60dbe4228
F20101209_AAAQIB havemann_s_Page_157.tif
58581dd9b3e8791dd7d685ef1a85b4b6
a37bd68973a8d86aac79c2ec1a7b83b2407eeb84
F20101209_AAAQHN havemann_s_Page_143.tif
27e3da19d2166b656abd9635c7874d42
83eca8b1751f67115a13ddc4cce06ae94fabe58d
F20101209_AAAQGZ havemann_s_Page_129.tif
78be601bc95ad67de01173381ce1ae33
8745e8721df4481756d54d339fe99e963c01c31c
F20101209_AAAQIC havemann_s_Page_158.tif
1a043fd54e36090fc0394e20462d960e
066d2cb822ccc65a81cc4cdbff4eeb1cf2550f54
F20101209_AAAQHO havemann_s_Page_144.tif
20a337ddd4527504ef49d1cc9173000b
04e68f4bb6aa036cf850bf34ec0f943e760d5778
F20101209_AAAQID havemann_s_Page_159.tif
dae41be5c0c4b66bb3c4c695106dc48a
64f9c992b89c943c98c789c63e8ca7529e0dbc59
F20101209_AAAQHP havemann_s_Page_145.tif
778cc2dd8b307c2f16e077500e2abd92
d8251d0da3c993233febe6e4d9cab31da6148da2
6996 F20101209_AAAQIE havemann_s_Page_001.pro
d2f3778e6794507abe9c69df09396223
a3bed5b76712052e8b6bacc3d09a6431ff20e7d3
F20101209_AAAQHQ havemann_s_Page_146.tif
0696bb40f38343f9cf56104e6ecb943e
44b0d2cfb0309aa358096d5c799a2dee69096e2b
1356 F20101209_AAAQIF havemann_s_Page_002.pro
4654fbcee26d2c2a3cd77a83493a9544
e851002507e09352c57e277887c27d91af13953e
F20101209_AAAQHR havemann_s_Page_147.tif
c766e180e7a3fcda69d1d8a4b43f97d6
f4be89dd62b5d87d2f850984ee9057eb65aaddf5
F20101209_AAAQHS havemann_s_Page_148.tif
204480eceac52407288c87babaab2100
d11b4f53e3070fc2089461a452f9ae4efe24579a
3317 F20101209_AAAQIG havemann_s_Page_003.pro
87a876410b45628188372b05a73e2563
69ceb6d237d7fd56c937cf9245bad04b4e619b8f
F20101209_AAAQHT havemann_s_Page_149.tif
7818352c8a4eb1a92300583dd75c4d60
41e0d520db431f3eaadc0e1bb1bdd75bc29747cf
53885 F20101209_AAAQIH havemann_s_Page_004.pro
42ee12a01ad80ac7772544f4f2b17739
17ba8be4310837e22b4c22d325240963254c248c
F20101209_AAAQHU havemann_s_Page_150.tif
80ec82af0e0677b7f99ad6d683a65d0d
6841c60686bdda22e52a9f4af50fc53d597d8950
8598 F20101209_AAAQII havemann_s_Page_005.pro
ee5de6131034d9d3c936f029505708e7
0336172828a6a9fc6f8407d5f4ebd1c0cdfa241f
F20101209_AAAQHV havemann_s_Page_151.tif
7be12ea385e5d66c6fbaa361f9c0026b
75e715e934d4505c47a0f5e6426edbf6d52839a8
70086 F20101209_AAAQIJ havemann_s_Page_006.pro
c9f77066fd5554849ebb513f2d1401b4
a39442b2ffdfa72f93a8d6ae3b3e871b86c115a5
F20101209_AAAQHW havemann_s_Page_152.tif
bd927b645501687052c44201995bd3b3
beba4d35285dfb90b9633e76dfe83689a6d920de
56694 F20101209_AAAQIK havemann_s_Page_008.pro
1d34519cc4aca4f4584192f1444b9ccf
1c96d91913c5e20080e61215d792ecb0139b7fbc
F20101209_AAAQHX havemann_s_Page_153.tif
f6cfc6302b326ccbc4e0e9ca0e0dbc59
7f7da1cbd28d17abfe17120ebf5e3863a81d3e9c
56070 F20101209_AAAQJA havemann_s_Page_029.pro
a9fb54e65fd6c7e6b26b2ce4bc8bf93e
bf5c60a982960a8f009d9d1f80f1585207186dc4
59979 F20101209_AAAQIL havemann_s_Page_010.pro
afe71ddccaffc4b58053de1c9da2a2a7
f1b86ab79457b3a08c48e4da0bde0e861bad9224
F20101209_AAAQHY havemann_s_Page_154.tif
f7c9f18cdc2398037e2a6852979b6b5d
73fc4e2894a6dca81705371ed4ce9c31abb884f3
53450 F20101209_AAAQJB havemann_s_Page_030.pro
19bfff2f917ee24ddcbebefe8cee0d93
a5899ff3cb27ab4574e4d795a6406ceeeee0556c
61335 F20101209_AAAQIM havemann_s_Page_011.pro
6e645f56035e86ec998d9175358a58fe
6f92bda097b1366f26bc86975807d94bc192bb84
F20101209_AAAQHZ havemann_s_Page_155.tif
11229202b31698ea808c0e409e3fec74
8db43d545eb25b004ffcd6d9b462e677450f476f
55428 F20101209_AAAQJC havemann_s_Page_031.pro
37648e5d424cd97a2fae277fa4d4bfaf
42a52b57905befb24285869b2089505899189204
17703 F20101209_AAAQIN havemann_s_Page_012.pro
1143bbae5a5cbe1d78d1245fc46c553b
5b02b86d85fd3059a5b49e2bd76b5c47d6a95a54
53037 F20101209_AAAQJD havemann_s_Page_032.pro
b4585802245e922fa84fb0ee5cb2e496
941c7f178c9328524f3c8c38d48aa3ca2601db15
18087 F20101209_AAAQIO havemann_s_Page_013.pro
8c52f062b3895a45358435919acf578c
10a19f71af77d1e35200aa3d88bb2d636927e964
53498 F20101209_AAAQJE havemann_s_Page_033.pro
7e932d1aff2bfa4a28b719379b6487c6
a362b62d86bc3967d94b1125ee188b75e3f4db59
14978 F20101209_AAAQIP havemann_s_Page_014.pro
c691c237d454877fe203a90c12d6a973
b26d3c8b95398b01c10a9e314dbdb4c3b54f0951
54235 F20101209_AAAQJF havemann_s_Page_034.pro
291c96748e12171d9fcd9ca5c160cd35
11c79c9c380ed07d9ec5c12aef2ef2a34da9fe85
26201 F20101209_AAAQIQ havemann_s_Page_016.pro
76683c9c2cfe9256f5e48454a91cfb50
301c7121b87928fa8ab783f90206258e302878b1
53704 F20101209_AAAQJG havemann_s_Page_035.pro
afa7a9ca4b1695d6f3741d507dce0f89
e6b8a4e000a735e4ea9459c114e06b61e9096abf
54197 F20101209_AAAQIR havemann_s_Page_019.pro
cb054a9a35c3e5b9f033a892489c9040
41f4a852e90018987e72d4ca42aa3ce32e29a65b
50691 F20101209_AAAQIS havemann_s_Page_020.pro
58d85d97e4b729bfc8283a6c8fca521d
fba80bf94e0fab8539de11752350a69b1a9ced47
51597 F20101209_AAAQJH havemann_s_Page_036.pro
97693e8a2828beccde2aa2be1ec0a9f2
22d5503944986496461f89ef44d7af5cf8b502d2
52504 F20101209_AAAQIT havemann_s_Page_021.pro
524a8bbdd788d3ed7611c24a010b6648
b008bb275ccc80d500c84f97ae214066724e9bc9
20327 F20101209_AAAQJI havemann_s_Page_037.pro
0456211cd51dd3d771f243ac74c5352d
b98471149a6c701aad1212a8799523f228688cea
49707 F20101209_AAAQIU havemann_s_Page_022.pro
7059dcbd1dbccae0ee8010ebf0748d0b
b477bffae70ad607977ee85ba034cb15023549d4
18592 F20101209_AAAQJJ havemann_s_Page_038.pro
56e8f33fbce87f89f1123f47e13b94cc
4c55e67de66a0fa6fd9b85f26d1de81854651ab7
51854 F20101209_AAAQIV havemann_s_Page_024.pro
bd68235cab5dd9c074ee443fd6519c43
9a2475533bec4dc83d7d2ee9ee3bfd5c5bc4316c
27589 F20101209_AAAQJK havemann_s_Page_039.pro
764c364364cebdb4a191a11452714a3c
c6a5d929959060cec8a586726cff9a97232f12a6
53727 F20101209_AAAQIW havemann_s_Page_025.pro
0f47f4d9ecce8ab8649ffc2fde8a0a7a
e27ce3a0ed626584243b76597bf1232a72955b0c
46076 F20101209_AAAQKA havemann_s_Page_055.pro
7c73346309a7f76a07458de1f6079158
de586834b864ae7917546aa3409656f65917c8f2
25861 F20101209_AAAQJL havemann_s_Page_040.pro
48941cde3ce22187022a6d94b4312d18
48c49227c09fad9c428d6789849642f713e14936
52537 F20101209_AAAQIX havemann_s_Page_026.pro
5d143ff98d9690c5f24341f769ca2114
107db017bfd703ea13228498cd6d884b59903142
46805 F20101209_AAAQKB havemann_s_Page_056.pro
1f26944e5bd78401425cb1f6af1ebc34
6a2ed682facb1caf16549bdadf89434457d905b6
13106 F20101209_AAAQJM havemann_s_Page_041.pro
d9085870d35eda2de112eb5b75754371
060f29e6f8af7fabc1f294c901476ce87f72d28c
55651 F20101209_AAAQIY havemann_s_Page_027.pro
2d9d6303501874677ee07d691357713d
abe1785722e152be1a9ce51f0851187a9fa09ea7
51433 F20101209_AAAQKC havemann_s_Page_057.pro
e6bb0f415b35c8fe03588a2991876592
01388e543524082a2f0abbd831cf58309d3684a0
15495 F20101209_AAAQJN havemann_s_Page_042.pro
c3185439e78baf204437f46a61ef7dfd
fd0462f060274344669138a1bead0bd349dec58c
55471 F20101209_AAAQIZ havemann_s_Page_028.pro
c537486a14083018108d733531cf98be
ff5be751915d20753fd83654265bfd40fcf9ac42
50971 F20101209_AAAQKD havemann_s_Page_058.pro
a635328367ece4a74b71c415cc9849ec
be12ec3b504f95ccc8d0b1c565ce2d0511d31649
33132 F20101209_AAAQJO havemann_s_Page_043.pro
2828cf8c3a4321b8b8f271f4ecbeae5c
1e9a77a0efb8b01051c5c1f969f63a849b7abcea
57219 F20101209_AAAQKE havemann_s_Page_059.pro
d0d085e400c29094825d3a8520097d45
0f25264bfcaa5e35367ac42ce9aeb9d6e344ea3a
37904 F20101209_AAAQJP havemann_s_Page_044.pro
48523d768b66ba6102f78083c19155f7
642c0ff62c1cc3dfc9a84125589def62c506b254
53956 F20101209_AAAQKF havemann_s_Page_060.pro
f06be9d40a0c721c2b2a6ee8ac2044af
2c19d95784a251013573c7b77dec4e048e02a95c
35560 F20101209_AAAQJQ havemann_s_Page_045.pro
9217d9ae4fe37ecab82c782f368ae26c
f9a5b168fa8314aad671fac54f1f0ce57650d5b4
11524 F20101209_AAAQKG havemann_s_Page_061.pro
d05beb6f5fe9f431674ef0d753e90cef
de6aad47400e0c917a657fff81214dff9373cec0
17401 F20101209_AAAQJR havemann_s_Page_046.pro
4bca1014c0b910076b9c448a8e9e40bc
89836dd714bc9de517f203ffac48a64e9aaa7c4a
6272 F20101209_AAAQKH havemann_s_Page_062.pro
bcf546dc4b0fe73843ac1eae30d7d718
e9fbdc4aded5eb6c21c83371e4898174a3866283
18567 F20101209_AAAQJS havemann_s_Page_047.pro
5b4fb1383a2ffe92cd78457e616c2c69
8611e529d6a577df189c5cc882ec63fb06869db3
40882 F20101209_AAAQJT havemann_s_Page_048.pro
6a96220b986a338e63716349c9abbd15
e0d751d5b037bcdab1f229f62537f46511bdc482
30030 F20101209_AAAQJU havemann_s_Page_049.pro
291224f969cb8f48b5c309affdac9c07
715ba846a6a5536cbb12a06cc3a57eef918130be
9584 F20101209_AAAQKI havemann_s_Page_063.pro
552fa2785c61a6efa8e445edce719ae4
e074be200ef29f09637cc82dbbe9f771ab0f12c3
49068 F20101209_AAAQJV havemann_s_Page_050.pro
3a0d20ad3bd7e235a44f3f1e9f3ca178
978ee7d1576ef2f5ef19f56ba9f3e95a7fc6f157
8317 F20101209_AAAQKJ havemann_s_Page_064.pro
71c852e24726922832d1d46eb42371b5
59eaa981461648991d565277332ead9396399a06
52420 F20101209_AAAQJW havemann_s_Page_051.pro
ccac7b5bb79f8da16fd6ba216ccf6400
32ba9277aa3d1f6ec60a7d33b640a9f7e28d8d0d
25831 F20101209_AAAQKK havemann_s_Page_065.pro
4c13e466f90bda27c7334e275e56ac5a
c12ccd8700e468d8fb0e9be98890c8aae2552b04
51285 F20101209_AAAQJX havemann_s_Page_052.pro
adecf4de942980e42eab6c4dfe625eaa
a87da3d45892a9253d7487b788b496609c03f153
50532 F20101209_AAAQLA havemann_s_Page_081.pro
6e045a576254a5d5ffffa6ba53728adb
b001229e2b84ac722bfe89087139fae4394dd126
15104 F20101209_AAAQKL havemann_s_Page_066.pro
b2f6fe98d42a962008d9d6bc0fd2ffba
42013daa6e52fb46d012709cabc9e2cef9fbd843
41797 F20101209_AAAQJY havemann_s_Page_053.pro
094cf7090df9fd67eaf33752e2480bed
7d4c58ceeba4c6e748e800b1d51ac34c51f8f919
49024 F20101209_AAAQLB havemann_s_Page_082.pro
2fc0f08091e396bce3d8f0fec980aacd
cb5cb3d0fbe87c6fc98896e842e584d978e98bbd
56471 F20101209_AAAQKM havemann_s_Page_067.pro
cf29efc72bc397b3c98389adc74c7ac7
f7730b6692189176167c0de1023b1cdbef45efcd
50596 F20101209_AAAQJZ havemann_s_Page_054.pro
d589f37ea95538cf40847dbdbc505349
513daaf174f23f5456a9fdf2d745b484de32de54
49245 F20101209_AAAQLC havemann_s_Page_083.pro
8208b52d405e180de846fbad6a86d0a1
cc4092ae9448aa2633b61668695f4e660d6aa104
64129 F20101209_AAAQKN havemann_s_Page_068.pro
d62f1299a7d5857468186e55a04e43c6
26bc903395805a0c21aa87b9488f727ec71187ea
53004 F20101209_AAAQLD havemann_s_Page_084.pro
ec558986e097ed835e8b872c8743ef91
e5b286f1b1f86fc7e69131f7febfe044271457a8
40195 F20101209_AAAQKO havemann_s_Page_069.pro
0ca1ab2cfedbcccb815801d06a706b8f
ab272a8425e7ed9d18e20e70f6e76f2cbf03f731
51053 F20101209_AAAQLE havemann_s_Page_085.pro
08ea4aa17789b9a154122aefd33ac0a5
261844ee0d0ef39696004cd20986eab9d52323a9
50816 F20101209_AAAQKP havemann_s_Page_070.pro
0022c4e52f6b156497fa38aa198b8fe1
d78f26f396db5bb9387851c9749bae81adbb024b
52813 F20101209_AAAQLF havemann_s_Page_087.pro
decee98805ed8fd5b5a69273726b027e
f4277ba7e464d320b33b45a99b260df82b1eba6e
52276 F20101209_AAAQKQ havemann_s_Page_071.pro
3e5e1f8bc969e2ba13bef4c5af666201
fd14e19236109c2e44f077dbc9ad07fd4c8614dc
18044 F20101209_AAAQLG havemann_s_Page_088.pro
5a36ed07bef1bfebcaf8a9c1ce5e2c3d
4429f1859cafc340e102128bfbf26ba775a38f0c
54264 F20101209_AAAQKR havemann_s_Page_072.pro
83ecfe5af86c09ccade3a8259630f03d
0760414497282f0897bce7e9eca4e7b33109b282
14830 F20101209_AAAQLH havemann_s_Page_090.pro
d3200d52c1d6c777344b0f5f3ccf8b3f
70fb13b21caa4d1d8d24805184149139a522a041
52836 F20101209_AAAQKS havemann_s_Page_073.pro
cee010e9f8e0cf963429ed9b68fa1a3d
f9d7f6304c762d5213cae6f35a5cbee26762597f
14472 F20101209_AAAQLI havemann_s_Page_091.pro
2bfdd4c0afa6f14cde3700360a561860
35883a0d77dcd3796cf54b023e830d576cf81d65
50128 F20101209_AAAQKT havemann_s_Page_074.pro
31c71f732769ecf9de29ee0328eb8008
42e384ecc1ce4e48df19348a595d06cdac2cd060
54039 F20101209_AAAQKU havemann_s_Page_075.pro
1f7a34cda9a96218a913d91c73909f4b
cb34acd5cce8ae8221f1b027dae5ab6794d1ba27
15915 F20101209_AAAQLJ havemann_s_Page_092.pro
8be60c7e194f57b49647fc589a392e40
5374eb4e227c132dec189bf92481fd8e02c6576c
51659 F20101209_AAAQKV havemann_s_Page_076.pro
792ee25dfb98981407cc186d90b854e2
c42c53e9a1410248414dd7f0c168d9948af5c292
15443 F20101209_AAAQLK havemann_s_Page_093.pro
2e38696b90af115441e258e22f249ddc
930e755b5ec5b0369bc4d678bfbe921e9a1eee84
53048 F20101209_AAAQKW havemann_s_Page_077.pro
80c67c6942668e84d4c4ee8dd93faa37
07d9c65e6c29ce49bab9d239e971b2834ae8e522
52613 F20101209_AAAQMA havemann_s_Page_111.pro
37defd586701d75a111773a99a9442ed
96ea6024806768efeefdc6ef9f82f7087c103bbd
14138 F20101209_AAAQLL havemann_s_Page_094.pro
ed46270b45b1ce1f9111abc34d61d046
fc7814f96f8d0ce3570c4c659f7a0e6f8b9b7ab9
51739 F20101209_AAAQKX havemann_s_Page_078.pro
5f42f51b9f43003ec5ca47df40647e64
edd178cb84ab13e534a89196cd8e5770ea276ee7
50860 F20101209_AAAQMB havemann_s_Page_112.pro
a446f47273bc405d6e68d98eba50c14a
09e1ba69df7392cd31713a1ad5eaaf3ed915b15f
34722 F20101209_AAAQLM havemann_s_Page_095.pro
33b70f66670a5705c50071db894b6924
0e4ccbc2344df249c1b9293272c731b050b1da12
50123 F20101209_AAAQKY havemann_s_Page_079.pro
fdebe4c1aa3a9601932c7695910fa776
3446a07a2e868ac48e97f1ef5c910f4990a70490
49699 F20101209_AAAQMC havemann_s_Page_113.pro
14070e40bb32e08f44f90e27b6b70d91
7f7981b46f12c196f90d63018836f7778654564a
233578 F20101209_AAAQLN havemann_s_Page_096.pro
097702289ad7effdd054fb77c72451cc
a988e6831614934770cc9c57a68d8482f82a82c9
49304 F20101209_AAAQKZ havemann_s_Page_080.pro
032432697ccd569d3aac4b64702c17ba
08b7c4eacffe0ad08e5ddc1d67df5ec4e7362388
53348 F20101209_AAAQMD havemann_s_Page_115.pro
cae3015b7eee994b155621107163726f
7014115f4d970867beee3af5a091336f82c5806d
49607 F20101209_AAAQLO havemann_s_Page_097.pro
d60f4447b289072e6944a6f7a8feab7d
ddc7238099de4bd1fff1c14dd6bcfe749f207b1a
54604 F20101209_AAAQME havemann_s_Page_116.pro
33736dbe9a238e9e06b9936def73a672
5dffdf8b8ee26844ae812bf59b4d3a67abea6219
25750 F20101209_AAAQLP havemann_s_Page_098.pro
310b681e53dc2fdde0e5222b1f994b8b
949d13b1d19e7c54b409fbcfef69d6638b408f43
54873 F20101209_AAAQMF havemann_s_Page_117.pro
05a8b5b6274677eaba26b47ebecf526e
028fb710c873bdf8855affd058f1769c2013033f
32190 F20101209_AAAQLQ havemann_s_Page_099.pro
c26ddb7c4f2f016fb8abe7f5787bba9e
7b454824ca786f9b011a4718ad4567f2d443df67
265203 F20101209_AAAQMG havemann_s_Page_120.pro
7980489b001aa351e1733ee3c80bcef5
1480b349240400d12cb3b31d4bff29b4c9c17da9
28019 F20101209_AAAQLR havemann_s_Page_101.pro
1a6b6a1ad62117ef01d7d1c08aadbc6a
072f61ed1b32ea2fd32fe3da7bd9bf81fec86525
73441 F20101209_AAAQMH havemann_s_Page_121.pro
54970dbf65ab37b32f70316fa627bb0c
8399cb79d116e0a04a521ad0ca05553a3884ba80
72087 F20101209_AAAQLS havemann_s_Page_102.pro
20ef2196fbe55c937788fa26d5e80998
61c357b4358c212a361053a491c23573d7292e10
24042 F20101209_AAAQMI havemann_s_Page_122.pro
7766db4eeee1ea3c9fe0eed4568c43a2
507d42c98a1bea5c087e0acda471753e472a5069
49711 F20101209_AAAQLT havemann_s_Page_103.pro
d619e3feb35a75713483edc58c0ce409
e28c124c0059d5fe36bbc16a94176646ff90dfe8
78732 F20101209_AAAQMJ havemann_s_Page_123.pro
2a0abf6ee02ec2a00faff4dfdd349598
a48ca858a6c27002f9b9ac0fe3756f9404465831
50909 F20101209_AAAQLU havemann_s_Page_104.pro
daa2e8348ea7554c9536bc17f0a80e22
c88332fc9f03aeb1284d6b1c3213c38ac94b090c
48669 F20101209_AAAQLV havemann_s_Page_105.pro
d346ca8b8c544ee9aa98897b44861f85
368a8eb36175ef798c4f05288b40c58d95cb1a55
62869 F20101209_AAAQMK havemann_s_Page_124.pro
6fac1fc514126ea672572203916f481d
4cda9bace20c9c840626cbc16161c05e5a405c55
48925 F20101209_AAAQLW havemann_s_Page_106.pro
af01310deb1f580c221f4aaf17ffd71f
931ad9e002980f8d2e9e73aa9fefacd7c00f82a8
13498 F20101209_AAAQML havemann_s_Page_125.pro
242d8aaf691648621b7857a83f102225
357092568e7db642c9e75a0ec37e42ec6cf12e35
53307 F20101209_AAAQLX havemann_s_Page_108.pro
d37443b945739024ca7116eb8408649e
7f234b9bcf0c8567a4033ee7402f07fb2e9672bf
48705 F20101209_AAAQNA havemann_s_Page_143.pro
67f1024796a0051ad703b3c11cfbadf9
c3838e03daef014637d4fa912b45c1d0d3a27bd9
34976 F20101209_AAAQMM havemann_s_Page_126.pro
1bf633a96202128069a6a25c48798576
902f866cc325b0c422552969f6c23dd165c2929d
42817 F20101209_AAAQLY havemann_s_Page_109.pro
e018348d6091376cd6f76e59a3336a2f
0d81c85826e2bb2dee5e256a030e91c3f07ccb3a
49660 F20101209_AAAQNB havemann_s_Page_144.pro
c012b65794822a8bf27e3504860da6ff
6db0fd7b24d816d28ac7ddc7191182b654f93988
32954 F20101209_AAAQMN havemann_s_Page_128.pro
1ef33cb8487fee0dcc85b6738341405a
7ec8b3d548fc99134cbe6c817394af9453f80ed1
48323 F20101209_AAAQLZ havemann_s_Page_110.pro
0e62357745f00cddd200bd1a9686eb87
e9a4a9e7e325da305f24815bdfb89ac09227a248
19636 F20101209_AAAQNC havemann_s_Page_146.pro
f030f3890a96893ff99d4c3e94a51235
87bbcdcab574cf5fcbec8ea74225dc348f01a838
40296 F20101209_AAAQMO havemann_s_Page_129.pro
eacb64e378ced60330a97d4753398217
c720a91e5df0c065273315263176fb14cc79b266
24162 F20101209_AAAQND havemann_s_Page_147.pro
f6069a8fc8a6860039ff7bb216440895
50bd77669245a336b445de2825177b7f27fc5903
35341 F20101209_AAAQMP havemann_s_Page_130.pro
312036e892816c4de2166953c6e14cfa
936d451b62f28edce2edc327bf5a03910a4d1e1b
20016 F20101209_AAAQNE havemann_s_Page_148.pro
1f2f9e6914076cb8bab51242229b705a
3e6ee96ed8089587346a1a722aa642bb53cde85f
26186 F20101209_AAAQMQ havemann_s_Page_131.pro
48dbd1033399d46f20faa084bc841be1
c929b7655fece34f63910d230af64071a357800e
10411 F20101209_AAAQNF havemann_s_Page_149.pro
d4f8479df1634cd2fc920409354faab0
9d9920a6e22a306eb131dc601db972601e7ffdc5
51393 F20101209_AAAQMR havemann_s_Page_132.pro
280c492ef34d42329f759905f9107842
2e53410928a487145c5390ceb7e331e0dca64d3d
F20101209_AAAQNG havemann_s_Page_151.pro
b9f8c50e5833d4bcb1fb4f8a172e10a5
cefd772251276041337bfc130ab0f3aa03e46433
51015 F20101209_AAAQMS havemann_s_Page_133.pro
6a5572ec3195674637bbdc4bc8c29cf9
8f6c17e4d096f9ba45fc1b6d90d8e85a27f50666
52254 F20101209_AAAQNH havemann_s_Page_152.pro
4987a0a2cce74aed0413fe68fe090392
69393ec059f85fd84d971e3d65e9a6589513d181
47096 F20101209_AAAQMT havemann_s_Page_134.pro
f2192c6d0956b2de7c0a02abefb63513
7d083f432e55f529b4796af88d13c1f478cdc305
57298 F20101209_AAAQNI havemann_s_Page_153.pro
eee3fcd6387b119cb905e6d551c86747
bc0bc24c071aaefd7aa83b21a268b43ecdd99a3f
53321 F20101209_AAAQMU havemann_s_Page_136.pro
ad585edff2393d58b252a9b6999c61b3
16ab1b324a8a11bb476ff1c00a62729a800e9aae
54127 F20101209_AAAQNJ havemann_s_Page_154.pro
898d4774723f77e4a3f1ee3d5ff85be2
38d28f723368941a6d13cb5ef7a393ff2d7dad45
56409 F20101209_AAAQMV havemann_s_Page_137.pro
f421b02d86c96990e22c5ff79d630cc4
a6aafd41fd3ed481afbd4aadf6127bb997549dc4
54229 F20101209_AAAQNK havemann_s_Page_155.pro
57d41f177092f70ed5101644c62cb210
b0ce1dc6b08f2c44b3b30a69dcf200852c1ce0f1
53610 F20101209_AAAQMW havemann_s_Page_139.pro
55de596368559182519d3b9a6bc29e73
e74a243be16803dad6159d2a3e8bab7386917458
53140 F20101209_AAAQMX havemann_s_Page_140.pro
c5599ecc7bd40b21f79e65be39e70df7
fbcac990960172152f0ab0adaa3b550d3d296b33
2069 F20101209_AAAQOA havemann_s_Page_015.txt
9a81cca2218edb03e96532881ab7890f
c22bee6e84a48f65b5467099c968554577f37857
51858 F20101209_AAAQNL havemann_s_Page_156.pro
bcca8591d24948801f406c02c4d0a499
0359f5896cbee676f0796fbc265eeb430a999c69
47254 F20101209_AAAQMY havemann_s_Page_141.pro
412a5a24e726f03dd1c197a73925b824
116bba0e104947f6a732fae0fcf8fa0934616c3e
1044 F20101209_AAAQOB havemann_s_Page_016.txt
9ae7a6e6e311502b883bfd9e2f6c2db7
3afc2c4d47ef52d27d9f35d35666f190941750d6
48825 F20101209_AAAQNM havemann_s_Page_157.pro
84d21da07b264620fff656a12922c6bb
fca7043fdd47b8273ff81e6574f8a1efdc26f668
51396 F20101209_AAAQMZ havemann_s_Page_142.pro
c72ef48568b81ff43e301e088a9f31b2
8804ee517fe1ce48b2dc708571c0f715477941db
1982 F20101209_AAAQOC havemann_s_Page_017.txt
1d326ed0d82b7f10be35393be533bdf3
c4b283c28d6ef8813b195fb78c1ef1e8cdc791aa
4382 F20101209_AAAQNN havemann_s_Page_159.pro
80b911ffe3357d87e6f7f86265702bbc
fecb6346a6a1e1de86d30e9e752e2a44d674857c
2191 F20101209_AAAQOD havemann_s_Page_018.txt
9cb588f4fc59a9edd5aab7bfef394908
554e8f3be02c5b7c8417d279c1c64d82995b7486
398 F20101209_AAAQNO havemann_s_Page_001.txt
c8078c7f578bf354898f004b706ab429
3fabc538ef8b43b18fd411105435308160cfd241
2134 F20101209_AAAQOE havemann_s_Page_019.txt
c8ff4883725f1a2c832c1e129163ef20
148d8cc02b2fa51fcd0ba21d6142ec68b252608e
209 F20101209_AAAQNP havemann_s_Page_003.txt
0bd7fa4aa3758f868443b0588aeaed41
956ffbc5d2f7c9aa36181d5784714206ae094e7c
2038 F20101209_AAAQOF havemann_s_Page_020.txt
5c8806996b6d52b86543b1bbc451f5ee
01f9ff0bf80f2f6b66c48578e39356eac80bf901
2159 F20101209_AAAQNQ havemann_s_Page_004.txt
d01e69ba8169985bc25df36f13bff061
8d4681f37d3fb0643a2e6abc2970ca95e5e5a150
2077 F20101209_AAAQOG havemann_s_Page_021.txt
fd301c25db858beaaa078f4216965d6d
2cca11a7761734b812b5959612399380421acfff
349 F20101209_AAAQNR havemann_s_Page_005.txt
462f061e8122d3265e5b2848eac065a7
9e7fff5c76ee4ea5d476f83324895bc8d04cafd6
1963 F20101209_AAAQOH havemann_s_Page_022.txt
b96a55156520fa11456bc2f9a026040e
0bffbd6e72bb8a8eee98dde66ce4a0543ae06edc
3093 F20101209_AAAQNS havemann_s_Page_006.txt
8abe44265de5aa2376d2287a7b0ea8fd
342523f3434a5710b1b135aea789a6cbaf8839a5
2133 F20101209_AAAQOI havemann_s_Page_023.txt
293aabcdacf07507afde2e9aa38c4e71
35f5a57384aa5b61ce21f6fccfe6b37bf840c83c
3903 F20101209_AAAQNT havemann_s_Page_007.txt
0678501eba57e535cd2f95a80c3144a2
b9e5381f09301c9833500a1359e422aa09c858a7
2040 F20101209_AAAQOJ havemann_s_Page_024.txt
a7a2bdf6451ea76bee651bba13d57ff9
06bcb14b64dce3c3d402343a18f9546542afe807
2001 F20101209_AAAQNU havemann_s_Page_009.txt
b4b13863508edacb877c804978976ac4
4c8208e5ce473ef42525bdc5770eb5735abba592
2076 F20101209_AAAQOK havemann_s_Page_026.txt
952d11da68c3d0ba601f4aec12bd6293
4aab203f2c0a5ff45e829134fe2584547d58f323
2498 F20101209_AAAQNV havemann_s_Page_010.txt
e8940729fae3a70b8da094e2e12a869f
378944d89391422b1c224f349e29e3bdeb0e579c
2197 F20101209_AAAQOL havemann_s_Page_027.txt
6b1630198522dfca747357396ae12314
55bba258d69eaca6b27d4047f7989d90cd71d3d0
2533 F20101209_AAAQNW havemann_s_Page_011.txt
e44022589b8445910d8748a0db9aa641
382afd32a79103223cae6c600f6083ed1955c4a0
1471 F20101209_AAAQPA havemann_s_Page_044.txt
a886893814f36b05de8620fecb4c23da
f106b0e39d690307e002fd5df9636ebbf9752ba2
787 F20101209_AAAQNX havemann_s_Page_012.txt
4b71919da41f4e84409d22a233ebe094
078c1e0efc3690088ebcbde8a9e7a7d17c6472a9
1456 F20101209_AAAQPB havemann_s_Page_045.txt
450b637b070de3c00f5a21d25fa34a02
e869fef1e01c6567b15085d4be4514cfe95aeafc
2177 F20101209_AAAQOM havemann_s_Page_028.txt
270f77c76212c5fdba90a745e9ee12a7
7385922275a8995ccb07da3fd65c9eed0b631385
747 F20101209_AAAQNY havemann_s_Page_013.txt
772856177ec9c881483c9cad4de1a7a7
0c079e002a9ac616e0d48ec2ccc5427a33ec3d00
871 F20101209_AAAQPC havemann_s_Page_046.txt
f621f68274cc797c7f816e960d13871b
5569070e54468d94df79cafe5745354d22ac89fb
2239 F20101209_AAAQON havemann_s_Page_029.txt
6e628213e0e064670f7b71fb4216d217
3023bb11b1d87b3130c56eb422f0fc0676467c97
688 F20101209_AAAQNZ havemann_s_Page_014.txt
adbf2d83ae385e9222ec02f3cec7ce51
226b4d4c8e0fcb878d8537974a124b22f2855cd6
749 F20101209_AAAQPD havemann_s_Page_047.txt
b99497ab3867a9ee4134af055afab69b
ade2c05a3efded78c9b34cbd498fb7890d4f1972
2104 F20101209_AAAQOO havemann_s_Page_030.txt
283d70776120b755b83ebdb6ebe78975
30edbf21f1ef88a88f72a420383cbd292097fe4f
1829 F20101209_AAAQPE havemann_s_Page_048.txt
a37179fe9dddb2191d1a8ba6f7742698
74cd92da2a4173fd131e8a3351830bf19367f1dc
2176 F20101209_AAAQOP havemann_s_Page_031.txt
44393ce5e800e130264b59646f046015
e92108ba88a59e5248de1483aa1b52b90e90f45b
2081 F20101209_AAAQOQ havemann_s_Page_032.txt
1ad19c539b8e9faac26fa30ab88fcb53
2269bdb249bf871a96a6f1f7de75a675a86650ad
1305 F20101209_AAAQPF havemann_s_Page_049.txt
d3c0e8de045e5a0e421d7ab7cc714914
b743bf947588329fdb7c874b41052022591b8a16
2107 F20101209_AAAQOR havemann_s_Page_033.txt
8a528b49c9e18e055411b735028b3d30
414cfc56e77da02ac801fc5dee3154d3eadc2914
2018 F20101209_AAAQPG havemann_s_Page_050.txt
db3b759fec0b6a706d82182c5988db33
d944d8a856e4f1fe459b4aebb6db98874f0b605e
2164 F20101209_AAAQOS havemann_s_Page_034.txt
fd11360afdd1f34318a6007c61caff35
5fcfb52ab9effe2b046cc324e214f2115608d364
2071 F20101209_AAAQPH havemann_s_Page_051.txt
1508f2a93266e57ecdff09a3c6968e8f
e5201f3b523396ddb2aae3b77646ee6809d642fd
2114 F20101209_AAAQOT havemann_s_Page_035.txt
e66be3db725d709f2b29d1fbc349d6da
2380f1ef9211919602a70439a74979ba238094b4
2047 F20101209_AAAQPI havemann_s_Page_052.txt
1e8366b7d916df051171583d70cd1092
191fee4b3d8adfd72f1621109fbc7e31350be1fb
2039 F20101209_AAAQOU havemann_s_Page_036.txt
c9e7c192128b0f94c4595af8c26e890b
e86c09186d9ff850292df259fae613ce4363cf05
1668 F20101209_AAAQPJ havemann_s_Page_053.txt
1e33ad480ff96b7b9c5e3919b03a1e08
5f0ec9ada4bc9bfaa0919d502b776e21e6cc9e01
834 F20101209_AAAQOV havemann_s_Page_037.txt
fc13014ab9811fbe9d08b31e10428cd2
aa9e97bb49706ba6e9f3480a866da4674b992d3e
1995 F20101209_AAAQPK havemann_s_Page_054.txt
ef6015c213e1354bb72182e93992e2b4
4dd09b4e4ea0efe3ce8edaa9fe26dc1d69377b78
1248 F20101209_AAAQOW havemann_s_Page_039.txt
af3e8c5f2152271c5a3e724baebcbe85
a5ba11e5a4b46468888a7af9fc489221620a16a9
1830 F20101209_AAAQPL havemann_s_Page_055.txt
deb94dbdf61038f244f216baf99e9f2a
d0a13eea8889e00d55c5e7942ced98852bda6552
1117 F20101209_AAAQOX havemann_s_Page_040.txt
0a81e3d8ab40629c3236fc49a2a97b8c
715ee1c7a3bcf6e273a95ec884d7f7f1f31adb84
F20101209_AAAQQA havemann_s_Page_074.txt
d2102f222fa7f5fe6ecf447dddd4b4d3
8d311ae0054f911d8a9206ab22dbbf8d49123d8b
1896 F20101209_AAAQPM havemann_s_Page_056.txt
246b2256d39b6f8bb41d20d56a6a0b63
04bec687cfbf08eefcaace5d8f80749217c5b150
608 F20101209_AAAQOY havemann_s_Page_041.txt
d3efaadad776b1617cf46556b81d1258
d2755e023919d65e9ac565e7ec7705d5dafa1ba0
2125 F20101209_AAAQQB havemann_s_Page_075.txt
0f38c791ad192d6dcb8012848c00c787
bb353d3e6364d64a73985775581c5c6c3732bd9f
1033 F20101209_AAAQOZ havemann_s_Page_042.txt
78a0786520eb193b5336b0a50a967ac4
a44c679c944c14bfe777ef27332fb4eb045c0f6c
F20101209_AAAQQC havemann_s_Page_076.txt
16a26046ddae7875ca3f51b8fa96f13a
10c41ce57f271d88cbb3f855daa2f4727860da46
2022 F20101209_AAAQPN havemann_s_Page_057.txt
ac6fa48cfc7e4c1055ed0fbb11aa1dc7
9355050a48941a253f872114bcd3356381d78836
2093 F20101209_AAAQQD havemann_s_Page_077.txt
132b323e3e214c5ca17488061ff56290
06776cd45e9da24b4c65a18d9a8ad88a910eab28
2250 F20101209_AAAQPO havemann_s_Page_059.txt
14720f00ba11c5d3020f631f7ee4d8a7
fa3567937f0f91cd3ed937e1a6be3933ff068af1
2035 F20101209_AAAQQE havemann_s_Page_078.txt
27baa9f1c009bcc81208ebc1e33826bc
a6eaf89712be3516fbebc91029180cc28ab8d52f
461 F20101209_AAAQPP havemann_s_Page_061.txt
08cd4b74dd96c81d1e71d1d2f8bd1c00
cc4dca2d9a162c9399ab13cc1ec8adc57095934c
1979 F20101209_AAAQQF havemann_s_Page_079.txt
01edc2e20ac14807c324f53711f946db
d1bd601c01e8323281293ee5336eb56c8bb38728
565 F20101209_AAAQPQ havemann_s_Page_063.txt
788b556da1ea1fb6a7e69c43765e3775
af14666b9fac8b352cb56cc66aa3b8141a45540d
1946 F20101209_AAAQQG havemann_s_Page_080.txt
e6e197903fd1872dd9ba5aa8217a0b96
fd5bb7ea5f72ae3ea20b55f84eb55d615dda33cd
328 F20101209_AAAQPR havemann_s_Page_064.txt
fc54dd0d596837b9796aac6cbf614a91
90f7be06f1990a4cf1ceaf66c7236ff25960ca98
1997 F20101209_AAAQQH havemann_s_Page_081.txt
703372980a6e503cac9422df3bf21930
aa99048a830709517646912aced6fcfd7ef93e0d
1154 F20101209_AAAQPS havemann_s_Page_065.txt
d26461eb2518eaa9be3b340e7633d756
36cb7cb6cf8c012aeb6675181c9b1487aa4e97f6
1968 F20101209_AAAQQI havemann_s_Page_082.txt
b92fd90675aec4c46867c18a5c7e7f22
f74058f512c2adb0dfa0ab46f432c1ca94736cf8
1325 F20101209_AAAQPT havemann_s_Page_066.txt
2488ac9c51b71757f54ccbe071b668ed
79f3be06cd83bf0a00cac06baba73a32b943d090
1948 F20101209_AAAQQJ havemann_s_Page_083.txt
2ce90ba9f61c368e9c772d0fca236afb
665fe39659eff3180dc0049e93fae05d08da62bf
2738 F20101209_AAAQPU havemann_s_Page_067.txt
e4b8003df82c084cb0d7e12c6930afda
478739cc927517636cf7fc3fdf4fdec44178dec4
2087 F20101209_AAAQQK havemann_s_Page_084.txt
2c33af51a51a2287e3b2c0e52544a924
746419661fe9a1e09867dd5610a3a5b1652b8322
2730 F20101209_AAAQPV havemann_s_Page_068.txt
73468777c1e595422c2c26c5b69825fb
c982600c539179c164a807c39ebadade961d7b8f
2045 F20101209_AAAQQL havemann_s_Page_086.txt
fad91b660f1260c74de8a9b1cc229280
54609786bec62cad5aa38ea999cc58abc614d3bf
F20101209_AAAQPW havemann_s_Page_070.txt
9b20ea4b4b7e39905cb63dab1b97171a
b305ba80b5ae7f6d08f1034925f0b0bc5cdfcaa4
F20101209_AAAQRA havemann_s_Page_103.txt
030e92279e512c8c425ee304975fdb44
161255754cf2873989e6fbcaa16c702d4a3cf4be
717 F20101209_AAAQQM havemann_s_Page_088.txt
6215edf06bd26ce3fcc750fb5380d30b
67c36a700efdd11a4bb8e7a94e4cc01729123603
2067 F20101209_AAAQPX havemann_s_Page_071.txt
c968bb27921a14271be8bd26971fe325
0358c55d0a89c55c9eff3c19ef4b2aaf9d1e0c92
2019 F20101209_AAAQRB havemann_s_Page_104.txt
e95b407906d3b2fe12e0c47e24b60e89
47bdbcb41611864d63cfaa75ab41e27ef62da61b
591 F20101209_AAAQQN havemann_s_Page_089.txt
093b29d990972a0ba65e01941ebc31d0
8d5ca547a95e3be5d55db64ebd1d6665d7a3889e
2130 F20101209_AAAQPY havemann_s_Page_072.txt
558075cbeeba8313eff5a2b4382d098f
2d5c0085c905bf6b689b1328e586f02415f8c581
50536 F20101209_AAAPOA havemann_s_Page_107.pro
a8927220dfd25b105075b70bc39aa09b
2a3099660eeeef0fd0d953cf4d973bec53586621
1953 F20101209_AAAQRC havemann_s_Page_105.txt
a4cfa8a55c2ccd99be731ea427e28d85
af7e831bbd84f3ad555b0723377c234969939079
F20101209_AAAQPZ havemann_s_Page_073.txt
82ec3b7024e041537d0091bb4e02e952
11dc8a37edc22014ad81a2db6d8804c8f4ff1994
56231 F20101209_AAAPOB havemann_s_Page_138.pro
996429946451aa27c4bfc859e8971c4c
2adacedafeb10bf26aef86638f7dd8c5dab7652f
1926 F20101209_AAAQRD havemann_s_Page_106.txt
d96213811059674cab47e774b6cff840
b817b92e59103af9a49e969044a29e508e7aeda0
647 F20101209_AAAQQO havemann_s_Page_090.txt
3ff4edd5e64860286a2dfcb9154603bc
998e9d198189b07146c62e99f84996f063f77ddf
77778 F20101209_AAAPOC havemann_s_Page_025.jpg
9e4e45ccf3e49d69a685054ad19a4259
6e53ff4e9cd2141cd735946112ca2048db75cf09
1993 F20101209_AAAQRE havemann_s_Page_107.txt
73dcce74df7924a4770b45e76df1b167
f3b8876390a3a671a5f665e7812c6c5dbbcdbdd6
602 F20101209_AAAQQP havemann_s_Page_091.txt
916c544da40b4ccfce63acca1614eba3
ba0bf6f37a95d93b615887ce2016f65c3c732c22
122 F20101209_AAAPOD havemann_s_Page_002.txt
979449ae708d371a3bbf247612a54384
b6af800b2e177aeffeed2c381cffe3ab94ee1cd4
2100 F20101209_AAAQRF havemann_s_Page_108.txt
a4b69e0f81dab5f879b5bf70c017a3b0
d57ca2e4196612da58ae6bf0856153311ba5ff40
843 F20101209_AAAQQQ havemann_s_Page_092.txt
d7657940e2c051a06138b5b36db270f8
2471e379db9748c4c40b5375439a450547d6b2c7
8878 F20101209_AAAPOE havemann_s_Page_089.pro
d92ee4dbe02130b5fa229ea8ee60befe
aab85ab2f26a89b9c6c6f11c9a8af6198a964f21
1705 F20101209_AAAQRG havemann_s_Page_109.txt
fadec96d56fddebde352243fa0b323a8
67d932bbbcb0686b0d240743fcf9b7a122c01125
F20101209_AAAQQR havemann_s_Page_093.txt
4a1c89928e6134a0b554a4231d2ef53d
2540e1bda916f117e4370d113857a70ec79c551f
7557 F20101209_AAAPOF havemann_s_Page_061.QC.jpg
05e1e2976af74538e333150d52dcf20d
edf1f5aeaacb8c260f49a84697be7e403a2e60df
1955 F20101209_AAAQRH havemann_s_Page_110.txt
0c76eda28a89df993aaf9a807dab9c11
26d8b70c3be264685a5f5260222c054ef5edae88
676 F20101209_AAAQQS havemann_s_Page_094.txt
a7b510c38615946bfd75c4de3d9d97d5
e6d3f3c9afaaef9df259f3354cdcc4174dc4a70a
3658 F20101209_AAAPOG havemann_s_Page_063thm.jpg
9f61df0baf5617471483982292f7f81e
179794812037f949e5c2b24f47637661d1f3ebc4
2074 F20101209_AAAQRI havemann_s_Page_111.txt
1c253303850d695705e0a3c9832e2e34
14a0ecc5798342eaf491d6acd3d365eb595110ca
2143 F20101209_AAAPNR havemann_s_Page_025.txt
bb458873953a603c25dc9e4f1716c13a
3479ab0d79d80626cf0ebee30fb0389e430bb631
1623 F20101209_AAAQQT havemann_s_Page_095.txt
bcb3eec9123ac9bf9349788280afef50
68e7bd715570b062dea9ab1d2c42a072cf630fbd
110126 F20101209_AAAPOH havemann_s_Page_144.jp2
972b978654c5ee32a37d131ba8cc5c6a
dbc6ef6c9ca908a677a83ba7aa3f8e39715552e4
2003 F20101209_AAAQRJ havemann_s_Page_112.txt
aa86d11e7afa72ea0277aeebf2bbef43
df012031d86c5073c2121e5b416c317f0f795141
25391 F20101209_AAAPNS havemann_s_Page_023.QC.jpg
fdf181ecf983082b140978fbcace10e0
39dc38114bf260cb2e2182e11409377293e57aff
9439 F20101209_AAAQQU havemann_s_Page_096.txt
1c079deda7558d2349e3857d7ac58f8c
307b369ad3a11833729334a6cb94f5591b948c1f
1672 F20101209_AAAPOI havemann_s_Page_069.txt
d6fc95cd11a0e644701048f0d7be2377
b1faa267c616a23b8f6ee98a658b8ec7b900467a
1967 F20101209_AAAQRK havemann_s_Page_113.txt
e8d798404bb1c10b8ea885179638c723
c5356c013315d060ccdf47783e5441acbbc7245b
2119 F20101209_AAAPNT havemann_s_Page_060.txt
e6b29bd1a0972684db9480d7771b8a7f
f451ed8474bfe6bfcbfeeae576e49f49091f55dc
2042 F20101209_AAAQQV havemann_s_Page_097.txt
3ed2fbe9e336e5e4845d6f1538206e83
2f6731b33da3b833bf84a97e8f9f2a3fa361bc72
74318 F20101209_AAAPOJ havemann_s_Page_085.jpg
89ea9dc3ac5288ddacb331396fd2a152
ccf90cd3d337235819871ffeea6aa98110b13a63
2098 F20101209_AAAQRL havemann_s_Page_114.txt
e04cf16939db92d15ffaf104e1613b7f
5406d20bafe3af6930d8f48f5f9f95d5c96fe2c5
116475 F20101209_AAAPNU havemann_s_Page_033.jp2
05c1b4ac8047d5c0a235bd5c8f658594
399a15130c7f712b89fc96f9481120e964cdfeb3
F20101209_AAAQQW havemann_s_Page_098.txt
044f6692ab9e66c067c8d588829787de
5905a6b91e6994bceb3fac77378547be6d8118cd
1639 F20101209_AAAQSA havemann_s_Page_129.txt
703ded3b328a3ba11c925206afc0f499
ce3b376a2e841c785b477c37ce0b0ac112e5591a
2054 F20101209_AAAPOK havemann_s_Page_058.txt
3bdf3a20997ea7e0db3d96e5f8875d33
8a1e2bbcb76ec83333e860fb9454858c4581d1de
2109 F20101209_AAAQRM havemann_s_Page_115.txt
b1535aa4e6e5406ebea4c4d33e506caa
1639a0617c42f70cf36b86bbe720d986b13b2933
72943 F20101209_AAAPNV havemann_s_Page_083.jpg
de9a69501fa32d95c117e6e599bd71f0
8cb66291412a2cfb86d298bf571c706710cb4f2c
445 F20101209_AAAQQX havemann_s_Page_100.txt
bf8d232159652391b6ed4e088fa28584
55077408af5d3b1f6dda8c6359c0dcfd384ad4a2
1546 F20101209_AAAQSB havemann_s_Page_130.txt
9d072603a6db6775eac676007ef167c4
344622154eceac76d99eb15fc0eb1095f8207742
76439 F20101209_AAAPOL havemann_s_Page_077.jpg
8233f08845e8cfc29ee72d8dfc06a3cf
e82212419bec9efba922900b927cfc3dfa3f3f0e
2144 F20101209_AAAQRN havemann_s_Page_116.txt
5812325ef8151057f9d4155a13ded56c
1217a3277d338c2d06c5e5277385e71c31e40f7e
4190 F20101209_AAAPNW havemann_s_Page_145.pro
0b8506eaf615e3dde957a1bd08e504eb
0d0a5aa7e3518a218b1ed3c4675c553024a15d6b
1348 F20101209_AAAQQY havemann_s_Page_101.txt
3aa86f22333f92863653b29848abb9b9
0ae7ce00ba446ce8743641a09372572bdc4449cc
1031 F20101209_AAAQSC havemann_s_Page_131.txt
654051504f01ef6b6f740cb1459c2a9f
3c9876985a5378f0dae542c13ddb8412b3e2716b
16562 F20101209_AAAPOM havemann_s_Page_150.QC.jpg
882d3ccef3caee5b4778ee24cda3a144
57d8b8700ca7796542af0b60a3ae5fd1aebdaf2b
2163 F20101209_AAAQRO havemann_s_Page_117.txt
f34c1dd8a65c55a0f9bd1bc21cbd5fee
92039af15053cf29d45bfd411ac54b156cd0e98f
1506 F20101209_AAAPNX havemann_s_Page_099.txt
23977769f560f337b83e447023e7fa61
4f168a47fc3eadfabe24cf343ecba5357cfb687e
4755 F20101209_AAAQQZ havemann_s_Page_102.txt
38fc82783ee2b926567dae3d4232a07b
dc29d2da99b1fe4b5f40c5630275d08522831d3d
1051945 F20101209_AAAPPA havemann_s_Page_067.jp2
12175571ad13281f6a8afc0d44ccb756
11ede7e4571aca13af95981626c740b232174fa4
2126 F20101209_AAAQSD havemann_s_Page_132.txt
3717616f19c6c60b183fe1765e8d3b4f
12f95f3edd479dfe5f202e78a27ff5a59067da43
F20101209_AAAPNY havemann_s_Page_006.tif
ad3dc2b0e32e677d311ccacbfffa3c4d
0dd02ecb43cd3306feb017fcd3d9e940c9914438
47466 F20101209_AAAPPB havemann_s_Page_017.pro
041d10fa7f8bb54e17961159345a9e88
b9d74af7ca727997ff525744943c8d33a126657c
2029 F20101209_AAAQSE havemann_s_Page_133.txt
6fe4834ad5b745e0d5127d3190be9693
7b6f16609df8f1d46f9633fdf3655c4809fb4e2f
5925 F20101209_AAAPON havemann_s_Page_046.QC.jpg
aff62e5e49b2a6490b5828080c60c965
bc8bcb0223c09c5ccd4a5b90a98c337d5ac35a53
1666 F20101209_AAAQRP havemann_s_Page_118.txt
fd77466c7f7121a61ead3e436316a469
01baeceb9e6c4d8c852acaa60ebcd8ec670c895a
23469 F20101209_AAAPNZ havemann_s_Page_157.QC.jpg
b56fa648a82949bb77a89fcdfe20daf3
ecd342f2e5e4114953375d0ba00493e8f1e221f2
24269 F20101209_AAAPPC havemann_s_Page_073.QC.jpg
6a446ff41a8ebe603d48cd691deceaca
2c071a25f53345788ca40c20b865d250e40d94b7
1920 F20101209_AAAQSF havemann_s_Page_134.txt
02599daca6a97d925306009be11c21f6
b5aa50913b502ba2a7c0c017e24101e13de31f87
1584 F20101209_AAAPOO havemann_s_Page_043.txt
8e8656dac3bcc9399c676de368f738db
318c751408e34f0e9a1a91316a9433e7d3f383ee
568 F20101209_AAAQRQ havemann_s_Page_119.txt
9dde115e79f8f94ea957b90a7af7de4d
418af0098283c37acd1b1e6a99ce6322c60ffbce
F20101209_AAAPPD havemann_s_Page_055.tif
a7ff678c603bb20a9aeb98109549b993
e795bd7605244ac4c0c612a437df5bc273a63573
F20101209_AAAQSG havemann_s_Page_135.txt
675c60642b44c737d2c989c4d9076bcb
1105741687f44256dcb3f94ab73cd7b53fa23e2e
67332 F20101209_AAAPOP havemann_s_Page_109.jpg
8071ddfbcb0ee6f5c3a2191fa1dc6056
aec87cde57b55e65299cb616735aa407147f738b
10632 F20101209_AAAQRR havemann_s_Page_120.txt
261bd80b78695e1a0df61686fc7cab2a
9ad724d37e9c8294023de9f908c6ee59bcf09bde
91214 F20101209_AAAPPE havemann_s_Page_007.pro
387f1d010e2a413084aceedad999302c
cb6142f1e088be6c9f55f2336c37492867557395
2116 F20101209_AAAQSH havemann_s_Page_136.txt
274617ccaf8a7ba76d973777fb944f2b
854597f14cb9737c660593bc4e92d533c65c0ba7
2373 F20101209_AAAPOQ havemann_s_Page_008.txt
661272997f55d503aae93040912b7512
5d7315f3bc3df8c5f53dd3f4ea95d10f979e83e2
3316 F20101209_AAAQRS havemann_s_Page_121.txt
d6e8584ae498cd0d0d3d5d23f084c215
41e1f744bed442706203f26111a8df7d3ac0bd4e
28950 F20101209_AAAPPF havemann_s_Page_150.pro
f746c5b72f879dcf24ce0f874ae392cf
f074bbe731a81901737597c5e8af7b8bf7f0e0c9
2226 F20101209_AAAQSI havemann_s_Page_137.txt
c65244dd99f5e0498abe0ed86f24586a
2feab569d9c2a516138bd076919edcfca4a37347
2051 F20101209_AAAPOR havemann_s_Page_085.txt
97f89dd6002bdd12b348bd9c5cb7ff13
784a34e93d9a42b6f782bd6a40140656b1fe11b2
1056 F20101209_AAAQRT havemann_s_Page_122.txt
19419082728203cebf35c94658a5288b
f0b3f32792504204fc52b162c5dd9c8ba6e705f4
72043 F20101209_AAAPPG havemann_s_Page_110.jpg
72ff16dd62a578144148578df8e8fdad
adde6e03986a3ea58afd0017b1b9d4544a554a0f
2218 F20101209_AAAQSJ havemann_s_Page_138.txt
df9bd2d1ac93e00b1f8c5160cc538845
10a96c50b2fff985220e846533c3683615f38fbb
3363 F20101209_AAAQRU havemann_s_Page_123.txt
9b6418f2253642147ff6e7b01e399a34
dff8a149b5c3fda0c810534b5cf8f194adc68428
7039 F20101209_AAAPPH havemann_s_Page_019thm.jpg
4739bf7b7cb78226b9808a385f28c46a
0ceb12a3a0385271e7cdd0bc0a8bd4240a195600
48333 F20101209_AAAPOS havemann_s_Page_009.pro
58e37e11c469183223b344baf617c456
e853c1cfe05999d9a4e10fdbd3ddbb69c9447671
F20101209_AAAQSK havemann_s_Page_139.txt
d37a5cc7be8cdf622fc8d51809cf2c25
93d67a5c170e1e2a82d08d538bd66e75b2d8eed8
2819 F20101209_AAAQRV havemann_s_Page_124.txt
6ea4191a12768c27b815e73578bd0c67
d21530a0fe203ff24fd54911014773408b525384
1051970 F20101209_AAAPPI havemann_s_Page_008.jp2
13b6473e20748a20c86ef3c2a31ab0d2
a4ea0f2b7ed1bec5a9b8d098f9c859b2aed028c6
55736 F20101209_AAAPOT havemann_s_Page_018.pro
f5f3a1318f7d092457b0d0c95736c4b9
cb9e98fe096279ed94029055ee21367558bdfef7
F20101209_AAAQSL havemann_s_Page_140.txt
ea87b9557b12724ff7c50ef741d521f6
b646232f4819e71836769739e35b97bf56f5fd0f
532 F20101209_AAAQRW havemann_s_Page_125.txt
60e176dd376a267e0f90448cf80ee1c4
3ce363b64a73d175749e84576ce10a784c9d34e8
34772 F20101209_AAAPPJ havemann_s_Page_127.pro
40b48a41bf786a352ecd4b194bf5e387
085309b5ce7c6c7c800abae92d6f16d12c70c202
12283 F20101209_AAAPOU havemann_s_Page_092.QC.jpg
938831ff0284fea8d7ccb708e5f62a45
088b860014f884443fe7855fb9e4bd94a49a3865
2089 F20101209_AAAQTA havemann_s_Page_156.txt
c087268ea92b208d53ab74fee2a33649
130f1ed73c9752c8107e348283a2409c3864c929
1905 F20101209_AAAQSM havemann_s_Page_141.txt
b7c0070bc305c384b730077aef7189a5
5881cad8e484b75ffe2f1f91aeb9e6fa67e7fc07
1495 F20101209_AAAQRX havemann_s_Page_126.txt
0a5d60a38208738c66eadc9539140c98
5b052d4737049106fca800153d9a6d619ddaa45a
51688 F20101209_AAAPPK havemann_s_Page_086.pro
40b0fb5c989df3dcfc406a9b2818831b
8c08bd5db42b417832c7cf7d652d660e8e9a0dc1
112946 F20101209_AAAPOV havemann_s_Page_133.jp2
6c2cfe61b1b104bbd029902468156c6b
360c59f975abe4b1adbce81785fe40f510422126
F20101209_AAAQTB havemann_s_Page_157.txt
d0246e5aef2e20cddd6b4c00dc9ac8bb
5fbf3a48a4e961ed5ab93b18d26d1d0340abec24
2031 F20101209_AAAQSN havemann_s_Page_142.txt
f7296c96e499ec65b75825e6e807ec33
ef2fd40b1069f006870bc24f69b16b400a9a24d9
1520 F20101209_AAAQRY havemann_s_Page_127.txt
4c3ed7680429f5a89088269e8ea4f25e
e69c6d52db7a8223e47c775500045c24ca889689
42640 F20101209_AAAPPL havemann_s_Page_088.jp2
b22bdcdf97f8164556546260280637e2
93baad05d62eae17f1ac80c73755513a9c59afbd
26135 F20101209_AAAPOW havemann_s_Page_135.QC.jpg
9c7435e84f338beac43738e2ce53f718
aaadf8173dd43f4ef7d2d71d5ff586a1b0457dbf
2080 F20101209_AAAQTC havemann_s_Page_158.txt
1e367045dd4096cb23a9872a415faa10
e1d07253a832b7c02d6171fcf194c567bafa48e2
1932 F20101209_AAAQSO havemann_s_Page_143.txt
e76aa5789ce21c9a5476c6bfef7e39f8
58ad9ceed4fd4ba28e57f6a59ba57b6efdd5b257
1388 F20101209_AAAQRZ havemann_s_Page_128.txt
41232fee6a0eedb77a290d5e6feb8693
95528bed14350efa11b1565ade48d8e1c3d9621f
36970 F20101209_AAAPQA havemann_s_Page_041.jpg
dd133b2f56115ff568c1a89553622dc4
2d27847fd041a465ce351fdb69f680732c400e90
352 F20101209_AAAPPM havemann_s_Page_062.txt
6b09a9ee0985e57432122fa0d1a4ee15
3f81c9841b97c8960eea85ee0415b65936da5d01
54147 F20101209_AAAPOX havemann_s_Page_023.pro
a57a79bb9ce650ee716b699d7ac5bf6b
6496d5769f76a32229d605d91a08268842b25b70
177 F20101209_AAAQTD havemann_s_Page_159.txt
3fdc68333e4b12fd1420e8ff891312b1
a65b9fa9d298c02b3af585a9efacaa35bbd6daea
170 F20101209_AAAQSP havemann_s_Page_145.txt
226de69be172f279741f5f99846f5c8d
299c7d35206fa644efb647762fdb469d4ce69d73
51960 F20101209_AAAPQB havemann_s_Page_158.pro
f9866cbdfab03e62026d3d71a0076aed
f08d27a91fc2cb6d61715841de82e143dc921689
F20101209_AAAPPN havemann_s_Page_127.tif
46a533c26c6b1572b085101fe5bdd05c
041e4a6a19e5c4ff8d43aef5c1f85b883e8e0bcc
F20101209_AAAPOY havemann_s_Page_072.tif
9408dda5fa1f156e4ed27017555ee4d4
1ead3a895af07318d458a243a1a851a3b31181e0
2330042 F20101209_AAAQTE havemann_s.pdf
e01994b0be1b52a3a1ee03603beb9952
cb2c2768012a576535820daa417f32f703c2f07b
6943 F20101209_AAAPQC havemann_s_Page_036thm.jpg
89cf8cba6069f2b5763c6a6fe1c00ad6
5f592ad59d535c5fac839368085eb884cbe10c68
F20101209_AAAPOZ havemann_s_Page_010.tif
3e11e99bde3bf7260814536ba4abbd47
b7d3e9db234b9c40e04dfbee4ef5237e8262b8f6
6978 F20101209_AAAQTF havemann_s_Page_029thm.jpg
58052cd04b09e28cb94c47f7c377a161
1d1ef053b67e20c6aaa5cb576252683725a7430b
1001 F20101209_AAAQSQ havemann_s_Page_146.txt
145436deebfddc1a5a6c682277fb9990
d46d6a3214ffb5737d09ce08a81fe77a2d2d4100
3103 F20101209_AAAPQD havemann_s_Page_088thm.jpg
737e12635845b8fb922ae334702d90bb
b80b872f1d52dbb04c40cbab905e867c70095b43
25197 F20101209_AAAPPO havemann_s_Page_087.QC.jpg
2b7f064d50a901c297e4aee3762dda1c
d5330614b6c7af21a622701086b8880a69d0be93
6692 F20101209_AAAQTG havemann_s_Page_087thm.jpg
d3d066482b53bb9d23525fb43825a371
a30621b615d45166b317b722a76d6eb4dd1572db
1310 F20101209_AAAQSR havemann_s_Page_147.txt
a15e504876ea0778951f5a94a2be07aa
3c49a2e083b1b913beb5a28658ac83abadd61973
77636 F20101209_AAAPQE havemann_s_Page_060.jpg
df8a9d8519ebaa0ac328d01e8fdd1a95
8fea4ae4660f623d5f781a82f84218d3183d409a
6723 F20101209_AAAPPP havemann_s_Page_132thm.jpg
9d0b006d65874deea75493bc15118904
03a4a6e8f16af3f90a441793cb81d04efce93326
25396 F20101209_AAAQTH havemann_s_Page_054.QC.jpg
0a7f5caece324197d4b41a6a35d800e4
01e6c014f5b9df907639420f8ad0ee5ee0ef3974
1251 F20101209_AAAQSS havemann_s_Page_148.txt
8934a9ba6461b1a11b101958f15cf211
116d31913dd966af4796ecd3316c574ac9756cca
120192 F20101209_AAAPQF havemann_s_Page_027.jp2
bdc7dad54a412982579207f9596fb3e4
ee0b60926dcf678a4818242c14cc64effbbc5f66
76181 F20101209_AAAPPQ havemann_s_Page_132.jpg
def3b075c6162a368eb01efcf8fc3e01
cf786307ef130b45fcac53f7ff5f024f6b3b6ca7
6745 F20101209_AAAQTI havemann_s_Page_113thm.jpg
095f3b8c8e69ba0e347d0b30baafa727
5d9d26ca6253405294b2717eb2f57017d4072a63
468 F20101209_AAAQST havemann_s_Page_149.txt
f52ceb4cfcf9e65df89818cdd943b93f
2c8d001eed46b8b326209e2a0b895fc0e999acc3
114971 F20101209_AAAPQG havemann_s_Page_052.jp2
5533809786abcd3fcd038c82650492ee
0af85dfc8ae27ab64e696068fe920ec64f0fc2e8
102379 F20101209_AAAPPR havemann_s_Page_055.jp2
12f25e5efa1a9fe8bcc0529f12597ff2
afd7f1f0fecb6ab868f15656428625743a5f4274
6933 F20101209_AAAQTJ havemann_s_Page_076thm.jpg
207e32928aad4b8b9932e7aac29a6ec6
8bdad44d667710f13476092800fc36817a3686ca
1365 F20101209_AAAQSU havemann_s_Page_150.txt
b6113f322901f41ac6d3be6d52bd7de3
352d74199af38d3c3728290843e6df6f37206adb
4779 F20101209_AAAPQH havemann_s_Page_126thm.jpg
aab1855e8f2790410aae2481a81c74aa
f1a5109772adf48bc39558ee9d0e8fe80ad9f257
55839 F20101209_AAAPPS havemann_s_Page_135.pro
e4f3ed56a4db33ee8de69b7d7ecb585d
9f09983e1be4d2662bb265a9e67e9de3c1b7d51d
2893 F20101209_AAAQTK havemann_s_Page_062thm.jpg
525d32b4bd29895bebefa17398942fcf
2c36ab04429f3b379678d1ad6ba61b95e159e9a6
2141 F20101209_AAAQSV havemann_s_Page_151.txt
1c2966f5289d581294908139d4fea2ca
b172ed90a3a0146e840517e71d0ab48d92500da5
41775 F20101209_AAAPQI havemann_s_Page_118.pro
74b3b4f3624da2790ef783963e1b018c
04c3d0b8ba4f28b541c4f4c7279071fc167ae96b
F20101209_AAAPPT havemann_s_Page_040.tif
4b9aa2634517d0897ce803fa1cb16e7b
933aeccd1f5935410ac941d8b6015df3429853af
21531 F20101209_AAAQTL havemann_s_Page_006.QC.jpg
854ffd3b9fe1e51130205ef845a30446
798a7bb6bda9ce224b3fc7b7414fefd9726a869a
2112 F20101209_AAAQSW havemann_s_Page_152.txt
215b2dd8033129a386ccc76edadf1e7a
d465157eaffa8854cdf21a7e21b610ce243d8680
25364 F20101209_AAAPQJ havemann_s_Page_004.QC.jpg
85ae455f043a5569077bd64d983e8ac9
1d28fb0dda0fbc1a0654357f2c81c9b43d08ea6e
8039 F20101209_AAAPPU havemann_s_Page_100.pro
41cb5bb36d46dafb0e5dc379e98d7d67
c2795f266ab367883a5c0c0b911696421e93e35c
10294 F20101209_AAAQUA havemann_s_Page_088.QC.jpg
8042f5fb4f201028de5a61d2db056c23
c35171ca56e2309f07b189b1c818fd37ec38f459
4403 F20101209_AAAQTM havemann_s_Page_047thm.jpg
baff81254202a6d353288a34c51f3730
5c96af50a7ff445e101bd8e42f9ebb30fefd2066
2306 F20101209_AAAQSX havemann_s_Page_153.txt
9cd647193a8f0b92dd0315c594b71825
cfabc0efb584df6e2b20ace6b310b9c3e7c4e203
66945 F20101209_AAAPQK havemann_s_Page_128.jpg
d0aa2e80cd5b9e700a5ad29458b039f0
3a45c32cd58c45d4df6941731e22a07cb61d3850
2085 F20101209_AAAPPV havemann_s_Page_087.txt
67c0e92a82f93cbe6385f70c84560064
a3388d1fe897ba72f8d28e80deaac3038c84225e
24870 F20101209_AAAQUB havemann_s_Page_081.QC.jpg
7531bebc1038c98228ba5890b304a31d
3609d6bc4c7c72458e0400a30bb0611b3f4b3ace
25203 F20101209_AAAQTN havemann_s_Page_032.QC.jpg
796c5b23cb99135581ca6f43409d7c0e
36e54cea518567e4f7ec90debb7b667fb478b95d
F20101209_AAAQSY havemann_s_Page_154.txt
45c32203b1e9978cad98feac2efbf335
022cb441f78c834227417192328b3721b2b2098a
74379 F20101209_AAAPQL havemann_s_Page_050.jpg
fb72dbf26107ff16b13d46f944fab14d
24a1c597e5e71da9247e88795076f5f04d201333
19308 F20101209_AAAPPW havemann_s_Page_005.jpg
83b0c03ce41716244a3ff86162634d8a
30b9e18a78bafabe950f95e549212cd70aa0d57e
5026 F20101209_AAAQUC havemann_s_Page_048thm.jpg
2487964a27f99b9a3f3995c821ff57da
653ac33388b78c8b29b21548b6daa6d1496e8bbf
13133 F20101209_AAAQTO havemann_s_Page_102.QC.jpg
f843ab13772ed98ef2edf6ff925a6981
8773cfa378e15c91d05355da98e9c262c27380f4
2183 F20101209_AAAQSZ havemann_s_Page_155.txt
69c4f07b616fcc004e78e774b6d64fe7
6f407da4788589e24961d43e45bbe9a8a56a40cb
47395 F20101209_AAAPQM havemann_s_Page_013.jp2
40bc385f3315cd093a9cb5c68784ac83
4b89ab131a4047ac719a93e2447273511bb49fc6
947 F20101209_AAAPPX havemann_s_Page_038.txt
52bfae0897f4c4c55f5935d295eac183
d4b5e85fb6af50e077634bc11162a67eed6ba628
101641 F20101209_AAAPRA havemann_s_Page_064.jp2
5aca0b576a6a96a08f00915e8cda59c4
009553f9c69e93bf449b3b035077c7b0a9e031f5
6864 F20101209_AAAQUD havemann_s_Page_073thm.jpg
dc28538daebbaec3ce898c41181b47f4
c2811a223a141acd7f4f826aa5729542be9e7b9b
17976 F20101209_AAAQTP havemann_s_Page_048.QC.jpg
04ffeb32fdc40866046536d873f450dc
a6ca4f835e84242f182533f70f2fd4d3f25743c1
F20101209_AAAPQN havemann_s_Page_105.tif
a0ac65d162b9a35432f2418f360bd434
eed767f89331adec4216844917bd700fcf6157de
3827 F20101209_AAAPPY havemann_s_Page_102thm.jpg
b637f99ff070f1997a90c06c10845d1f
8c0f34404ae1880285c09e2971a2997bb9601c19
5798 F20101209_AAAPRB havemann_s_Page_096thm.jpg
bdd7c283a9bb3f608b6f2338eb49f9d8
0c3114987ab52a40174672e7dd0e7185a5a89469
26170 F20101209_AAAQUE havemann_s_Page_068.QC.jpg
fd56c2c6d2cd2c50a25bc13ea001f3d6
514b380c1dd591f9c92452a11c67f7d4af7348ca
6247 F20101209_AAAQTQ havemann_s_Page_010thm.jpg
76c63c0033c5b1f64308d6a5faa6e952
2da1ecfb590a094d10d0bf95fa7b876ecdc81aee
72656 F20101209_AAAPQO havemann_s_Page_103.jpg
816a91e9c27f3763ccff50bba4257009
1b827be2c5dd488438f2458addedfb6844f394d6
52316 F20101209_AAAPPZ havemann_s_Page_114.pro
0cdc4430f7e51951917d496a7b1942ed
9153e0a01c51346a8a8d74cb9ed709d092a494a0
3735 F20101209_AAAPRC havemann_s_Page_013thm.jpg
fd4e8085144951023923cab24c45412e
ce24db39491b5f3156feeea9cb63eb59222015ae
22962 F20101209_AAAQUF havemann_s_Page_017.QC.jpg
9fd0e474a256818c08fd8ee89c656135
49a8819246cf0fa1457e628cc4f4f950dc0485e6
184622 F20101209_AAAPRD UFE0017563_00001.mets FULL
ae32b087e8b592dd9d6cbdf7419c2552
2fe16893b13cca3f8659647db0290563c068b5c7
14886 F20101209_AAARAA havemann_s_Page_047.QC.jpg
9563c738fb0350520038ef6a2eeca213
f49aac9651f85aad354dfb62e2850cfa51059d09
19076 F20101209_AAAQUG havemann_s_Page_065.QC.jpg
b7c61bf41d7168520287a50060c988b9
00c4c86b96c27b5d9159593f75c31adedaabc939
25163 F20101209_AAAQTR havemann_s_Page_057.QC.jpg
343b7d1707db321194659ff6fbf58db7
c752a66b9947382b9a834c65f480453005223f11
39590 F20101209_AAAPQP havemann_s_Page_014.jp2
c88271dbbd0ec653a428a36b26134049
d16522adb56b000da9ea741570390526ea75b43c
4757 F20101209_AAARAB havemann_s_Page_049thm.jpg
20b44acbb582a672770f5eb3aa5899ac
d44844b6f2f381ed4c95dd086cd415ba2d683d30
3858 F20101209_AAAQUH havemann_s_Page_093thm.jpg
acd2e9e71303b4ab42658d2fbed1e880
ba5544ed9c8874f606b4b05d374f209de1740443
25791 F20101209_AAAQTS havemann_s_Page_106.QC.jpg
0ceabe94c779af34188d2d3d62c53451
b47aec5cb055f4da9eeb9f9733cc1f965d610ac7
6677 F20101209_AAAPQQ havemann_s_Page_111thm.jpg
a9782232c0744f97ee346ea36c118ffa
b4dc96348544286169e4cd875c1f11475f9014f2
25124 F20101209_AAARAC havemann_s_Page_051.QC.jpg
ae3ff49e8b825ada7d56a32cf6ce88e4
1d4e79efe5b701b351efdfc3c6892e0048a6a658
10024 F20101209_AAAQUI havemann_s_Page_101.QC.jpg
efc4ae04f56568abbad14a3f1298c321
8212b808e1540a11a3aa428d9633d2f434869bfd
4955 F20101209_AAAQTT havemann_s_Page_145.QC.jpg
5a8a61773d0db7d21942de1a1a663e05
7d97b42b63c0f5b4397bfcd9ccba4820ba355b4a
F20101209_AAAPQR havemann_s_Page_118.tif
9293d5068f508783adb792f47a9a9121
02638c9aac8879490956f67ae9df04a436239752
21199 F20101209_AAAPRG havemann_s_Page_001.jpg
8fb9a774f3ff1b77304f8aed3502f73b
440599b9d3eb7d7374cc9d980f41f48b7d403941
6865 F20101209_AAARAD havemann_s_Page_051thm.jpg
6db636cedb7e4a0c50f759b28c34084a
50daa46da64772466856f98831572a8ca9140dea
2883 F20101209_AAAQUJ havemann_s_Page_129thm.jpg
408a2d2b00d8486691b2436e91586a31
6e7c2cdc0ec855029bcb39c5fd261c98fb1e16b2
25779 F20101209_AAAQTU havemann_s_Page_107.QC.jpg
67a06e81b171babab678cc5fa94c0e31
022cf5c72e4ca92d644edb46e3d4eb360f4506f6
9363 F20101209_AAAPQS havemann_s_Page_042.QC.jpg
3b1c1b4d728b77e3ce0e472c4de21a2d
ea9809fd64b708fde83c6bce095006ca78564e26
10687 F20101209_AAAPRH havemann_s_Page_002.jpg
31b2a33cdb4a6dba4ed0ad5e95495523
5714767fc11c5a9d909a01fe68dbb25fee2185e1
26065 F20101209_AAARAE havemann_s_Page_052.QC.jpg
75819671686a2af242638670817e66c1
e27be9465000678c65c8b41c0c28907240a18c94
25923 F20101209_AAAQUK havemann_s_Page_078.QC.jpg
f0a9f4e5614b3812659e391a87e512d0
5517cefc04b91af6844ffd74929db30cb5b96704
26126 F20101209_AAAQTV havemann_s_Page_139.QC.jpg
64e63485fb15dbe2f6a07a1885c3145b
2e4940d2f4f856652cae86706ceccff918c37f34
47698 F20101209_AAAPQT havemann_s_Page_015.pro
611a126ac8f10b89142d74382f648765
429999c06f4b578564e430c06250b40a94b45615
13413 F20101209_AAAPRI havemann_s_Page_003.jpg
c815b8d9bbf71c8ad779aeb7a4b6f424
472f1d42ab147440857318246882805505ab569d
6263 F20101209_AAARAF havemann_s_Page_053thm.jpg
0c20d871562e7a25f86f8d9479a865fd
1b98bb3112460dbb3da0369cfef6e3389a38e062
6967 F20101209_AAAQUL havemann_s_Page_054thm.jpg
14d3c419dbbc7eb8f5717ae6129acc27
d3c231a06bce58f8d0256a2327959dced5e59fec
22649 F20101209_AAAQTW havemann_s_Page_141.QC.jpg
837197f9428a092774000d47848fa178
136621357657890996e3692ab89820cea591df3e
F20101209_AAAPQU havemann_s_Page_045.tif
5b4c683329ae21e68ce8cc65fc219225
b51b5295afc9897502eece3bcf8761d1a0db17e0
78237 F20101209_AAAPRJ havemann_s_Page_004.jpg
17ac26c7b21f6f9ecf15c70cf5827248
092d36c1d1c67c11c5e7841429228597d9b33219
6609 F20101209_AAARAG havemann_s_Page_055thm.jpg
c54ac585534512cf762b6e66bfdecbf9
db01b8b7c61bdca55b251dddf37758c7cba48a47
6733 F20101209_AAAQVA havemann_s_Page_050thm.jpg
70e0c1fcfcd7c4375b3788abe739e574
45d26c586ca42a77ad0b3873adfbbae904fc2c1c
22201 F20101209_AAAQUM havemann_s_Page_015.QC.jpg
d618560fce69c524d2785b7d92681e7c
2d4e8f543df45d4fd54947b1e750f51495553c85
4793 F20101209_AAAQTX havemann_s_Page_150thm.jpg
937f28201c14591d84712057a7266880
f649b7ca7b128b14c2079becedd97849839e31c9
72871 F20101209_AAAPQV havemann_s_Page_020.jpg
a24e2d27bfdc2d7506943f275fce96af
d42c72ca69e65ed1edd1380c9a755c8014352b89
86175 F20101209_AAAPRK havemann_s_Page_006.jpg
962a4b23afa9a6b27695e2afe64b09ee
99c0a0de20cc070938394ea63744686367c40fd0
23055 F20101209_AAARAH havemann_s_Page_056.QC.jpg
5c4911866220061d9faa9b886d2e273a
defd7620354b3e0d653d311c2ffcaef0fbd6826c
8980 F20101209_AAAQVB havemann_s_Page_129.QC.jpg
d40f0ec34da5c91cce12d2afdb0f5da2
d071bf760466e4fb0d411c5c87c2553ab134752b
6492 F20101209_AAAQUN havemann_s_Page_105thm.jpg
e5525aa94baf9e41ea08926003f9eb5a
56492936a39109ae272900bfb788b07b611f2f57
25965 F20101209_AAAQTY havemann_s_Page_059.QC.jpg
658a6d15949f87b2e0035f60f101fafd
60791db9315ea786fc9da387a9beed1c8efee6b8
32512 F20101209_AAAPQW havemann_s_Page_012.jpg
1396da0c0907e6de4bfeb48d5e434a9e
68beecac6ea8c89f514bfe7a72a88b305a344d58
110320 F20101209_AAAPRL havemann_s_Page_007.jpg
8aff06ca138ab3334bcaa24f784476c0
d4e0bf000dc47f7184574f6b0c9c7aae86e2487f
6683 F20101209_AAARAI havemann_s_Page_056thm.jpg
d6c4db7f84f3df307cfb2ae29773aaa0
8a19a51ade7175a730751dabd7487c907ec83b8b
17699 F20101209_AAAQVC havemann_s_Page_126.QC.jpg
aadb5ce5ef61291ae03e99a57dd63e05
cf66ea5412b9b95e70adc15b3ba7524ecfc6da55
5272 F20101209_AAAQUO havemann_s_Page_128thm.jpg
9fe9881066200332816cdb555b6c3d55
d2e3666df6e97bddb71fc3e348d49acb3e160f6a
26475 F20101209_AAAQTZ havemann_s_Page_028.QC.jpg
774623dd5fff1cbfbe41375bda6651cd
ed25bbd60236f61ff850b252bba15ff2058569e3
2026 F20101209_AAAPQX havemann_s_Page_144.txt
4b4eecc8e763ca38d4c33f3d9a30963c
37817298006d58d51850df3c7149689446bf3363
75280 F20101209_AAAPSA havemann_s_Page_024.jpg
c0683aa67f00368bc796677e99e78c9d
d17c931edb4560f38f829dba8e4ca7cc9b335b14
77375 F20101209_AAAPRM havemann_s_Page_008.jpg
ce95dceba1ff2bed66186dd6c54581bc
0fcea96f311199bd92b77c346bf66ecf13ad268a
6904 F20101209_AAARAJ havemann_s_Page_057thm.jpg
ec0ba895bf4989806ad8223b3f314b0f
09a7251b49debee24aad06540e140bbeca98d846
24410 F20101209_AAAQVD havemann_s_Page_142.QC.jpg
8559751c40db97ffee43f0e2477f39eb
26a30bb2d9e5c7962bd3e67ca90c0f9bf9a69747
17434 F20101209_AAAQUP havemann_s_Page_049.QC.jpg
4a3e804ad69cf34b45755d3993eaacff
3098c78aa09034fcccd252b8125e674526ab892b
72828 F20101209_AAAPQY havemann_s_Page_104.jpg
80ce327a4a81a9b3a3e32747e923a2aa
f9027a1e6288f906f29e7486bba0cd3983878b03
75823 F20101209_AAAPSB havemann_s_Page_026.jpg
605723b4dd5573e631ffc724a2d7caf0
0cbf0614ef1fdba15c51c33361c2b5d3308a4800
66613 F20101209_AAAPRN havemann_s_Page_009.jpg
1a48bc6ca451ebc793af3ccbc13a5ebd
e6bfb9afb341c7de0cebd7adaf43e7ebbc3f6180
25429 F20101209_AAARAK havemann_s_Page_058.QC.jpg
1259b818154dcfae8b6e2d436fe525c9
ab53cdd4ed9af3bcdbdb93c3665e1ec9ed063c47
24657 F20101209_AAAQVE havemann_s_Page_071.QC.jpg
415b4d400f103d7b79e08dbf0283d87a
9771b0f594fc918cc7865fe8736e68cad859edbe
25321 F20101209_AAAQUQ havemann_s_Page_158.QC.jpg
be4f75de043c95709153abe3532b2cde
b46a2bc350bd31d64e91887801f93979c01909ae
11041 F20101209_AAAPQZ havemann_s_Page_119.pro
9b76a81f7558f5f498c9a8094140139b
c2ea2ebcdff61c1329fd1e8b508d5318dd183232
80416 F20101209_AAAPSC havemann_s_Page_027.jpg
01a292915aeb43c4867b64495a7f04bd
48e22f19026c8c5fb933da298876c1e5b8d98b7f
83903 F20101209_AAAPRO havemann_s_Page_010.jpg
15505aab3e93ea8d72a4fd12580dfa31
2e963b9651b8ab114c34daad3653269bc4eb3d63
25722 F20101209_AAARAL havemann_s_Page_060.QC.jpg
ff312eb26882543ba9343a23554e390e
4d8fa4415c1876381cccc749b92c0bec01099c97
25156 F20101209_AAAQVF havemann_s_Page_035.QC.jpg
72f6f398aeb8f7bf0e514438e2f6ac62
6d50686e71b299bafc6fa65394d9b7ac54d8cb03
6815 F20101209_AAAQUR havemann_s_Page_032thm.jpg
a3589354ce45938701939161f345c391
f0e97d14f144a1096be0e4550ab239c8547e9ef6
79172 F20101209_AAAPSD havemann_s_Page_028.jpg
d9a7cb4286b45db29610887efa595343
90572a1a72b1e5158d53ba450a4eb2c88b6c1a32
84272 F20101209_AAAPRP havemann_s_Page_011.jpg
6e3fba20cca9ff7ec0250c0bb3dd1ecc
601df1bc8df93afac0ada5139875423c727fb8b1
6936 F20101209_AAARBA havemann_s_Page_072thm.jpg
f32c5ad5179f3c2b993d8c5fc0e5b25b
1fe09a628db73374fc5e95e890a3454f57e86e01
7167 F20101209_AAARAM havemann_s_Page_060thm.jpg
e7548ca8c294f47baa6e45f94d9cb230
7c5888a65333af58851cecf2c17a274ea257320d
24219 F20101209_AAAQVG havemann_s_Page_156.QC.jpg
126f93ffaf0012997840d1063589456f
7edd597261fed2e04d1149236b26a6df06cc4d00
78663 F20101209_AAAPSE havemann_s_Page_029.jpg
6653623bcc2b95dff7c6bbe37dae0d3f
0a235ce2d38823311124177e3eda69e6385da1a5
24143 F20101209_AAARBB havemann_s_Page_074.QC.jpg
effc6e2e625a193051d065f71638e9a7
6b245fe66ac69919bed259015ab9db4b018d4566
8302 F20101209_AAARAN havemann_s_Page_062.QC.jpg
848b3ccaeb60913b12a4eb3d030eb703
ee1728fd7474f4cd2fb021c843ac37ffffa88ca2
6429 F20101209_AAAQVH havemann_s_Page_157thm.jpg
bd744166aea603a86d09ea3ab0a8fa2d
fd8ac66314a3240015d96422c1a7b33d357a0256
6926 F20101209_AAAQUS havemann_s_Page_004thm.jpg
08598d506e58868df2b3cb1a6b57617c
d95027a53ede6834b0fbbe29d6daf28abbab31f9
76807 F20101209_AAAPSF havemann_s_Page_030.jpg
71bd8fbf4cf50809d5162d7f3ee1bb4c
55910d51d076e3f3257d1033c13fe7376d72968b
33231 F20101209_AAAPRQ havemann_s_Page_013.jpg
bf9c3d01baf9acc190b4a360eb2630b5
2c5c5291bbd966db770502a057942c3aae7b54e0
26271 F20101209_AAARBC havemann_s_Page_075.QC.jpg
029c74529dc0ec367f69d0c7e0e11ef0
7843f8cddf2ff22a869dfe7c358df9ebca8fd9bf
11424 F20101209_AAARAO havemann_s_Page_063.QC.jpg
0c516a6dd8801a790c88c998a54300a0
c6fa3d905a42f46a9345f966996c64c479423995
7014 F20101209_AAAQVI havemann_s_Page_114thm.jpg
a2e118b3fda01ee396a35c21af01a30e
3bb739a3067f64d2aac265ad184b903451826930
F20101209_AAAQUT havemann_s_Page_131thm.jpg
be89d62d671f6440edcd6b42e744152f
9aad4ce0282a3dba8d720b37dad77fbcaaa35bdc
78013 F20101209_AAAPSG havemann_s_Page_031.jpg
c8c5a473206820c426e5bd18ddf1abf5
6b326945e75dacbc88c8af9135271aeae47484aa
28463 F20101209_AAAPRR havemann_s_Page_014.jpg
690d8750e96e6a1c47fa33f9b7349a7e
565aa9a61740d0a6fe60a0c199ec35a61c91443d
6902 F20101209_AAARBD havemann_s_Page_075thm.jpg
064b84d7b33ea82c8032486f7db02891
ff88ad2b58928f77c70deb7342bcdc8897a9b02b
16757 F20101209_AAARAP havemann_s_Page_064.QC.jpg
53555e88e49969e7ab27c74b68dbbe9c
8acba896d7e012ba8b964bfea0b2d415cc90193c
3952 F20101209_AAAQVJ havemann_s_Page_016thm.jpg
00c0c72a2b289bfdf4f22e1a97aa66c0
650f1fe39450a4883430f435bd0a2da0bacfeca8
5169 F20101209_AAAQUU havemann_s_Page_044thm.jpg
894fd0c1ddb847cfb88760b047f96ccd
2869c25938e8af564285222db5ab1c30c15e117c
76350 F20101209_AAAPSH havemann_s_Page_032.jpg
cd9af9faf5dfb03da16452fcae486a5d
90413cbe3b8827f11383f36f404e70a7a252c5fd
70865 F20101209_AAAPRS havemann_s_Page_015.jpg
7d0819e2f449d8910cd85190dcd2aeaa
dd50358255e16ec9452df1223bf586a28c0ad222
25598 F20101209_AAARBE havemann_s_Page_077.QC.jpg
82c4aabd864f481f6a92db6d58d5c509
982580b7428f6e21977678e414cf1c5c0064a651
4054 F20101209_AAARAQ havemann_s_Page_064thm.jpg
eb07ff437be1019cf62f3d7c39627e2e
70fd42583a9276b261e1d405efee1b13036d6582
23978 F20101209_AAAQVK havemann_s_Page_022.QC.jpg
7a10d547d30468d02749d24006feabeb
b5cd9999f1ea9bb634b64269c1e6c5317f42abe3
9522 F20101209_AAAQUV havemann_s_Page_014.QC.jpg
38dde07fe44f8eb0df997e04404b840d
a099a242010335b5adc49927d96d28a13df1120d
75953 F20101209_AAAPSI havemann_s_Page_033.jpg
f609139b366a61d46d2de850e50ac36b
fc99aaa9a09c1cd89ac51cf290c057de90d2763e
42119 F20101209_AAAPRT havemann_s_Page_016.jpg
68e9ba6d1698a4e8486033f47983f3b9
2b6533ebfb6e93ee241c91e88224bb5aa9c484de
7243 F20101209_AAARBF havemann_s_Page_077thm.jpg
006ca7bf2d2a5cb7bfacbae4ae3c91c5
eaab722755940dd6d71e58566a9d65281283dab8
5191 F20101209_AAARAR havemann_s_Page_065thm.jpg
275e2dc40230e75c8337eedaf9ee26d5
df021a0535a3dcdbd94594d18cd1aa2aeff9ffa3
5538 F20101209_AAAQVL havemann_s_Page_006thm.jpg
9ad60407a7b56992c5a86c09b8fd04a6
f409e285c101f96fbe0d37089cefb73e22278259
1732 F20101209_AAAQUW havemann_s_Page_159thm.jpg
4ca3dfba3253c5d60ce2d302617a7d44
0eaf49d200255be7d3f06150bb193057c314bbef
77014 F20101209_AAAPSJ havemann_s_Page_034.jpg
c44e221e56a9a49e59f9831e2e38c22e
cd2b15214dd4fd72f0a36c6f44cf7e7fe136486f
70594 F20101209_AAAPRU havemann_s_Page_017.jpg
d4812cb422eb9ff4d1deb3a9093f8d9b
2ebd7e271b46cdbffe9181280a3eba4ac16ad93a
7089 F20101209_AAARBG havemann_s_Page_078thm.jpg
e9e9ccddff55151130818aa501d139a8
a6d62c91bafe2e0bde47427980a71c7fd3bc4ec8
9314 F20101209_AAARAS havemann_s_Page_066.QC.jpg
b535758698fe8abaa718f0496b94bcfd
7619cf89a947fa38c3aaa0436d67e43c8b44a879
21909 F20101209_AAAQWA havemann_s_Page_097.QC.jpg
e9facb07f8668045a06e733405451a59
b6f1832e38cdf114db6ac37f0c88ea7ccfac44b1
25748 F20101209_AAAQVM havemann_s_Page_025.QC.jpg
8780f9039bd5f14d66de1c9509f5afd6
3c64cd6e63f202b2ed0322192c4b511cfed3d902
24899 F20101209_AAAQUX havemann_s_Page_033.QC.jpg
38ed4e86a404d8f99776eb19feceac67
aa6607caa48b0d72a2908b950ccc430bbcb0f4f6
75213 F20101209_AAAPSK havemann_s_Page_035.jpg
84460222fcfc7140c6791b882f2d44f3
4095af20875e52d62716ac0514333fc7a385a36c
79815 F20101209_AAAPRV havemann_s_Page_018.jpg
d2d679568f895e457c8a9cb59929ec99
8c9e719b76eda39e81ae3c96403a195de7bc2118
7134 F20101209_AAARBH havemann_s_Page_079thm.jpg
84f5ea234d61020168dd64b080d39a7d
e4b2a53f08582179e0efa28ec7b1cc29f306b556
3064 F20101209_AAARAT havemann_s_Page_066thm.jpg
7c36f723b05c56f40d8777d981d900ee
0421438a2fbfafa43994ca9925f32a0655eba073
8218 F20101209_AAAQWB havemann_s_Page_091.QC.jpg
d335c7f57a78ab4c6a7185101af4275d
c79e6d2ee835351fc051a566acfeba3b72bc2830
20875 F20101209_AAAQVN havemann_s_Page_053.QC.jpg
3548cce6dbd3b23f0cb2ab77324677e1
9a46a770410fa5e84ff64337162a0628bde9cc46
2437 F20101209_AAAQUY havemann_s_Page_061thm.jpg
8d190393723cde5fd060c5970ea49e00
06cbb56142ecbe185fd3a043eabc0a4092944233
75172 F20101209_AAAPSL havemann_s_Page_036.jpg
b94c1a3f0e3a3e9fa06cdfe8ea16e51c
0278fc2e779a5181b909d7733364f1ae61b2d15a
79084 F20101209_AAAPRW havemann_s_Page_019.jpg
b229fc12120ade54f6f3cacfd7d90113
244eaf1cb4fccc678b902955f0333308643552ad
26226 F20101209_AAARBI havemann_s_Page_080.QC.jpg
dafadf7fc241e408016540992b285d57
120a599a2c27f682b4e3a3eba96f2b6c2654d54e
6751 F20101209_AAARAU havemann_s_Page_068thm.jpg
ad95d9f433e8e546d230df135ceac3b9
59c13394e85e0bc31951fb874944accd48c66f7e
24912 F20101209_AAAQWC havemann_s_Page_034.QC.jpg
96490b302d30524277c165072fffec25
b2b4b4c35aa061bba12dac894a97bc021020cf27
7269 F20101209_AAAQVO havemann_s_Page_137thm.jpg
bfb6be62bb4534d0ef38eb909b6d673a
7936d7f976e6f50183803e5f11e9328bcbc68fff
7045 F20101209_AAAQUZ havemann_s_Page_153thm.jpg
fd4881da0ccb5762f58ac1fa90f5fd67
0f255f2b6da5f9cb8ff806fe1d65d8e3d56ddcc5
63703 F20101209_AAAPTA havemann_s_Page_053.jpg
295a0b9a2052c7006e71bdf326fdc9fc
69212657bc62bb14765ede9c070b44506be67803
42384 F20101209_AAAPSM havemann_s_Page_037.jpg
42cbc85a6a04ca4743589a0de256190f
f62d152ff05b199cee06639c5aca068a0ba05dc2
75450 F20101209_AAAPRX havemann_s_Page_021.jpg
c90f5df601ee80c4a185b5dbf315a8d0
f1a71691ffb12265e0f2322cd99c7d59f73f5d6b
6953 F20101209_AAARBJ havemann_s_Page_080thm.jpg
ed5dbe8920aae9abd76d5cb795159807
669e34590bfa6f8ab33398a5c2977d12e4e2c860
5043 F20101209_AAARAV havemann_s_Page_069thm.jpg
e0994e3c70f2d6557aaada22099cabd9
beb8fc0cbb6e42373d420084e847e2210713a662
24701 F20101209_AAAQWD havemann_s_Page_154.QC.jpg
dfc1d3dbec37f52aeb0c445e5cb63111
a6b77524150432bca02579c429841b0707e7bf21
6972 F20101209_AAAQVP havemann_s_Page_023thm.jpg
4c90644d9f5749552128e333e98f4312
59eef71206720573c695423a7a55f28d15f97f39
76861 F20101209_AAAPTB havemann_s_Page_054.jpg
4c7ad3fafc5a94ad451e3845377d8bad
1351d0e3ae723eae6f75d91cb179d0ea6664068e
37548 F20101209_AAAPSN havemann_s_Page_038.jpg
421b9decb443e2249646ae73605131bd
24eafe58ff6ad30dad0852fd77e116ba1d077129
72372 F20101209_AAAPRY havemann_s_Page_022.jpg
4adef58637010affa63beb026e63119c
422dcf310b71ece5a79dc01f540ef013f8210f08
6888 F20101209_AAARBK havemann_s_Page_081thm.jpg
3df3c671be72ac94e2ed1e817b8ccd5a
1269b39ab0d4529cfd1004988e866671460b6a15
24302 F20101209_AAARAW havemann_s_Page_070.QC.jpg
4489453979934651959fe0a6aa2f66a8
8ee4ff22c8330c95d09d274c1201efd72fce1a35
6403 F20101209_AAAQWE havemann_s_Page_017thm.jpg
079f1f5bf762746a85083e5cb166106d
b27844570e8adf238e6727e376ed439ab8513a58
7185 F20101209_AAAQVQ havemann_s_Page_059thm.jpg
1dc11566bfe9529c20ce2720145a6c6c
1dd611d7754f94012ec9f3153b8e33f3e3dd914f
70976 F20101209_AAAPTC havemann_s_Page_055.jpg
74c0b52405fabc475c6d733c3c617742
04090836f01298b840beb14af9be9d8ba268ffa4
54818 F20101209_AAAPSO havemann_s_Page_039.jpg
1e178b25aa2c5117454d5255d7645478
e023cf2423e475b6783be5c427b19cc018a1b2e9
78590 F20101209_AAAPRZ havemann_s_Page_023.jpg
b45a707de1fda7457fb3265d351b29f5
48b003b856f02d58a32466cf26afa8ee10b5ab77
6371 F20101209_AAARBL havemann_s_Page_082thm.jpg
b6a390c8ab4253c73bce127f1fb88bdc
13209ed0e67922e4534e78680460967fd551b3be
6995 F20101209_AAARAX havemann_s_Page_070thm.jpg
c5b3106bcfbc94b25ea1cbfe8779c14a
21a74836249990008371b97233d097abbadcc4f8
22568 F20101209_AAAQWF havemann_s_Page_134.QC.jpg
010c0f70484fdf4f069d0b8bff23bb92
f36c36c4e818f845ce4dfc1a8c23fe43d2244fbc
6506 F20101209_AAAQVR havemann_s_Page_005.QC.jpg
82e41454ceaf182518e67217adc2525e
76a568ae9c61ac6581ac059264b04055b6a9e041
68143 F20101209_AAAPTD havemann_s_Page_056.jpg
a0896fa8bdd4c2cff50bfca0b2ad9b67
14df2037a1d6aa8a6f7b28e29f0b4b9204f759bb
58191 F20101209_AAAPSP havemann_s_Page_040.jpg
6dca2c417011bddca8a2134998eaf71f
f53c5265775215ca822ee7eb2a57f827cbb13607
21948 F20101209_AAARCA havemann_s_Page_095.QC.jpg
0d534936933c93ea6d42c2be5c9b1497
8c77dcdd489a299887b2e7ca5fc6c09c48e49953
23946 F20101209_AAARBM havemann_s_Page_083.QC.jpg
02b5cff8183a6b9fc7b4ef3bbb3722e0
7129b6d617246edb34ebb76ba44b42474da02979
6511 F20101209_AAARAY havemann_s_Page_071thm.jpg
ac547452f148ab1668a27eda7f51fd31
498cea0acbde7fd1088624a61ffffe1a203c5982
6963 F20101209_AAAQWG havemann_s_Page_058thm.jpg
0887f3bb29825a57b31ee2f1a6869877
6c3ccbba521bf957c05c1f3388e6a353dd8f0177
6794 F20101209_AAAQVS havemann_s_Page_033thm.jpg
49ace16c87b1001d19173a318861bdd5
f942ba778b525b80058e40091610c7dd9f93697a
75765 F20101209_AAAPTE havemann_s_Page_057.jpg
92d82e1267e37b8f040357b9b0ca7cda
1898325e2b64dc8e3705b8a39fc4ebf8aded9800
30317 F20101209_AAAPSQ havemann_s_Page_042.jpg
50877b8ee4134f1a1ecf8efe56242c9f
945747a5d06726b067927788927cd35a495eb545
26860 F20101209_AAARCB havemann_s_Page_096.QC.jpg
f2fb34d43023780d7e411e382bd959cb
cfac2813f2ad8285ebc8df3883327cf51780fd6e
6739 F20101209_AAARBN havemann_s_Page_083thm.jpg
968bbb462a81f6bbc0f8ebc73a503ad8
d1493830ae8be10a0f5dd23eda29bfe920322b5d
25607 F20101209_AAARAZ havemann_s_Page_072.QC.jpg
dccbed0403ed0e36359c2fea4cb26b73
5af75115e189c63d03b75f35d3307463ac372161
6605 F20101209_AAAQWH havemann_s_Page_103thm.jpg
2a37ee0d35ad796b1ce609507ebb7990
2c7b7085acd0e31244f96da60c9bf1a4feb6106c
74065 F20101209_AAAPTF havemann_s_Page_058.jpg
0a6c84de123f6100ca733bf094dec72f
329f415314eb34831ed9a283a19f608abe05322a
5602 F20101209_AAARCC havemann_s_Page_097thm.jpg
d54484a4d41a8f56aa5e1c9a02e42b39
8cbaea1a399774dc82b9f1f6ce26944050b7024a
25267 F20101209_AAARBO havemann_s_Page_084.QC.jpg
5cc088d0d66dbc5d0ed6c5849ff15d24
d5a89b161b4b4c187d4006c2f526eb9674ddb5f1
6082 F20101209_AAAQWI havemann_s_Page_067thm.jpg
4154d5afbc25d2bcfecec1113395b3fe
7e507667377da9aa254582495341f3b3d218e398
23869 F20101209_AAAQVT havemann_s_Page_010.QC.jpg
0dc88f63dffcf97e09c4f3043883d8e4
27768b84241bd157b615baf6eb65d5e88ee08d8d
80013 F20101209_AAAPTG havemann_s_Page_059.jpg
b64e1495647cac548d2e1c04d8c1cac5
dd007230041c1e39885c3450489575d01c81a3dc
58713 F20101209_AAAPSR havemann_s_Page_043.jpg
fe978ae644d18e48e99dec300583114d
f3004318791f7a44dd629c0956b2faf8c7f37ab4
13442 F20101209_AAARCD havemann_s_Page_098.QC.jpg
0fe77fb4f1b7574100e36b97e978e5c3
0bdbbb1cea4f9d8ad66157badbc8ea93882fa37a
7099 F20101209_AAARBP havemann_s_Page_084thm.jpg
4ac4d1159406a7869dc793fbffbfd361
449b1f5b4fcbd7790df07eeff869610d25b0bba1
6866 F20101209_AAAQWJ havemann_s_Page_074thm.jpg
c39f368e8693175a1b769931c5b3ad36
b690c73a9aead5a065466afa60db88fde7a5072d
16882 F20101209_AAAQVU havemann_s_Page_043.QC.jpg
e73835593033c22b42138b2f3a2a1b64
1da1fa663cda8a2596defcd9d8bf6bf296a59ab4
22759 F20101209_AAAPTH havemann_s_Page_061.jpg
e6ba7072878945d8c5ea3160eff3e33b
e5bd9cae690d697a5f3e8a63afabc773a7c52e1b
71936 F20101209_AAAPSS havemann_s_Page_044.jpg
84fc5a3af1510d3aaa4d40c5fa257dc4
ff508adb289c82c090f0b56cff7b4c3e48975a1c
21423 F20101209_AAARCE havemann_s_Page_099.QC.jpg
727abc9869388da7d7afb4c1be2e82f7
5d6c840d11f92f850d7b40fe9c6621d0d4618344
24018 F20101209_AAARBQ havemann_s_Page_085.QC.jpg
6607e57384c6e39c8128320dc1cccd65
c8f3e0473550331dc57aa1707b206847fac47c8c
5314 F20101209_AAAQWK havemann_s_Page_127thm.jpg
ecca36cd32708d0fb5f52b16151c72b9
10e714f10c58055dd9748180529aba028baa14be
6296 F20101209_AAAQVV havemann_s_Page_015thm.jpg
a2243ebd72ab49c02a7edf3c69c0a37f
374970ac9d20033ee848c0af8318f538d35fcf3c
26293 F20101209_AAAPTI havemann_s_Page_062.jpg
1b0bcad2c64851dc1f0ab643c92277e1
f1c53b5cc0745018632083f467ee72c2390d4f01
77684 F20101209_AAAPST havemann_s_Page_045.jpg
fc8118b01a5fef15c063dae6d4cebe16
a8374ae8724c731b97687ce2ab8eef5b45dfbffd
5760 F20101209_AAARCF havemann_s_Page_099thm.jpg
cc8a9b307d091b535ea24cf3a3004f6e
25e79d2af5a7c5ba60cc50b44830c4c68dfef94f
6758 F20101209_AAARBR havemann_s_Page_085thm.jpg
de4d09d1f29a8f787cb953d6d71db85c
2d37f8139b1c57cebba32a437c51c6ab9263cd00
26240 F20101209_AAAQWL havemann_s_Page_027.QC.jpg
907c159f2182e673e96d63320e2c7256
9b791adc04b28bf42db7965cbbaf82f1aea2459a
4039 F20101209_AAAQVW havemann_s_Page_098thm.jpg
09501bf1d34c57626f2a1e7fe61cefe1
3ffab84396348434733b2a12e6a1d63054061381
34977 F20101209_AAAPTJ havemann_s_Page_063.jpg
42beba0f41dc1a2e57f626cba0abaf35
a6194f4d8e0a613befef930d9c7c3b9c914c66fa
17516 F20101209_AAAPSU havemann_s_Page_046.jpg
f81917f6985600ba00b5e5c9d205e1d4
4344dfc2a31156cac187beecea674554a09739d8
9507 F20101209_AAARCG havemann_s_Page_100.QC.jpg
cd77d78756b301de8375dcca86649ebb
b4f493b681d307add574e80748297ea5b93b95c3
24534 F20101209_AAARBS havemann_s_Page_086.QC.jpg
f4aab3546a3bde388732f20b666d94f4
b5733bbdcbf79bc697acbe8ecf47e3d9f9a94e39
6770 F20101209_AAAQXA havemann_s_Page_144thm.jpg
36c82b585546b3a0ba6228773d7441dd
f1f1bde9eff155ae366b958512c36992fe0f908e
11838 F20101209_AAAQWM havemann_s_Page_094.QC.jpg
a79245916e01c55e964c7cad5f4f2a2f
ce1547bd71dd5fda75ae1082a462df2d6f99683a
18651 F20101209_AAAQVX havemann_s_Page_127.QC.jpg
a12588207fd8b3bf6624c83e69504ba4
b0f0add07d7e776571313e349a0c099017422620
67817 F20101209_AAAPTK havemann_s_Page_064.jpg
ddc632d160d468a73e571948eb5fd9d6
19772db2ed9dc78da0009b75c5c74eaac7132b9e
47709 F20101209_AAAPSV havemann_s_Page_047.jpg
a16e39d17aaae7ee30dde27f61a1553a
95aece403be7409664cebc9d5bbf57bafe114729
2877 F20101209_AAARCH havemann_s_Page_101thm.jpg
10c50884fb51adca70c6d5920446d2db
5b76e1cb4824f2657f7174a58404a57e02ce929b
6754 F20101209_AAARBT havemann_s_Page_086thm.jpg
a1491fa8bc6578175211fd055a44ad2b
f47718584b5b9f1897405e96c596970b53b465a9
17857 F20101209_AAAQXB havemann_s_Page_040.QC.jpg
33748eedaadf4f2b164ce314510d54c8
044b892eb2b856f0df2c825e9d25bfd661ab2c59
20180 F20101209_AAAQWN havemann_s_Page_045.QC.jpg
f9207f07ddae71c3cabbae1e756e7aab
d1d9df3e06af1d2179dbb2df49048070d0d9950c
21614 F20101209_AAAQVY havemann_s_Page_131.QC.jpg
12be3b9aaf79305001054bbb24939c0a
419929d676ba386aba7a8aaadbdf8d64c5414f3b
67003 F20101209_AAAPTL havemann_s_Page_065.jpg
1b89619eb5077d931a91ccf4a39807ce
d22e5b68019f243775a6974c001e7b082da8f4d7
67131 F20101209_AAAPSW havemann_s_Page_048.jpg
099f95ea958746ca46e3f5d9ba6ca6d2
7680cd636ff187669933fcf193f8bf9ff8451eae
24119 F20101209_AAARCI havemann_s_Page_103.QC.jpg
56fc84eb949cfb046e60d14f9cbad6f1
5c6b307c9e2362146ddc4a8b92de1b291f0f5951
14198 F20101209_AAARBU havemann_s_Page_089.QC.jpg
cd8be2056ca1952e065c7444b51c3cf3
a9160a3cab31cd37b4b7c34eb1c3edd7755e37d1
26234 F20101209_AAAQXC havemann_s_Page_138.QC.jpg
2c1f56d373ce8ecd0b6c36953b158624
e2fb16a948f371bb6c711530429c3e1f1d029697
3498 F20101209_AAAQWO havemann_s_Page_148thm.jpg
0cff2427b3a7848a0f05e75f3b5c6986
b5712a4516d47c69159ca63bed0a18e6eeca60e4
23495 F20101209_AAAQVZ havemann_s_Page_067.QC.jpg
7815d25884dc726d93de511186ca2174
a00b1d353a20c156f6944f3e0b27bbcc798fd841
32976 F20101209_AAAPTM havemann_s_Page_066.jpg
00be52376c6e202b15ccf67197a1b73e
7ceed35838a6f664006e1f4f818c300bd517c617
59978 F20101209_AAAPSX havemann_s_Page_049.jpg
659cb0b70fb8272207936b040131c900
dd368f4790d3a2cdad2331fb02cfc7c553c9f340
74258 F20101209_AAAPUA havemann_s_Page_081.jpg
a65aa825dbf7e7a1c47d951262e9d1a5
40d69fe45cb27230bff3c5e11e0fa92f874a20a1
24255 F20101209_AAARCJ havemann_s_Page_104.QC.jpg
158740407c3156d5097e28b1c1e5e3b1
01321abbe3f9229c501fe4fbed778714d66a0a76
5566 F20101209_AAARBV havemann_s_Page_090thm.jpg
2e113b212e8871e4f9baa0d3ffb8c934
9c4e48334b3466c658a16cd0810859512a53e822
4003 F20101209_AAAQXD havemann_s_Page_146thm.jpg
aa4417014f786e7dd8ea482c8f414e7a
a6c505c4060a5e24c46a6bbb3cba04bd57c9472c
3633 F20101209_AAAQWP havemann_s_Page_012thm.jpg
cc5749ea3e8b52cf8ca18ec8f41988e5
5a111ce8b37d85b74569c09f89b453f3d65cbfed
86483 F20101209_AAAPTN havemann_s_Page_067.jpg
0bd5a90e2c242ebc0ed95acd4e5a888b
067a72e5a955a05948443b8993e1ecf80895c92a
75340 F20101209_AAAPSY havemann_s_Page_051.jpg
59237ec076ab983b2be0aa9644b3a3af
5fb9205fd2261399a7d64bddae80fb28960733fb
71908 F20101209_AAAPUB havemann_s_Page_082.jpg
f2649dd200b4652eaba3f6f8d9dedf3a
c04c979035f037f151a8c4dfa7265af6a461f104
6870 F20101209_AAARCK havemann_s_Page_104thm.jpg
988c0fbd6792c55b02c14cbc8254aad5
722f92013a30bb751c556c60a6594d9d4b36e3eb
2397 F20101209_AAARBW havemann_s_Page_091thm.jpg
7b2bf5a58130842959d4dca94a2d710c
9c5c19bffab901b197b720ed661f4f7b462c6a4b
5112 F20101209_AAAQXE havemann_s_Page_009thm.jpg
10b0e5c98be8d120b79ed524f139d32b
53a7a7e7faa3c80ff073cf3c87a09af1d9697352
24085 F20101209_AAAQWQ havemann_s_Page_050.QC.jpg
895fb52a1b2b950f92728e70ee4b03ac
eb2e2c63b44eb0e9c3b8a35d919bcf9329facf83
95706 F20101209_AAAPTO havemann_s_Page_068.jpg
5308028541672b6aa27d220c888e9227
a71a77f50c297b6b99f53ee5df46d1f5939246c0
77703 F20101209_AAAPSZ havemann_s_Page_052.jpg
a512de2efb0c4259d51821aa524fc7c4
9749b9768473bcd771dc341b684e01bcab42e480
77479 F20101209_AAAPUC havemann_s_Page_084.jpg
e659dca25fb7978c27c4ec32c43bf42d
ccfdb5b55568825da6a5664af7a3eb74907bc475
24260 F20101209_AAARCL havemann_s_Page_105.QC.jpg
a1772538a0460a6f89f5fb91d8235582
35adaa9fcd3c5b07079a703a0efc53d3bdefa74d
3976 F20101209_AAARBX havemann_s_Page_092thm.jpg
319bc1cece0ac74d75775165171335cc
5be0fbb6718eee1353d4e0eaee33404a6dbf1a33
25253 F20101209_AAAQXF havemann_s_Page_111.QC.jpg
25d76db34f1b7c3684cba0cc8765b785
971159c15e8006e6b6610c6e89a9c8ffce9fcca3
5376 F20101209_AAAQWR havemann_s_Page_130thm.jpg
f6fe5223594b1534dd4dc7a39d4b3f6d
024624f562b380e03f1ac14215af98e5a476d159
68730 F20101209_AAAPTP havemann_s_Page_069.jpg
6fa26538b338f7e92616539915933d65
d1b5986462053c91fd609b8b87b2c1c47a8215d3
72611 F20101209_AAAPUD havemann_s_Page_086.jpg
ac08a7a5b861534cc9513960f2212b71
c40b5a4987b973e6569bd9057f3bbcebff9818c1
7003 F20101209_AAARDA havemann_s_Page_117thm.jpg
8d95bf5226cc0ee6c7fd466cc6208fb7
3a0f2ee2b7beeefceb4bd3795918b837015faffb
6721 F20101209_AAARCM havemann_s_Page_106thm.jpg
dff724c04b9c980747c21b7227d9491d
6bdf9a124ca77be3fa7295ce1bb8d0fe3c7379cb
12049 F20101209_AAARBY havemann_s_Page_093.QC.jpg
8bdd3c3120d13442a92b9df584356cd7
6cb74ff32d82723d15499f2b970d1092b884ddd7
24196 F20101209_AAAQXG havemann_s_Page_152.QC.jpg
4a1601c0e486bff7561601afb0a82d2d
ab06555c381d3021fd199ddc902b9e2afd06bc7d
6946 F20101209_AAAQWS havemann_s_Page_052thm.jpg
8e247a7b5fce9806e19bef2726687778
5dbd74dba58efefaeeb08674fa66a59bf436c50d
75461 F20101209_AAAPUE havemann_s_Page_087.jpg
13c6032e172d542ad338414b32ebe6ae
99d21bf0f0089ab46c9e51b7bae68af245db8285
75050 F20101209_AAAPTQ havemann_s_Page_070.jpg
9670c1e9b7c100527dcd0ac3024fee33
ec83f6903b6f504d991322ef47da9955b42299da
20792 F20101209_AAARDB havemann_s_Page_118.QC.jpg
5ccb9c7347110a6bdb44acd3660472fd
983e98478ce99b38113f81d2767e854603822e94
6892 F20101209_AAARCN havemann_s_Page_107thm.jpg
6cee770eb87b3dfd0cc1c72864ae0dea
5815092c788d795c438ee4f4ff07ccda548f322e
3835 F20101209_AAARBZ havemann_s_Page_094thm.jpg
3afa6968904ee5c6a72cc3adfaf3a1c0
7a0bcda9058c9eecb58948f8c8a60f92f42b4be4
22729 F20101209_AAAQXH havemann_s_Page_109.QC.jpg
645ee004c4690ab8b810be2c38c6480d
1a85406cd65bd931b74a89db63e8f6de79fe314a
25552 F20101209_AAAQWT havemann_s_Page_031.QC.jpg
eb66888452ad52e4e1f09de87bca8771
629ca0f13db0ac59ba9c0904c52f9f9c5971d61c
31237 F20101209_AAAPUF havemann_s_Page_088.jpg
164cc2d594e1ac24806863df40ba3f5c
dd34a774727e523310e7a76b50448575e6375b4b
74440 F20101209_AAAPTR havemann_s_Page_071.jpg
225f5d6bb23f7070cd472901977c174c
fda93101c5edf745137af7cb479609c03e11a935
5731 F20101209_AAARDC havemann_s_Page_118thm.jpg
36b26807764233fb1e62a9ae5131702c
44833c9150d38a1edf9b7f603398b0631ce84fb7
7094 F20101209_AAARCO havemann_s_Page_108thm.jpg
cbe0e8968598e4d4fa35a5d22dabe1d3
e233f6efe31d9d8f10c67ce6182682262e37f718
1648 F20101209_AAAQXI havemann_s_Page_003thm.jpg
4ce3a0e54d7ba798c80d41b0d77dc2a5
8428b7c9bd47c46f2b06c31cb62d20091c92d0a6
236528 F20101209_AAAQAA havemann_s_Page_096.jp2
b962269dba2b6b2cf24a5e625a0e339a
a48a133f95849d62719c3323d4db5fa08b8c1859
47036 F20101209_AAAPUG havemann_s_Page_089.jpg
6061d23329ecd1ae9a284fb1d787a007
99c5cdcbd6efc74c7fdf20e4f30c9d0482af1219
8512 F20101209_AAARDD havemann_s_Page_119.QC.jpg
c9f999e966af0e309ec34dd61f746e95
4724aa107eb19ebb16818a9821f4b72913e68270
23343 F20101209_AAARCP havemann_s_Page_110.QC.jpg
e46e56201ba829ce3f599d556e068ed9
aec924de8a03e802d9fee4aa6c2f4ea475d8ccbc
24564 F20101209_AAAQXJ havemann_s_Page_151.QC.jpg
34424eb6bdf9594b39bad49fcd6dc6fc
34fdc37b10353ceb0c2a5f64dd59e6a5d53ea9d0
5186 F20101209_AAAQWU havemann_s_Page_147thm.jpg
874ad52c8ef410d48c47cea1367aff8e
532ebffc51353f33b9f10172622a0329a7483069
1051979 F20101209_AAAQAB havemann_s_Page_097.jp2
40f3c4ed0e2e83276ee36714ed8e2eb5
06b5c2cc5a1831f3718c6f5c31d5db6e5cfe09ee
52440 F20101209_AAAPUH havemann_s_Page_090.jpg
3fdf6eb85b1a95a20e708f864cd5511a
594b2bf4ba656a118c5721a1fb9f2f37fefb2b85
77634 F20101209_AAAPTS havemann_s_Page_072.jpg
90474de0cfd8eb3646a55e818ae6a43e
d7c3bbd49fc60ed8eb0e3b1844a860a8fb8b9266
2687 F20101209_AAARDE havemann_s_Page_119thm.jpg
544993818f5985050bb28a1b911884c1
39c7e483949ac18a6aff2c8b4714ee5f3439c77e
6728 F20101209_AAARCQ havemann_s_Page_110thm.jpg
d67446a7cdfd27ad6776bd6a88d3e198
618052d70f23d479904002a6d02b88cf72c8e9d4
6783 F20101209_AAAQXK havemann_s_Page_025thm.jpg
98f9614b10cdffb46fc7b5f3624d8d72
1cf07c3edbfae0193d81dc02c20e048dc6b0307d
6466 F20101209_AAAQWV havemann_s_Page_109thm.jpg
8046afd9d1c22706c5e590cfec2b2bc2
15878c15e5aac1db99c1150684a15ff7449b07ac
758172 F20101209_AAAQAC havemann_s_Page_098.jp2
cf0473f0d3ab09485121223bb4dec503
deff94e9dd4bb1ec6b9a3a6fc30d26cb026bae0f
27698 F20101209_AAAPUI havemann_s_Page_091.jpg
2e8f2a588e9778dae4560351c8a9b629
674277ff09e8a4c29fffdc8b849f28c982f750d0
76344 F20101209_AAAPTT havemann_s_Page_073.jpg
ea10785500a4ca6612c7cd1b97cc3dc5
94302928e253130a83df81f0ccba20bd06353fc7
25102 F20101209_AAARDF havemann_s_Page_120.QC.jpg
2fa7242f8a3161326d79ff8b90562aa1
5c4bdd221a21c692bfeb14acb48c688559671274
24500 F20101209_AAARCR havemann_s_Page_112.QC.jpg
fb8e1308084d922e880bb561f9198f16
e97311ae8bd3b265809732ffe56e423e38a1ba3c
6982 F20101209_AAAQXL havemann_s_Page_139thm.jpg
7b4efdae1d5cb313fa12388a50db716b
a0b45db6ea41c91f381d05959b23df8791ce2b85
5387 F20101209_AAAQWW havemann_s_Page_095thm.jpg
84beb59ce693a4d97025bff02eda7498
86488c6d8a2f20b95d017606b01a7ecb89279548
1051985 F20101209_AAAQAD havemann_s_Page_099.jp2
490e9b1f9b56293fa1331e96768e7f4c
a47606b3db76a4b1e67f4434522b3fcf54ac1301
40725 F20101209_AAAPUJ havemann_s_Page_092.jpg
8bf5465d92ee6b2ab196b60dd2c18b0c
277ad43ec7eca52b6b1a4ea402bb6285005a7abd
74498 F20101209_AAAPTU havemann_s_Page_074.jpg
70190c6a4e9a4107254cc0be6eaf31a6
b9ca11452513537c4a73649faa5801f98371bd09
5310 F20101209_AAARDG havemann_s_Page_120thm.jpg
20106104645100d20c79c3b241c41c7d
faaa771a673107fba34bcdbc3543dd1fc5f0ade6
6965 F20101209_AAARCS havemann_s_Page_112thm.jpg
ed55aca082b29cd8e9800b74a91159d2
db4a050eced2fc5a0bc9c90c539c266fae1f297a
18644 F20101209_AAAQYA havemann_s_Page_069.QC.jpg
bd3b4396ae4f73e49bba88d538589957
3c3dbfe9b79d4f8d85061a571e063346630c58d9
13524 F20101209_AAAQXM havemann_s_Page_121.QC.jpg
069cca5cf34fd0ab65d5ea35c12262af
d229e4456197926ea81b693b7376e530378fb3cb
23678 F20101209_AAAQWX havemann_s_Page_082.QC.jpg
2761f76430f5c7ce07fc37c48d7a99e1
46f0cc7dc0f31738125e359297903c4613580fe9
1051938 F20101209_AAAQAE havemann_s_Page_100.jp2
89686cb4a30f28b792f6a27384446dd5
222deb58c9aa9e7a8fb7c4501a39f23f11be6c32
39776 F20101209_AAAPUK havemann_s_Page_093.jpg
6c4b88e9390ad7a5405a613d9db857c2
9ff4c5447260c387a7ee736cff8c91f8293ca803
79408 F20101209_AAAPTV havemann_s_Page_075.jpg
5fec2ac5867394c6fd56dbe6d9dc9232
45a389ea9bf3df34039b1cc22382f82c6404beb2
3409 F20101209_AAARDH havemann_s_Page_121thm.jpg
b4d2e6cef39ff7e0bb6a08c5e0e82358
fd12d36b8e74beb70976efba946e26c0719f59b6
F20101209_AAARCT havemann_s_Page_113.QC.jpg
cb3d151c173a1d7607e02728c394164f
bc9b31f5522e96eb217236088f52424ccf845f5c
7010 F20101209_AAAQYB havemann_s_Page_018thm.jpg
b7093c02d3c5490b93a62af85d833e30
b99dcfc61df85cc8c1b34e78ba414ebafb348062
7180 F20101209_AAAQXN havemann_s_Page_136thm.jpg
e951d18a7a8c766144f780ef11652bd0
e317837f0d8a044fc6ce74816d1940079ae9327a
15992 F20101209_AAAQWY havemann_s_Page_090.QC.jpg
96aee3c1036ee5fb790f6545068e3c8f
42a005ad8f8620cd15b58f142a4324285ac6a860
46463 F20101209_AAAQAF havemann_s_Page_101.jp2
e008ed97dc2d66909310fbaeab9314ff
9a943a474eac1fde796ef497c7e49ccca078f8bf
39407 F20101209_AAAPUL havemann_s_Page_094.jpg
31ab4c553d494c4b64deed6bcdeb5405
96e7cc5b757937fd86559827b81a019028900649
76355 F20101209_AAAPTW havemann_s_Page_076.jpg
9a4a7733b47267be81d50f016b3372b9
39277879d2ac1b52af08573437b472da8610d296
6025 F20101209_AAARDI havemann_s_Page_122thm.jpg
f0e3cd5d93c799b9e79186901ed451ea
885536f6a167e18d12ccf103bdfeb0ca67b37f70
26011 F20101209_AAARCU havemann_s_Page_114.QC.jpg
414dff7be8d2db2c594070f2760e9788
b3bf2b0d9d55d8cfd1d82d18f90631e58a103f66
25555 F20101209_AAAQYC havemann_s_Page_124.QC.jpg
fbaa8d7eae4c5aacfcc18dbecf44ada3
12cce434927a4b3dd297003ebadf143917fa143d
11053 F20101209_AAAQXO havemann_s_Page_013.QC.jpg
fa2e0cf74b9a6579f961dc90259af9af
66ea9caa0c227e799291501a80e51e5e8800fa1b
24875 F20101209_AAAQWZ havemann_s_Page_021.QC.jpg
3f37266ab9c5add1411e480bfbbaa259
43e9adca8bcf03bc95973f43bd813cfbae33baaa
61383 F20101209_AAAQAG havemann_s_Page_102.jp2
3ea061c3fd7a2d10f7fc810ae6679aea
6f1c3303e0331464d0b5676b5962a70b90697abd
73458 F20101209_AAAPVA havemann_s_Page_113.jpg
9aa82ee51922ba43155b5120acb84465
3fe88e9456c59de0ea32df9b05b1f8960d3ef172
86635 F20101209_AAAPUM havemann_s_Page_095.jpg
b64ff034f02f51965bee3e262024b19d
a221f60e8e93f1cbdf7c2d2005d0a6724c130a2d
78727 F20101209_AAAPTX havemann_s_Page_078.jpg
162458e2949d4b9de3ae023c8d9b2cd4
c95eb1f136b02d13fb0c82084c383bf7be928cc0
22553 F20101209_AAARDJ havemann_s_Page_123.QC.jpg
575edf4eeb4602e108b05007964d87c2
5536dd8f46d44e285bf04afece875627c70007d5
25200 F20101209_AAARCV havemann_s_Page_115.QC.jpg
f863ec027b43b93221b9bf404af72b6f
3d122e43eff8e36a177bfc520581a160ac231cb9
25682 F20101209_AAAQYD havemann_s_Page_108.QC.jpg
662a7f3aa4d62367d3756fe2ae69f13d
7cfabbc8bb29904f3fe232b10f7c9b11e4c91b51
19074 F20101209_AAAQXP havemann_s_Page_147.QC.jpg
042a0b5f1d2dc1f711bcf9c88e8c4e73
c78caed0ba25ac851bab8983261c705539498ea3
108530 F20101209_AAAQAH havemann_s_Page_103.jp2
a8f8461dbc684c6886355c49661d12fd
497ca40ea97fb9708cd9ee7f624bb2e792407c7e
75856 F20101209_AAAPVB havemann_s_Page_114.jpg
a6382e30dcf8d2aac1bc16fcc935d7f0
fca22dabb882b1e1571810e673db3dc773315772
117866 F20101209_AAAPUN havemann_s_Page_096.jpg
882d7cac885eebb3012db32d33fdd393
32f8290a8ef94b91a9093b99b71cdddea515b487
75832 F20101209_AAAPTY havemann_s_Page_079.jpg
4c26d6fee6a555d450edf312131e9ac7
b70561e1eb88d28953b3b3f66a05731d50b2f53d
5534 F20101209_AAARDK havemann_s_Page_123thm.jpg
1b43bda034c2096ea63b5b26c75ef7b2
c519387018e86cccc27856405100f0cf26336d64
6662 F20101209_AAARCW havemann_s_Page_115thm.jpg
1ed604143fcbe16d19631522f8ca4111
c04cad5f8346b50f733241b33c9b53c45bbb71df
6684 F20101209_AAAQYE havemann_s_Page_133thm.jpg
2c82a45504472c8b86f73be98f802896
0938bd7ecd3f6609f1004d3c68dc9dedf1cc93eb
26192 F20101209_AAAQXQ havemann_s_Page_079.QC.jpg
e30c165c417e68f6849a58166bd2bacf
3753f3278576a375be1cb7ffbcf83f64b5ee8951
111051 F20101209_AAAQAI havemann_s_Page_104.jp2
67fa73cf4045fa634e04413033c8d0f8
90ef64cc7ff7a1b93b8524509002db8b794bac70
76636 F20101209_AAAPVC havemann_s_Page_115.jpg
9be09c35503c87b085602b7f64213f16
c4f2d5a7736f084a7f095bbd7a8ec11810b3291c
82038 F20101209_AAAPUO havemann_s_Page_097.jpg
def3310b8cdebf02d673e3a0bb6f6840
fbf8540ed74a1e8ba967b8414589a6d27519f958
75068 F20101209_AAAPTZ havemann_s_Page_080.jpg
50ba97b9829c074fdc424f45b17f9834
a18e28e32c6ef2c77b886ae5f9a3d9af642a4c19
6634 F20101209_AAAREA havemann_s_Page_142thm.jpg
e0a9261ffcb9dfd582b747b07ccc0346
3ccca2bd7b51a7739419440ed824acdaf8658fbd
6273 F20101209_AAARDL havemann_s_Page_124thm.jpg
3313551cea50ae6cc91520564edf0773
5f80b9c25769c9a18815d20d2ac68eafb8ada324
25549 F20101209_AAARCX havemann_s_Page_116.QC.jpg
0a7340870038797a94fcab13efbe908a
32d56723ddf4012388177739865cdb33dc103762
239526 F20101209_AAAQYF UFE0017563_00001.xml
da363d39278f70c6a6439c7144c618e0
dff8d7405d0c7e49c0ce557873593f7859704a8a
19971 F20101209_AAAQXR havemann_s_Page_044.QC.jpg
db1417c98f69e1b5da77d85c31cdaf56
6dc70f28342238d6bc390fc118d0dd210e91092c
108656 F20101209_AAAQAJ havemann_s_Page_105.jp2
d45f03e410e7f0772944ef0dd3326e4a
f781bcf59a9589c8f76aadb33a70d845a836ae1c
78621 F20101209_AAAPVD havemann_s_Page_116.jpg
67dd8278b7792074b74ae565b15c4e1b
1dd0214350c25f861fcf9c3f98cb5cfe5951fb35
49016 F20101209_AAAPUP havemann_s_Page_098.jpg
31a85287e55583071572a6895491402c
7ca34b129879d6edd0b6df302d01ded1e782f379
13894 F20101209_AAARDM havemann_s_Page_125.QC.jpg
c514b6b0f102d638c69040fcd45a791d
c462445a2702d5275e825665725845f5678d6839
7123 F20101209_AAARCY havemann_s_Page_116thm.jpg
5f06b101d31541bf449536a5b30d7ea5
ea416aeaeba7c8bdda7e309d4d7256fd09610831
6704 F20101209_AAAQYG havemann_s_Page_001.QC.jpg
90e23a61109328abd4d67257ade9715b
cb7c9440c0e0ca652af637fa73daad110d41dfa3
20322 F20101209_AAAQXS havemann_s_Page_122.QC.jpg
24119aa001d7a9ed59441c9734316c45
2404c924a0497aded04c3f5e2ac049e15d8f4889
110761 F20101209_AAAQAK havemann_s_Page_106.jp2
40f091de9c4e62151ebc95477336e2eb
83d5baa2cd0fd2b9b43b52da1021190257af1746
77863 F20101209_AAAPVE havemann_s_Page_117.jpg
9d0bad90a4bb1fade9880f05cd6a04bd
633526050f856534a12293e25b532e32e172f823
74160 F20101209_AAAPUQ havemann_s_Page_099.jpg
3ee3d64d2a7e5440fcce71b478565c34
6b06f3f828da65f967bf90b915765e7670fdf308
23042 F20101209_AAAREB havemann_s_Page_143.QC.jpg
71be25236e6a139aeec8596d65edc091
f24125d2451905874818b096092c80fa50594ef1
4827 F20101209_AAARDN havemann_s_Page_125thm.jpg
9142bfd79611ae4bab32185bf111caf3
7def9c5a2f9739edc3f09d67753cce7379f65425
26482 F20101209_AAARCZ havemann_s_Page_117.QC.jpg
127de4e3f4ab04f567a883ff0a660263
5f7e8fc7d5680197789e90edb89d910b8a91850d
2179 F20101209_AAAQYH havemann_s_Page_001thm.jpg
60691b808b323febe8d597ed37d0d060
6e19cfde6d5855e055f1faece49283fc4bc3387d
4169 F20101209_AAAQXT havemann_s_Page_089thm.jpg
190b25613e387958bfead2d8292c6df3
ec5644fc261511083b8d4835f5b325f6cc4be6a0
112211 F20101209_AAAQAL havemann_s_Page_107.jp2
1ff8d4614eb8476c3e5381ecf3f9d9e5
f8b8f2db890c4325b288cfb7a7c6c0ef930b1206
61995 F20101209_AAAPVF havemann_s_Page_118.jpg
3f3c421cb4bc03a96e5a1e542ec92ece
ffcdd828f1e71da83d6b4f914e5c36717a03e712
34001 F20101209_AAAPUR havemann_s_Page_100.jpg
0e6d29c496f8d56df1359427279131d8
5f4d66b4ef3b02ec27eb1d7a1cdcaa4ab76b78fa
6271 F20101209_AAAREC havemann_s_Page_143thm.jpg
9a252b3a7bccb138f10954cdadd949c3
93e04677d3674e11f9e662430cabfa66be69725c
19087 F20101209_AAARDO havemann_s_Page_128.QC.jpg
ce454ef0d6c021bd00e9c2c92541f121
ee9a1c5b316026a8f2824d435e9bf92bbec4c721
3440 F20101209_AAAQYI havemann_s_Page_002.QC.jpg
2c8694df040968fcec1ef11b751b6aea
bbed265f945a40a9f77db71bd97bd6f4cbb64981
26298 F20101209_AAAQXU havemann_s_Page_019.QC.jpg
5dcacd64940d36ae26ef822d44689cde
824649c6f0f8e9bfec72a700cc535f138a36353a
115750 F20101209_AAAQAM havemann_s_Page_108.jp2
cff9fb3bda7dd8924c9fc95193f1e140
a43c8560f44b39358a33534d3030cfc6cbd3fc12
26907 F20101209_AAAPVG havemann_s_Page_119.jpg
a2172b7604b145b54ca7c305bc75977d
2b394b11c92df5088f77bd5d3c1c8d0071e8f051
33641 F20101209_AAAPUS havemann_s_Page_101.jpg
1f10d164d10030a4ce66656df1fd24a9
1ea8990b6d885a7341cfade1fbb6eee758a2349d
994511 F20101209_AAAQBA havemann_s_Page_122.jp2
d9f9820ec5c739f10c64505a27b288aa
a9910f44a2f2aace7d7a88d7c7a92afae34c7cbd
25179 F20101209_AAARED havemann_s_Page_144.QC.jpg
a6210e5c1082f5010549e06b5b25e57c
aace03713584e5060fe2475abaeece7215c08365
18690 F20101209_AAARDP havemann_s_Page_130.QC.jpg
95fba16288c672ed8e988f7ad73d6d9d
755f5eb48f15045460288bdf506a260f005f4c09
1405 F20101209_AAAQYJ havemann_s_Page_002thm.jpg
9036cd818fd3cc53d7fcbf9e4689acdc
aa93bd810acb60b5e2b2ec18cdb637ac7ea365d7
110100 F20101209_AAAPVH havemann_s_Page_120.jpg
e5c082c5b11c4cf274b66e7c224b508b
6026c939e1298c8662f309df6aa59ed48297d500
1051980 F20101209_AAAQBB havemann_s_Page_123.jp2
596333d553310d3ddda5774d8cdc8c5f
89f72e466e8e41828773ef703a0d7029a33e2b44
96154 F20101209_AAAQAN havemann_s_Page_109.jp2
6a164e04e90a3fc315ce72d732030a0f
dbea183e8254c9da396333e2d87f005784f358db
1754 F20101209_AAAREE havemann_s_Page_145thm.jpg
7fff017533342bd6d2d18dba27eaf56a
31efb4c56b7d09bcdd9eefa676af0c5f56584e5f
24525 F20101209_AAARDQ havemann_s_Page_132.QC.jpg
ec70d0727ee82b62d4b5068533d1b334
5b4fefa110d6146457c9d72c964cb5d133da279e
3939 F20101209_AAAQYK havemann_s_Page_003.QC.jpg
82bfd7d69fba328ace50aed77f3efccf
7ffd98a349a0f1fab75531cddb2365ef9f4aac55
25191 F20101209_AAAQXV havemann_s_Page_076.QC.jpg
f45f2e235a95d81baf8ad248dd5df5a2
404c84c9076b914a9dc50aaa73678f7d1c62c4a0
51467 F20101209_AAAPVI havemann_s_Page_121.jpg
d9e7086628ad537a65d5863e46a416da
1332ba709c820c2d1709059affb217fed917a7f7
41425 F20101209_AAAPUT havemann_s_Page_102.jpg
5bf3d0ed833a3222c9d08c69dfdba07f
29fc9c0d5980864dafda1bcecf5a1a0d52271b27
1051923 F20101209_AAAQBC havemann_s_Page_124.jp2
d61f7d598013ab5a67c0a394a1909543
0b57574830e234ed4d49ddb916140193698449cc
106290 F20101209_AAAQAO havemann_s_Page_110.jp2
51ad27d373a09ed4b31b99144f88028f
79dacd719de334b80acfe5dcf715bd3355778944
11137 F20101209_AAAREF havemann_s_Page_148.QC.jpg
addf1bd121f676f22539af1e91a2424e
efb53c1d99185f52f605df290516de8ba04239b0
24266 F20101209_AAARDR havemann_s_Page_133.QC.jpg
43abd144e4cfba5f6c26c037e0257928
9d614e6819dd58cf7e83bc56f50ac56cf1c998a3
2224 F20101209_AAAQYL havemann_s_Page_005thm.jpg
dfb0efb98de1bc59f24e88a7f553ef4f
42bde204bcda230db137b49f278d1cc0b9245d80
24092 F20101209_AAAQXW havemann_s_Page_055.QC.jpg
c798d2bce5847d31ca6b33b23be37c13
d4ee8ca1a54a92da97512e58c86e62d1bfb8c0fb
66242 F20101209_AAAPVJ havemann_s_Page_122.jpg
b5ce5a08872bbb8e408a01304f239b01
d5a8e12de070af6951e8b885e0a550595cf4001f
72357 F20101209_AAAPUU havemann_s_Page_105.jpg
e5f5c1c4c5e75071594e06a044c5a3a4
3a5495491e1b630f4bc2e2081f65fa7a85226653
520025 F20101209_AAAQBD havemann_s_Page_125.jp2
9bf1b6211c86de5944c5f41c1909338f
ed050c9a6aae478010116ae9e0ba80dd5df09a17
113355 F20101209_AAAQAP havemann_s_Page_111.jp2
46396de42c8e8629411568be0cbd3b07
9b5eb865f87ce00112de7c34ca0a7b1e9dff275b
9065 F20101209_AAAREG havemann_s_Page_149.QC.jpg
557f1b2bdd7fa6f715f83a9bbfa081bc
626b420db3b02ebb9ebd15489b910892ecc707cc
6541 F20101209_AAARDS havemann_s_Page_134thm.jpg
9bcfa0f77032ad9e0b5825727e036ec5
1e0afc15531fe454529e943894f4a08a1fcd970e
6535 F20101209_AAAQZA havemann_s_Page_022thm.jpg
87fc03d5f8a37970f50cd3b0e45417df
c2eb09aa11c64a8f5b5fc5b2a141d910beda8bd1
28140 F20101209_AAAQYM havemann_s_Page_007.QC.jpg
12041a1296e65c3e7c313e667151f6f0
a4ba89692714b2b5b630c479d428492594e1b4c5
2987 F20101209_AAAQXX havemann_s_Page_100thm.jpg
22aff37410384057217b9b3f15ce3bd8
6aa0853271d7b964bc13c05f43dfc47f9af439c9
80503 F20101209_AAAPVK havemann_s_Page_123.jpg
78cccd1bf28b50776ff623abfb77b598
3d033dc437ddec14ccaa354dbea777f5f4097b2e
75519 F20101209_AAAPUV havemann_s_Page_106.jpg
4a5ff0ff810aa6be236f90cf87907a4c
110763525842c86a9937241e882301e4b118f6dc
1051949 F20101209_AAAQBE havemann_s_Page_126.jp2
69d3394f66bda7c2cfcd642e0703c542
2f4f27a4bc6e24423cbcd2b5c4f67b342d218a94
111258 F20101209_AAAQAQ havemann_s_Page_112.jp2
0b5594d9107e6f1707240022e26a2d68
7344d22e19232cf3fec707404f76dce0cdc15492
3022 F20101209_AAAREH havemann_s_Page_149thm.jpg
edbcea077866163d676996a1a45f963c
077768efb23fed250c8cc337bcbef6e17eb03602
7034 F20101209_AAARDT havemann_s_Page_135thm.jpg
bf55019edf5f6f983dbadbf06d935275
9d3f99a894d7e3fc5b1624ef11ea71f9d37e7516
24524 F20101209_AAAQZB havemann_s_Page_024.QC.jpg
7bb7adc3670d4b513f8d19bfca78d13f
8ddfce8803e4996bcaac910e5b5421b0f7e19c70
6868 F20101209_AAAQYN havemann_s_Page_007thm.jpg
3d8b8504204b794a695172351270fa83
94e4f87aa0fd1b1d83ec559a1cdc271c1ce680d7
12401 F20101209_AAAQXY havemann_s_Page_146.QC.jpg
27ee10f8abf72e3e210740fddea85134
84327406e4202985e9e3cedf8ef5873aeb631e11
91296 F20101209_AAAPVL havemann_s_Page_124.jpg
cd000bd995d8fa482625a8331cfef5c4
cf69bedc2b96316bd614a93f43f93fd124a7b687
76376 F20101209_AAAPUW havemann_s_Page_107.jpg
5c27cd0f7b2342d810e4ee8ac17d4985
9170ed8da5ec33c6ceee7cc7f1c106acb57f7d1d
972366 F20101209_AAAQBF havemann_s_Page_127.jp2
83bf3cefb5f5ad66ba4da50c2514defc
d1a784d1dde015dbc2d0570f873aa49fa826b723
109315 F20101209_AAAQAR havemann_s_Page_113.jp2
5ee6de1bd07cd936e228f602430004c3
c318fc6d445bfc3c331c012559498c0350c16adb
6840 F20101209_AAAREI havemann_s_Page_151thm.jpg
c61aa3e99556f5f4c55428edfa804320
d4d32a1754b2898e7cb45e617bda678014ca7f10
25707 F20101209_AAARDU havemann_s_Page_136.QC.jpg
3554c8f9b875cc70bc8cc6040de43533
bda2e6337ab03c6ad89ac05cc1b7b97eed45c83b
6895 F20101209_AAAQZC havemann_s_Page_024thm.jpg
c3483730671dc6117489c2b8afc25521
033e51c17e736e48af01c8ee386afb9239e8a545
20671 F20101209_AAAQYO havemann_s_Page_008.QC.jpg
0cfdd6891d45560cbde038eee159c920
ff624c7722e06192a76dcd5801a3eef480cbde35
13102 F20101209_AAAQXZ havemann_s_Page_037.QC.jpg
e0588a08f91d5e0f49a2060e0c9fabf8
f446eb80c6525a56e6a38934892ce39f96706c5b
43646 F20101209_AAAPVM havemann_s_Page_125.jpg
8228c0902da6fb26e59f1843c0b11a9b
a6609e7f9cb7653b38aa2da3461ba77296b9d975
78790 F20101209_AAAPUX havemann_s_Page_108.jpg
fe20aa3991e0ffee34d5f8dbaa65e796
37e549557640305d7e04898dc1295887250c3524
1011057 F20101209_AAAQBG havemann_s_Page_128.jp2
a81ab98ded075035768242645cbfdf5f
14ddd74ec3b6b479e00decaaea8e0daf82bd4d1c
69591 F20101209_AAAPWA havemann_s_Page_141.jpg
42aea80eff140185087b20415f9d57af
9a71382e8ba0a40a819702fc3248a7952e6d957a
112259 F20101209_AAAQAS havemann_s_Page_114.jp2
b1d962fcdc7957dc972c3ec62a45f60f
12bc0f0fda9f3ad1679dfe9977a401225399ac7b
6761 F20101209_AAAREJ havemann_s_Page_152thm.jpg
2a24e38c859846b9b6e6012868f66a96
d1fa4fb24d601ef476ae52deba38f6823e590c7a
26770 F20101209_AAARDV havemann_s_Page_137.QC.jpg
277095c470c91f73b92d69554a028060
af246bb21b33a0c81469acb873697cdecdc7dc77
25087 F20101209_AAAQZD havemann_s_Page_026.QC.jpg
a5ce151aed61cbe6472b4ad68dc99426
4e16b9468963e3ee53b8bbbaaea2f0154a290a02
5440 F20101209_AAAQYP havemann_s_Page_008thm.jpg
265593c25986d088069328617d375581
d8e40ab20d10adb47c6d573e4472f078cbf16f4d
65461 F20101209_AAAPVN havemann_s_Page_126.jpg
fc2630ca271d0164c4f79154c010c7ea
af2cdb5dcc7b8504e002ec4159d51295dcb4cee0
76276 F20101209_AAAPUY havemann_s_Page_111.jpg
1ad13e9fde04ec09ee748235fcc8439e
2f1332c431f4662cb30a5bb53f01f03fa15a99f3
74415 F20101209_AAAQBH havemann_s_Page_129.jp2
41f02e821ebae17d48bebbe0cb6a2bbc
891ad2b9975b45f8d1a12d94099fc23f266c32b0
74221 F20101209_AAAPWB havemann_s_Page_142.jpg
6b0c1d2d8f7354821494dc299737db6a
41ec3c2bf33afb1e9a6a738203c4487dc98739f2
113722 F20101209_AAAQAT havemann_s_Page_115.jp2
b9a093ca1b3878cbb9bf6cf822983a81
3ee83d72bb050859bbd28fa7bb57a2afb475f957
26104 F20101209_AAAREK havemann_s_Page_153.QC.jpg
7777b73037b7dbcd77cf178abe1a104c
e27bd5d0d5a2fb9dad33a4c13255fa6ad6f91e9e
7307 F20101209_AAARDW havemann_s_Page_138thm.jpg
3d24f0777b521407b848af132b8fa15d
3737769e71838b8bb9adb5c9a6c34788f7d4d625
6753 F20101209_AAAQZE havemann_s_Page_026thm.jpg
ba8d916c502b48437b54b8b0822c05aa
17279930128e170fe0832062ef65832f0d1e6446
19511 F20101209_AAAQYQ havemann_s_Page_009.QC.jpg
ab9ff52ed39e86bbedcdc4338053abe9
49c78e24d3f2c0b61aab515fbf71071d29807fd5
64828 F20101209_AAAPVO havemann_s_Page_127.jpg
3063f6d19993fc853895b0ad1bee8e56
a0a0c924b4d2286f549c87860fbe43526e59a1a6
74092 F20101209_AAAPUZ havemann_s_Page_112.jpg
2748b47a75590b73377b901127e54836
f4f1b85596f4d720dea88c7e8dce58955859be84
977664 F20101209_AAAQBI havemann_s_Page_130.jp2
b4a4f4913e78c9c8e7afe6ecba4d560b
32204d7b9d23d32226eb5336a18df65363007d50
70051 F20101209_AAAPWC havemann_s_Page_143.jpg
ce69e6655acadafc061aca8d9ee6e4b4
7da0b4c47644c74e0dda19d84193e70c224187c9
117042 F20101209_AAAQAU havemann_s_Page_116.jp2
f484096e4826f31306c9d76e7918e5c6
6158070cb6d14f09337297076f9636f1222ab92b
6922 F20101209_AAAREL havemann_s_Page_154thm.jpg
c2289bb79500885930253140f341aa6c
ba0a31f129bfcdf107ebab05ba0121c6f7e60a80
25809 F20101209_AAARDX havemann_s_Page_140.QC.jpg
ba10f8b1380ee2bd946e9c9c4a92d02a
a2b31bc22172e114b82a9e601f44da82a468b993
7057 F20101209_AAAQZF havemann_s_Page_027thm.jpg
67019873381230c6608e7bd96ca36487
5600cac13d057675e325d7f3815e6de76dc82bbc
24797 F20101209_AAAQYR havemann_s_Page_011.QC.jpg
73a29b1d38f638ccc7993f3e956fe693
ee0e6aa82021a325f1e6da62c6386c5f855293dc
31800 F20101209_AAAPVP havemann_s_Page_129.jpg
9689cf14149c6ae37cb4f9cafe1a690f
c358bea7502b212291587c58294189fc85cebf57
1051966 F20101209_AAAQBJ havemann_s_Page_131.jp2
517fa74757310c291f0b855b676abcaf
d77a6b9a63325d1af6dbab32e036a712de93c10b
75827 F20101209_AAAPWD havemann_s_Page_144.jpg
6ac7e4e50f28b45e0e19b3b29991fdd5
56dc9cf5ce004b70ed13223d5bdfed5e36b3102f
116095 F20101209_AAAQAV havemann_s_Page_117.jp2
69e63462e6531e0a27f78f6c18d7c2ce
608c4be9e3e28b31290056a450ceab8397a994f7
25262 F20101209_AAAREM havemann_s_Page_155.QC.jpg
760315f23131f0bf8413c7cc3eeb8fec
50db3615da237d999e750b990893aad57c3d74f3
7126 F20101209_AAARDY havemann_s_Page_140thm.jpg
1ffc2800356292120d018d23add24159
3267076944411b59c7106a726a3d81c8a95da5ef
7117 F20101209_AAAQZG havemann_s_Page_028thm.jpg
ba7e24b8af83a57e22d89eca7efcca20
f947c2156ebaec6434d9478b533008aba255d799
6461 F20101209_AAAQYS havemann_s_Page_011thm.jpg
1b6eae2543d8af5c5a1c710b7f984406
82873ef670b90706e30530c30e2f07d3ccf21da8
65030 F20101209_AAAPVQ havemann_s_Page_130.jpg
f7ca17812862a7822545e8d07db21687
b01c18df268f2ba278de0d9c66590dfeae9782d9
113014 F20101209_AAAQBK havemann_s_Page_132.jp2
de265218003a584868b4f1b1119dce5b
ed12bdc1bd6d387f4bf1632b35f0aa86a6dc9742
14087 F20101209_AAAPWE havemann_s_Page_145.jpg
46135b7244ffc0ce170b03c32149d6bd
68faca2bddbaad21e339ce6ee2818498cfe38948
91505 F20101209_AAAQAW havemann_s_Page_118.jp2
4228f716ea781bc72c7314e79b17baa9
2854938c98e0aba43003c4a874254314793cdc0d
6977 F20101209_AAAREN havemann_s_Page_155thm.jpg
88e088432d52faff753cc1006a2fd9d7
51d2169110ff0bf6690e4ea8129980f34f93622b
6300 F20101209_AAARDZ havemann_s_Page_141thm.jpg
27ed70e889c708a1e991efb92d38b885
fd87e113e52a7532285be5f98922ea578a5f05c3
26090 F20101209_AAAQZH havemann_s_Page_029.QC.jpg
197373bc3d97e572bc02dfdb95a1af9b
5a4b12a7708314930bf8deec8ea7b49d38e11073
10449 F20101209_AAAQYT havemann_s_Page_012.QC.jpg
95646edff213f986943039695d1c057e
760a7d14c39ca433e59bb8f46578976c2e2d2e5b
74326 F20101209_AAAPVR havemann_s_Page_131.jpg
86d3cd2dc56cae11f386196d17d729d0
2a36450e64cb5db87072ccfd5fc2abc15ce9d46a
103457 F20101209_AAAQBL havemann_s_Page_134.jp2
b7d3d2974047b7f840fda86a4d695a03
d72ba2d41cab0f3423914856f7c2ada85e6c86e8
37882 F20101209_AAAPWF havemann_s_Page_146.jpg
043e6aa4c6e88eeba4b95d17cc20307a
99e8176ce6b94af96b998e80a1c78108583f5bee
32198 F20101209_AAAQAX havemann_s_Page_119.jp2
9741bc5aec4d2c6cb76653111c00d5a8
738d1847cb91abb37c6b9197a31bdf37275e0734
6650 F20101209_AAAREO havemann_s_Page_156thm.jpg
ea2583533694ef22073159794f169b07
7b1ee34aa6e283be4150588114c12fdfa3ff24cd
25510 F20101209_AAAQZI havemann_s_Page_030.QC.jpg
30ae162ec73178abeb4d74eacc07676a
47f3ce84474f7fe61c731348420e69e767acf445
3249 F20101209_AAAQYU havemann_s_Page_014thm.jpg
34beec76c70348b946b27a50409e0d64
e3280b722e29b06e65087e84098cd3b8b10ec641
74657 F20101209_AAAPVS havemann_s_Page_133.jpg
930ebfd3c5ed8bb69ac8918b07b7a613
97ff1d78fefd6edeb305bdd6b2a564ca6337eed8
922001 F20101209_AAAQCA havemann_s_Page_150.jp2
1882384b8560097a91d4b70266692a0a
d0344fdb7d7e8e9e34dcc54c8a543fdd777fef74
121168 F20101209_AAAQBM havemann_s_Page_135.jp2
1d79b05f6ef9ac1095e1a09473ba4d8a
1b35c9cf9f29e576f7539aa13b503c59bfc2def3
59742 F20101209_AAAPWG havemann_s_Page_147.jpg
f9706c0d0bb35a0b03d7d880b7f416a7
767621d0a680e5c91dc30abea39cd3936781ac99
224821 F20101209_AAAQAY havemann_s_Page_120.jp2
cc0bbb48ff85f2e69f500721ddb44c1b
30c1aa599bdc52fed82488b5fa4f0234bb66875f
6601 F20101209_AAAREP havemann_s_Page_158thm.jpg
04fb8d03004357919fa23ec5a0edd0ee
4035091d266bada1aef6897b335cce7031bf4700
6837 F20101209_AAAQZJ havemann_s_Page_030thm.jpg
c47d6f7e9d7bf2d9ede51252d53d05d4
680dc31fee65266aa89ac83b22cf8bb42d4f8247
13895 F20101209_AAAQYV havemann_s_Page_016.QC.jpg
62bd9b5e1b5e8969a7faa29971a2692b
18f822a5b69ce82cba1dc3cf4deb4fac91624363
67359 F20101209_AAAPVT havemann_s_Page_134.jpg
2c1cac4252b04259bb7502b9daa3b8c8
3d7cb1c0aa65b62ea29beabf3f402c9ce1be5b28
116854 F20101209_AAAQCB havemann_s_Page_151.jp2
135dc53630b0529dd3a6904bb300551d
7c6e8dc465169032e5c9579f00dd2fe3a3092e03
115591 F20101209_AAAQBN havemann_s_Page_136.jp2
bb8ab671ae1d6fbdd02a34adfb4fda90
a79e4158d459d7b4c8008de04f56fa6a483a1782
37834 F20101209_AAAPWH havemann_s_Page_148.jpg
bb7ab4b556bd9bf03c3a805236731ac2
3d95686faf59b6a5ff6fc5caed6b677061164fa4
78120 F20101209_AAAQAZ havemann_s_Page_121.jp2
ee63054549b281593585e1fe2250a3c9
c1d4959aac58b074db8c9c31337e98f65c589bc4
4848 F20101209_AAAREQ havemann_s_Page_159.QC.jpg
3b4e0b5cffa85b31aecc7f1b095ff6a3
468a99f868e2e2079132510b4f259219ded4ab9b
6804 F20101209_AAAQZK havemann_s_Page_031thm.jpg
d8a0f403ea55bc49cda34620022fa2dc
d31010c116c9e29aa151ef4fc821c06c6c98f365
115140 F20101209_AAAQCC havemann_s_Page_152.jp2
fbbd96b11e8d15da1b32cbc3c5b53c94
b875426658dd7b02fd6e9ef5131db8d934055a4b
121646 F20101209_AAAQBO havemann_s_Page_137.jp2
7b0840f41ededb576bc8d1b5e5da1494
d7ee3f4b04e61eb7bb9b7fd27d9ca089dd2d593f
30933 F20101209_AAAPWI havemann_s_Page_149.jpg
0705e3b3768b02655fd7ddaa932020f8
7709bcb5fe73fe71750eac2c7f0bfe895da0daa3
6843 F20101209_AAAQZL havemann_s_Page_034thm.jpg
af8b582411bfaa29f060cff0bdf883d7
a3907c85fe70a3dd3dec16be8293bebdd68ea9f1
26236 F20101209_AAAQYW havemann_s_Page_018.QC.jpg
29ecc091af299ff8af14137b76dc2e23
2e838c6b19962abf0eda12689f29324a750415d0
78118 F20101209_AAAPVU havemann_s_Page_135.jpg
7c821190e9ee3c026e96d21a2c7849ad
f9d2830c6c02fd80442adeb6c774dd3455f077ff
124893 F20101209_AAAQCD havemann_s_Page_153.jp2
8b3b189d62544843ad9f3bd8e0ccf856
a81cd331102cfe87ee19fbc4d86e107bb366d5b6
118396 F20101209_AAAQBP havemann_s_Page_138.jp2
4f0a2c0a1b75a8a3583d70f338b7ce07
053863dc01fd7de7cfcac3bbce5c03723a75d1c5
56184 F20101209_AAAPWJ havemann_s_Page_150.jpg
c1716d34e638b347192a5d47845cb399
8e22dfdc27f50254def4dbeafe0e3b3669718570
6941 F20101209_AAAQZM havemann_s_Page_035thm.jpg
3ad4860ba8aeb80693c3bc5f1bbec30c
9ee11fa713ce144b79f53a5af03197169ee31ef7
23564 F20101209_AAAQYX havemann_s_Page_020.QC.jpg
8b7f809055ac49be4ce490157013403c
dbda53da2af9630a657d0d132f6ceae2d72a55ea
75637 F20101209_AAAPVV havemann_s_Page_136.jpg
1e68fdb2e9ea8d19866e971512b9518b
694231ddbd031df984dfa59b87e475374145ed35
114154 F20101209_AAAQCE havemann_s_Page_154.jp2
fa7ff1dec4923dd5635f02c39ebb6712
ba2fc6824c2368b94bbe150b8648ec5a63f6e506
116124 F20101209_AAAQBQ havemann_s_Page_139.jp2
326a7cc5bdfa2fe87107c88e314e8a50
75615ab157c1542d9d8fd3a6d45c504b52ea7141
76253 F20101209_AAAPWK havemann_s_Page_151.jpg
8caf4448af9e98c8e01268eaae227d37
8d525c9393e1262095727d644d57f42517292a59
24777 F20101209_AAAQZN havemann_s_Page_036.QC.jpg
aeae498c3e99cfbd289989e51502f5d0
5de16d9fdd7a44256dd0e833f199c716bbf0ef7f
6521 F20101209_AAAQYY havemann_s_Page_020thm.jpg
f4fa016d545709eb9e062fa9c22e76c8
5b438e39023b4821ec6fa48042ddcbff8117b978
81487 F20101209_AAAPVW havemann_s_Page_137.jpg
c45cae977ecad53b20aeb4ecfb85996b
613b293163efcdaa61260e63d1783108c0c49803
117807 F20101209_AAAQCF havemann_s_Page_155.jp2
917048c6d24753f7fe258a9c81368eba
6c968888b563a9ae9efb3674ac9f840809d95f80
114066 F20101209_AAAQBR havemann_s_Page_140.jp2
f6c0d12cb2b93f1b469bcc529d20a2b9
063e1643938c08a80337a518d407b53813edc54d
74673 F20101209_AAAPWL havemann_s_Page_152.jpg
21e617d691ba28a316b4454e135c7cc1
11bcdce9d068352c100d6cab3e8ec1d9f9246313
3788 F20101209_AAAQZO havemann_s_Page_037thm.jpg
cc7dc1398bb0d751db5e5c7a87de6d0c
291e966e5cee3418af135a5dae22ddedd8410ccf
6763 F20101209_AAAQYZ havemann_s_Page_021thm.jpg
d716af88fb2c9f3cabc9f8df753327f0
a120f7655acdd73ec4114b48914ae344e516e1fa
79061 F20101209_AAAPVX havemann_s_Page_138.jpg
2285c3a1ff8caa166b2ae3a58a374108
cdacd68b212431da60b2911c86865a5d7dde4796
111382 F20101209_AAAQCG havemann_s_Page_156.jp2
302517e20b51e8aa66d733c87fe9bf0f
cf8a39ec6a2007d77c7b28a8b33a2e39d7e2deb6
F20101209_AAAPXA havemann_s_Page_009.jp2
3c9956011b430a5caa72c53796db2f97
36c563fe620dff7d341420040e017859df158d17
104258 F20101209_AAAQBS havemann_s_Page_141.jp2
bb646b7a538255f1e466587fd4b3e3c8
702ffa7ba781b586456f019960a23a89e7eb3f2c
82473 F20101209_AAAPWM havemann_s_Page_153.jpg
65a8a8fbfe1ad1b91e880efd137a0062
37b2affbad780a8c855408297446acf66648264a
13238 F20101209_AAAQZP havemann_s_Page_038.QC.jpg
c2df0308e2f2a9b3202429f3534a6bd2
724443929581fb547dc6b7207d1ab6780080ef9e
75575 F20101209_AAAPVY havemann_s_Page_139.jpg
aa04209456a9ce7ad9c8bb6866068883
062294f00c256a5b0f45427b6c94f6bb20f96e24
108110 F20101209_AAAQCH havemann_s_Page_157.jp2
ebe0222e1d647805e2dccfb785a46c81
fa862bc6acbb8e3c727219a7374e97c2a09c6c79
1051973 F20101209_AAAPXB havemann_s_Page_010.jp2
184e454fb446f6e7a22b6dcb6227c2f8
51f6172f8f869b21a494cca33be5ecc9b023dcdb
110546 F20101209_AAAQBT havemann_s_Page_142.jp2
cb4a339888cd013a805e2dbec07d7a1a
3b3616968a5cbeea43d48ed964e9e6072e7fbdbd
75939 F20101209_AAAPWN havemann_s_Page_154.jpg
caf860739e6dad2040974a21b7647df2
3dfef3be22ed89de5545958be72fd3da74ee9824
4062 F20101209_AAAQZQ havemann_s_Page_038thm.jpg
3f2222f16df6ab1ba61e17e357471d85
f3cf4dfac5012418cb1e5f8bd3ad6b614bd3fc1e
76435 F20101209_AAAPVZ havemann_s_Page_140.jpg
530774e89f345383dcbe4687d1327541
7b9a0bd8fff152ae1a382aeeb4bf4091005de18f
114356 F20101209_AAAQCI havemann_s_Page_158.jp2
af922a1e08b59fd98b86065c59a416a6
09e9abfe69bd572847bce1129d4e87ecf051a768
1051972 F20101209_AAAPXC havemann_s_Page_011.jp2
ffdb862807a9e2d3910def36c53da87b
b6f28bd2fd982eefb5ac0f40b1922181d4b3ca70
105510 F20101209_AAAQBU havemann_s_Page_143.jp2
c5bb1e53656344eaef0fae1361875359
7e018c90a6739405f6d57b8b54097bf1b50bac42
78180 F20101209_AAAPWO havemann_s_Page_155.jpg
cc06ba2ec80612b0b239e86747dde295
38002fecbac6abc5fb0eb85c926af316ca246e1b
15570 F20101209_AAAQZR havemann_s_Page_039.QC.jpg
d9a630b36003741a7087da86371b4149
4163d7c4f58f8883f2662c344adee07a28f430e6
13083 F20101209_AAAQCJ havemann_s_Page_159.jp2
e3b5b9065b06ef9ef78fe0b137387076
268225f9819a41b43de6e31bdd29ca260658e704
45354 F20101209_AAAPXD havemann_s_Page_012.jp2
e91d7e7dbe801866ff71cc78b82664e0
47b3b1b30030eac1f1370caabd5e1907081e5c4b
11506 F20101209_AAAQBV havemann_s_Page_145.jp2
52da814afef3cb9616c1e9d613a0dc01
bcb42dc0ed265ff47de81941d2bdd62ef8851a57
73680 F20101209_AAAPWP havemann_s_Page_156.jpg
5e9e00a02b6a79d03d5b58d965ba77c6
adaad9e234b2e473d38f21ceac5e506b958ea04e
4578 F20101209_AAAQZS havemann_s_Page_039thm.jpg
911006e4503fcbbd9bb475bad871bc8a
a671e976d90ecebf329ea06531792ca6e576836e
F20101209_AAAQCK havemann_s_Page_001.tif
1b3cdb1c977bbb76127123bd9e278b20
8e7f54f356ffd4539eb87e957f4c67850f9a8018
105190 F20101209_AAAPXE havemann_s_Page_015.jp2
c41fc108af67ca128c1441edaaeeb10c
7221b97e46174014c209b3fa8177317e51f0c6e7
54206 F20101209_AAAQBW havemann_s_Page_146.jp2
afe32de8cbe1cc6d4d35602ce38e1788
52e7ca9d8519259e173141d92f36d41d958fc9ac
71553 F20101209_AAAPWQ havemann_s_Page_157.jpg
57e908dd15cd1c1d014ef9dfec21060d
7c29c92bf06e6dd7f58847da8ddd15fe365a3288
4949 F20101209_AAAQZT havemann_s_Page_040thm.jpg
ec2e0fcdfc88bbceb2c78b4bfdd624c3
63c9192ac0874964bff82c293799e951e4b48f80
F20101209_AAAQCL havemann_s_Page_002.tif
5cff903606f88eb67ff02f1fd8b14dfd
d8ddd27daa91a0bc5ab9fc3815e7037fa3a040d7
59076 F20101209_AAAPXF havemann_s_Page_016.jp2
58f1a819f5d612959873c0b62300483e
4c93f9b1b28ffae83b62238cd38172bdbb6d0eb9
794611 F20101209_AAAQBX havemann_s_Page_147.jp2
f5a26a48cfc4593fe3aa8c468d25fdbb
f117c0f784b72492e7a931dd24a1cd518ae61c17
76740 F20101209_AAAPWR havemann_s_Page_158.jpg
45754cd8980cdf45c2e9ff90a61e6327
ee79ae988b5543432c2394264108919813de298d
F20101209_AAAQDA havemann_s_Page_019.tif
50d5ae47ac3a609b0269e318e57dec11
427dd78fb801712b338c776a47c6cb6e328dfa2a
10521 F20101209_AAAQZU havemann_s_Page_041.QC.jpg
70707b332e76e3a53f50db32b23f728b
037f346bd9a13bdf144d26f8587eef7e4c74e6e5
F20101209_AAAQCM havemann_s_Page_003.tif
ac4c586d6282f7e18c2b998b4391ff5e
ee66ca4e894851dc45480c520afc48afb4883f6f
103567 F20101209_AAAPXG havemann_s_Page_017.jp2
dfa86069ce21cca9df55e7256606c19c
4179f3b3a66f642abbb2f3ccd01492cb247877bd
491248 F20101209_AAAQBY havemann_s_Page_148.jp2
fb970aa93ae5ca6f1d1b67d335c46a85
bf324834b70249b33fa103450258589210f1f8ff
14209 F20101209_AAAPWS havemann_s_Page_159.jpg
068a0f3936f041d8c12e55f4f3d8f0c7
6e590b0fe41c71caba39465ba626bd51b6f30b61
3185 F20101209_AAAQZV havemann_s_Page_041thm.jpg
36d7e9ac7579247d1ec8b4d9c912096c
f519909d63f8c40887bc2f021e2bd96ea03d049c
F20101209_AAAQCN havemann_s_Page_004.tif
45d810aca5f13632231ac3b70352eb3e
ce446a148d395bb9b8f50cb7639b0a702c5c6e30
119427 F20101209_AAAPXH havemann_s_Page_018.jp2
d5bf901c5023b9d2227e9f426707994d
485a326a6f78919f0f3435b225dc4c4a6135f9fc
383216 F20101209_AAAQBZ havemann_s_Page_149.jp2
f89a4fbc196f57bcd9c8ea7519aad3bb
e6ed66f5dfc7649d2cbb69e8abefa67eb4eb4618
23028 F20101209_AAAPWT havemann_s_Page_001.jp2
dbf863c42cbded421fd5b9cf7b7abeb5
a076d1a1838a35bd6be14ac8ebbde9aa487a598a
F20101209_AAAQDB havemann_s_Page_020.tif
98f5d440acd1135581501ec775c4db69
0012fc6f4857f303c6d356170ce077759f75871b
3128 F20101209_AAAQZW havemann_s_Page_042thm.jpg
51dc096ebfe926570ba53c1d3801153d
4d7fc0de0f38b7efc7d7ae7d47275a6c805b8561
F20101209_AAAQCO havemann_s_Page_005.tif
787508c3f15605bf56821e0645e16c89
70df02a40e7ceadbe7c6b166877b4ce64842da02
118561 F20101209_AAAPXI havemann_s_Page_019.jp2
33ac89ebf2f9515d8d95cbe56f609e6c
d8e561bec0def3b8e5e78bd86b5d2e6aac3383be
6451 F20101209_AAAPWU havemann_s_Page_002.jp2
3447edd07be5f22105ce7e6ca3fd8e86
c6f136671a2f37e30a1a1880e6fd46148c7f2f02
F20101209_AAAQDC havemann_s_Page_021.tif
d503b96083b6d6904b9bd0c1a315bd67
7211564ba6e28563c2b0a8777e6072d5ecd803d2
F20101209_AAAQCP havemann_s_Page_007.tif
04b7c31b49911205834cda3c98e1c240
1ec8d53aa22d228c81d15e143d6fe7ed5deec518
110405 F20101209_AAAPXJ havemann_s_Page_020.jp2
beb494648144cf56de2bb154201989bc
45db7289f73cf3ae9f503f13237261a4127c0cdf
F20101209_AAAQDD havemann_s_Page_022.tif
ba6c4b0b62569ba3816d1533d5d47c02
e947c02425672750c46869af22e860ab329a9780
4857 F20101209_AAAQZX havemann_s_Page_043thm.jpg
c98e4370fea32a0a3484adc6e64dded7
a931b4e7861285fe819b2d3659999927ce95c6f3
F20101209_AAAQCQ havemann_s_Page_008.tif
bc12a4f672784e142817980aa6b9a0ab
98a8bd366bb8dff8db130d9799d8d1ce576e7802
114286 F20101209_AAAPXK havemann_s_Page_021.jp2
d0dde85e42f1c96ff8865df94501ff14
bc3d75ddedd433dedd175dbcb4ec06f0aaf8c04e
10459 F20101209_AAAPWV havemann_s_Page_003.jp2
d6122ef43ea7739d7143ff622eb538f1
0cb60b3d933a9cf883f28b4af93c006b7d372d3e
F20101209_AAAQDE havemann_s_Page_023.tif
e0d65401c3c8c43404ce8d5880421580
ff5355262ac5726a49f72c40b49dc8fcdf87c331
4958 F20101209_AAAQZY havemann_s_Page_045thm.jpg
b3980a289bdcf899ca15da79ab1411c9
cc4e10755c9ed9f5631544319eb8a3a29a29a1af
F20101209_AAAQCR havemann_s_Page_009.tif
89b873ed325a38f7cd6580bc932e0b9f
cbc5cfadaf1a4c9a287a77c77a2dda4873ef53b4
109961 F20101209_AAAPXL havemann_s_Page_022.jp2
ead02106fab259fdeecaade127da879f
ec1c4c8219147cce3ed79f500e76d79c2faeb3eb
117013 F20101209_AAAPWW havemann_s_Page_004.jp2
32660245cd801b5cf1ad0558d53a19de
888a0947a766eee9fbb4d69685cd404b346a72ef
F20101209_AAAQDF havemann_s_Page_024.tif
58b15ac0a45d298e0ebdd2add7b68621
a983cfeab12f7c8f8ad4797ec608f27dc6ab3255
F20101209_AAAQZZ havemann_s_Page_046thm.jpg
099791a5c58f1e0e2aaad616b4a08edf
abd02b852d6e979aac85abde3f631558b986bb4d
873845 F20101209_AAAPYA havemann_s_Page_039.jp2
07942bce2cbe38a0112fd3c9798deb73
bbbd565ab6f48bdddde13dbc4e0812604929f30f
F20101209_AAAQCS havemann_s_Page_011.tif
bf7cbd3b5d502ab6bd910b00ac47e326
9f6361067221f8a033dcbe846c91ce84a8b1ad94
117600 F20101209_AAAPXM havemann_s_Page_023.jp2
ebf12d5383c2d03bd168ac441b99c902
50f70c861cd0c3aeb5a486d0b3fc87cdbaae4c79
21730 F20101209_AAAPWX havemann_s_Page_005.jp2
1dbbb7caa0e8abb74e8e3941eb67978a
04120ca6925e56c8dc39419ef53d8aeed698cbe2
F20101209_AAAQDG havemann_s_Page_025.tif
4c1bae4820a86f6116a9b681feffa25c
02ae7ceed6887d33c4df0551cc31fc07154d2897
972950 F20101209_AAAPYB havemann_s_Page_040.jp2
c3011fa51c0424ea5b417d6b0f63ccf0
d736d6924c4307c9eda48bf003569d21d65a1a5f
F20101209_AAAQCT havemann_s_Page_012.tif
81508051dc142e862d7f4bd9b5f8dff2
050c778c111fcd8e4825cd8723c70034744be065
111786 F20101209_AAAPXN havemann_s_Page_024.jp2
eea565663536c3a5c1a6c13e034a1998
020195ad70685eec9ee1cd19f3e0bd3fb0c29ac7
F20101209_AAAPWY havemann_s_Page_006.jp2
94ab781aa5812f36c7a31f24f98d91bc
ff673fc8ae35a5280b03f36cdd98b41ed518728a
F20101209_AAAQDH havemann_s_Page_026.tif
d17ce3c7410ff21651f0885af79ea04f
300c11b5e46bcdfa06bb56196244aa26ab933dce
436497 F20101209_AAAPYC havemann_s_Page_041.jp2
b34463c8ef451a4a3c108d3406458fa8
4228e7967288f8918e3b4f3e6d6e03df8aa75254
F20101209_AAAQCU havemann_s_Page_013.tif
f5f0fe1fa451f4b2e92764787ec3612e
f0b2f5c130a9de065d8fcc69e4946436e24fe511
116603 F20101209_AAAPXO havemann_s_Page_025.jp2
ab90536839462cd2ef25814e90674fb3
156a747992154be20b9e251300de7d93c9cf9ab1
1051967 F20101209_AAAPWZ havemann_s_Page_007.jp2
187b0ef737368fd9661692b3ca99aabc
84fe13c1fa66d0cc944c0a89848034a6285f3b2d
F20101209_AAAQDI havemann_s_Page_027.tif
d7d022f9cab1de28a6331f368c64f05e
7dc9d23806143d9872a55745acaa01d8e942629b
39605 F20101209_AAAPYD havemann_s_Page_042.jp2
dc0b1a2c0eb84f4fa02b20bb24657ccb
f9d816afc24327eba032eedc8d9022da26f997d0
F20101209_AAAQCV havemann_s_Page_014.tif
4eed1cdedd218f3263dd034c060fe180
5edb4e0291dc24f3ef3a461bfe8dd1cf1f4b6550
113660 F20101209_AAAPXP havemann_s_Page_026.jp2
b97a0700c8ffe4cd62c143d8fa1606dc
8c66f019f0b0ade33ea889d84a03758c96f3cba0
F20101209_AAAQDJ havemann_s_Page_028.tif
eead39a8f1e099c157af74db1669f7a0
36bcf9bd38ccc01ce8ae3e794cce177f6da4cb1e
82781 F20101209_AAAPYE havemann_s_Page_043.jp2
327f6e98308cbe2cb055f7efb704eaf9
633e7cff50252b68e8745550d2abc11d416fa156
F20101209_AAAQCW havemann_s_Page_015.tif
9c61d5248c830bf8c2debd557098acb1
8fa28cd5b1281aaafa623b6cd1fedef26444b931
121201 F20101209_AAAPXQ havemann_s_Page_028.jp2
2de0645c4b4b11325168b9d3a871f093
4e92fe6a71020dbd101ec864d2f4ba66a93c3490
F20101209_AAAQDK havemann_s_Page_029.tif
469163744408973e94749a08c6e2fb53
e817c8bfeef9bdeed6199ca3a8a914d9931f2297
1017465 F20101209_AAAPYF havemann_s_Page_044.jp2
9aada076aa88070148c3fd950dcfd2af
3628e4f2a02e0e4206fba7fb5ed0cc9e96160d19
F20101209_AAAQCX havemann_s_Page_016.tif
10d56e077f6d6f9831400c814b172c34
a037fad77db46ff0085f3b8ebf1addaecd0e63c4
120747 F20101209_AAAPXR havemann_s_Page_029.jp2
13180ac1ba711fc37308ba55aca1d7d7
517211989102e71b6ef933d670dcd899274349bb
F20101209_AAAQEA havemann_s_Page_047.tif
b520c1fd99a9197dfb898d21ddf2fc1a
4e321f1e2d094e667f9670b78f053e864b58cf21
F20101209_AAAQDL havemann_s_Page_030.tif
b81eb004e8d4ec68322ee6b7a1e0f8b3
077cf75134bc8e97b3a09ccfa8b0e4522f29d628
1051965 F20101209_AAAPYG havemann_s_Page_045.jp2
6a96d84201a68fc70eedef2fa5628695
f5d69801200d7334d030433cfbc59acb5a19cf28
F20101209_AAAQCY havemann_s_Page_017.tif
68369c20c8a8f1891b31908d884217f2
0c7a25c787373c03924b0b20a36a9d83d6d3dfb9
114663 F20101209_AAAPXS havemann_s_Page_030.jp2
8d4b1dd1d19cb3f4c91227aade68849b
0af2161597e375e8b234f3fa4799eb2c82a79e86
F20101209_AAAQEB havemann_s_Page_048.tif
d57fc937cddae99d36a7beea42bf3a5a
c0c23bcc2eb7dea82db868e31340607049fea2ca
F20101209_AAAQDM havemann_s_Page_031.tif
860bddb8854561eb4649fe498e482a88
40e34a513017b5b21b0c57c8842c68d0b8db4f8f
32687 F20101209_AAAPYH havemann_s_Page_046.jp2
84a6523fb26bc88465550dc6b50c46e3
0ec999912703e40eae4756f12e598f0d0c989ef5
F20101209_AAAQCZ havemann_s_Page_018.tif
050bc3188fe5ecd2eccf5e020391da2c
3f7d18332e04c200e03a01c3b1b0b16cea038450
117171 F20101209_AAAPXT havemann_s_Page_031.jp2
232ca618f7789cab2c7871d2364854bb
c6d91783c554d88374861fd62969c92cece49f46
F20101209_AAAQDN havemann_s_Page_032.tif
98734d8b14e1297f9b998dce767114e7
50942ddcfd8cc000d819cdce8de01c7d0ad55ce3
519685 F20101209_AAAPYI havemann_s_Page_047.jp2
55cf5c3edfbc9fbd79c3377ace051598
01462f33ffa8057234c2d3f5c55e7a6809712108
116385 F20101209_AAAPXU havemann_s_Page_032.jp2
aa7c97cc6bc9e0b8ae1b6a55b01315aa
892b1e7c723497c9d55b58b18c19d8fe50c2956b
F20101209_AAAQEC havemann_s_Page_049.tif
796b1d1bd5fc57f0046674851d1f4541
65b4cecd3de7e84727ee345343abf04cb37762a0
F20101209_AAAQDO havemann_s_Page_033.tif
8841643404daff738cdea9967bd46ca2
ea0d528c81bc3ef2db9178075d3597ffc67a5981
968146 F20101209_AAAPYJ havemann_s_Page_048.jp2
e532c0a3db13d342e780ebf898edf630
517de24320ff9da66f8365c9f9bc3078f8b74b67
117808 F20101209_AAAPXV havemann_s_Page_034.jp2
321e2c029c8bb298bc6ac91b28b35849
19d68d9a05b712f15486595c3c4c45130510b8d0
F20101209_AAAQED havemann_s_Page_050.tif
a7366b6092728b59dde99828b1125fa2
82068c1f1179eae81db09f4d05456c377b11fff1
F20101209_AAAQDP havemann_s_Page_034.tif
bccc6f5627afff5890edb1e2aeb8c5e0
d205e13a62d800b654c206d2871e9136fc700863
818918 F20101209_AAAPYK havemann_s_Page_049.jp2
c5d3922081fbc7a2b087b8902fa4b4f4
aaf7e38b23728ae8b5033ec2eb05dd292fe91004
F20101209_AAAQEE havemann_s_Page_051.tif
8002997ee7a529d524fde41734d1dd88
1b3956de81ac4a41d522373458389fec43131871
F20101209_AAAQDQ havemann_s_Page_035.tif
bd604c79287fa2632e1fa948adf4e5c3
6988834fd3d29032b8d7e1dd1f45b33654f32144
109752 F20101209_AAAPYL havemann_s_Page_050.jp2
32369b9db7b42dc6ffb5b5134efe8ba4
c0fd4cfe37bde14868a667cb770fce0cb8d734e3
115353 F20101209_AAAPXW havemann_s_Page_035.jp2
99749bc38f3414fa8fe244b0cf0cc688
d8549f57d6302fa851fa189c5eccd4cd582fc4fd
F20101209_AAAQEF havemann_s_Page_052.tif
def8c36d515583edf2ff052bc0ed337d
8dcb8513fc4d932b93e4f6328677047faaa5bc2e
F20101209_AAAQDR havemann_s_Page_036.tif
8bb1b73438ba44e72fcbc4cd7b28968b
f60c5f5393e9f687daba538199f27606c8352eb2
115075 F20101209_AAAPYM havemann_s_Page_051.jp2
1f541fb9ff8d91ce0ae5dc3d54c7515f
623231b4adfae9aa799bbddede08cab84e846cea
112585 F20101209_AAAPXX havemann_s_Page_036.jp2
96d334cccd3553ad628aac4fb8cda123
1cb965d42619c0a148196168ee9ab12d7479bf21
F20101209_AAAQEG havemann_s_Page_053.tif
73481a01d32057bb1e45743bc0c85bb7
77363474b013022906cea4039c570373c0fd7684
1051960 F20101209_AAAPZA havemann_s_Page_069.jp2
b94714ed79560c3e5c33fcbefdfb13a5
840b89012923ea0b1dd7077cf070df3136c92c9f
F20101209_AAAQDS havemann_s_Page_037.tif
fc241da827d1558d1ee57cc076c062d2
38d822120bb0e9990fbf1df6c240f02c46d779ab
59566 F20101209_AAAPXY havemann_s_Page_037.jp2
c6313f11e301d2e0ddafa73249be71fc
5b0a5b0a3d0c007fbba06136dc16524da7280af2
F20101209_AAAQEH havemann_s_Page_054.tif
91060cebd2eb2bdfa501b150a17acb75
b54b15981f236674f8f44d203f8918e0784c9b4c
112854 F20101209_AAAPZB havemann_s_Page_070.jp2
d0089515919892d35f9ae7e246d2968a
34679d2456498445fc733e4738d6a83cb2ccb049
F20101209_AAAQDT havemann_s_Page_038.tif
d2269ab5e9c7811608a6e81aebebc054
c23242739898db9e9f1cf6aa5524ed48cf5cd414
93164 F20101209_AAAPYN havemann_s_Page_053.jp2
c7b18818e973981281f375d8a2a12b79
9c730bb2579068cf69164ce2312ca91fb8c0f136
47801 F20101209_AAAPXZ havemann_s_Page_038.jp2
881e6070a952d532becee845861a5ae4
0cc0d998ca39bfeca6f6d961bd5d742e16c32968
F20101209_AAAQEI havemann_s_Page_056.tif
743d681eb58a631bd7238386502bcc12
0198fe6d2245b7b628c3cbbdddd6e314dabddfd9
112462 F20101209_AAAPZC havemann_s_Page_071.jp2
fdc9a92dc464f02f9ed7d9949746367e
afefa05dd6bfc932f7fc903a84713333e39b9d7b
F20101209_AAAQDU havemann_s_Page_039.tif
d8b530139e802ca8bf0469dc67377d44
80423c65a7e71203591744d1872f81fd7f4bc9e7
112750 F20101209_AAAPYO havemann_s_Page_054.jp2
3cef34aa19f1d239c4999369fd4a0ff8
0d046bf6cbce3c7410a7aed9c394b5371536caca
F20101209_AAAQEJ havemann_s_Page_057.tif
db586f566c65a5e453ad091c97cba78e
15c77cf1cd8b8279cf763812d79f4d237b4a790b
117358 F20101209_AAAPZD havemann_s_Page_072.jp2
7c9eb0ee31e0a60bc85f64f78ac0ad82
9ee6a24c946bb71ea04b6f1fbca51f3056d09477
F20101209_AAAQDV havemann_s_Page_041.tif
d2254b7dda1f2b9ce5f89cf84b012b39
692fd6b8facd4681244fd7d7ddaa779cc5f4eaaa
102861 F20101209_AAAPYP havemann_s_Page_056.jp2
21ffeccc01920fa82f7056ae7aea3ed5
7a4ead3b5d5ac1eb7c22898cfcaba205c98bb4b9
F20101209_AAAQEK havemann_s_Page_058.tif
60dd1bd86fc21e4a4558156e764b3122
6060a47cd4e199868fe9a697d533abf03a79f3be
114950 F20101209_AAAPZE havemann_s_Page_073.jp2
1b80bcf4948c9f535ce920c5396658d5
27d4db3f7d241e05a5e0593cb1b1b773cc4555e7
F20101209_AAAQDW havemann_s_Page_042.tif
0af39fb85c1e18e5b726a7b52bf4a200
b1312bc1287bf7d2de9ea9bdd1e1606c91272ba0
112631 F20101209_AAAPYQ havemann_s_Page_057.jp2
53bf714a8c76e6751f77ddc462b8c451
c6cf4d148b448de419b11121e1817c8ca0a7a8e6



PAGE 1

1 DIRECTED EVOLUTION OF DNA POLYMERASES By STEPHANIE ANN HAVEMANN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

PAGE 2

2 Copyright 2007 by Stephanie Ann Havemann

PAGE 3

3 To my family and my husband. Without your co nstant and vigilant s upport, I would not be where I am today. Thank you!

PAGE 4

4 ACKNOWLEDGMENTS I would like to begin by thanking my advisor, Dr Steven Benner for all of his wisdom and guidance; it has been an honor a nd a privilege to study under his tu telage. His passion for all facets of science, and how they can be intert wined, should serve as inspiration to us all. I would like to thank the rest of my committee: Dr. Tom Lyons, for always having an open door and an receptive ear when I had questions ; Dr. Nemat Keyhani, whose enthusiasm for science was contagious and whose knowledge of microbial genetics was extremely valuable; Dr. Nicole Horenstein, whose constant support a nd knowledge helped guide me throughout my graduate career; and Dr. Rob Ferl whose eagerness to learn and sh are information about various aspects of astrobiology helped me determin e the field of study I wish to pursue. Special thanks go to Dr. Eric Gaucher, Dr. Ryan Shaw, and Dr. Nicole Leal, all of whom have worked closely with me over the past fe w years and who have assisted me in various experimental designs and implementations. Er ic performed the rational design of the Taq mutants and was my source of knowledge for all th ings dealing with evol utionary biology. Ryan and I worked closely to discern the best method of creating and isolating DNA from oil-in-water emulsions; his idea of changing th e composition of the oil layer dr astically improved our yields. Nicole assisted me in performing some of my pr imer-extension assays and was a valuable source of information and never-ending support. I am extremely grateful to Dr. Daniel Hutter for the synthesis of the 2deoxypseudothymidine-5-triphosphate, to Dr. Shuichi Hoshika for the synthesis of the pseudothymidine precursor, and to Dr. Ajit Kamath for the synthesis and purification of the pseudouridine-containing oligonucleotides. Speci al appreciation also goes to Dr. Michael Thompson for providing the wt taq gene and his suggestions for the purification of the polymerase, and to Gillian Robbins for assisting on the growth curve studies.

PAGE 5

5 I am also thankful for the assistance of Dr. Art Edison and Omjoy Ganesh for their assistance in the circular dichroism experiments. Finally, I would like to thank all the members of the Benner group for their advice and discussions over the years, and Romaine Hughes, without whom, our group would be in total chaos.

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES................................................................................................................ .......10 LIST OF ABBREVIATIONS........................................................................................................12 ABSTRACT....................................................................................................................... ............15 CHAPTER 1 INTRODUCTION..................................................................................................................17 What are Nucleic Acids?........................................................................................................17 Rules of Complementarity...............................................................................................17 DNA Helical Conformations...........................................................................................18 Central Dogma of Molecular Biology.............................................................................19 What is AEGIS?................................................................................................................. ....20 Use of AEGIS Components.............................................................................................20 Problems with AEGIS Components................................................................................22 C-Glycosides...................................................................................................................23 Pseudouridine...........................................................................................................23 Pseudothymidine......................................................................................................24 DNA Polymerases................................................................................................................ ..25 General Structure of Polymerases...................................................................................25 Polymerase Families........................................................................................................27 Taq Polymerase...............................................................................................................28 Directed Evolution............................................................................................................. .....29 Mutagenic Libraries.........................................................................................................30 Systems of Directed Evolution........................................................................................32 Phage display............................................................................................................32 Compartmentalized self-replication.........................................................................33 Research Overview.............................................................................................................. ...34 2 POLYMERASE INCORPORATION OF MU LTIPLE C-GLYCOSIDES INTO DNA: PSEUDOTHYMIDINE AS A COMPONENT OF AN ALTERNATIVE GENETIC SYSTEM......................................................................................................................... ........50 Introduction................................................................................................................... ..........50 Materials and Methods.......................................................................................................... .52 Synthesis of Triphosphates and Oligonucleotides...........................................................52 Circular Dichroism..........................................................................................................53 Standing Start Primer-Extension Assays.........................................................................53

PAGE 7

7 Polymerase screen primer-extension assays.............................................................54 Taq polymerase primer-extension assays.................................................................55 Results........................................................................................................................ .............56 Circular Dichroism..........................................................................................................56 Polymerase Screen Primer-Extension Assays.................................................................56 Taq Polymerase Primer-Extension Assays......................................................................57 Discussion..................................................................................................................... ..........58 3 CREATION OF A RATIONALLY DESIGNED MUTAGENIC LIBRARY AND SELECTION OF THERMOSTABLE PO LYMERASES USING WATER-IN-OIL EMULSIONS...................................................................................................................... ...70 Introduction................................................................................................................... ..........70 Materials and Methods.......................................................................................................... .74 DNA Sequencing and Analysis.......................................................................................74 Construction of Plasmids.................................................................................................74 Construction of pSW1..............................................................................................74 Rationally designed mutagenic lib rary (RD Library) creation.................................75 Growth Curves and Cell Counts......................................................................................75 Purification of His(6)wt Taq Polymerase........................................................................76 Incorporation of d UTP by RD Library.........................................................................79 Selection of Thermostable Muta nts Using Water-In-Oil Emulsions..............................80 Water-in-oil emulsions.............................................................................................80 Re-cloning of selected mutants................................................................................81 Results........................................................................................................................ .............82 Growth Curves and Cell Counts......................................................................................82 Purification of His(6)wt Taq Polymerase........................................................................83 Incorporation of d UTP by RD Library.........................................................................83 Selection and Identification of Ther mostable Mutants Using Water-In-Oil Emulsions.....................................................................................................................84 Discussion..................................................................................................................... ..........85 4 DISTRIBUTION OF THERMOSTABILITY IN POLYMERASE MUTATION SPACE.103 Introduction................................................................................................................... ........103 Materials and Methods.........................................................................................................105 DNA Sequencing and Analysis.....................................................................................105 Bacterial Growth Conditions and Strains......................................................................105 Synthesis of Triphosphates and Oligonucleotides.........................................................106 Random Mutagenic Library (L4 Library) Creation.......................................................106 Incorporation of dNTPs by RD and L4 Libraries at Various Temperatures.................108 Incorporation of d UNTPs by RD Library at Optimal Temperatures..........................108 Incorporation of d UTP and d TTP by coTaq Polymerase at Various Melting Temperatures..............................................................................................................110 Results........................................................................................................................ ...........110 Random Mutagenic Library (L4 Library) Creation.......................................................110 Incorporation of dNTPs by RD and L4 Libraries at Various Temperatures.................111

PAGE 8

8 Incorporation of d UNTPs by RD Library at Optimal Temperatures..........................112 Incorporation of d UTP and d TTP by coTaq Polymerase at Various Melting Temperatures..............................................................................................................113 Discussion..................................................................................................................... ........114 5 CONCLUSIONS..................................................................................................................132 DNA Helical Structure in the Presence of C-Glycosides.....................................................132 Polymerase Screen for the Incorporation of C-glycosides...................................................133 Taq Polymerase Primer-Extension Assays...........................................................................134 Growth and Purification of Taq Polymerase........................................................................134 Creation of coTaq Polymerase Mutant Libraries................................................................136 Creation of the Rationally Designed Mutagenic Library (RD Library)........................136 Creation of the Random Mutage nic Library (L4 Library)............................................137 Preliminary Studies of the Incorporation of d UTP by the RD Library..............................137 Incorporation of dNTPs by RD and L4 Libraries at Various Temperatures........................138 Incorporation of d UTP by the RD Library at Optimal Temperatures...............................139 Incorporation of d UTP and d TTP by coTaq Polymerase at Various Temperatures......140 Selection of Thermostable RD Muta nts Using Water-In-Oil Emulsions.............................140 Future Experimentation........................................................................................................141 APPENDIX A SYNTHESIS OF PSEUDOTHYMI DINE AND PSEUDOTHYMIDINECONTAINING OLIGONUCLEOTIDES............................................................................144 B PHYLOGENETIC TREES OF FAMILY A POLYMERASES..........................................147 C GENETIC CODE AND AMINO ACID ABBREVIATIONS.............................................150 LIST OF REFERENCES.............................................................................................................151 BIOGRAPHICAL SKETCH.......................................................................................................158

PAGE 9

9 LIST OF TABLES Table page 1-1 Comparison of the structural geom etries of A, B, and Z-DNA forms...............................38 1-2 Characteristics of the various polymerase families...........................................................46 2-1 Oligonucleotides used in this study...................................................................................64 3-1 Oligonucleotides used in this study...................................................................................91 3-2 Rationally Designed (R D) Mutant Library........................................................................95 3-3 Bacterial strains used in this study.....................................................................................96 3-4 Incorporation of d UTP at 94.0 C by RD Library........................................................100 3-5 Mutations present after sele ction for active polymerases................................................101 3-6 Breakdown of types of mutati ons present after selection................................................102 4-1 Additional bacterial stra ins used in this study.................................................................120 4-2 L4 Mutant Library.......................................................................................................... ..121 4-3 Generation of full length PCR products from dNTPs by individual polymerases from the rationally designed (RD) Library at the indicated temperatures................................123 4-4 Generation of full length PCR products from dNTPs by individual polymerases from the randomly generated (L4) Library at the indicated temperatures................................124 4-5 Incorporation of d UTP by RD Library at optimal temperatures...................................126 4-6 Incorporation of d UTP and d TTP by coTaq Polymerase at various temperatures...129 C-1 The Genetic Code........................................................................................................... .150 C-2 Amino acid abbreviations................................................................................................150

PAGE 10

10 LIST OF FIGURES Figure page 1-1 The standard deoxyribonucleotides...................................................................................37 1-2 Puckering of the furanose ring of nucle osides into various envelope forms.....................39 1-3 The central dogma of molecular biology...........................................................................39 1-4 The six hydrogen bond patterns in an arti ficially expanded genetic information system (AEGIS).................................................................................................................40 1-5 The Versant branched DNA assay.................................................................................41 1-6 An example of non-standard nucleobas es coding for a non-standard amino acid.............42 1-7 Pseudouridine and pseudothymidine.................................................................................43 1-8 The polymerization reac tion of deoxyribonucleotides triphosphates catalyzed by DNA polymerases..............................................................................................................44 1-9 Kinetic steps involved in the nucleotide incorporation pathway.......................................44 1-10 Locations of active site residues in Taq polymerase.........................................................45 1-11 The staggered extension process (StEP) fo r rediversification of mutant libraries.............47 1-12 Phage display selection scheme.........................................................................................48 1-13 General scheme for CSR....................................................................................................49 2-1 A schematic representation of the CD spectra of Aand B-DNA forms...........................62 2-2 The base pairing interactions between a st andard A-T base pair and the non-standard T-A and U-A base pairs................................................................................................63 2-3 Representative CD Spectra................................................................................................65 2-4 Depiction of primer-extension assa ys used in the polymerase screen...............................66 2-5 Family A polymerase screen..............................................................................................67 2-6 Family B polymerase screen..............................................................................................68 2-7 Incorporation of one to twelve consecutive dT, d T, or d U residues by Taq polymerase..................................................................................................................... ....69 3-1 A phylogenetic tree of the Family A polymerases.............................................................89

PAGE 11

11 3-2 Locations of the 35 rationally designed (RD) sites in the Taq polymerase structure........90 3-3 View of the pASK-IBA43plus plasmid.............................................................................92 3-4 View of the pSW1 plasmid................................................................................................93 3-5 View of the pSW2 plasmid................................................................................................94 3-6 Growth curves, cell counts and expression of various E. coli TG-1 cell lines.................97 3-7 Purification and activity of His(6)wt Taq polymerase.......................................................98 3-8 Representative gels showing the amount of full-length PCR products generated with different dNTP/d UNTP ratios and the indicated polymerases........................................99 4-1 Epimerization of 2-deoxypseudouridine........................................................................119 4-2 Representative images of ethidium-bromi de stained agarose gels resolving products arising from PCR amplification using standard dNTPs and three different polymerases.................................................................................................................... ..122 4-3 Number of active RD and L4 mu tants at various temperatures.......................................125 4-4 Generation of full le ngth PCR product at 86.3 C using d UTP by the coTaq polymerase and the RD polymer ase in the SW29 cell line..............................................127 4-5 Generation of full le ngth PCR product at 94.0 C and 86.3 C using d UTP by the RD polymerase in the SW8 cell line................................................................................128 4-6 Generation of full le ngth PCR product at 86.3 C by coTaq polymerase using various TTP:d UTP and TTP:d TTP ratios..................................................................130 4-7 Graphical comparisons of the ba nd densities listed in Table 4-6....................................131 A-1 Synthesis of pseudothymidine precursor.........................................................................146 B-1 A seed alignment of the Family A polymerases..............................................................147 B-2 Inset of the phylogenetic tree of Family A polymerases (from Fig. 3-1) showing the location of Taq polymerase..............................................................................................148 B-3 Inset of the phylogenetic tree of Family A polymerases (from Fig. 3-1) showing the location of some viral polymerases..................................................................................149

PAGE 12

12 LIST OF ABBREVIATIONS A adenosine AEGIS artificially expanded genetic information system Amp ampicillin APS ammonium persulfate ATP adenosine triphosphate bp base pair Bst Bacillus stearothermophilus C cytosine Ci Curie (1 Ci = 3.7 x 107 Bequerel) CD circular dichroism Cfe cell-free extract cfu colony forming unit CPM counts per minute CNT counts CSR compartmentalized self-replication DMSO dimethyl sulfoxide dN deoxyribonucleoside (dA, dG, dC, T, T, U, etc.) DNA deoxyribonucleic acid DNase I deoxyribonucleic acid specific endonuclease ds double-stranded nucleic acid chain DTT 1,4-dithio-DL-threitol E. coli Escherichia coli

PAGE 13

13 EDTA ethylendiamino tetraacetate exolacking 3 5 exonuclease activity FLP full-length product G guanosine HIV human immunodeficiency virus type-1 hr hours HPLC high performance liquid chromatography isoC deoxyisocytidine isoG deoxyisoguanosine LB Luria-Bertani medium min minutes M-MuLV moloney murine leukemia virus mRNA messenger ribonucleic acid MWCO molecular weight cut-off NMR nuclear magnetic resonance NSB non-standard nucleobase OD optical density PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction Pfu Pyrococcus furiosus PMSF phenylmethylsulfonyl fluoride PNK polynucleotide kinase REAP reconstructing evolutionary adaptive paths

PAGE 14

14 RNA ribonucleic acid RNase A ribonucleic acid specific endonuclease rRNA ribosomal ribonucleic acid RT reverse transcriptase SDS sodium dodecylsulfate s seconds StEP staggered extension processes T thymidine T pseudothymidine Taq Thermus aquaticus DNA Polymerase I TBE Tris / borate / EDTA buffer TEMED N,N,N,N-tetramethylethylenediamine Tet tetracycline Tris tris(hydroxymethyl)aminomethane Triton X-100 octyl phenol ethoxylate tRNA transfer ribonucleic acid Tth Thermus thermophilus U uracil U pseudouridine UV ultraviolet wt wild type

PAGE 15

15 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DIRECTED EVOLUTION OF DNA POLYMERASES By Stephanie Ann Havemann May 2007 Chair: Steven A. Benner Major Department: Chemistry To achieve the long-term goal of the Benner research group to crea te a synthetic biology based on an Artificially Expanded Genetic Inform ation System (AEGIS), polymerases that are able to incorporate non-standard bases (NSBs) into DNA must be id entified. In this dissertation, a polymerase from Thermus aquaticus ( Taq Polymerase) was identified that was able to incorporate non-standard nucleotide analogs that contain a C-glycosidic linkage. This activity was limited, meaning that the polyme rase needed modification to s upport this goal. Further, we asked whether sequential C-glycosid es destabilized the duplex and al tered its structure, to better understand whether a synthetic biology based on C-glycoside nucleotides was possible. To this end, two libraries of polymerases were created to identify mutations necessary to alter the polymerases ability to withstand hi gh temperatures. One library was created by the random mutagenesis of the taq gene, the other was rationally de signed based on previous studies. Seventy-four mutants from each library were scr eened for their ability to generate a full-length polymerase chain reaction (PCR) product using sta ndard nucleoside triphosphates at various temperatures; the library of random mutants cont ained more thermostable polymerases than the library obtained by rational desi gn. Water-in-oil emulsions were then tested to determine whether these, as artificial cells might deliver thermostable polym erase variants from those used

PAGE 16

16 in the screen. This identified difficulties in tools used to analyze th e output of the library, suggesting solutions that will guide future work. We also tested the individual components of the rationally designed libr ary for their ability to incorporate Cglycoside triphosphates in a PCR. Structural studies with synthe tic DNA containing multiple, consecu tive C-glycosides showed no change in conformation, at least not one th at is detectible by circular dichroism. These results represent a step towards the goal of creating an AEGIS-based synthetic biology, an artificial ch emical system that mimics emerge nt biological behaviors such as replication, evolution, and adapta tion. In addition, the mutant polymerases created in these experiments are an inventory of polymerases useful in biotechnology, possibly allowing the development of new, as well as improving on existing, clinical diagnostic t echniques and helping to facilitate a better understanding of polymerase-DNA interactions.

PAGE 17

17 CHAPTER 1 INTRODUCTION What are Nucleic Acids? Deoxyribonucleic acid (DNA), one of the fundamental constituents of life, serves as a key component for the storage and transfer of genetic information. It is built from four building blocks, adenosine, guanosine, cytidine, and thymidine, all of which are comprised of a nucleobase attached to a 2-deoxyribose molecule (Fig. 1-1). Similarly, ribonucleic acid (RNA) is also built from four building blocks, except that thymidine is re placed by uridine and the sugar moiety is a ribose. When a phosphate group replaces the 5-hydroxyl group of these molecules, they become acids that can be linked by their phos phate groups, resulting in the formation of the backbone of a nucleic acid strand. Genetic info rmation is commonly stored in a double stranded (ds) helix, which is formed when the nucleoba ses are paired by hydrogen bonds. These helical duplex strands are aligned so that the chains are anti-parallel to one another; in other words, one strand lies in the 5 3 direction and the complement is in the 3 5 orientation. Rules of Complementarity Watson and Crick proposed that the interactio ns between nucleobases are governed by two rules of complementarity: size compleme ntarity and hydrogen-bonding complementarity (Watson and Crick, 1953a, Watson and Crick, 1953b) Size complementarity means that a large purine, such as adenosine or guanosine, pairs w ith a small pyrimidine, like cytosine, thymidine, or uridine. Hydrogen-bonding complementar ity means that hydrogen bond donors from one nucleobase pair with the hydrogen bond acceptors from another. With these rules, it is expected that in the formation of nucleic acid duplexes, guanosine must pair with cytosine and adenosine must pair with either thymidine or uridine.

PAGE 18

18 DNA Helical Conformations The conformation of a DNA duplex is often assu med to be described using one of three abstract models: A-DNA, B-DNA, or Z-DNA (Saenger, 1984). The most common form of DNA found in living organisms is presumed to be the B-DNA helix. A-DNA is the common helical structure whose geometries are described in Table 1-1. It is also in teresting to note that many other minor helical conformations of dsRN A or dehydrated DNA, but it can also be found when certain DNA sequences repeat (Ghosh and Bansal, 2003). The only left-handed helix known is the Z-DNA conformation, which appears to be a characteristic of alternating GC-rich sequences that may help stabilize DNA duri ng transcription (Rich and Zhang, 2003). Many other helical conformations of D NA are possible, of course. I ndeed, over twenty-six different forms have been described in the literature to date (Egli, 2004, Ghosh a nd Bansal, 2003, Saenger, 1984). Nevertheless, for this work, we w ill reference the A-, B-, and Z-DNA models. In actuality, the conformation of a DNA mol ecule must be described by examining the structure atom by atom. Terms used to abstract the results of such an examination are described in Table 1-1. Thus, the different types of helic es are characterized by different geometries, such as the number of base pairs per turn, the height of a turn, the rotation per base pair, the size and depth of the major and minor grooves, and the type of sugar pucker. The sugar pucker refers to the conformation of the sugar, which can ex ist in one of four envelope forms: C2-endo, C2-exo, C3-endo, and C3-exo (Fig. 1-2) (Saenger, 1984). In some cases, helical structures can be tr ansformed from one conformation into another simply by the modification of the humidity of the environment (for fibers) and/or the concentrations of salt in the so lution (Saenger, 1984). Helical structures ca n also be changed by altering the chemical structure of the constituents. The conf ormation of the sugar pucker can alter the helical form of the DNA by increasi ng or decreasing the distances between the

PAGE 19

19 phosphate groups, thereby changing the number of base pairs per turn and the size of the grooves. The C2-endo conformation is usually found in B-DNA, while the A-DNA prefers the C3-endo pucker. The major and minor grooves f ound in B-DNA can act as binding pockets for polymerases, since they allow for the pres entation of nucleobase hydrogen bond donors and acceptors (Garrett and Grisham, 1999). The grooves presented by A-DNA are more symmetrical, making it difficult for polymerases to gain access to these potential hydrogenbonding sites (Garrett and Grisham, 1999). The conformation of a DNA helix can be assesse d in several ways. X-ray crystallography is, of course, the best way to identify the posi tion of individual atoms, with nuclear magnetic resonance (NMR) emerging as a preferred choice in solution. The general overall conformation can be extimated, however, by circular di chroism (CD) (Ghosh and Bansal, 2003). Central Dogma of Molecular Biology Nucleic acids maintain genetic information inside a cell by means of replication and transcription; translation uses this genetic inform ation to create proteins. This sequence has been called the central dogma of mo lecular biology by Crick (Fig. 1-3) (Crick, 1970). DNA is transcribed into messenger RNA (mRNA) using RNA polymerases, which is then translated into proteins. The translation of the mRNA uses a combination of ribosomes, which are composed of ribosomal RNA (rRNA) and proteins and transfer RNA (tRNA), wh ich carry amino acids to the ribosomes. In situations where the genetic material is stored as RNA, such as in viruses, the information is first converted back into DNA by enzymes known as reverse transcriptases prior to being translated. DNA can replicate itself by employing enzymes known as DNA polymerases, and RNA replicates itself using RNA polymerases. This feature of life raises an obvious question: Which came first, nucleic acids or proteins? At first glance, the answer appe ars to be nucleic acids, sin ce proteins cannot store genetic

PAGE 20

20 information. Upon further study, one realizes that without proteins, the genetic material could not be replicated. One possible answ er to this question is that the nucleic acids were once able to act as both storage molecules and as proteins that could ca talyze their own replication. The discovery of ribozymes and deoxyribozymes lends support to this theory by showing that nucleic acid molecules are not limited to the ability to stor e genetic information, they can catalyze reactions both within their own structure or upon ot her structures (Muller, 2006, Emilsson and Breaker, 2002, Paul an d Joyce, 2004). Many of these nucleic acid catalysts have been created using non-standard nucleobases (N SBs) to add additional functionality to the nucleic acid molecules (Muller, 2006). What is AEGIS? Using Watson and Cricks rules of comple mentarity and the requirement that the nucleobases be joined with three hydrogen bonds, it is feasible to create an artificially expanded genetic information system (AEGIS) containing ei ght additional base pairs (Fig. 1-4), thereby expanding the genetic alphabet from four to twelve letters (Switzer et al ., 1989, Piccirilli et al., 1990, Geyer et al., 2003). Since these bases retain the Watson and Crick geometry, they can be incorporated into growing DNA strands via synthe sis, primer-extension experiments, or by the polymerase chain reaction (PCR), which can subse quently be used in a variety of different techniques. Use of AEGIS Components The importance of AEGIS components has already been illustrated in many ways. It has been used in clinical diagnos tics, to expand the genetic code to understand DNA and polymerase interactions, and has even been implicated as a factor for evolution of life on Earth. These components have also been used in the first su ccessful six-letter PCR r eaction, lending support to the development of a synthetic biology.

PAGE 21

21 The powerful Versant branched-DNA assay, used to monitor the viral load of patients infected with HIV, Hepatitis B, or Hepatitis C viruses, requires the use of at least two nonstandard nucleobases (NSBs) (Collins et al., 1997). This assay uses 5-methyl-2'deoxyisocytidine (isoC) and 5-me thyl-2'-deoxyisoguanosine (isoG) to decrease the non-specific binding of a nucleic acid probe (F ig. 1-4), thereby increasing signa l amplification relative to noise by eight-fold over previous systems used (Fig. 1-5) (Huisse, 2004, Collins et al., 1997). EraGen Biosciences (Madison, WI) is now using these AEGIS components in a similar multiplexed system to identify newborns with cystic fibrosis (Johnson et al., 2004). These assays have barely begun to scratch the surface of the potential clinical diagnostic uses of this expanded genetic alphabet. The current genetic code uses 64 three-letter codons to encode for the incorporation of 20 canonical amino acids (Appendix C); use of all twelve AEGIS nucleotides would allow for 1728 three-letter codes, and if the AEGIS compone nts were functionalize d, the possibilities are seemingly nearly endless. AEGI S components have been already been used to encode for the incorporation of non-standard am ino acids in ribosome-mediated translation. For example, in 1992 Bain et al. used isoC and isoG in a codon-anti-codon pair to generate peptides containing the non-standard amino acid L-iodotyrosine (Bain et al., 199 2). More recently, Hirao et al. used the 2-amino-(2-thienyl)purine and pyridine -2-one in a codon-anti-codon pair in an in vitro transcription study to generate peptides containi ng 3-chlorotyrosine (Fig. 1-6) (Hirao et al., 2002, Hirao et al., 2006). Some of these AEGIS components have also been used in the characterization of the kinetic parameters of polymerases (Joyce a nd Benkovic, 2004, Sismour and Benner, 2005), and in the first six-letter PCR, which was catalyzed by a mutant of the HIV-reverse transcriptase

PAGE 22

22 (Sismour et al., 2004). AEGIS components have also been us ed to better understand the interactions between polymerases and DNA (Lutz et al., 1998, Joyce and Benkovic, 2004, Hendrickson et al., 2004, Delaney et al., 2003). For example, studi es have been performed using variety of different NSBs, such as those lacki ng minor-groove electrons (Hendrickson et al., 2004) and those with a C-glycosidic linkage (Lutz et al., 1999), in order to identify characteristics of nucleobases that are esse ntial for correct incor poration by polymerases. Problems with AEGIS Components Although the AEGIS components retain Watson and Crick geometry, it is possible that some of the features present on the NSBs, such as the absence of minor groove electrons or the presence of C-glycosidic linkages, may presen t a challenge to polymerases. The ability of polymerases to function in the ab sence of an unshared pair of el ectrons in the minor grove of dsDNA, as seen in the pyDAD-puADA base pair (Fig. 1-4), was previously examined by Hendrickson et al (Hendrickson et al., 2004). In those studies, Hendrickson discovered that the presence of electrons in the minor grove ma y only be necessary for exonuclease activity of polymerases, and not for incorporation (Hendric kson et al., 2004). This, however, presents a problem when trying to incorporat e NSBs with efficiency and fi delity, since the polymerase has no proofreading ability. Lutz et al. examined the ability of polymerases to function in the presence of nucleosides exhi biting a C-glycosidic linkage, a carbon-carbon bond between the nucleobase and sugar as seen in the pyDAD, py AAD, and pyADD nucleosides (Fig. 1-4) (Lutz et al., 1999). He also reported that polymerases with exonuclease activity were less likely to accept the C-glycoside than were those lacking the proof reading ability, making replication with fidelity difficult.

PAGE 23

23 C-Glycosides An N-glycoside is a nucleoside with a carbon -nitrogen bond linking the nucleobase to the sugar; all standard nucleosides are therefore N-glycosides. However, three of the AEGIS nucleosides use a carbon-carbon bond to join the nucleobase to the sugar, making these nucleosides C-glycosides by de finition (Fig. 1-4). This car bon-carbon linkage can cause a structural change in the sugar puc ker of the nucleoside, making it a C3-endo pucker instead of a C2-endo pucker, possibly changing the form of the DNA from B-DNA to A-DNA (Davis, 1995). Wellington and Benner detailed strategies by which these molecules can be chemically synthesized in a current review article (Wellington and Benner, 2006). C-glycosides have also been found in vivo in various types of RNA, howeve r (Charette and Gray, 2000). These Cglycosides are of great interest, not only because of their presence in the AEGIS nucleosides, but also for their clinical uses; many naturally occu rring C-glycosides are antibiotics or antiviral agents (Michelet and Genet, 2005, Zhou et al., 2006) More generally, C-glycosides can be used in gene therapy (Li et al., 2003, Li et al., 2004). Pseudouridine Pseudouridine ( U), the 5-ribosyl isomer of uridine (Fig. 1-7A), is present in both tRNA and rRNA and is vital to the fitn ess of organisms (Raychaudhuri et al., 1998, Charette and Gray, 2000). This modified nucleoside, found in all th ree domains of life, wa s the first naturally occurring NSB discovered (Charette and Gray, 2000) and is introduced into the RNA sequences by the posttranscriptional modification of uridin e (Argoudelis and Mizsak, 1976, Grosjean et al., 1995). Pseudouridine has been reported to have a propensity to adopt a syn conformation around the glycosyl bond when in solution, although the data supporting this are questionable; it is, however, found only in the anti conformation when in a nucleic acid strand (Fig. 1-7B) (Lane et al., 1995, Neumann et al., 1980). The anti conformation allows the coordination of a water

PAGE 24

24 molecule between the 5 phosphate group of the U residue, the 5 phosphate group of the preceding residue, and the N1-H of the U residue (Fig. 1-7C) (Arnez and Steitz, 1994). The coordination of this water molecule results in an enhanced base stacking ability and a reduced conformational flexibility of the RNA molecule, thus increasing the local rigidity of the RNA (Charette and Gray, 2000, Davis, 1995). Pseudouridine is thought to play several roles in Nature, as described in the review by Charette and Gray (Charette and Gray, 2000). In tRNA, it is thought to play a critical role in the binding of the tRNA to the ribosome during transl ation because it stabilizes the tRNA structure, allowing tighter binding to occur, thereby increas ing translational accuracy. Pseudouridine also has been implicated in alternat ive codon usage in tRNA, and as a player in the folding of rRNA and ribosome assembly by its contributions to RNA stability. Pseudothymidine Pseudothymidine ( T), or 1-methylpseudouridine (Fig. 1-7D ), was originally isolated from Streptomyces platensis in 1976 by Argoudelis and Mizsak (Ar goudelis and Mizsak, 1976). This naturally occurring C-glycoside, found in R NA, is also thought to be created by a posttranscriptional modification of uridine (Limbach et al., 1994). The first successful in vitro transcription of T was performed by Piccirilli et al. using T7 RNA polymerases with a template containing T and standard ribonucleoside s (Piccirilli et al., 1991). Further studies, conducted by Stefan Lutz, observed the ability of DNA polymer ases to not only incor porate this NSB into a growing DNA strand in primer-exten sion assays, but also challenged a polymerase to use T in a PCR reaction that required the successful incorporation of up to three consecutive d T residues. (Lutz et al., 1999). Si nce then, no further studies requiri ng the incorporation of this Cglycoside into nucleic acid s have been performed.

PAGE 25

25 DNA Polymerases DNA polymerases are the enzymes that perfor m template directed DNA synthesis from deoxyribonucleotides and an existing DNA templa te. These enzymes, essential for the replication of the genetic information carried in all living organisms, were originally discovered in 1956 by Arthur Kornberg (Kornb erg et al., 1956), for which he was awarded a Nobel Prize in 1959. The synthesis of the complementar y DNA strand always occurs in the 5 3 direction through the addition of incoming nucleotides triphosphate group onto the 3-OH group of the preceding nucleotide, releasing a pyrophosphate gr oup in the process (Fig 1-8) (Garrett and Grisham, 1999, Lewin, 1997). After the successful replication of a DN A strand, the new strand is complementary to the template (leading) strand, and identical to the la gging strand. Since all DNA polymerases function in this manner, it is easy to comprehend that their structures are also generally conserved. General Structure of Polymerases All DNA polymerases share a common structural framework that is commonly referred to as a right hand comprised of three subdomains: th e fingers, the palm, and the thumb. The fingers domain is responsible for nuc leotide recognition and binding, the thumb domain binds the DNA substrate, and the palm domain is the catalytic center of the protein. It appears that this framework is the same in all DNA polymerase familie s. It is not clear wh ether this represents convergent or divergent evolution; there is no sequence similarity between, for example, Family A and Family B polymerases that makes a case for their distant homology (Rothwell and Waksman, 2005). In 1985, the laboratory of Thomas Steitz first solved th e crystal structure of the Klenow fragment, the C-terminal domain of the Escherichia coli DNA Polymerase I (Ollis et al., 1985). Since then, the crystal structure of ma ny different polymerases have been solved, not only in their nascent states, but some w ith DNA or dNTPs and pyrophosphate bound to the

PAGE 26

26 catalytic site (Rothwell and Wa ksman, 2005, Beese et al., 1993b, Beese et al., 1993a). It has also been determined that during polymerizat ion, divalent metal cations, such as Mg2+, are coordinated in polymerase activ e sites to help activate the 3-OH group for attack on the incoming nucleotide (Steitz, 1999). Features of polymerases that are not conser ved throughout the families include both the 5 3 and 3 5 exonuclease subdomains that allow for pr oofreading, and other subunits used for different types of repair. The exonuclease s ubdomains, when present, are the proofreading centers of the polymerase. The 5 3 exonuclease activity is usually involved in nick translation, or the synthesis of DNA at a location where there is a break in the phosphodiester bond of one strand (Perler et al., 1996). The 3 5 exonuclease activity is the true proofreading activity of the polymerase, res ponsible for the excision of a newly synthesized mismatch (Perler et al., 1996). The process by which a DNA polymerase adds an incoming nucleotide onto the 3hydroxyl group of the preceding nucleoside involves many steps, which are only now being fully understood. Figure 1-9 details the kinetic steps involved in this addition (Patel and Loeb, 2001, Rothwell and Waksman, 2005). In Step 1, the po lymerase (E) binds to the DNA primer:template complex (TP); the polymerase then binds the incomi ng nucleotide triphosphate (dNTP) in Step 2. The polymerase then undergoes a co nformational change (E) in St ep 3 that brings the various components into positions that can support the chemis try of this reaction; this is the rate-limiting step of polymerization. The polymerase perfor ms the addition of the nucleotide, remains complexed with the pyrophosphate, and undergoes another conformational change in Step 4. The pyrophosphate group is released in Step 5; in Step 6, the polymerase can dissociate from the DNA or translocate the substrate fo r another round of synthesis.

PAGE 27

27 Polymerase Families Based on sequence similarity, seven major fam ilies of homologous polymerases have been classified (Patel and Loeb, 2001, Rothwell and Waks man, 2005): A, B, C, D, X, Y, and RT. The most extensively studied are t hose of the Family A and Family B polymerases, but Table 1-2 identifies characteristics and representative polymerases of all seven families. Polymerases behave differently not only between the families, but also within the families themselves, based on their ability to repair, their processivity, and their fidelity. Processivity is defined as the ability of the polymerase to continue catalysis without dissociating from the DNA (Kelman et al., 1998); this is important when dealing with AE GIS components since it has been previously shown that polymerases tend to pause, or fa ll off the DNA, after the incorporation of a NSB (Lutz et al., 1999, Sismour and Benner, 2005). Fidelity is the ability of the polymerase to select and incorporate the correct complementary nucl eoside opposite the template from a pool of similar structures (Beard et al., 2002, Cline et al., 1996); this is importa nt to AEGIS components to guarantee that the newly replicat ed DNA contains the correct sequence. Family A polymerases, which contain some of the prokaryotic, eukaryotic, and viral polymerases, are best known for the E. coli DNA Pol I, Thermus aquaticus ( Taq ) Pol I, and the T7 DNA polymerases (Perler et al., 1996). The E. coli DNA Pol I and Taq polymerases are known as repair polymerases since they contain the 5 3 exonuclease domains, while the T7 is known as a replicative polymerase since it has a strong 3 5 exonuclease activity (Rothwell and Waksman, 2005, Kunkel and Bebenek, 2000). Family B polymerases contain representatives from prokaryotic, eukaryotic, archaeal, and viral polymerases, this is the onl y family of polymerases with me mbers from all four of these populations (Patel and Loeb, 2001). This family of polymerases is predominately involved with DNA replication, as opposed to repa ir, and exhibit extremely strong 3 5 exonuclease

PAGE 28

28 activities. In eukaryotes, thes e polymerases carry out the rep lication of chromosomal targets during cell division. The most well known of the archaeal pol ymerases from this family, Pyrococcus furiosus ( Pfu ) DNA Polymerase, has the lowest know n error rate of all thermophilic DNA polymerases that can be used for PCR amp lification (mutational fr equency/bp/duplication is 1.3 x 10-6 ) (Hogrefe et al., 2001, Cline et al., 1996). Family C polymerases contain the bacterial chromosomal replicative polymerases, and Family D polymerases are suggested to act as ar chaeal replicative polyme rases (Patel and Loeb, 2001, Rothwell and Waksman, 2005). Family X polymerases are found in eukaryotes, and are believed to play a role in the ba se-excision repair path way that is important for correcting abasic sites in DNA (Patel and Loeb, 2001, Rothwell an d Waksman, 2005). Family Y polymerases, found in prokaryotes, eukaryotes, and archaea, are part of a re plicative complex, and function by recognizing and bypassing lesions cr eated by UV damage so that replication of the DNA is not stalled (Zhou et al., 2001, Rothwell and Waksma n, 2005). The last characterized family of polymerases, the reverse transcriptases (RT), found in eukaryotes and viruses, catalyze the conversion of RNA into DNA, but they can also replicate DNA templates as well (Najmudin et al., 2000, Goldman and Marcy, 2001, Rothwell and Waksman, 2005). Taq Polymerase Thermus aquaticus an organism found in thermal spri ngs, hydrothermal vents, and even hot tap water, was first isolated by Brock and Freeze in 1969 (Brock and Freeze, 1969). Taq polymerase, a 94 kDa protein, was isol ated from this organism by Chien et al. in 1976 (Chien et al., 1976), and belongs to the Family A polymerases. This thermophilic polymerase has 5 3 exonuclease activity, but lacks the 3 5 exonuclease activity required for the proofreading ability, therefore giving this polymerase a low replication fidelity of about 8 x 10-6 (mutational frequency/bp/duplication) (C line et al., 1996). However, Taq is fairly processive with an

PAGE 29

29 average incorporation of 40 nucle otides before dissociating fr om the DNA, and it has a quick extension rate of about 100 nuc leotides per second (Pavlov et al., 2004, Perler et al., 1996). Taq polymerase, one of the most extensiv ely studied polymerases, was the first thermostable polymerase to be used in PCR; thereby eliminating the need to add additional polymerase after every round of PCR as was necessary when E. coli DNA Pol I was used for thermocycling experiments (Saiki et al., 1988). In 1995, the Steitz laboratory was the first to crystallize nascent Taq polymerase (Kim et al., 1995), and ha ve since crystallized the polymerase with DNA at the active site (Eom et al., 1996) These, and other studies, have allowed researchers to identify the active site of the polymerase and the specifi c residues which contact the DNA, the incoming nucleotides, or are involve d in metal ion chelation (Eom et al., 1996, Fa et al., 2004, Li et al., 1998b, Li et al., 1998a, Kim et al., 1995, Suzuki et al., 1996). Due to Taq polymerases lack of proofreading ability, it has been identifie d previously as a candidate for replication of DNA containing non-standard nucl eosides (Lutz et al., 1999). Taq has been used to incorporate and/ or replicate NSBs exhi biting C-glycosidic linkages (Lutz et al., 1999), NSBs lacking an unshared pa ir of electrons in the minor groove (Hendrickson et al., 2004), and nonpolar nucleoside isoteres (Morales and Kool, 2000). Directed evolution has created Taq polymerase mutants that have been used to incorporate an even larger repertoire of NSBs (Henry and Romesberg, 2005). Directed Evolution A recent review by Griffiths and Tawfik discus sed the application of techniques developed for the in vitro evolution of various proteins to increase their rate of catalysis, perform different functions, and accept new substrates (Griffiths a nd Tawfik, 2006). These procedures all select for desired enzyme characteristics from pools of millions of genes with schemes designed to link genotype to phenotype. This pr ovides a great advantage over th e older methods of screening

PAGE 30

30 mutant library members individually, because th ese approaches use a one-pot technique that allows for the testing of a la rge number of variants (2 x 108 or more) at once (Griffiths and Tawfik, 2006) Other common features of these directed evol ution systems include the development of a mutagenic library, expression of this librar y, a high-throughput assa y designed to identify individuals with the desired ch aracteristics, and a means for re shuffling mutants between rounds of selection (Brakmann, 2005, Lutz and Patrick, 2004, Arnold and Georgiou, 2003a). The most challenging part of any selection experiment is the design of the technique that will be used to isolate variants with the desired character istics (Brakmann, 2005), because you get what you select for. In other words, sc ientists may want to select for a specific characteristic of an enzyme, but if the technique is not designed corr ectly, they may end up selecting for an enzyme with a different characteristic. Mutagenic Libraries The first step in any directed evolution experi ment is to create a large library of mutant enzymes. There are many ways to accomplish th is task, varying from the rational design of mutations at selected sites to the random mutagenesis of re sidues along the length of the sequence. Francis Arnold co-authored a book with George Georgiou that gave detailed instructions on how to perform ni neteen different techniques to generate libraries for directed evolution (Arnold and Georgiou, 2003b). This book gave attention to stan dard error-prone PCR techniques that use MnCl2 instead of MgCl2 in PCR reactions catalyzed by a polymerase with low fidelity, such as Taq and to methods that could be used for the rediversification of libraries between rounds of selection, such as the staggered extension pr ocess (Fig. 1-11). An important consideration when creating a true random library of mutants is the bias of some techniques to create certain transitional or transversional mutations preferentially.

PAGE 31

31 Transitional mutations occur when one purine-pyrimidine pair is replaced with another purinepyrimidine pair; this creates four possible transi tion mutations with the standard nucleotides. Transversional mutations occur when a purinepyrimidine pair is replaced by a pyrimidinepurine pair, creating eight possibl e transition mutations when using standard dNTPs. When creating an unbiased library, someti mes it is necessary to use two or more methods in order to allow for the same approximate percentage of transitional and transversional mutations to occur. The use of the MnCl2 and Taq polymerase in an error-prone PCR allows for all four transitions and all eight transversions to occur, however the A-T to T-A transition and A-T to GC transversion tend to be more prevalent when using this technique (V artanian et al., 1996, LinGoerke et al., 1997, Arnold and Georgiou, 2003b). Biases such as this can be altered by increasing or decreasing the concen trations of some of the nucle otides in the reaction. This technique can be performed on a low budget, and can be easily modified to increase or decrease the frequency of mutagenesis by altering the concen tration of dNTPs or the number of PCR cycles (Arnold and Georgiou, 2003b). Another method of creating muta genic libraries is by rational design. The random library approach generates a large, diverse repertoire of polymerases, but a low nu mber of active clones. Guo et al. has shown that at least one-third of all random amino acid changes will result in the inactivation of a protein (Guo et al., 2004), so it is likely that a protein with more than a few random amino acid changes will be inactive. Furthermore, Guo et al. also calculated that approximately 70% of random mutations in the active sites of polymerases will result in an inactive polymerase variant (Guo et al., 2004). A desirable library for directed evolution experiments would optimally have a large, dive rse number of proteins with a high number of active clones (Hibbert and Dalby, 2005). To genera te a library such as this, the reconstructing

PAGE 32

32 evolutionary adaptive paths (REAP) approach can be used (Gaucher, 2006); this approach allows researches to modify only the sites where func tional divergence occurred within a family of polymerases. In other words, sites that, in the hi storical evolution of the polymerase, had a split conserved but different pattern of evolutionary variation, are chosen for modification. In theory, this technique has a high probability to generate new activities and functions (Gaucher, 2006). Systems of Directed Evolution Some of the more common methods used in di rected evolution experiments include phage display (Fa et al., 2004), ribos ome display (Yan and Xu, 2006), complementation (Arnold and Georgiou, 2003a), and compartmentalized self-rep lication (CSR) (Ghadessy et al., 2001, Tawfik and Griffiths, 1998). Two of these techniques, pha ge display and CSR (Henry et al., 2004), were applied to the evolution of pol ymerases to increase thermostability (Ghadessy et al., 2001), permit activity in the presence of an inhibitor (Ghadessy et al., 2001), and allow incorporation of non-standard bases (Ghadessy et al., 2004, Fa et al., 2004, Xia et al ., 2002). Both phage display and CSR systems have been successfully used to evolve Taq polymerase in vitro (Ghadessy et al., 2001, Ghadessy et al., 2004, Fa et al., 2004). Phage display The phage display directed evolution system was developed by atta ching a fragment of Taq polymerase and an oligonucleotide primer substrat e to the exterior of a phage particle via its minor phage coat protein pIII (Fa et al., 2004). Si nce there are approximately five of these coat proteins per phage, all localized to one area on the phage coat researchers were able to successfully link phenotype to ge notype (Fig. 1-12). The mutant polymerases were challenged to add non-standard nucleosides and one biotinyl ated nucleoside onto the oligonucleotide primer by template directed synthesis; those polymerases with the ability to do so were immobilized on

PAGE 33

33 streptavidin beads, and were recovered. Th e genes encoding the active polymerases were identified by sequencing, or rediversified and s huttled into another round of selection. This technique, while excellent for identifying polymer ase mutants able to incorporate a small number of non-standard bases, does not require the pol ymerase to perform a PCR; this would not be conducive to the design of an AEGIS based synthe tic biology that require s the polymerase to replicate its own gene. Compartmentalized self-replication Compartmentalized self-replication makes use of water-in-oil emulsions as a way to link genotype to phenotype, and require s polymerase mutants to replic ate their encoding gene in a PCR reaction (Tawfik and Griffiths, 1998, Ghad essy et al., 2004, Ghadessy et al., 2001, Williams et al., 2006), theoretically an excellent technique for devel oping polymerases for a synthetic biology. A library of polymerase gene va riants is cloned and ex pressed in cells (Fig.113A); the bacterial cells containing the polymerases and thei r encoding genes are then suspended in aqueous droplets in an oil emulsion. Each of these droplets, on average, contains one cell as well as the primers and dNTPs/NSBs required for PCR (Fig. 1-13B). The thermostable polymerase is released from the cell during th e first denaturing cycle of PCR, allowing replication of its encoding gene to proceed. P oorly adapted polymerases fail to replicate their encoding gene, while better-adapted polymerases succeed in replication (Fig. 1-13C). The resulting polymerase genes are then released from emulsions by extraction with ether; those encoding the most active polymerases dominate th ese clones. A run-o ff PCR using standard nucleotides prepares the DNA for recloning, which can then be subjected to another cycle of selection (Fig.1-13E). CSR has been previously used to generate Taq polymerase variants that are more thermostable (Ghadessy et al., 2001), have an incr eased resistance to inhi bitors (Ghadessy et al.,

PAGE 34

34 2001), and are able to incorporate various non-st andard bases (Ghadessy et al., 2004). More recently, Philipp Holliger and co-workers, w ho originally performed the aforementioned selections, have modified this technique to chan ge a selected region of the polymerase sequence, and replicate that region in CSR reacti ons (Ong et al., 2006). This short-patch compartmentalized self-replica tion reaction (spCSR) has alre ady been used to develop Taq polymerase variants able to func tion with both NTPs and dNTPs, a nd variants that are able to incorporate NSBs with 2-substitutions. This tec hnique allows the researcher to mutate only the active site of the polymerase, and then challenge s the polymerase to amplify the region encoding the active site; this makes it easier for polymerases with the ability to in corporate NSBs, but who lack the catalytic efficiency and processivity, to be isolated from a pool of mutants. By reducing the stringency of the initial selections, more cl ones can be isolated with the desired traits; catalytic efficiency and processivity of the polymerase can be selected for later using the polymerase sequence of the desired variant under normal CSR conditions. Research Overview To create an AEGIS, the first step should be to create or identify polymerases with the ability to incorporate multiple, consecutive NSBs into a growi ng strand of dsDNA, efficiently and faithfully. Rather than challenging a polymeras e with a gamut of NSBs containing different unique features, we decided to focus on one uniqu e characteristic of AEGIS nucleosides, the Cglycosidic linkage. Previous studies have shown that poly merases have a difficult time incorporating the non-standard ba se pairs containing a C-glycosid ic linkage (Switzer et al., 1993, Sismour et al., 2004), therefore representati ve C-glycosides, 2-deoxypseudouridine (d U) and 2-deoxypseudothymidine (d T), that could base pair with a canonical nucleotide, in order to decrease the strain on the polymerase, were selected for study (Lutz et al., 1999).

PAGE 35

35 The research presented here began with the determination of the effect of multiple, sequential C-glycosides on duplex DNA structur e, to better understand the obstacles a polymerase would have to overcome in order to incorporate bases exhi biting C-glycosides. Next, a screening of a variety of Family A and Family B polymerases, identified Taq as a polymerase that exhibited a limited ability to in corporate non-standard bases that contain a Cglycosidic linkage. However, fu rther modification of the protei n sequence of this enzyme was needed to identify a mutant Taq polymerase with an increased ability to incorporate multiple, sequential C-glycosides NSBs more efficiently. To achieve this, the second part of this dissertation focuse d on the creation of a rationally designed (RD) library of 74 mutant Taq polymerases. Variants were screened for the ability to incorporate d U in a PCR amplification of their encodi ng gene. None of these variants were shown to produce more full-length PCR product than the wild type Taq polymerase. Only 18 variants showed any activity at all in this fi rst test, even with standard dNTPs, under these reaction conditions. A rationally designed library wa s then used to perform an initial selection, by using water-in-oil emulsions to select for the active mutant polymerases we identified in our initial screen. It was postulated that the low number of activ e variants in our RD library was due to a decrease in the thermostability of the enzyme. After altering the PCR reac tion conditions to test this hypothesis, we were able to identify 33 active mutant polymerases in this library. Since this library was rationally designed, it was interesting to speculate as to whether a randomly created library of polymerase clones would tend to have increased or decreased thermostability when compared to the number of active clones in our RD library. A random library (L4) was created for this purpose, and was screened for activity at various temperatures in PCR reactions; 39

PAGE 36

36 clones were found to be active. This comparis on of the thermostability of the two libraries shows that the randomly created library has an enhanced ability to retain polymerase thermostability when compared to our rationally designed library. The RD library was designed to identify mutant s able to incorporat e non-standard bases, and not to have a high degree of thermostabilit y. Optimal temperatures for function in a PCR were determined for each of the RD variants, and the mutants were then screened for their ability to incorporate various concentrations of d U at that optimal temperature. One mutant in the pSW27 plasmid, containing the A597S, A740R, and E742V residue changes, was identified with the ability to generate, on aver age, 72% more product at all d U concentrations tested, than wt Taq polymerase at a temperature of 86.3 C. While d U is a C-glycoside with the ability to pair with 2-deoxyade nosine, it has been shown to epimerize (Wellington and Benner, 2006, Cohn, 1960, Chambers et al., 1963). Since d T cannot epimerize, due to the presence of the extra methyl group, we performed a comparative analysis between wt Taq polymerases ability to cope with d U and d T in various concentrations and at different temperatures in a PCR. Results indicated that it may be the epimerization of the nucleotide hindering the incorporation of d U, and therefore it should not be used as a model C-glycoside for directed evol ution studies. These results presented in this work represen t a significant step towards the long-term goal of creating an AEGIS-based synthe tic biology. In addition, the repe rtoire of mutant polymerases designed and created in these experiments will a ssist in creating an inventory of polymerases useful in biotechnology, possibly allowing the de velopment of new, as well as improving on existing diagnostic techniques and helping to facilitate a bett er understanding of polymeraseDNA interactions.

PAGE 37

37 N N N N NH2 O H OH H H H H HO O H OH H H H H HO N N NH2 O O H OH H H H H HO N NH O O NH N N O NH2 N O H OH H H H H HO 2'-deoxyadenosine2'-deoxyguanosine 2'-deoxythymidine2'-deoxycytosine Figure 1-1. The standard deoxyrib onucleotides. The nucleobases pair based on the two rules of complementarity: hydrogen-bonding co mplementarity, when the hydrogen bond donor from one nucleobase pairs with the hydrogen bond acceptor from another, and size complementarity, when a large purine (top row) pairs with small pyrimidine (bottom row) (Watson and Crick, 1953a, Watson and Crick, 1953b). Therefore, 2deoxyadenosine joins with 2 -deoxythymidine and 2-de oxyguanosine joins with 2deoxycytosine. When a phosphate group replaces the 5-hydroxyl group of these molecules, they become acids and can be linked by their phosphate groups to create a DNA strand.

PAGE 38

38 Table 1-1. Comparison of the structural geometries of A, B, and Z-DNA forms. GeometryA-DNAB-DNAZ-DNAHelical SenseRight-hande dRight-handedLeft-handed Helix diameter2.6 nm2.0 nm1.8 nm Repeating unit1 base pair 1 base pair2 base pairs Rotation per base pair34 36 60 /2 Rise per base pair0.256 nm0.338 nm0.38 nm Base pairs per turn111012 Pitch per turn of helix2.82 nm3.38 nm4.56 nm Major Groove Very narrow and very deep Very wide and deep Flat Minor Groove Very broad and very shallow Narrow and deep Very narrow and deep Sugar Pucker C3-endoC2-endo C: C2-endo & G: C2-exo *Data adapted from Saenger and Garrett & Gris ham (Saenger, 1984, Garrett and Grisham, 1999).

PAGE 39

39 O C' 5 N B)1' 2' 3' 4'O C' 5 N D)1' 2' 3' 4'O C' 5 N C)1' 2' 3' 4'O C' 5 N A)1' 2' 3' 4' Figure 1-2. Puckering of the furanose ring of nucle osides into various envelope forms. In the envelope form, four of the five atoms are coplanar, the remaining atom departs this plane: A) a C2-exo sugar pucker, B) a C2-endo sugar pucker, C) a C3-exo sugar pucker, and D) a C3-endo sugar pucker. B-DNA has a C2-endo pucker, while ADNA exhibits a C3-endo pucker (Saenger, 1984). Figure 1-3. The central dogma of molecular biology (Lewin, 1997, Crick, 1970). Genetic material, in the form of DNA, is first tran scribed into RNA and then is translated into proteins. On the occasion that gene tic material is stored as RNA, it first undergoes reverse transcripti on to create DNA before it is shuttled back into the system.

PAGE 40

40 Figure 1-4. The six hydrogen bond pa tterns in an artificially expanded genetic information system (AEGIS). These patterns are c onstrained by Watson and Cricks rules of complementarity and by the requirement th at the nucleobases be joined by three hydrogen bonds (Switzer et al., 1989, Pi ccirilli et al., 1990, Geyer et al., 2003, Benner, 2004, Watson and Crick, 1953a, Wa tson and Crick, 1953b). Purines are denoted by pu, pyrimidines by py, hydrogen-bond acceptors by A, hydrogen bond donors by D, and R indicates the point of attachment of the backbone. Note the presence of a C-glycosidic li nkage in the pyDAD, pyADD, and pyDDA nucleotides. N N N N N R H N H H H N N O O H R N N N N O R N H H N N N O R H H H N N N N N R H H N N O N R N NH N N O R N N N N R H H H H H O H O H H N N N N O R N N N O R N N N N N R N N O N R N O H H H H H H H H H H T aminoA acceptor donor acceptor donor acceptor donor pyA D A puD A D C G donor acceptor acceptor acceptor donor donor pyD AA puA DD X donor acceptor donor acceptor donor acceptor pyD A D puA D A isoC isoG acceptor acceptor donor donor donor acceptor pyAA D puDD A acceptor donor donor donor acceptor acceptor pyA DD p uD AA acceptor acceptor donor donor donor acceptor pyAA D p uDD A

PAGE 41

41 Signal Molecules Solid Support NSBshere improvethe signal-to-noise ratio AnalyteDNA CaptureStrand BranchedDNA NSB-containingDuplex Figure 1-5. The Versant branched DNA assay. This assay exploits the pairing of nonstandard bases (NSBs) to reduce the signa l to noise ratio 8-fold over a previous version of the assay that did not use NS Bs (Huisse, 2004, Collins et al., 1997). The branched DNA assay is used to monitor th e viral load counts of patients with the HIV, Hepatitis B, or Hepatitis C vi ruses (Collins et al., 1997).

PAGE 42

42 N N N N Ribose S N H H N H O Ribose H 2-amino-6-(2-thienyl)purine(s) pyridin-2-one(y) A) B) CUsNH2CH C H2C O O OH Cl sTC 3' 5' yAG 5' 3' Transcription Translation DNA mRNA tRNA 3-chlorotyrosine Figure 1-6. An example of non-standard nucle obases coding for a non-standard amino acid. This shows the transcription and translation (seen in B) of the nonstandard base pair (seen in A and denoted as s and y) to ge nerate a protein containing the non-standard amino acid 3-chlorotyrosine. This picture is adapted from Hirao et al (Hirao et al., 2002, Hirao et al., 2006).

PAGE 43

43 NH O O N O OH OH H H H H HO HNNH O O O OH OH H H H H HO 1 3 5 6 5 6 13 HNNH O O O OH OH H H H H HO NH HN O O O OH OH H H H H HO 5 6 1 3 6 5 3 1AntiSynH NNH O O O OH OH H H H H NNH O O O OH OH H H H H HO 5 6 13A) B) C)O P O O O O H H O P O O O D) Figure 1-7. Pseudouridine and pseudothymidine. A) This naturally occurring C-glycoside, found in RNA, is thought to be created by a posttranscriptional isomerization of uridine (Argoudelis and Mizsa k, 1976, Grosjean et al., 1995 ). B) Pseudouridine has a propensity to adopt a syn conformation around the glycosyl bond when in solution, but it is only found in the anti conformation when in a nucleic acid strand (Lane et al., 1995, Neumann et al., 1980). C) The anti conformation allows for the coordination of a water molecule be tween the 5 phosphate group of the U residue, the 5 phosphate group of the precedi ng residue, and the N1-H of the U residue (Arnez and Steitz, 1994). The coordination of this water molecule results in an enhanced base stacking ability and a redu ced conformational flexibility of the RNA molecule, thus increasing the local rigi dity of the RNA (Charette and Gray, 2000, Davis, 1995). D) The structure of pseudothymidine (1-methylpseudouridine). This naturally occurring C-glycoside, found in R NA, is also thought to be created by a posttranscriptional modification of uridine (Limbach et al., 1994).

PAGE 44

44 N O O N O O N N N N N O O O H H H P O O P HO O P O HO O N O O N O O N N N N N O O H H H PH O O HO O P O O N O O N O O N N N N N O O H H H PH O O HO O P O O O P HO O O DNA Polymerase Figure 1-8. The polymerization reaction of deoxyribonucleotid es triphosphates catalyzed by DNA polymerases. The triphosphate of th e incoming group is linked to the 3hydroxyl group of the preceding nucleoside releasing a pyrophosphate in the process; therefore DNA synthesis requires synthesis of new molecules in the 5 3 direction (Garrett and Grisham, 1999). E+TPE-TPE-TP-dNTPE'-TP-dNTPE-TP+1-PPiE-TP+1+PPiE-TP+1 123456 Figure 1-9. Kinetic step s involved in the nucleotide incorpor ation pathway. The kinetic steps involved in the addition of a nucleotide ont o a growing DNA strand (Patel and Loeb, 2001, Rothwell and Waksman, 2005). In Step 1, the polymerase (E) binds to the DNA primer:template complex (TP); the polymerase then binds the incoming nucleotide triphosphate (dNTP) in Step 2. The polymerase then undergoes a conformational change (E) in Step 3 th at brings the vari ous components into positions that can support the chemistry of this reaction; this is the rate-limiting step of polymerization. The polymerase performs the addition of the nucleotide, remains complexed with the pyrophosphate, and underg oes another conformational change in Step 4. The pyrophosphate group is released in Step 5; in Step 6, the polymerase can dissociate from the DNA or tr anslocate the substrate for another round of synthesis.

PAGE 45

45 1 MRGMLPLFEP KGRVLLV DGH HLAYRTFH AL KGLTTSRGEP VQAVYGFAKS 51 LLKALKEDGD AVIVV FDAKA PSFRHE AYGG YKAGRAPTP E DFPRQLALIK 101 ELVDLLGLAR LEVPG YEADD VLASLA KKAE KEGYEVRIL T ADKDLYQLLS 151 DRIHALHPEG YLITP AWLWE KYGLRP DQWA DYRALTGDE S DNLPGVKGIG 201 EKTARKLLEE WGSLE ALLKN LDRLKP AIRE KILAHMDDL K LSWDLAKVRT 251 DLPLEVDFAK RREPD RERLR AFLERL EFGS LLHEFGLLE S PKALEEAPWP 301 PPEGAFVGFV LSRKE PMWAD LLALAA ARGG RVHRAPEPY K ALRDLKEARG 351 LLAKDLSVLA LREGL GLPPG DDPMLL AYLL DPSNTTPEG V ARRYGGEWTE 401 EAGERAALSE RLFAN LWGRL EGEERL LWLY REVERPLSA V LAHMEATGVR 451 LDVAYLRALS LEVAE EIARL EAEV FRLAGH PF N L NSR D Q L ERVLFDELGL 501 PAIGK TEKT G KR STS AAVLE ALREAH PIVE KILQY R ELT K LK STY IDPLP 551 DLIHPR TGRL HTRFNQT ATA TG R L SSSD P N LQ NI P VR TPL GQRIRRAFIA 601 EEGWLLVAL D YSQIELRVLA HL SGDENLIR VFQEGRD IHT ETASWMFGVP 651 REAV D PLMR R AAK T IN FG VL Y GM S AH R LSQ ELAIPYEEA Q AFIERYFQSF 701 PKVRAWIEKT LE EGRRRGYV ETLFGRR R YV PDLEARV K SV R E AA ERM AF N 751 MPV Q GTAADL MKLAMVKLFP RLEEMGARML LQ VHDE LVLE APKERAEAVA 801 RLAKEVMEGV YPLAV PLEVE VGIG EDWLSA KE Figure 1-10. Locations of active site residues in Taq polymerase. Residues shown in blue are involved in contacting the DNA during polymerization; thos e shown in red indicate residues involved in metal ion coordination (Eom et al., 1996, Fa et al., 2004, Li et al., 1998b, Li et al., 1998a, Kim et al., 1995, Suzuki et al., 1996).

PAGE 46

46Table 1-2. Characteristics of the various polymerase families. FeatureABCDXYRTDomains Containing Polymerase Prokaryotes, Eukaryotes, Viruses Prokaryotes, Eukaryotes, Archaea, Viruses ProkaryotesArchaeaEukaryotes Prokaryotes, Eukaryotes, Archaea Eukaryotes, Viruses Representative Polymerases E. coli DNA Pol I; Taq Pol I; T7 DNA Pol Pfu DNA Pol I; Eukaryotic DNA Pol a E. coli Pol III(a) Pfu DNA Pol IIEukaryotic DNA Pol b E. coli DNA Pol IV; E. coli DNA Pol V HIV-RT; M-MuLV-RT; Eukaryotic telomerases General UseRepairReplicativeReplicativeReplicativeRepairReplicative/RepairReplicative FidelityGoodExcellentExcellentExcellentN/APoorGood

PAGE 47

47 A B C D E Figure 1-11. The staggered extens ion process (StEP) for rediversif ication of mutant libraries. This process has already been successfully used to rediversify libraries between rounds of selection in CSR reactions (Arnold and Georgiou, 2003b, Zhao et al., 1998, Ghadessy et al., 2001). A) Denature d template genes are primed with the same primer. B) Short fragments are pr oduced by brief primer-extension. C) In the next cycle, fragments randomly prime the templates and extend further. D) This process is repeated until full-length genes are produced. E) Full-length genes are then purified, amplified, and reclone d into a vector for another round of selection.

PAGE 48

48 Taq gene AcidPeptide Taq polymerase pIII pIII Taq gene (P60)TAGGG (T28)ATCCCA(n)GGCTCC Basicpeptide-DNAduplex (P60)TAGGG (T28)ATCCCA(n)GGCTCC Taq gene (P60)TAGGGU(n)C (T28)ATCCCA(n)GGCTCC dATP,dGTP, Biotin-16-dCTP Biotin Taq gene (P60)TAGGGU(n)C (T28)ATCCCA(n)GGCTCC StreptavidinCoatedBeads Biotin Streptavidin Taq gene DNAseIcleavageA) B) C) D) E) Figure 1-12. Phage display selection scheme. Th is details the scheme used in the directed evolution of a Taq polymerase fragment to incorp orate non-standard nucleosides into a growing DNA strand (Fa et al., 2004). A) A phage particle is displaying an acidic peptide and a mutant polymerase on the pIII minor coat protein of the phage. These coat proteins are localized to one area on the phage molecule, allowing genotype to be linked to phenot ype. B) The primer-template complex is attached to the phage particle via a basic peptide, whic h links with the acidic peptide displayed on the coat protein. C) The polymerase in corporates modified nucleotides in a primer-extension assay, which terminates with the addition of a biotinylated standard nucleotide. D) Th e biotin tag is captured by streptavidin and the entire complex is immobilized on magnetic b eads, allowing those phage particles displaying inactive polymerases to be wa shed away. E) DNase I is used to dissociate the phage complex from the DNA strands, allowing the phage displaying the active polymerase to be captured in an elution. The genes encoding the active polymerases can then be identified by se quencing and/or rediversified and shuttled into another round of selection.

PAGE 49

49 waterdropinoil plasmid plasmid PCRprimers E.coli cell E.coli cell waterdropinoil plasmid PCRprimers waterdropinoil plasmid Manycopiesofgene (ifpolisactive) Manycopiesofgene (ifpolisactive) Suspendin water-oil emulsion HeatinginfirstPCR cyclelysescell PCR temperature cycle Extract oilaway Run-offwithstandard nucleotides&reclone fornextroundof selectionA) B) C) D) E)dATP, dCTP, dGTP,TTP, and/or d UTP dATP, dCTP, dGTP,TTP, and/or d UTP Figure 1-13. General scheme for CSR. CSR allo ws for the selection of polymerases with an ability to incorporate an unnatural nucleotide using wa ter-in-oil emulsions. ) A library of polymerase gene varian ts is cloned and expressed in E. coli Spheres represent active polymerase molecules inside of a bacterial cell. B) The bacterial cells containing the polymerases and their encoding genes are suspended in aqueous droplets in an oil emulsion. C) The thermostable polymerase enzyme and encoding gene are released from the cell during the first denatu ring cycle of PCR, allowing self-replication to proceed. D) The resulting mixture of polymerase genes is released by extraction with ether. E) A single run-off PCR with standard nucleotides prepares the DNA for recloni ng and another cycle of selection.

PAGE 50

50 CHAPTER 2 POLYMERASE INCORPORATION OF MU LTIPLE C-GLYCOSIDES INTO DNA: PSEUDOTHYMIDINE AS A COMPONENT OF AN ALTERNATIVE GENETIC SYSTEM Introduction Each of the four standard nucleobases found in natural DNA (adenine, guanine, cytosine, and thymine) is joined to their sugar via a carbon-nitrogen bond. This, by definition, makes standard nucleotides N-glycosides. The nature of the glycosidic linkage is believed to have consequences on the detailed conformation of the nucleoside, including through the operation of the anomeric effect. In particular, the nature of the glycosidic bond may influence the puckering of the sugar. Unlike the standard nucleotides, the nucleotides that allow artifici ally expanded genetic information systems (AEGIS) to be created are frequently C-glycosides, which have a carboncarbon bond between the nucleobase and the sugar. This is exemplified in the case of nonstandard pyrimidines that present DonorDonor-Acceptor, Donor-Acceptor-Donor and Acceptor-Donor-Donor hydrogen bonding patterns s een in Figure 1-4. If replacing the Nglycosidic linkage by a C-glycos idic linkage changes features of the nucleoside that are important specificity determinants for polymeras es, problems are created for those seeking to expand the genetic alphabet artificially and develop a synthetic biology from an expanded genetic alphabet. Reverse transcriptases have an ability to process both DNA and RNA, whose sugars have different conformations. Reverse transcriptases therefore, should be able to accept components of an artificially expanded genetic information syst em that incorporate C-glycosides. Perhaps it is not surprising that the first reported exampl e of PCR amplification of a six letter genetic alphabet, where one the extra two letters was a Cglycoside, exploited HIV-RT (Sismour et al., 2004).

PAGE 51

51 When attempting to develop a synthetic biol ogy using C-glycosides, the physical structure of the DNA must be considered, especially since the presence of multiple, sequential Cglycosides can possibly alter the structure and stability of duplex DNA. Previous studies have shown that poly(U) poly(A) helices favor th e A-DNA form while poly(T) poly(A) helices display perfect B-DNA struct ure (Ivanov et al., 1973, Saenge r, 1984, Chandrasekaran and Radha, 1992). Circular dichroism was employed to infer the secondary structure of our DNA, since the spectra generated by ADNA and B-DNA are quite different (Fig. 21) (Ivanov et al., 1973). Duplex DNA containing one to twelve consecutive dA-d U base pairs was studied and it was determined that all remained in the B-DNA form. To take the next step towards a synthetic biology with an expande d genetic alphabet, it would be desirable to have DNA polymerases that accept multiple C-glycos ide nucleotides. To determine whether natural DNA polymerases have th is capability and the extent to which this capability is conserved, four Family A DNA pol ymerases and four Family B DNA polymerases were screened for their ability to incorporate multiple 2-deoxypseudothymidine-5-triphosphate (d TTP) and 2-deoxypseudourid ine-5-triphosphate (d UTP) across from template dA. These C-glycosides are steric analogs of thymidin e-5-triphosphate (TTP) and present the same hydrogen bonding pattern to a complementary stra nd as TTP (Fig. 2-2). Consequently, they should serve as a relatively specific probe fo r this non-standard structural feature. In these experiments, all of the polymerases tested were able to incorporate both Cglycosides to an extent; but there was room for improvement in some, such as Taq To determine the extent of Taq polymerases ability to incorporate the C-glycosides, it was screened for its ability to incorporate anywhere from one to twelve consecutive d TTP or d UTP across from template dA.

PAGE 52

52 Materials and Methods Synthesis of Triphosphates and Oligonucleotides Dr. Shuichi Hoshika, from the Foundation for Applied Molecular Evolution (FfAME, Gainesville, Florida), synthesized the pseudothymid ine precursor as described in Appendix A. Dr. Daniel Hutter (FfAME) synthesized 2-deoxypseudothymidine-5-triphosphate (d TTP) as described in Appendix A. 2-D eoxypseudouridine-5-triphosphate (d UTP) was purchased from TriLink BioTechnologies (San Diego, California). Standard deoxynucleotide triphosphates (dNTPs) of 2-deoxyadenosine-5-triphosphate (dATP), 2-deoxycytidine-5-triphosphate (dCTP), 2-deoxyganosine-5-triphosphate (dGT P), and thymidine-5-triphosphate (TTP) and were purchased from Promega Corporation (M adison, Wisconsin). Triphosphate solutions identified as d TNTPs were comprised of dATP, dCTP, dGTP, and d TTP, while those acknowledged as d UNTPs were contained dATP, dCTP, d GTP, and d UTP. The oligonucleotides used for these experiment s are listed in Table 2-1. Those sequences containing only standard nucle otides were commercially obtained from Integrated DNA Technologies (Coralville, Iowa) as desalted or PAGE (Polyacrylamide Gel Electrophoresis) purified oligonucleotides. Thos e oligonucleotides containing d U were synthesized by Dr. Ajit Kamath (University of Florida, Gainesville, Florida) and we re prepared using standard monomers and reagents (Glen Research, St erling, Virginia) on an Expedite 8909 DNA Synthesizer (PerSeptive Biosystems, Inc., Fram ingham, Massachusetts). The crude products were digested, with agitation, in 1 mL of concentrated ammonium hydroxide at 55 C for 16 hrs to release and deprotect the o ligonucleotide (Sambrook et al., 1989). The mixtures were briefly centrifuged and the supernat ants were passed through 2 m cellulose acetate syringe filters. The residual products were washed three times with 1 mL portions of sterile water. The combined

PAGE 53

53 filtrates were lyophilized to dryness and were purified by polyacrylamide gel electrophoresis (PAGE) and isolated by reversed-phase chromat ography on a silica gel as described previously (Sambrook et al., 1989). Circular Dichroism Each template, containing one through twelve consecutive dA or d U residues (T-13 through T-22 or T-23 through T-34, respectively), was annealed to its complement template, containing consecutive dT or dA residue s (T-35 through T-46 or T-47 through T-58, respectively). Reactions containe d 5 nmol of each template and 290 L of CD buffer (1 M NaCl, 10 mM Na2HPO4, 1 mM Na2EDTA at pH 7.0) for a total volume of 300 L. The mixtures were incubated for 5 min at 96 C and allowed to cool to room temperature over the course of 1 hr. The CD spectra from 200 to 320 nm, using a wave length step of 1 nm, were measured in a nitrogen atmosphere at 25 C in a 0.1 cm pathlength cuvette, us ing an Aviv Model 215 Circular Dichroism Spectrometer (Proterion Corporation, Inc ., Piscataway, NJ). Scans were performed in triplicate for each sample mixture and the data was averaged. Standing Start Primer-Extension Assays Radiolabeled primer was prepared by incubating 0.5 nmol P-1, 100 Ci -32P-ATP, 1X T4 Polynucleotide Kinase (PNK) Buffer, 50 U T4 PNK (New England BioLabs, Beverly, Massachusetts), and sterile dH2O in a final volume of 100 L, for 1 hr at 37 C. The radiolabeled primer was purif ied using the QIAquick Nucleo tide Removal Kit (Qiagen, Valencia, California) and eluted from the column in 100 L Buffer EB (10 mM Tris-HCl, pH 8.5).

PAGE 54

54 Radiolabeled template, to depict the locati on of full-length product (FLP), was prepared by incubating 50 pmol T-4, 10 Ci -32P-ATP, 1X T4 PNK Buffer, 25 U T4 PNK, and sterile dH2O in a final volume of 50 L, for 1 hr at 37 C. The radiolabeled T-4 was purified using the QIAquick Nucleotide Removal Kit, and eluted from the column in 50 L Buffer EB. 200 L DNA PAGE Loading Dye (98% formamide, 10 mM EDTA, 1 mg/mL xylene cyanol, and 1 mg/mL bromophenol blue) was added to the 1 M radiolabeled T-4 for a final concentration of 0.2 M radiolabeled T-4. Radiolabeled 10 base-pair (bp) ladder was prepared by first incubating 1.95 g 10 bp DNA Step Ladder (Promega Corporation), 30 Ci -32P-ATP, 1X T4 PNK Buffer, and sterile dH2O in a final volume of 27 L, for 1 min at 90 C. Immediately following, 30 U T4 PNK was added and the mixture was incubated for 30 min at 37 C. The radiolabeled 10 bp ladder was purified using the QIAquick Nucleotide Removal K it, and eluted from the column in 30 L Buffer EB. 120 L DNA PAGE Loading Dye was added to the 65 ng/ L radiolabeled 10 bp DNA Ladder for a final concentration of 13 ng/ L radiolabeled 10 bp DNA Ladder. Polymerase screen primer-extension assays Klenow Fragment (3 5 exo-), Bst DNA Polymerase (Large Fragment), Taq DNA Polymerase, VentR (exo-) DNA Polymerase, Deep VentR (exo-) DNA Polymerase, and Therminator DNA Polymerase were purch ased from New England BioLabs. Tth DNA Polymerase was purchased from Promega Corporation. Pfu (exo-) DNA Polymerase was purchased from Stratagene (La Jolla, California) Buffers used in these experiments were supplied by the manufacturer as follows: reactions using Bst Taq Tth Vent (exo-), Deep Vent (exo-), and Therminator were performed in 1X ThermoPol Buffer (20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100); Klenow (exo-) reactions were

PAGE 55

55 performed in 1X NEBuffer 2 (10 mM Tr is-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol); and reactions using Pfu (exo-) were performed in 1X Cloned Pfu Buffer (20 mM Tris-HCl (pH 8.8), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 0.1 mg/mL nuclease-free Bovine Serum Albumin). Optimal temperatures for polymerase function were 37 C for Klenow (exo-), 65 C for Bst and 72 C for Taq Tth Vent (exo-), Deep Vent (exo-), Pfu (exo-), and Therminator. T-4 Primer-Template complex was prepared by mixing 25 pmol ra diolabeled P-1, 200 pmol non-radiolabeled P-1, and 300 pmol non-ra diolabeled T-4, in a final volume of 15 L. The mixture was incubated for 5 min at 96 C and allowed to cool to room temperature over the course of 1 hr. For primer-extension assays, 1.5 L of the primer-template complex, 1X of the appropriate manufacturers supplied buffer, 1 U/ L of the appropriate polymerase, and sterile dH2O were used in a final volume of 9 L. Reactions were then incubated at the appropriate temperature for 30 s. Each reaction was initiated by adding 1 L of one of the followi ng: 1 mM dTTP, 1 mM d TTP, 1 mM d UTP, 1 mM dNTPs, 1 mM d TNTPs, or 1 mM d UNTPs, and incubated for two more minutes at the appropria te temperature. Reactions we re immediately quenched with 5 L of DNA PAGE Loading Dye. Samples (1 L) were resolved on denaturing PAGE gels (7 M Urea and 20% 40:1 acrylamide: bisacrylamide) and analyzed on a Molecular Imager FX System (Bio-Rad, Hercules, California). Taq polymerase primer-extension assays Primer-Template complexes were prepared by mixing 25 pmol radiolabeled P-1, 200 pmol non-radiolabeled P-1, and 300 pmol of non-radiolab eled template (T-1 through T-12), in a final

PAGE 56

56 volume of 15 L. The mixtures were incubated for 5 min at 96 C and allowed to cool to room temperature over the course of 1 hr. For primer-extension assays, 1.5 L of the appropriate primer-template complex, 1X ThermoPol buffer, 1 U/ L Taq Polymerase, and sterile dH2O were used in a final volume of 9 L. Reactions were then incubated at 72 C for 30 s. Each reaction was initiated by adding 1 L of one of the following: 1 mM dNTPs, 1 mM d TNTPs, or 1 mM d UNTPs, and incubated for two more minutes at 72 C. Reactions were immediately quenched with 5 L of DNA PAGE Loading Dye. Samples (1 L) were resolved on denaturing P AGE gels (7 M Urea and 20% 40:1 acrylamide: bisacrylamide) and analyzed on a Molecular Imager FX System (Bio-Rad). Results Circular Dichroism Duplexes were formed by annealing each template (T-13 through T-34) to its complement sequence (T-35 through T-58) creating twelve co ntrol helices containing only thymidine and twelve helices containing pseudouridine. Figure 2-3[A-E] shows a repres entative set of these spectra, specifically the spectra of duplexes containing 1, 3, 6, 9, or 12 AU base pairs. When compared to the spectra seen in Figure 2-1, all spectra are consiste nt with B-DNA being the overall conformation of all duplexe s. In addition, th e spectra representing the oligonucleotides containing the dA-d U base pairs are similar to the patter ns of the spectra containing the dA-dT base pairs. Polymerase Screen Primer-Extension Assays Four Family A and four Family B polymeras es were screened for their ability to incorporate non-standard bases e xhibiting a C-glycosidic linkage with efficiency. Polymerases were tested in both 4-base and 13-base extension assays, and were challenged to incorporate (4-

PAGE 57

57 bases) or incorporate and extend beyo nd (13-bases) four consecutive dT, d T, or d U residues across from template dA under the polymerases optimal conditions (Fig. 2-4). Reactions used TTP, d TTP, or d UTP in the 4-base extensions and either dNTPs, d TNTPs, or d UNTPs for the 13-base extension reactions. Family A pol ymerases (Fig. 2-5[A-B]) were represented by Klenow (exo-), Bst Taq and Tth ; Family B polymerases (Fig. 2-6[A-B]) were represented by Vent (exo-), Deep Vent Exo-, Pfu (exo-), and Therminator. Pfu (exo-) was the only polymerase that was not able to generate FLP when challenged to incorporate and extend beyond both of the non-standard bases. All other Family A and Family B polymerases were able to incorporate the four consecutive non-standa rd bases (NSBs) and extend beyond them, to some measure, to generate FLP. Bst and Therminator polymerases appeared to have consumed almost all of the pr imer in the course of their reactions, generating large amounts of FLP, with all of the different NTPs tested. Kl enow (exo-) and Vent (exo-) also did an exceptional job at incor porating the NSBs, but the rema inder of the polymerases did appear to have difficulty given th e intensity of the paus e sites relative to the intensity of the FLP bands. Taq Polymerase Primer-Extension Assays To replicate its own encoding polymerase gene, Taq polymerase would be required to incorporate and extend beyond four consecutive dT, d T, or d U residues. In these experiments, Taq polymerase was challenged to in corporate and extend beyond twelve consecutive dT/d T/d U residues opposite template dA. From these results (Fig. 2-7[A-B]), it was determined that Taq appears to have some difficulty incorporating twelve consecutive dT residues, as evidenced by the pausin g in those lanes, but it is stil l able to generate FLP (N+13). It is also apparent that Taq has difficulty incorporating multiple consecutive residues of C-

PAGE 58

58 glycosides, since it was not able to generate FL P when forced to incorporate five or more d T or d U residues. However, it does, generate a small amount of FLP when challe nged to insert four consecutive dT, d T, or d U residues, and therefore should be able to replicate its own gene using a C-glycoside substitute for TTP. Discussion It was first necessary to determine if the presence of multiple d U residues in doublestranded DNA would perturb the he lical structure to a point wher e there is a phase transition from B-DNA to A-DNA, perhaps making it difficult for polymerases to replicate the DNA. It is well known that poly(U) poly(A) favors the A-helicies, while poly(T) poly(A) favors B-DNA helicies (Ivanov et al., 1973, Saenger, 1984, Chandrasekaran and Radha, 1992). The distinctive differences in the CD betw een the canonical A-duplex and the canonical Bduplex structures involves a shift of the positiv e potion of the spectrum to shorter wavelengths, to 267 nm for the A-form compared to 275 nm for the B-form (Ivanov et al., 1973). A similar shift with a similar magnitude is seen in th e negative portion. Further, the Q-DNA shows a stronger Cotton effect than the B-DNA. Theref ore, to determine whether the addition of Cglycosidic units tends to drive the conformation of the duplex from B towards A, we look for an increase in the Cotton effect and a shift towards shorter wavelengths. Circular dichroism was performed on 24 duplex DNA molecules co ntaining anywhere from one to twelve consecutive d U dA or dT dA base pairs. The observed spectra (Fig. 23[A-E]) were compared to those in Figure 21, the reference spectra for canonical A and B duplexes. In all spectra containing d U, the wavelength shifted marginally (ca. 4 nm) towards longer wavelengths. This shift does not displa y a trend, however. The shift is the same no matter how many d U units are incorporat ed into the strand.

PAGE 59

59 The only possible trend is a cha nge in the relative intensity of the positive (at 275 nm) and negative (at 264 nm) band in tensities (Ivanov et al., 1973). Here the intensity of the 246 nm band and the 275 nm band both decrease. As concen trations were carefully controlled, we do not believe that this reflects a cha nge in the concentration of the oligonucleotides. This is also suggested by the intensity of signals at lo wer wavelengths, although these are notoriously compromised by any trace of impurity. Disregarding this detail, the trend is the opposite of what one expects for the conversion of the duplex structure from canonica l B to canonical A. These results provide no ev idence that addition of d U units causes the duplex structure to change from a B-DNA to an A-DNA conformation. Thus, there was no evidence to suggest that there would be a conformational problem with th e duplex structure when incorporating multiple, sequential C-glycosides. It should be mentione d, however, that CD is indicative only of the gross properties of the sy stem; it does not provide information a bout detailed structure. It is conceivable that the conformation is cha nged in a different way, or some subtly. Nevertheless, these results encouraged us to te st polymerases for their ability to work with C-glycosides. Polymerases that already display some of the desired catalytic activity, in this case the incorporation of the C-glycos ides, should facilitate in the e volution and/or creation of an AEGIS. Previous studies have shown that poly merases are able to incorporate up to three Cglycosides, but have not tested their ability to incorporate more than three multiple, sequential Cglycosides that would be required for an AEGI S (Lutz et al., 1999, Sismour et al., 2004, Piccirilli et al., 1991). Accordingly, four Fa mily A polymerases, Klenow (exo-), Bst Taq and Tth and four Family B polymerases, Deep Vent (exo-), Vent (exo-), Pfu (exo-), and Therminator, were screened for the ability to incorporate TTP, d TTP, and d UTP across from template dA in both 4-base and 13-base primer ex tension assays (Fig. 2-5[A-B] and Fig. 2-6[A-B]). In the 4-

PAGE 60

60 base extension assay, polymerases were challe nged to incorporate f our consecutive TTP, d TTP, or d UTP across from template dA during tw o-minute incubations at the optimal temperature for each enzyme. The 13-base assa y, incubated as descri bed above, took place in the presence of dCTP, dGTP, dATP, and TTP, d TTP, or d UTP, and required incorporation and extension beyond the f our consecutive TTP, d TTP, or d UTP. The Bst and Therminator polymerases appeared to have worked extremely well and consumed almost all of the primer in th e course of all of their reactions, while Pfu (exo-) did not appear to generate any 13-base FLP when presen ted with either of the two NSBs. All other polymerases generated varying amounts of FLP w ith both of the NSBs, suggesting that any of the aforementioned polymerases c ould be potential candidates for adaptation to an AEGIS, based on the qualification that the polymerase must alre ady be able to incorporate C-glycosides. However, two of these polymerases, Klenow (exo-) and Bst are not thermostable, and thus could not undergo PCR and, according to the manufactur er, Therminator is not recommended for any applications except DNA sequencing and primer-e xtension reactions, thereby making these three polymerases unlikely candidates for future studies. As in previous studies, Taq was selected as the best polymerase candidate to undergo furt her testing since it so readily accepted the consecutive non-standard ba ses (Lutz et al., 1999). In an AEGIS system, a polymerase would be required to replicate its own encoding gene with efficiency and fidelity. In order for Taq to replicate its encoding polymerase gene, it would be required to incorporate four consecutive d T or d U across from template dA. Since we have already shown that Taq can in fact incorporate and extend beyond four consecutive Cglycosides, we next tested its ability to incorporate and extend beyond up to twelve consecutive d T-dA or d U-dA base pairs. Primer extension experiments were performed under optimal

PAGE 61

61 polymerase conditions using templates T-1 throu gh T-12. Based on the results of the study (Fig. 2-7[A-B]), Taq polymerase will not readily incorporat e and extend beyond more than five consecutive C-glycosides to gene rate FLP. If this polymerase is to be used as a potential candidate for an AEGIS system, it must be modifi ed, possibly by directed evolution experiments, so that it can incorporate more of these non-standard bases.

PAGE 62

62 260300 220 0 A-DNA B-DNA Wavelength (nm) 260300 220 0 A-DNA A-DNA B-DNA B-DNA Wavelength (nm) Figure 2-1. A schematic representation of the CD spectra of Aand B-DNA forms. The dotted line indicates the pos ition of the absorption maxima (adapted from Ivanov et al. 1973 (Ivanov et al., 1973)).

PAGE 63

63 Figure 2-2. The base pairing inte ractions between a standard A-T base pair and the non-standard T-A and U-A base pairs. Note the C-glyc osidic bond (shown in blue) between the base and the sugar in both T and U.

PAGE 64

64 Table 2-1. Oligonucleotid es used in this study. OligoSequence (5' 3' Direction)Purification P-1GCG TAA TAC GAC TCA CTA TAGPAGE T-1GTT CCT GTG TCG ACT ATA GTG AGT CGT ATT ACG CDesalted T-2TTC CTG TGT CGA ACT ATA GTG AGT CGT ATT ACG CDesalted T-3TCC TGT GTC GAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-4CCT GTG TCG AAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-5CTG TGT CGA AAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-6TGT GTC GAA AAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-7GTG TCG AAA AAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-8TGT CGA AAA AAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-9GTC GAA AAA AAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-10TCG AAA AAA AAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-11CGA AAA AAA AAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-12GAA AAA AAA AAA ACT ATA GTG AGT CGT ATT ACG CDesalted T-13 CGG CGT AAA CTA TAG TGA GTC GTA TTA CGC Desalted T-14 GGC GTA AAA CTA TAG TGA GTC GTA TTA CGC Desalted T-15 GCG TAA AAA CTA TAG TGA GTC GTA TTA CGC Desalted T-16 CGT AAA AAA CTA TAG TGA GTC GTA TTA CGC Desalted T-17 GTA AAA AAA CTA TAG TGA GTC GTA TTA CGC Desalted T-18 GAA AAA AAA CTA TAG TGA GTC GTA TTA CGC Desalted T-19 GTT CAA AAA AAA ACT ATA GTG AGT CGT ATT ACG C Desalted T-20 GTC AAA AAA AAA ACT ATA GTG AGT CGT ATT ACG C Desalted T-21 GCA AAA AAA AAA ACT ATA GTG AGT CGT ATT ACG C Desalted T-22 GAA AAA AAA AAA ACT ATA GTG AGT CGT ATT ACG C Desalted T-23CAG AGA CG CTA TAG TGA GTC GTA TTA CGCPAGE T-24CGG ACG A CTA TAG TGA GTC GTA TTA CGCPAGE T-25CGG CGA CTA TAG TGA GTC GTA TTA CGCPAGE T-26GGC GA CTA TAG TGA GTC GTA TTA CGCPAGE T-27GCG A CTA TAG TGA GTC GTA TTA CGCPAGE T-28CGA CTA TAG TGA GTC GTA TTA CGCPAGE T-29GA CTA TAG TGA GTC GTA TTA CGCPAGE T-30G CTA TAG TGA GTC GTA TTA CGCPAGE T-31GAA C CT ATA GTG AGT CGT ATT ACG CPAGE T-32GAC CT ATA GTG AGT CGT ATT ACG CPAGE T-33GC CT ATA GTG AGT CGT ATT ACG CPAGE T-34G CT ATA GTG AGT CGT ATT ACG CPAGE T-35 GCG TAA TAC GAC TCA CTA TAG TCG ACA CAG Desalted T-36 GCG TAA TAC GAC TCA CTA TAG TTA CGA CCG Desalted T-37 GCG TAA TAC GAC TCA CTA TAG TTT ACG CCG Desalted T-38 GCG TAA TAC GAC TCA CTA TAG TTT TAC GCC Desalted T-39 GCG TAA TAC GAC TCA CTA TAG TTT TTA CGC Desalted T-40 GCG TAA TAC GAC TCA CTA TAG TTT TTT ACG Desalted T-41 GCG TAA TAC GAC TCA CTA TAG TTT TTT TAC Desalted T-42 GCG TAA TAC GAC TCA CTA TAG TTT TTT TTC Desalted T-43 GCG TAA TAC GAC TCA CTA TAG TTT TTT TTT GAA C Desalted T-44 GCG TAA TAC GAC TCA CTA TAG TTT TTT TTT TGA C Desalted T-45 GCG TAA TAC GAC TCA CTA TAG TTT TTT TTT TTG C Desalted T-46 GCG TAA TAC GAC TCA CTA TAG TTT TTT TTT TTT C Desalted T-47 GCG TAA TAC GAC TCA CTA TAG ACG TCT CTG Desalted T-48 GCG TAA TAC GAC TCA CTA TAG AAT CGT CCG Desalted T-49 GCG TAA TAC GAC TCA CTA TAG AAA TCG CCG Desalted T-50 GCG TAA TAC GAC TCA CTA TAG AAA ATC GCC Desalted T-51 GCG TAA TAC GAC TCA CTA TAG AAA AAT CGC Desalted T-52 GCG TAA TAC GAC TCA CTA TAG AAA AAA TCG Desalted T-53 GCG TAA TAC GAC TCA CTA TAG AAA AAA ATC Desalted T-54 GCG TAA TAC GAC TCA CTA TAG AAA AAA AAC Desalted T-55 GCG TAA TAC GAC TCA CTA TAG AAA AAA AAA GTT C Desalted T-56 GCG TAA TAC GAC TCA CTA TAG AAA AAA AAA AGT C Desalted T-57 GCG TAA TAC GAC TCA CTA TAG AAA AAA AAA AAG C Desalted T-58 GCG TAA TAC GAC TCA CTA TAG AAA AAA AAA AAA C Desalted *The represent the incorporation of a pseudouridine residue.

PAGE 65

65 Figure 2-3. Representative CD Spectra. Circular dichroism sp ectra of select double stranded templates with their complements cont aining varying amounts of dA-dT or dA-d U base pairs at 25 C. All of the spectra above ar e indicative of B-DNA (Ivanov et al., 1973). Note that the conformation does not dramatically change as the amount of U is increased. (A) The spectra of duplex es containing 1 dA-dT base pair vs. 1 dA-d U base pair. (B) The spectra of duplex es containing 3 dA-dT base pairs vs. 3 dA-d U base pairs. (C) The spectra of duplex es containing 6 dA-dT base pairs vs. 6 dA-d U base pairs. (D) The spectra of dupl exes containing 9 dA-dT base pairs vs. 9 dA-d U base pairs. (E) The spectra of dupl exes containing 12 dA-dT base pairs vs. 12 dA-d U base pairs. -20 -15 -10 -5 0 5 10 15 20 200220240260280300320 Wavelength (nm)ABS 1 A+1 T 1 A+1 pseudoU A -15 -10 -5 0 5 10 15 20 25 200220240260280300320 Wavelength (nm)ABS 3 A+3 T 3 A+3 pseudoU B -20 -15 -10 -5 0 5 10 15 20 200220240260280300320 Wavelength (nm)ABS 6 A + 6 T 6 A + 6 pseudoU C -20 -15 -10 -5 0 5 10 15 20 200220240260280300320 Wavelength (nm)ABS 9 A+9 T 9 A+9 pseudoU D -20 -15 -10 -5 0 5 10 15 20 200220240260280300320 Wavelength (nm)ABS 12 A + 12 T 12 A + 12 pseudoU E

PAGE 66

66 Figure 2-4. Depiction of primer-e xtension assays used in the polym erase screen. In the 4-base extension assays, polymerases were challenge d to incorporate up to four consecutive dT, d T, or d U residues across from template dA. In the 13-base extension assays, the polymerases were forced to incorporate and extend beyond those first four residues.

PAGE 67

67 Figure 2-5. Family A polymerase screen. Unexte nded primer is at position N; N+4 is the fulllength product (FLP) for the 4-base exte nsion assays; N+13 is the FLP for the 13base extension assays. Final concentrations: TTPs/d TTPs/d UTPs/dNTPs/ d TNTPs/d UNTPs (100 M), radiolabeled P-1 (2.5 pmol), non-radiolabeled P-1 (20 pmol), non-radiolabeled template T-4 (30 pmol), and appropriate polymerase (1 U). The mixtures were prewarmed to th e polymerases optimal temperature for 30 s and initiated with the appropriate NTP mixtur e. The mixtures were incubated at the polymerases optimal temperature for 2 mi n and immediately terminated with DNA PAGE Loading Dye (formamide, EDTA, and dyes). An aliquot (1 L) was loaded onto denaturing polyacrylamide gels (20% 7 M urea) and resolved. A) The incorporation and extension of dT and d T by various Family A polymerases. All polymerases were able to incorporate a nd extend beyond the four consecutive A-T or AT base pairs to generate some FLP in both the 4-base and 13-base extension assays. Klenow (exo-) and Bst most likely generated higher amounts of T containing FLP since their optimal temp eratures are lower than that of Taq and Tth B) The incorporation and extension of dT and d U by various Family A polymerases. All polymerases were able to incorporate and extend beyond the four consecutive A-T or AU base pairs to generate some FLP in both the 4-base and 13-base extension assays. Klenow (exo-) and Bst most likely generated higher amounts of U containing FLP since their optimal temperatures are lower than that of Taq and Tth A) B)

PAGE 68

68 Figure 2-6. Family B polymerase screen. Unexte nded primer is at position N; N+4 is the fulllength product (FLP) for the 4-base exte nsion assays; N+13 is the FLP for the 13base extension assays. Final concentrations: TTPs/d TTPs/d UTPs/dNTPs/ d TNTPs/d UNTPs (100 M), radiolabeled P-1 (2.5 pmol), non-radiolabeled P-1 (20 pmol), non-radiolabeled template T-4 (30 pmol), and appropriate polymerase (1 U). The mixtures were prewarmed to th e polymerases optimal temperature for 30 s and initiated with the appr opriate triphosphate mixture. The mixtures were incubated at the polymerases optimal temperature for 2 min and immediately terminated with DNA PAGE Loading Dye (formamide, EDTA, and dyes). An aliquot (1 L) was loaded onto denaturing polyac rylamide gels (20%, 7 M urea) and resolved. A) The incorporati on and extension of dT and d T by various Family B polymerases. All polymerases, except Pfu (exo-), were able to incorporate and extend beyond the four consecutive A-T or AT base pairs to generate some FLP in both the 4-base and 13 -base extension assays. Pfu (exo-) was able to generate FLP in the 4-base assay, but not the 13-b ase assay. Therminator was extremely adept at incorporating the d T residues, as depicted by the low levels of unextended primer remaining in those lanes. B) Th e incorporation and extension of dT and d U by various Family B polymerases. All polymerases, except Pfu (exo-), were able to incorporate and extend beyond the four consecutive A-T or AT base pairs to generate some FLP in both the 4-ba se and 13-base extension assays. Pfu (exo-) was able to generate FLP in the 4-base assa y, but not the 13-base assay. Therminator was extremely adept at incorporating the d U residues, as depicted by the low levels of unextended primer remaining in those lanes. A ) B )

PAGE 69

69 Figure 2-7. Incorporation of one to twelve consecutive dT, d T, or d U residues by Taq polymerase. Unextended primer is at pos ition N; FLP is denoted by N+13 in all of these assays (see Table 2-1 for oligonucle otides used). Final concentrations: dNTPs/d TNTPs/d UNTPs (100 M), radiolabeled P-1 (2.5 pmol), nonradiolabeled P-1 (20 pmol), non-radiolabel ed templates T-1 through T-12 (30 pmol), and Taq polymerase (1 U). The mixtures we re prewarmed to 72 C for 30 s and initiated with the appropriate NTP mixture. The mixtures were incubated at 72 C for 2 min and immediately terminated w ith DNA PAGE Loading Dye (formamide, EDTA, and dyes). An aliquot (1 L) was loaded onto denaturing polyacrylamide gels (20%, 7 M urea) and reso lved. A) The incorporati on and extension of 1 to 12 dT or d T residues across from template A by Taq polymerase. It appears that very little to no FLP is generated after the inco rporation of five or more consecutive d Ts. B) The incorporation and extension of 1 to 12 dT or d U residues across from template A by Taq polymerase. It appears th at very little to no FLP is generated after the inco rporation of five or more consecutive d Us. A) B)

PAGE 70

70 CHAPTER 3 CREATION OF A RATIONALLY DESIGNED MUTAGENIC LIBRARY AND SELECTION OF THERMOSTABLE POLYMERASES USING WATER-IN-OIL EMULSIONS Introduction To create synthetic biology us ing an artificially expanded genetic information system (AEGIS), a polymerase that is capable of incorporating non-sta ndard nucleotides (NSBs) is needed. Unfortunately, studies have not found an extant thermostable polymerase able to incorporate a variety of NSBs with efficiency and fidelity. Polymerases usually perform more efficiently with one type of NSB, than they do with another (Hendr ickson et al., 2004, Leal et al., 2006, Roychowdhury et al., 2004). Directed evolution may help to rectify this situation and allow us to mutate an existing polymerase to generate one with an increased abili ty to incorporate a variety of NSBs (Ghadessy et al., 2001, Ghadessy et al., 2004). Therefore, we became interested in directed evolution as a way to modify Taq polymerase to better incorporate NS Bs, specifically ones exhibiting a Cglycosidic linkage. Taq polymerase, a member of the Family A pol ymerases, has already been successfully evolved under direction to incorporate various ot her NSBs using directed evolution (Ghadessy et al., 2001, Ghadessy et al., 2004, Fa et al., 2004). Ghadessy et al. provided a procedure for doing so using water droplets in oil (Ghadessy et al ., 2004, Ghadessy et al., 2001); these served as artificial cells. They began with large, diverse rando m libraries of the Taq polymerase, with approximately 7 amino acid residue replacements. Ghadessy et al. found that three to four rounds of selection was sufficien t to identify a polymerase able to incorporate various NSBs using these random libraries. This result was initia lly surprising, as Guo et al. has shown that approximately one-third of all random multiple amino acid changes will result in the inactivation of a protein, and that 70%

PAGE 71

71 of random changes in the active si te of a polymerase will also re sult in inactivation (Guo et al., 2004). This implies that a protein having more than a few random amino acid changes has a high likelihood of being inactive. One might have expected that a very large fraction of the variants created by Ghadessy et al. would have been inactive, es pecially at high temperatures, and this expectation is consistent with results reported below. This raises a general question: What is th e likelihood that a librar y contains a protein having a novel but desirable property? A desirabl e library for directed would optimally have a large, diverse number of proteins with a high nu mber of active clones (H ibbert and Dalby, 2005). One approach to achieving this goal involves the se lection of sites to in troduce replacements. For example, if replacements th roughout the protein are equally lik ely to lower thermal stability, while replacements in sites near the active site are more likely to change catalytic behavior, it makes sense to focus randomization in residue s near the active site (Arnold and Georgiou, 2003b, Arnold and Georgiou, 2003a, Fa et al., 2004, Miller et al., 2006, Ghadessy et al., 2004, Ghadessy et al., 2001). An alternative approach recognizes that natu ral history has already explored polymerase sequence space. Much of this natural history is available to us in genomic sequence databases. This permits an approach, origin ally called evolutionary guidance, that extracts information from that history to identify sites that are more likely to influence behavior in a way that is desired, and less likely to damage the enzy me (Allemann et al., 1991, Presnell and Benner, 1988). Eric Gaucher, at the Founda tion for Applied Molecular E volution (FfAME), recently developed this approach a step further under the reconstructing evolutionary adaptive paths (REAP) rubric (Gaucher, 2006). He identified s ites where functional diverg ence occurred within

PAGE 72

72 a family of polymerases, but where natural hi story suggested that th e site was under strong selective pressure. In theory, this has the hi ghest probability to generate new activities and functions. Using the sites identified by the REAP appro ach, the Type II sequence divergence of the Family A polymerases was studied (Gu, 2002, Gu, 1999). In this approach, sites were identified that had a split conserved but di fferent pattern of historical evolutionary variation, and had been previously suggested to lead to a change in the function or behavior of the polymerase. Using Pfam (Fig. 3-2), a total of 57 amino acid changes across 35 sites within the 719 members of Family A polymerases that were available we re identified (Bateman, 2006, Finn et al., 2006). The 35 sites for mutational studies, distributed as seen in Figure 3-2, we re derived from these analyses, and from sequences discussed in a recent review by Henry and Romesberg on the evolution of novel polymerase activities (Hen ry and Romesberg, 2005). The 57 replacement amino acid residues were selected based on the Family A viral polymerase sequences at the 35 mutational sites. The viral sequences were exploited since literature has told us that viral polymerases are more adept at incorporating NS Bs than other polymerases (Sismour et al., 2004, Leal et al., 2006, Horlacher et al ., 1995), and ancient viruses have also been implicated in the origins of cellular DNA replication machinery (Forterre, 2006). The company DNA 2.0 created and synthesized the rationally designed (RD) library containing 74 different mutants using the 57 amino acid chan ges identified by REAP, in various combinations to yield three or four amino acid mutations per sequence. In addition to creating the library, DNA 2.0 also designed a nd generated a version of the wt taq polymerase gene that was optimized for codon usage in E. coli cells (co -Taq polymerase). The optimization of codon usage results in higher expression levels of the protein within th e cell (Gustafsson et al., 2004).

PAGE 73

73 Each of these 75 polymerases (co -Taq and the 74 mutants) were tested for their ability to incorporate increasing concentrations of a representative C-glycoside (Fig. 2-3), 2deoxypesuouridine-5triphosphate (d UTP). None were able to incorporate d UTP more efficiently than the coTaq polymerase, and only eighteen of the 74 mutants of the RD Library showed activity with the canonical dNTPs under the conditions with which they were presented. Selections require that some members of the library perform differently than the original protein of interest (Arnold and Georgiou, 2003a, Lutz and Patrick, 2004). We did not perform a selection to identify a polymerase with an increased ability to incorporate d UTP, since we determined there were no clones in the RD Libr ary that functioned with the NSB better than coTaq polymerase. In order to demonstrat e our laboratories ab ility to perform in vitro selections, we decided select for the eighteen mutant polyme rases that exhibited activity with dNTPs from the pool of 74 mutants. To perform our selection experi ments, we used a variation of the compartmentalized selfreplication (CSR) method developed in the laboratories of Griffith s and Holliger to create waterin-oil emulsions as a way to link genotype to phenotype (Miller et al., 2006, Tawfik and Griffiths, 1998, Ghadessy et al., 2001, Ghadessy et al., 2004). This method (Fig. 1-13) uses cells expressing the polymerase as the sole source of polymerase and plasmid template in a PCR reaction, which takes place inside the aqueous ph ase of the emulsion. Inactive polymerases fail to replicate their encoding gene, so they are e ffectively selected agains t after the extraction of products from the emulsion. After our selection, products we re recloned into the expression vector using a version of the megaprimer PCR method (Miyazaki and Take nouchi, 2002). As this protocol generated products that were crossover mutations, sequenc ing of the products provided a list of the

PAGE 74

74 mutations that survived the sele ction, without providing informa tion about which mutations were associated with each other. The megaprimer PCR is, nevertheless, an effective method for library rediversification be tween rounds of selection. Materials and Methods DNA Sequencing and Analysis DNA sequencing was carried out by the University of Florida Interdis ciplinary Center for Biotechnology Research, DNA Sequencing Core Facility using an ABI 3130 xl Genetic Analyzer (Applied Biosystems, Foster City, California) an d primers P-6 through P-9 (Table 3-1). BLAST 2 software was used for sequence similarity searching (Tatusova and Madden, 1999); Dertis Reverse and/or complement DNA sequences website was used to find the reverse complement of various DNA strands (Derti, 2003); and ExPASys translate tool was used to translate DNA sequences into their amino acid counterparts (Swiss Institute of Bioinformatics, 1999). Construction of Plasmids Construction of pSW1 The gene ( wt taq ) encoding wt Taq polymerase was cloned fro m a vector generously donated by Dr. Michael Thompson (UNC, Chapel Hi ll, North Carolina) using primers P-2 and P3. The product was digested with the Sac II and Nco I restriction enzymes (New England BioLabs, Beverly, Massachusetts) according to manufacturers protocol The restricted wt taq was then ligated into the identically digested pASK-IBA43plus vector (IBA GmbH, St. Louis, Missouri)(Fig. 3-3), using T4 DNA ligase (New England BioLabs) according to manufacturers protocol (16 C overnight with a 4:1 insert:vector ratio) to make the new plasmid pSW1 (Fig. 34), and adding an N-terminal hexahistidine tag onto the wt taq gene (His(6)wt Taq ). Plasmid constructs were verified by restrictio n digest analysis, using the enzymes Bam HI and Nco I according to the manufacturers protocol (New England BioLabs), as well as sequencing.

PAGE 75

75 Rationally designed mutagenic library (RD Library) creation DNA 2.0 (Menlo Park, California) synthesized a variant of the wt taq polymerase gene (cotaq ) that was optimized for the codon-usage of E. coli which was then used to construct the pSW2 plasmid (Fig. 3-5). Plasmids pSW3 pSW76 (Table 3-2) were designed by Dr. Eric Gaucher (Foundation for Applied Molecular Evol ution, Gainesville, Florida) and DNA 2.0 using the REAP approach. Sequence alignments a nd phylogenetic tree constr uction of 719 Family A polymerase protein sequences were generated us ing the Pfam website (Bateman, 2006, Finn et al., 2006). Type II functional divergence between the bact erial/eukaryotic Family A polymerases and the viral Family A polymerases was estimated with DIVERGE 2.0 software (Gu, 2002, Gu, 1999). The 35 sites for mutational stud ies were derived from these analyses, as well as sequences discussed in Henry and Romesberg (Henry a nd Romesberg, 2005); the replacement amino acid residues were selected base d on the viral sequences at those sites. The sites chosen are all located in or ne ar the active site of the polymerase. DNA 2.0 randomized the mutations throughout the 74 sequences so they were equally distributed (3 to 4 amino acid changes per gene). In addition to the synthesis of the genes, DNA 2.0 cloned all 75 of these plasmids (cotaq and 74 mutants) into the pASK-IBA43plus vector using the Sac II and Nco I restriction sites. Plasmid construc ts were verified both by restriction digest analysis, using the enzymes Bam HI and Nco I according to the manufacturers protocol (New England BioLabs), and by sequencing. Growth Curves and Cell Counts The bacterial strains used in this study are list ed in Table 3-3. The rich media used in these studies was Luria-Bertani (LB) medium (Difco Laboratories, Detroit, Michigan) (Miller, 1972). Ampicillin was provided in liquid or solid medium at a final concentration of 100 g/mL. Plasmids were transformed into the E. coli TG-1 cell line according to manufacturers protocol

PAGE 76

76 (Zymo Research, Orange, Californi a). Cell growth was determined by measuring optical density at 550 nm using a SmartSpec Plus Spectropho tometer (Bio-Rad, Hercules, California). Anhydrotetracycline (2 mg/mL stock in N,N -dimethylformamide) was used at a final concentration of final concentration of 0.2 ng/ L to induce expression. Inocula for the growth experiments were prepar ed as follows: bacterial strains were grown overnight (14.25 hrs) at 37 C and 250 rpm in LB medium (suppl emented with ampicillin, if applicable) in 14 mL 2059 Falcon Tubes (BD Biosci ences, San Jose, California). Cells (1 mL) from the 5 mL overnight culture were used to inoculate 100 mL LB or LB-Amp cultures in 500 mL baffled flasks. Cultures were grown at 37 C and 250 rpm for 8.75 hrs. Cell counts were measured by performing a dilution series using 10-fold dilutions of the cel ls in 0.85% NaCl. Dilutions were plated onto LB plates (supplem ented with ampicillin, if applicable), grown overnight at 37 C, and colonies were counted the next morning to determine the number of colony-forming units per milli liter of culture (cfu/mL). Samples of cells were taken at various time points to determine the levels of protein expression, before and after induction. 2X SD S-PAGE (62.5 mM, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 5% -mercaptoethanol (Laemmli, 1970))loading dye was added to the samples, and to 50 U Taq Polymerase (New England BioLabs). Samples were boiled for 8 minutes, then loaded onto a Tris-HCl Ready Gel (7.5%, Bio-Rad) and resolved for 45 min at 200 V. Gels were stained via the Fair banks Method (Fairbanks et al., 1971). Purification of His(6)wt Taq Polymerase The SW3 cell line was grown overnight in 5 mL of LB-Amp broth for 14.25 hr at 37 C and 250 rpm in 14 mL 2059 Falcon Tubes (B D Biosciences). Approximately 2 x 108 colonyforming units (cfu), roughly equal to 500 L of a culture with an OD550nm of 4.0, were used to

PAGE 77

77 inoculate two 100 mL cultures of LB-Amp in 500 mL baffled flasks. These cultures were grown at 37 C and 250 rpm for 3.75 hrs to an approximate OD550nm of 1.8, and were then induced by addition of anhydrotetracycline (0.2 ng/ L final concentration). The cells were allowed to grow for an additional 5 hrs to an approximate OD550nm of 3.5. Samples of the undinduced and induced cells were take n and stored at -20 C for further analysis. Cultures were then combined and the cells harvested by centrifuga tion (9000 rpm, 10 min, 4 C). The SW3 cells were washed in 40 mL of Cell Harvest Buffer ( 50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1 mM EDTA, 4 C) and centrifuged again (8000 rpm, 10 min, 4 C). The cell pellet was then resuspended in Cell Lysis Buffe r (20 mM Tris-HCl, pH 7.9, 50 mM NaCl, 5 mM imidazole, 1 mg/mL lysozyme, 5 g/mL DNaseI, and 10 g/mL RNaseI) at a concentration of 2 mL/gram of cells. The cells were gently lysed by rocking (Gyr oMini Nutating Mixer) at ambient temperature for 15 min, the proteins were th en denatured by heating to 75 C for 20 min. The lysed cells were centrifuged (39,000 x g, 10 min, 4 C) and the cell-free extrac t (cfe) removed and placed into a clean tube. The cfe was then sonicated with six 10 s bursts at 71% output with a 10 s cooling periods at 4 C between each burst (Model 500 Sonic Dismembrator with a 1/2 inch tapped horn with flat tip, Fisher Scientific Suwannee, Georgia). The cfe was centrifuged (39,000 x g, 10 min, 4 C) and the supernatant (cleared cfe) was removed. The cleared cfe was added to 1 mL of a 50% Ni-NTA slurry (Qiagen, Valencia, California) and incubated at 4 C for 60 min with gentle mixing (GyroM ini Nutating Mixer). The lysate-NiNTA mixture was loaded onto a Poly-Prep Column (Bio-Rad, Hercules, California) and allowed to settle for 10 min at 4 C. A portion of the flow-through (10 L) was then collected and saved for analysis. The column was washed twice with 4 mL of Ni-NTA Wash Buffer (20 mM Tris-

PAGE 78

78 HCl, pH 7.9), 50 mM NaCl, 60 mM imidazo le) and a portion of the flow-through (10 L) was saved for future analysis. The protein was elut ed four times (0.5 mL each) with Ni-NTA Elution Buffer (10 mM Tris-HCL, pH 7.9, 250 mM NaCl 500 mM imidazole) and portions of each (10 L) were saved for future analysis at -20 C. 2X SDS-PAGE loading dye was added to each of the samples mentioned above. Samples were prep ared, resolved, stained, as described in the previous section, and the elutions cont aining the majority of the purified His(6)wt Taq polymerase were identified. Elution fractions 2 4 were combined and loaded into a Slide-A-Lyzer 10K MWCO 0.5 3 mL Dialysis Cassette (Pierce, Rockford, Illinois) that was prehydrated in Taq Dialysis Buffer A (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.1 mM EDTA, 0.5 mM PMSF, 0.5% Nonidet-P40, 0.5% Triton X-100). The sample was dialyzed at 4 C for 4 hrs against 500 mL of Dialysis Buffer A. It was then dial yzed for another 4 hrs at 4 C against 500 mL of Taq Dialysis Buffer B (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.1 mM EDTA, 0.5 mM PMSF, 0.5% Nonidet-P40, 0.5% Triton X-100, 1 mM DTT). Finall y, it was dialyzed for 8 hrs at 4 C against 1 L of Taq Storage Buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mM PMSF, 0.5% Nonidet-P40, 0.5% Triton X-100, 1 mM DTT, 50% glycerol). The sample was removed, quantitated, and the prot ein concentration determined us ing the Bio-Rad Protein Assay Dye Reagent according to manufacturers instructions. The purified His(6)wt Taq polymerase and Taq polymerase (New England BioLabs) were used in separate PCR reactions. The same c oncentration of each polymerase (enough protein to equate to 3 U of Taq polymerase from New England BioL abs) were added to PCR reactions containing: 1X Modified Th ermoPol Buffer (2 mM Tris-H Cl, pH 9, 10 mM KCl, 1 mM (NH4)2SO4, 2.5 mM MgCl2, 0.2% Tween 20), 250 M dNTPs, 1.0 M P-4, 1.0 M P-5, and 1

PAGE 79

79 ng/ L pSW1. The PCRs (50 L) were run under the following conditions: 5 min, 94 C; (1 min, 94.0 C; 1 min, 55.0 C; 3 min, 72.0 C)x15 cycles; 7 min, 72.0 C. Products were analyzed by agarose gel electrophoresis and qua ntitated using the Molecular Imager Software (Bio-Rad). Incorporation of d UTP by RD Library 2-deoxypseudouridine5-triphosphate (d UTP) was purchased from TriLink BioTechnologies (San Diego, California). St andard deoxynucleotide triphosphates (dNTPs) were comprised of 2-deoxyadenosine-5-triphos phate (dATP), 2-deoxy cytidine-5-triphosphate (dCTP), 2-deoxyganosine-5-triphosphate (dGT P), and thymidine-5-triphosphate (TTP) and were purchased from Promega Corporation (Madison, Wisconsin). d UNTPs were comprised of dATP, dCTP, d GTP, and d UTP. Individual cultures (5 mL LB-Amp ) of the SW5 SW78 cell lines were grown for 14.25 hrs at 250 rpm and 37 C in 14 mL 2059 Falcon Tubes (BD Bi osciences). Approximately 2 x 108 colony-forming units (cfu), roughly equal to 500 L of a culture with an OD550nm of 4.0, were used to inoculate individua l 100 mL cultures of LB-Amp in 500 mL baffled flasks. These cultures were grown at 37 C and 250 rpm for 3.75 hrs to an approximate OD550nm of 1.8, and were then induced with anhydrotetracycline. The cells were allowed to grow for 1 hr longer to an approximate OD550nm of 3.0. Approximately 1 x 106 cfu (~2 L cells) were used as the sole source of polymerase and template in separate PCR reactions containing fi nal concentrations of these constituents: 1X Modified ThermoPol Buffer, 1.4 M P-4, 1.4 M P-5, 1.1 ng/ L RNaseA, and 6% DMSO. The final concentration of nucleotide triphosphates adde d to the reactions were one of the following: 500 M dNTPs; 500 M dATP/dGTP/dCTP; 500 M dATP/dGTP/dCTP + 450 M TTP + 50 M d UTP; 10 M dATP/dGTP/dCTP + 400 M TTP + 100 M d UTP; 10 M

PAGE 80

80 dATP/dGTP/dCTP + 350 M TTP + 150 M d UTP; 10 M dATP/dGTP/dCTP + 300 M TTP + 200 M d UTP; 10 M dATP/dGTP/dCTP + 250 M TTP + 250 M d UTP; 10 M dATP/dGTP/dCTP + 200 M TTP + 300 M d UTP; 10 M dATP/dGTP/dCTP + 150 M TTP + 350 M d UTP; 10 M dATP/dGTP/dCTP + 100 M TTP + 400 M d UTP; 10 M dATP/dGTP/dCTP + 50 M TTP + 450 M d UTP; 500 M d UTPs. The PCRs (50 L) were run under the following conditions: 5 min, 94 C; (1 min, 94.0 C; 1 min, 55.0 C; 3 min, 72.0 C)x15 cycles; 7 min, 72.0 C. Products were analyzed by agarose gel electrophoresis and quantitated using the GeneT ools Software, version 3.07 (SynGene, Cambridge, England). Selection of Thermostable Mutants Using Water-In-Oil Emulsions Water-in-oil emulsions The appropriate cell line was grown overnight in LB-Amp broth (5 mL) for 14.25 hr at 37 C and 250 rpm in 14 mL 2059 Falcon Tubes (B D Biosciences). Approximately 2 x 108 colonyforming units (cfu), roughly equal to 500 L of a culture with an OD550nm of 4.0, were used to inoculate a 100 mL culture of LB -Amp in 500 mL baffled flasks. These cultures were grown at 37 C and 250 rpm for 3.75 hrs to an approximate OD550nm of 1.8, induced with anhydrotetracycline, and allowed to grow for 1 hr longer to an approximate OD550nm of 3.0. The amount of culture containing 2 x 108 cfu was determined; that amount was centrifuged (13,000 rpm, 2 min), the supernatant removed, and the remaining pellet was stored on ice. The aqueous phase of the emulsions was prep ared by resuspending th e cell pellet in a 200 L solution containing: 1X Modi fied ThermoPol Buffer, 500 M dNTPs, 1.4 M P-4, 1.4 M P5, 1.1 ng/ L RNaseA, and 6% DMSO. For cont rol reactions, without cells, 1 ng/ L of pSW2 and 10 U Taq Polymerase were added to the aqueous pha se. Reactions were stored on ice until further use.

PAGE 81

81 To prepare the oil-phase of the emulsions, Arlacel P135 (Uniqema, New Castle, Delaware) was heated to 75 C, as was mineral oil (Sigma-Aldrich, St. Louis, Missouri). The mineral oil was mixed with the Arlacel P135 (1.5% v/v) in a 5 mL Corning Externally Threaded Cryogenic Vial (Corning, Acton, Massachuse tts) containing an 8 x 3 mm stir bar with pivot ring. The oilphase was stirred at 1000 rpm on ice while the 200 L aqueous phase was added drop-wise over a period of 2 minutes. The emulsion was stirred fo r 5 min longer, then subjected to PCR [5 min, 94 C; (1 min, 94.0 C; 1 min, 55.0 C; 3 min, 72.0 C)x15 cycles; 7 min, 72.0 C]. Products were extracted from the emulsions with the addition of two volumes of watersaturated ether. The ether and emulsions were mixed by vortexing, centrifuged (5 min, 8000 rpm), and the aqueous phases extracted. To rid the aqueous phases of contaminating enzyme, the products were subjected to a QIAquick PCR Puri fication Kit (Qiagen), an d products were eluted from the column in Qiagen Buffer EB (50 L). Products were separated using agarose gel electrophoresis; the product ba nd was extracted and then pur ified using a QIAquick Gel Extraction Kit (Qiagen). Samples were eluted in Qiagen Buffer EB (50 L), and product concentration was determined by measuring absorption at 260 nm. Re-cloning of selected mutants The final products of the emulsions were used in an adaptation of the Miyazaki and Takenouchi megaprimer PCR protocol (Miyazaki and Takenouchi, 2002). CSR products were digested with Nco I and Sac II according to manufacturers prot ocol (New England BioLabs). Digested samples (10 ng in 1 L) were added to a 49 L PCR mixture (1X Native Pfu Buffer, 100 ng pSW2, 500 M dNTPs, 6% DMSO). Mixture was heated to 96 C for 30 s prior to the addition of 0.05 U/ L Native Pfu Polymerase (Stratagene, La Jo lla, California). Samples were

PAGE 82

82 then subjected to PCR [2 min, 96 C; (30 s, 96.0 C; 10 min, 68.0 C)x25 cycles; 30 min, 72.0 C]. The template strands of DNA (pSW2 plasmid in the PCR) were digested with 2 U Dpn I (New England BioLabs) at 37 C for 2.5 hrs. Reactions were cooled to room temperature, purified using a Qiagen PCR Pu rification Kit, and eluted with Qiagen Buffer EB (30 L). Purified products were transformed into the E. coli DH5 cell line according to manufacturers protocol (Invitrogen, Carlsbad, Ca lifornia). Fifty isolated col onies were selected after the transformation (cell lines SW79 through SW128). Overnight 5 mL LB-Amp cultures (250 rpm, 37 C) were grown for each colony, and their pl asmids isolated using the QIAprep Spin Miniprep Kit (Qiagen). Plasmid c onstructs were verified by restri ction digest analysis, using the enzymes Bam HI and Nco I according to the manufacturers pr otocol (New England BioLabs), and mutations were determined by sequencing. Results Growth Curves and Cell Counts Growth curves, cfu counts, and protein expr ession studies were performed on the SW1 SW4 cell lines to determine the op timal times for induction (Fig. 3-6[A-C]). The optimal time (1 hr) for induction for both the SW3 and SW4 cell line s was found to be during late log phase at an optical density of approximately 1.8 at 550 nm. Th e optimal length of induction was 1 hr, due to the rapid death of the cells after the induction of the taq gene, as is evidenced by a drop in the cfu/mL counts (Fig. 3-6B). Induc tions longer than 1 hr, or induc tion at early to mid-log phases caused the cells to perish due to toxicity because of the over-expression of a polymerase in vivo (data not shown) (Moreno et al., 2005, Andraos et al., 2004). When the migration of the recombinant Taq polymerases (His(6)wt Taq and coTaq ) are compared to that of the Taq

PAGE 83

83 Polymerase purchased from New England BioLabs, they all appear to have the same observed molecular weight of 94 kDa on a Coomassie Blue stained SDS-PAGE (7.5%) gel (Fig. 3-6C). Purification of His(6)wt Taq Polymerase The His(6)wt Taq polymerase was purified from SW3 cells that were over-expressing the His(6)wt taq gene using nickel affinity chromatography. The polymerase was purified to a single band on a Coomassie Blue stained SDS-PAGE (7.5 %) gel (Fig. 3-7A), and elution fractions 2 4 were combined and concentrated via dial ysis to generate a working stock of His(6)wt Taq polymerase. The protein concentration was determined to be 0.744 g/ L, using the Bio-Rad Protein Assay Dye Reagent. To veri fy the ability of the purified His(6)wt Taq polymerase to amplify DNA in a PCR reac tion, similar to that of Taq polymerase (New England BioLabs), each of these polymerases were used in separate identical PCRs. The final concentration of polymerase (5.5 g/mL) in each reaction was kept constant Figure 3-7B shows the products of these PCRs, and after analysis it was determined that the densities of these two bands were almost identical. Incorporation of d UTP by RD Library In efforts to find a polymerase that can incorporate and extend beyond d Us with higher efficiency than the co -Taq polymerase, each of the mutant Taq polymerases in the RD Library were tested for their ability to incorporate d UTP across from template dA in PCR reactions containing varying ratios of TTP to d UTP. Reactions contained induced cells as the sole source of polymerase and template plasmid, so activ e polymerases were forced to replicate their own encoding gene (2603 bp). Figure 3-8[A-B] shows the difference between the PCR products from the co -Taq polymerase screen (Fig. 3-8A) and a representative (SW 21) of the RD Library (Fig. 3-8B). In

PAGE 84

84 both of these reactions, the polymerase could not produce full-length product (FLP) with concentrations of d UTP higher than 400 M (final concentration). Based on the product band densities, it was found that none of the active RD Library polymerases displayed a higher propensity for the incorporation of d UTP than the coTaq polymerase (Table 3-4). It was also noted that only 18 of the 74 mutant polymerase s tested showed activity with only dNTPs under these assay conditions (Tab le 3-2 and Table 3-4). Selection and Identification of Thermos table Mutants Using Water-In-Oil Emulsions We pooled all 74 RD Library strains to perfor m a selection in wate r-in-oil emulsions to isolate those 18 mutants that show ed activity. After the products we re isolated, they were used in a modified version of the Miyazaki and Take nouchi megaprimer PCR protocol (Miyazaki and Takenouchi, 2002), creating the full -length plasmid (pASK-IBA43plus with insert). Purified products were transformed into the E. coli DH5 cell line; fifty clones we re isolated, sequenced, and compared to the coTaq amino acid sequence (Table 3-5). Of these fifty clones, 22 showed no changes relative to the coTaq sequence, and the remaining 28 had at least one residue modified. Table 3-6 shows a breakdown of thes e mutations, and states whether they are random mutations or RD Library mutations. In the case of the RD Library mutations, it is indicated if they are true RD Library sequences, RD Libr ary sequences with additional mutations, RD Library sequences with reversions to the coTaq sequence, and/or crossovers between two or more RD Library sequences. In addition, only 5% of the mutations found in these sequences encode silent mutations (Table 3-6). As a control, the selection was also pe rformed using only cells expressing the coTaq polymerase. Five clones were submitted for sequencing following the megaprimer PCR protocol. Of these five, four were the correct coTaq polymerase sequence found in SW4, and

PAGE 85

85 the fifth contained only two amino acid mutations in relation to the coTaq sequence (data not shown). Discussion Previously, directed evolution experime nts have defined mutations that allow Taq polymerase, and other Family A polymerases, to be used in different situations; for example, a few allow for the incorporation of non-standard bases, others are more thermostable, and some are resistant to inhibitors (Ghadessy et al., 2001, Ghadessy et al., 2004, Henry and Romesberg, 2005). The design of our RD Library was based o ff mutations discussed in the review by Henry and Romesberg (Henry and Romesberg, 2005), and were carried out by using the REAP approach with the Family A polymerases. A library of 74 polymerases was designed, which contained three to four amino aci d mutations per polymerase out of a pool of thirty-five possible mutations, in an attempt to identify a polymeras e with the ability to incorporate non-standard bases, exhibiting a C-glycosidic linka ge, with efficiency and fidelity. It has been demonstrated previously that th e over-expression of a polymerase in a cell can cause toxicity problems and cause premature cel l death (Moreno et al., 2005, Andraos et al., 2004). To circumvent this problem, the gene encoding His(6)wt Taq polymerase was optimized for codon-usage in E. coli and cloned into a tightly-regula ted plasmid (Skerra, 1994) in an attempt to express the polymerase at higher levels only after induction. After appropriate expression conditions were found, the members of the RD Library were individually tested for their ability to incorporate d UTP, a representative non-standa rd nucleotide exhibiting a Cglycosidic linkage. The polymerases were chal lenged with increasing concentrations of the d UTP as the concentration of TTP presented was decreased. None of the RD Library polymerases were able to incorporate d UTP more efficiently than the codon-optimized Taq

PAGE 86

86 sequence. In the future, other possible mutation sites and combinations of mutations may need to be made and tested to find a polymerase that can accomplish this task. Interestingly, only eighteen of the 74 mutant polymerases tested sh owed activity with stan dard dNTPs under these assay conditions. Ideally, a selection would ha ve been performed using th e RD Library to identify polymerases able to incorporate d UTP with efficiency. Since none were able to incorporate the NSB more efficiently than the coTaq polymerase, as evidenced by the densities of the FLP bands, a selection was performed to identify thos e polymerases that showed activity with the dNTPs under these assay conditions. A water-in-oil emulsion system similar to that Ghadessy et al. described (Ghadessy et al., 2001), was used as a means to link geneotype to phenotype, forcing active polymerases to replicate their ow n genes in a PCR reaction. All 74 cell lines containing the RD-Library were used in equal pr oportions to perform su ch a selection. After products were extracted from the emulsion system they were recloned into a plasmid using a version of the megaprimer PCR (Miyazaki and Takenouchi, 2002). The megaprimer PCR method was chosen as the method for recombining the polymerase genes with the plasmid based on its one pot appr oach. After extracting th e final products from the emulsions, all further recloning can take plac e in one reaction vessel, and undergoes only one purification step prior to transf ormation into a cell line. Othe r methods, using digestions and ligations, require severa l purification steps between the vari ous procedures, resulting in low yields of final product. After sequencing, it was noted that 22 out of the 50 clones sequenced contained the original coTaq polymerase sequence; 15 ca rried partial forms of th e original RD Library sequences, and only four were true RD library sequences. The remaining nine sequences were

PAGE 87

87 random mutations most likely created during the P CR in the emulsions. This could be due to the fact that Taq polymerase has an error rate of approximately 8 x 10-6 (mutational frequency/bp/duplication) (Cline et al., 1996). It is also notew orthy that two of 50 sequences (SW119 and SW122) contained frameshift muta tions, which tend to occur once every 2.4 x 10-5 base pairs when using Taq polymerase (Tindall and Kunkel, 1988). Since the plasmid carrying the cotaq gene was only introduced during the megaprimer PCR, and the plasmid used as template was digested with Dpn I, it was determined that during the course of the megaprimer PCR reaction, recombina tions and reversions of the various sequences most likely occurred during this procedure. This would explain th e high number of coTaq sequences and the large number that contain various additions, reversions, and crossovers relative to the original RD Library mutations. This also accounts for the presence of the numerous coTaq polymerase clones identified after sequencing. Out of the four exact RD lib rary sequences that were r ecovered, only one coded for a mutant that was previously shown to have activity in the assay using d UTP. This could indicate that the emulsions are breaking, allowing active polymeras es to replicate the genes of inactive polymerases. Further tests could be perf ormed to confirm or deny this conclusion; an example would be using two different cell lines in an emulsion, one expressing active polymerase and one expressing inactive polymeras e. Identification of the final product would allow us to determine if indeed these emulsions ar e rupturing. If this is the case, modifications could be made to the oil phase of the emulsions such as increasing the percentage of Arlacel P135, to prevent this from occurring. We have determined that the megaprimer PCR method would be an efficient way of introducing diversity into a library between rounds of selecti on, but it is not an effective means

PAGE 88

88 for recloning if trying to identify specific products. Once the stability of the emulsion system is verified, and the recloning of the CSR pr oducts is performed using the standard digestion/ligation/transformation protocol (Sambrook et al., 1989), it is likely that we will be able to identify thermostable polymerases using this technique. The ne xt step would be using this method with a random library, instead of a rationally designed library, to identify thermostable polymerases and/or polymerases that can incorporate C-glycosides with efficiency and fidelity. After several rounds of evolution, we may be able to identify a polymerase capable of functioning with an AEGIS.

PAGE 89

89 0.1 ViralPolymerases Non-viralPolymerase s Figure 3-1. A phylogenetic tree of the Family A polymerases. This tree was generated using Pfam (Bateman, 2006, Finn et al., 2006), and analyzed for sites that underwent Type II functional divergence. Appendix B has part s of this tree expanded so that it is readable.

PAGE 90

90 Figure 3-2. Locations of the 35 ratio nally designed (RD) sites in the Taq polymerase structure. These held the mutations in the RD Librar y. There were 57 mutations made at these sites: sites in red were sites where th e natural amino acid was replaced by one different amino acid. Amino acids in blue indicate sites that were replaced by two different amino acids. Sites in green re present sites where three residues were substituted for the original amino acid. Im age created by Dr. Eric Gaucher using the PyMOL Molecular Graphic System (DeLano, 2002).

PAGE 91

91 Table 3-1. Oligonucleotid es used in this study. OligoSequence (5 3 Direction) P urification P-2GAT GAC CGC GGT ATG CTG CCC CTCDesalted P-3CAT TAC AGA CCA TGG TCA CTC CTT GGC GGA GDesalted P-4CAA ATG GCT AGC AGA GGA TCG CAT CAC CAT CACDesalted P-5CAG GTC AAG CTT ATT ATT TTT CGA ACT GCG GGT GGCDesalted P-6GAG TTA TTT TAC CAC TCC CTDesalted P-7CGC AGT AGC GGT AAA CGDesalted P-8GAA AAC CGC GCG TAA ACT GCDesalted P-9CCT GGA ACA CGC GAA TCA GGDesalted *All oligonucleotides were synthesized by In tegrated DNA Technologies (Coralville, Iowa).

PAGE 92

92 pASK-IBA43plus3286 bps 500 1000 1500 2000 2500 3000 Van 91I Msc I Xba I Nhe I Sac II Eco RI Ecl 136II Sac I Acc 65I Kpn I Sma I Xma I Bam HI Xho I Acc I Sal I Bsp MI Sbf I Pst I Nco I Eco 47III Bst BI Hin dIII Nae I Ngo MIV Psi I Ear I Xmn I Sca I Pvu I Fsp I Mun I I Bgl I Bpm I Bmr I Ahd I Nsp 10I Bpu NI Eco BI Sna I Nru I Bsm I Nsi 10I Ppu I Oli I Bsg I Nde I Spe NI Alw Promoter 6x-His Tag Strep-Tag f1 Origin AmpR Tet-Repressor Figure 3-3. View of the pASK -IBA43plus plasmid. This plas mid was purchased from IBA GmbH (St. Louis, Missouri) and it can generate an N-te rminal hexahistidine and a C-terminal Strep -tag. This high copy number plasmid is a tightly controlled tetracycline expression system conferring ampicillin resistance.

PAGE 93

93 pSW15723 bps 1000 2000 3000 4000 5000 HincII XbaI SacII AccI AscI BssHII Tth111I AatII Acc65I KpnI BspMI BstXI FseI PstI BamHI XcmI PshAI NcoI BstBI ScaI PvuI MunI BmrI AhdI SnaBI NruI BsmI NsiI Ppu10I NdeI SpeI AlwNI Promoter wt taq f1 ori AmpR Tet-Repressor Figure 3-4. View of the pSW1 plasmid. This is a ligation of the pASK-IBA43plus plasmid with the His(6)wt taq polymerase gene using the Sac II and Nco I restriction sites. This plasmid generates an N-terminal hexa histidine translated with the His(6)wt taq gene. This high copy number plasmid is a tightly controlled tetracycline expression system conferring ampicillin resistance.

PAGE 94

94 pSW25723 bps 1000 2000 3000 4000 5000 MscI HincII XbaI NheI SacII XcmI BglII Eco52I Bpu1102I SapI ClaI Ecl136II SacI BamHI EcoRV PmlI BssHII BseRI NcoI BstBI PsiI ScaI BglI AhdI NsiI Ppu10I OliI NdeI SpeI Promoter co taq f1 Origin AmpR Tet-Repressor Figure 3-5. View of the pSW2 plasmid. This is a ligation of the pASK-IBA43plus plasmid with the codon-optimized taq polymerase gene using the Sac II and Nco I restriction sites. This plasmid generates an N-terminal hexahistidine translated with the cotaq gene. This high copy number plasmid is a tightly controlled tetracycline expression system conferring ampicillin resistance.

PAGE 95

95 Table 3-2. Rationally Desi gned (RD) Mutant Library. Plasmid Name DNA 2.0 Gene ID # Mutations Present in RD Taq Library P lasmid Name DNA 2.0 Gene ID # Mutations Present in RD Taq Library pSW35339S573E,Y668F,A740SpSW405383S573H,D575T,L613I pSW45340Q486H,K537I,M670GpSW415384T541A,L606P,L613D pSW55342A605G,L613A,E739PpSW425385Y542E,V583K,A605E pSW65343D575F,L606C,A740SpSW435387E517I,F595W,A605E,I611E pSW75344T511V,R584V,I611EpSW445388T541A,D575F,L613A,D622A pSW85345N480R,F595V,E742HpSW455389T511V,A594C,L606S,A740R pSW95346E517I,V583K,A597SpSW465390Q486H,R533I,L606C,L613A pSW105347D575F,V583K,M670ApSW475391Q486H,F595V,D622A,F664Y pSW115348E517I,D607W,D622SpSW485393E517I,S573H,A605G,E612I pSW125349A594C,F664Y,A774HpSW495395Y542E,R584V,A605K,E612I pSW135350F595W,L606P,D622SpSW505396D575T,A605E,L606C,D622A pSW145351S573E,D575F,F595VpSW515397A594T,L613A,F664Y,E742H pSW155352S510I,A605K,L606SpSW525398D575F,N580Q,W601G,D622S pSW165353S573E,D622L,E742HpSW535399K537I,L606P,A740S,E742H pSW175356N480R,T511V,Y542EpSW545400A597S,W601G,L606S,F664H pSW185357A594C,F664H,M670GpSW555401S510I,E517G,D607W,I611E pSW195358Q486H,D575T,N580SpSW565402S510I,V583K,R584V,L606P pSW205359S510I,A605E,E612IpSW575405N480R,R533I,A597S,M670G pSW215360A594C,E612I,M670ApSW585408E612I,D622L,F664L,E739P pSW225361S510I,Q579A,I611QpSW595409I611Q,M670G,E739P,E742H pSW235363A594T,L606C,R657DpSW605410F595W,F664H,Y668F,E739P pSW245364T541A,A605G,L606SpSW615411A597S,A605G,D622A,F664L pSW255365E517G,K537I,L613ApSW625413L606P,I611E,E739R,R743A pSW265366K537I,Q579A,E742VpSW635414D607W,I611Q,R657D,E742V pSW275367A597S,A740R,E742VpSW645417T541A,I611Q,L613I,D622L pSW285368N580Q,A605E,L613IpSW655418K537I,S573H,N580S,D622S pSW295369N580S,F595V,A605GpSW665419N480R,S573E,D607W,A740R pSW305370N580S,D622L,A774HpSW675420D575T,L613D,E739R,A774H pSW315371R533I,R584V,F664LpSW685421Q579A,R657D,F664Y,A740R pSW325372Q486H,E517G,A605KpSW695422R533I,K537I,A605K,L613I pSW335375S573H,F664Y,R743ApSW705423T511V,E517G,L606C,F664Y pSW345376D575T,N580Q,R584VpSW715425D575T,F664H,E742V,R743A pSW355377T541A,F664L,R743ApSW725426A594C,I611E,F664L,A740S pSW365378T511V,R533I,D622ApSW735427N580S,L613A,A740S,R743A pSW375379A597S,I611E,Y668FpSW745428S510I,T511V,L613I,E739R pSW385381Y542E,F595W,L606CpSW755429V583K,E612I,L613D,Y668F pSW395382L606S,R657D,E739RpSW765430S573E,R584V,A594C,D622S *The pink cells denote the sequences of polymerases showing activity. The blue cells signify the sequences of polymerases that lack evidence of activ ity under these assay conditions. All are derivatives of the cotaq gene and inserted into the pASK-IBA43plus v ector. Mutations were designed by Dr. Eric Gaucher (Foundation for Applied Molecular Evolutio n) and were synthesized and assembled by DNA 2.0.

PAGE 96

96 Table 3-3. Bacterial stra ins used in this study. NameStrainGenotypeNameStrainGenotypeSW1 E. coli TG-1 FtraD36 lacIq (lacZ) M15 proA+B+ /supE (hsdM-mcrB)5 (rk mk McrB-) thi (lac-proAB) SW68 E. coli TG-1 SW1/pSW66 (pASK-IBA43+ with co taq mutant 5419, Apr) SW2 E. coli TG-1SW1/pASK-IBA43+SW69 E. coli TG-1 SW1/pSW67 (pASK-IBA43+ with co taq mutant 5420, Apr) SW3 E. coli TG-1 SW1/pSW1 (pASK-IBA43+ with wt taq insert, Apr) SW70 E. coli TG-1 SW1/pSW68 (pASK-IBA43+ with co taq mutant 5421, Apr) SW4 E. coli TG-1 SW1/pSW2 (pASK-IBA43+ with co taq insert, Apr) SW71 E. coli TG-1 SW1/pSW69 (pASK-IBA43+ with co taq mutant 5422, Apr) SW5 E. coli TG-1 SW1/pSW3 (pASK-IBA43+ with co taq mutant 5339, Apr) SW72 E. coli TG-1 SW1/pSW70 (pASK-IBA43+ with co taq mutant 5423, Apr) SW6 E. coli TG-1 SW1/pSW4 (pASK-IBA43+ with co taq mutant 5340, Apr) SW73 E. coli TG-1 SW1/pSW71 (pASK-IBA43+ with co taq mutant 5425, Apr) SW7 E. coli TG-1 SW1/pSW5 (pASK-IBA43+ with co taq mutant 5342, Apr) SW74 E. coli TG-1 SW1/pSW72 (pASK-IBA43+ with co taq mutant 5426, Apr) SW8 E. coli TG-1 SW1/pSW6 (pASK-IBA43+ with co taq mutant 5343, Apr) SW75 E. coli TG-1 SW1/pSW73 (pASK-IBA43+ with co taq mutant 5427, Apr) SW9 E. coli TG-1 SW1/pSW7 (pASK-IBA43+ with co taq mutant 5344, Apr) SW76 E. coli TG-1 SW1/pSW74 (pASK-IBA43+ with co taq mutant 5428, Apr) SW10 E. coli TG-1 SW1/pSW8 (pASK-IBA43+ with co taq mutant 5345, Apr) SW77 E. coli TG-1 SW1/pSW75 (pASK-IBA43+ with co taq mutant 5429, Apr) SW11 E. coli TG-1 SW1/pSW9 (pASK-IBA43+ with co taq mutant 5346, Apr) SW78 E. coli TG-1 SW1/pSW76 (pASK-IBA43+ with co taq mutant 5430, Apr) SW12 E. coli TG-1 SW1/pSW10 (pASK-IBA43+ with co taq mutant 5347, Apr) SW79 E. coli DH5 SW1/pCSRMut1 (pASK-IBA43+ with co taq CSR mut 1, Apr) SW13 E. coli TG-1 SW1/pSW11 (pASK-IBA43+ with co taq mutant 5348, Apr) SW80 E. coli DH5 SW1/pCSRMut2 (pASK-IBA43+ with co taq CSR mut 2, Apr) SW14 E. coli TG-1 SW1/pSW12 (pASK-IBA43+ with co taq mutant 5349, Apr) SW81 E. coli DH5 SW1/pCSRMut3 (pASK-IBA43+ with co taq CSR mut 3, Apr) SW15 E. coli TG-1 SW1/pSW13 (pASK-IBA43+ with co taq mutant 5350, Apr) SW82 E. coli DH5 SW1/pCSRMut4 (pASK-IBA43+ with co taq CSR mut 4, Apr) SW16 E. coli TG-1 SW1/pSW14 (pASK-IBA43+ with co taq mutant 5351, Apr) SW83 E. coli DH5 SW1/pCSRMut5 (pASK-IBA43+ with co taq CSR mut 5, Apr) SW17 E. coli TG-1 SW1/pSW15 (pASK-IBA43+ with co taq mutant 5352, Apr) SW84 E. coli DH5 SW1/pCSRMut6 (pASK-IBA43+ with co taq CSR mut 6, Apr) SW18 E. coli TG-1 SW1/pSW16 (pASK-IBA43+ with co taq mutant 5353, Apr) SW85 E. coli DH5 SW1/pCSRMut7 (pASK-IBA43+ with co taq CSR mut 7, Apr) SW19 E. coli TG-1 SW1/pSW17 (pASK-IBA43+ with co taq mutant 5356, Apr) SW86 E. coli DH5 SW1/pCSRMut8 (pASK-IBA43+ with co taq CSR mut 8, Apr) SW20 E. coli TG-1 SW1/pSW18 (pASK-IBA43+ with co taq mutant 5357, Apr) SW87 E. coli DH5 SW1/pCSRMut9 (pASK-IBA43+ with co taq CSR mut 9, Apr) SW21 E. coli TG-1 SW1/pSW19 (pASK-IBA43+ with co taq mutant 5358, Apr) SW88 E. coli DH5 SW1/pCSRMut10 (pASK-IBA43+ with co taq CSR mut 10, Apr) SW22 E. coli TG-1 SW1/pSW20 (pASK-IBA43+ with co taq mutant 5359, Apr) SW89 E. coli DH5 SW1/pCSRMut11 (pASK-IBA43+ with co taq CSR mut 11, Apr) SW23 E. coli TG-1 SW1/pSW21 (pASK-IBA43+ with co taq mutant 5360, Apr) SW90 E. coli DH5 SW1/pCSRMut12 (pASK-IBA43+ with co taq CSR mut 12, Apr) SW24 E. coli TG-1 SW1/pSW22 (pASK-IBA43+ with co taq mutant 5361, Apr) SW91 E. coli DH5 SW1/pCSRMut13 (pASK-IBA43+ with co taq CSR mut 13, Apr) SW25 E. coli TG-1 SW1/pSW23 (pASK-IBA43+ with co taq mutant 5363, Apr) SW92 E. coli DH5 SW1/pCSRMut14 (pASK-IBA43+ with co taq CSR mut 14, Apr) SW26 E. coli TG-1 SW1/pSW24 (pASK-IBA43+ with co taq mutant 5364, Apr) SW93 E. coli DH5 SW1/pCSRMut15 (pASK-IBA43+ with co taq CSR mut 15, Apr) SW27 E. coli TG-1 SW1/pSW25 (pASK-IBA43+ with co taq mutant 5365, Apr) SW94 E. coli DH5 SW1/pCSRMut16 (pASK-IBA43+ with co taq CSR mut 16, Apr) SW28 E. coli TG-1 SW1/pSW26 (pASK-IBA43+ with co taq mutant 5366, Apr) SW95 E. coli DH5 SW1/pCSRMut17 (pASK-IBA43+ with co taq CSR mut 17, Apr) SW29 E. coli TG-1 SW1/pSW27 (pASK-IBA43+ with co taq mutant 5367, Apr) SW96 E. coli DH5 SW1/pCSRMut18 (pASK-IBA43+ with co taq CSR mut 18, Apr) SW30 E. coli TG-1 SW1/pSW28 (pASK-IBA43+ with co taq mutant 5368, Apr) SW97 E. coli DH5 SW1/pCSRMut19 (pASK-IBA43+ with co taq CSR mut 19, Apr) SW31 E. coli TG-1 SW1/pSW29 (pASK-IBA43+ with co taq mutant 5369, Apr) SW98 E. coli DH5 SW1/pCSRMut20 (pASK-IBA43+ with co taq CSR mut 20, Apr) SW32 E. coli TG-1 SW1/pSW30 (pASK-IBA43+ with co taq mutant 5370, Apr) SW99 E. coli DH5 SW1/pCSRMut21 (pASK-IBA43+ with co taq CSR mut 21, Apr) SW33 E. coli TG-1 SW1/pSW31 (pASK-IBA43+ with co taq mutant 5371, Apr) SW100 E. coli DH5 SW1/pCSRMut22 (pASK-IBA43+ with co taq CSR mut 22, Apr) SW34 E. coli TG-1 SW1/pSW32 (pASK-IBA43+ with co taq mutant 5372, Apr) SW101 E. coli DH5 SW1/pCSRMut23 (pASK-IBA43+ with co taq CSR mut 23, Apr) SW35 E. coli TG-1 SW1/pSW33 (pASK-IBA43+ with co taq mutant 5375, Apr) SW102 E. coli DH5 SW1/pCSRMut24 (pASK-IBA43+ with co taq CSR mut 24, Apr) SW36 E. coli TG-1 SW1/pSW34 (pASK-IBA43+ with co taq mutant 5376, Apr) SW103 E. coli DH5 SW1/pCSRMut25 (pASK-IBA43+ with co taq CSR mut 25, Apr) SW37 E. coli TG-1 SW1/pSW35 (pASK-IBA43+ with co taq mutant 5377, Apr) SW104 E. coli DH5 SW1/pCSRMut26 (pASK-IBA43+ with co taq CSR mut 26, Apr) SW38 E. coli TG-1 SW1/pSW36 (pASK-IBA43+ with co taq mutant 5378, Apr) SW105 E. coli DH5 SW1/pCSRMut27 (pASK-IBA43+ with co taq CSR mut 27, Apr) SW39 E. coli TG-1 SW1/pSW37 (pASK-IBA43+ with co taq mutant 5379, Apr) SW106 E. coli DH5 SW1/pCSRMut28 (pASK-IBA43+ with co taq CSR mut 28, Apr) SW40 E. coli TG-1 SW1/pSW38 (pASK-IBA43+ with co taq mutant 5381, Apr) SW107 E. coli DH5 SW1/pCSRMut29 (pASK-IBA43+ with co taq CSR mut 29, Apr) SW41 E. coli TG-1 SW1/pSW39 (pASK-IBA43+ with co taq mutant 5382, Apr) SW108 E. coli DH5 SW1/pCSRMut30 (pASK-IBA43+ with co taq CSR mut 30, Apr) SW42 E. coli TG-1 SW1/pSW40 (pASK-IBA43+ with co taq mutant 5383, Apr) SW109 E. coli DH5 SW1/pCSRMut31 (pASK-IBA43+ with co taq CSR mut 31, Apr) SW43 E. coli TG-1 SW1/pSW41 (pASK-IBA43+ with co taq mutant 5384, Apr) SW110 E. coli DH5 SW1/pCSRMut32 (pASK-IBA43+ with co taq CSR mut 32, Apr) SW44 E. coli TG-1 SW1/pSW42 (pASK-IBA43+ with co taq mutant 5385, Apr) SW111 E. coli DH5 SW1/pCSRMut33 (pASK-IBA43+ with co taq CSR mut 33, Apr) SW45 E. coli TG-1 SW1/pSW43 (pASK-IBA43+ with co taq mutant 5387, Apr) SW112 E. coli DH5 SW1/pCSRMut34 (pASK-IBA43+ with co taq CSR mut 34, Apr) SW46 E. coli TG-1 SW1/pSW44 (pASK-IBA43+ with co taq mutant 5388, Apr) SW113 E. coli DH5 SW1/pCSRMut35 (pASK-IBA43+ with co taq CSR mut 35, Apr) SW47 E. coli TG-1 SW1/pSW45 (pASK-IBA43+ with co taq mutant 5389, Apr) SW114 E. coli DH5 SW1/pCSRMut36 (pASK-IBA43+ with co taq CSR mut 36, Apr) SW48 E. coli TG-1 SW1/pSW46 (pASK-IBA43+ with co taq mutant 5390, Apr) SW115 E. coli DH5 SW1/pCSRMut37 (pASK-IBA43+ with co taq CSR mut 37, Apr) SW49 E. coli TG-1 SW1/pSW47 (pASK-IBA43+ with co taq mutant 5391, Apr) SW116 E. coli DH5 SW1/pCSRMut38 (pASK-IBA43+ with co taq CSR mut 38, Apr) SW50 E. coli TG-1 SW1/pSW48 (pASK-IBA43+ with co taq mutant 5393, Apr) SW117 E. coli DH5 SW1/pCSRMut39 (pASK-IBA43+ with co taq CSR mut 39, Apr) SW51 E. coli TG-1 SW1/pSW49 (pASK-IBA43+ with co taq mutant 5395, Apr) SW118 E. coli DH5 SW1/pCSRMut40 (pASK-IBA43+ with co taq CSR mut 40, Apr) SW52 E. coli TG-1 SW1/pSW50 (pASK-IBA43+ with co taq mutant 5396, Apr) SW119 E. coli DH5 SW1/pCSRMut41 (pASK-IBA43+ with co taq CSR mut 41, Apr) SW53 E. coli TG-1 SW1/pSW51 (pASK-IBA43+ with co taq mutant 5397, Apr) SW120 E. coli DH5 SW1/pCSRMut42 (pASK-IBA43+ with co taq CSR mut 42, Apr) SW54 E. coli TG-1 SW1/pSW52 (pASK-IBA43+ with co taq mutant 5398, Apr) SW121 E. coli DH5 SW1/pCSRMut43 (pASK-IBA43+ with co taq CSR mut 43, Apr) SW55 E. coli TG-1 SW1/pSW53 (pASK-IBA43+ with co taq mutant 5399, Apr) SW122 E. coli DH5 SW1/pCSRMut44 (pASK-IBA43+ with co taq CSR mut 44, Apr) SW56 E. coli TG-1 SW1/pSW54 (pASK-IBA43+ with co taq mutant 5400, Apr) SW123 E. coli DH5 SW1/pCSRMut45 (pASK-IBA43+ with co taq CSR mut 45, Apr) SW57 E. coli TG-1 SW1/pSW55 (pASK-IBA43+ with co taq mutant 5401, Apr) SW124 E. coli DH5 SW1/pCSRMut46 (pASK-IBA43+ with co taq CSR mut 46, Apr) SW58 E. coli TG-1 SW1/pSW56 (pASK-IBA43+ with co taq mutant 5402, Apr) SW125 E. coli DH5 SW1/pCSRMut47 (pASK-IBA43+ with co taq CSR mut 47, Apr) SW59 E. coli TG-1 SW1/pSW57 (pASK-IBA43+ with co taq mutant 5405, Apr) SW126 E. coli DH5 SW1/pCSRMut48 (pASK-IBA43+ with co taq CSR mut 48, Apr) SW60 E. coli TG-1 SW1/pSW58 (pASK-IBA43+ with co taq mutant 5408, Apr) SW127 E. coli DH5 SW1/pCSRMut49 (pASK-IBA43+ with co taq CSR mut 49, Apr) SW61 E. coli TG-1 SW1/pSW59 (pASK-IBA43+ with co taq mutant 5409, Apr) SW128 E. coli DH5 SW1/pCSRMut50 (pASK-IBA43+ with co taq CSR mut 50, Apr) SW62 E. coli TG-1 SW1/pSW60 (pASK-IBA43+ with co taq mutant 5410, Apr) SW129 E. coli DH5 SW1/pCSRwt1 (pASK-IBA43+ with co taq wt mut 1, Apr) SW63 E. coli TG-1 SW1/pSW61 (pASK-IBA43+ with co taq mutant 5411, Apr) SW130 E. coli DH5 SW1/pCSRwt2 (pASK-IBA43+ with co taq wt mut 2, Apr) SW64 E. coli TG-1 SW1/pSW62 (pASK-IBA43+ with co taq mutant 5413, Apr) SW131 E. coli DH5 SW1/pCSRwt3 (pASK-IBA43+ with co taq wt mut 3, Apr) SW65 E. coli TG-1 SW1/pSW63 (pASK-IBA43+ with co taq mutant 5414, Apr) SW132 E. coli DH5 SW1/pCSRwt4 (pASK-IBA43+ with co taq wt mut 4, Apr) SW66 E. coli TG-1 SW1/pSW64 (pASK-IBA43+ with co taq mutant 5417, Apr) SW133 E. coli DH5 SW1/pCSRwt5 (pASK-IBA43+ with co taq wt mut 5, Apr) SW67 E. coli TG-1 SW1/pSW65 (pASK-IBA43+ with co taq mutant 5418, Apr) SW134 E. coli DH5 F 80d lac Z M15 ( lac ZYAarg F) U169 rec A1 end A1 hsd R17(rk, mk+) gal pho A sup E44 thi1 gyr A96 rel A1

PAGE 97

97 Figure 3-6. Growth curves, cell c ounts, and expression of various E. coli TG-1 cell lines. The SW3 (denoted SW3-I) and SW4 (denoted SW4-I) cell lines were induced after 3.75 hrs with a final c oncentration of 0.2 ng/ L anhydrotetracycline. A) Growth curves of various cell lines. Samples were grown in LB media, cultures SW2 SW4 were supplemented with ampicillin (100 g/mL final concentration), at 250 rpm and 37 C for 8.75 hrs. B) Colony counts (cfu/mL) of each of the cell line s in part A at the various time points. Cells were grown on LB or LB-Amp agar ove rnight at 37 C. C) Coomassie Blue stained SDS-PAGE ( 7.5%) gel showing protein expression of induced cells at various time points. U stands for uninduced cells, I-1 through I-4 indicate time-points at hours one through four after induction (t = 4.75 through t = 7.75 hrs), and NEB Taq depicts the migration of the 94 kDa Taq polymerase purchased from New England Bi oLabs. Since their genetic code has been optimized for use in E. coli cells, the SW4 strain, containing the cotaq gene, appear to grow to a higher OD550nm than the SW3 strain containing the His(6)wt taq gene. 0.000 1.000 2.000 3.000 4.000 5.000 6.000 7.000 012345678Time (hr)OD at 550 nm SW1 SW2 SW3-U SW3-I SW4-U SW4-IA) B) C) SW1SW2SW3-USW3-ISW4-USW4-I 01.36E+072.01E+076.10E+067.95E+066.00E+067.22E+06 15.40E+071.31E+071.22E+078.73E+069.53E+061.04E+07 21.80E+083.04E+078.14E+075.21E+072.65E+073.09E+07 36.48E+088.63E+072.87E+082.79E+083.88E+081.90E+08 3.751.47E+093.22E+086.83E+084.93E+084.09E+084.36E+08 4.752.73E+097.95E+081.08E+092.36E+088.26E+085.66E+08 5.752.94E+091.01E+091.08E+095.07E+071.10E+096.29E+08 6.756.06E+091.39E+091.88E+091.22E+071.53E+095.01E+08 7.753.08E+091.11E+091.77E+097.39E+062.23E+097.63E+08 8.753.14E+091.14E+091.55E+093.90E+062.15E+095.69E+08 Time (hr) Colony Counts (cfu/mL)

PAGE 98

98 Figure 3-7. Purificati on and activity of His(6)wt Taq polymerase. A) The purification of His(6)wt Taq polymerase from SW3 cells after five hours of induction. U uninduced cells, I-5 cells after 5 hrs of induction, L load from the Ni2+ column, W-1 and W2 wash fractions from the column, E-1 through E-4 elution fractions from the column. Elution fractions 2 4 were combined and subjected to dialysis. B) Products of PCRs comparing id entical concentrations of Taq polymerase (New England BioLabs) and His(6)wt Taq polymerase. The amount of product generated with each polymerase was almost identical considering the de nsity of the product band using Taq polymerase was 1980 CNT/mm2 and the density of the product band using His(6)wt Taq polymerase was 1925 CNT/mm2. B) A)

PAGE 99

99 Figure 3-8. Representative gels showing the am ount of full-length PCR products generated with different dNTP/d UNTP ratios and the indicated polym erases. Concentrations of dNTPs/d UNTPs listed are the starting concentr ations (see Materials and Methods for listing of final concentrations). All PCRs used 1 x 106 cfu of cells expressing polymerase as the sole source of polymeras e and template plasmid for the reaction. Polymerases were forced to replicate their own encoding gene (2603 bp). A) Incorporation of various dNTP/d UNTP ratios by coTaq polymerase. FLP is not generated beyond the ratio of 3 mM TTP/7 mM d UTP. B) Incorporation of various dNTP/d UNTP ratios by a representative of the RD Library (SW21). FLP is not generated beyond the ratio of 3 mM TTP/7 mM d UTP. A) B)

PAGE 100

100Table 3-4. Incorporation of d UTP at 94.0 C by RD Library. All dNTPs 9 mM dT/ 1 mM d U 8 mM dT/ 2 mM d U 7 mM dT/ 3 mM d U 6 mM dT/ 4 mM d U 5 mM dT/ 5 mM d U 4 mM dT/ 6 mM d U 3 mM dT/ 7 mM d U 2 mM dT/ 8 mM d U 1 mM dT/ 9 mM d U All d UNTPs SW4Codon-Optimized (co) wt Taq2244256200537119956491535822125537958963718875264360000 SW8D575F,L606C,A740S5857775405894073273076032027871187885249119033000 SW10N480R,F595V,E742H54728116477917088022128626568497901493210000 SW11E517I,V583K,A597S9193408367227471386209954120591624536291919455000 SW12D575F,V583K,M670A247942667915530200640000000 SW14A594C,F664Y,A774H66993312996714253399426591523136200000 SW17S510I,A605K,L606S325360000000000 SW21Q486H,D575T,N580S13448771310961117464999965064049733351111256948284000 SW25A594T,L606C,R657D5092412015401197216989446402000000 SW27E517G,K537I,L613A1371805337232796022605000000 SW29A597S,A740R,E742V766112500823350791263933233934774463873122923000 SW30N580Q,A605E,L613I284020000000000 SW31N580S,F595V,A605G431840000000000 SW34Q486H,E517G,A605K93870362565049336539590640112363888301270000 SW36D575T,N580Q,R584V407260346463592044580000000 SW41L606S,R657D,E739R469252304702968629736000000 SW47T511V,A594C,L606S,A740R66112265382744535249300212299100000 SW72T511V,E517G,L606C,F664Y499069309148155466970995598317934125530000 SW76S510I,T511V,L613I,E739R216700172538119075121527522902695500000 Raw Densities (CNT/mm2) Substitutions Cell Line

PAGE 101

101 Table 3-5. Mutations present afte r selection for active polymerases. Cell Line Mutations Present Cell Line Mutations Present SW79-SW104 P336L,M371T,N580S,L613A SW80 E517G,A597S,A605G,D622A SW105SW81 E468G SW106 E504G,R533I,R584V,F664L,F697S SW82 L11P SW107 G195D,L230P,P552Q,D575T,F664L SW83-SW108 L606P,I611E,E739R,R743A SW84 A594C,F664Y SW109SW85 Q579A SW110SW86-SW111 K257E,S510I,D575F,L606C SW87 F44S SW112SW88-SW113SW89 T31M SW114 M314I SW90-SW115 A201E SW91 V61L SW116 N480R,T511A SW92-SW117SW93 V646I SW118 Q486H,F595V,D622A,F664Y SW94 D575T,F664H,F721L,E742V,R743A,E817G SW119 S510I SW95 I60T SW120SW96-SW121 G327S,H330Y,N480R,R533I SW97-SW122SW98 R743A SW123 S510I,A605E,E612I SW99 I543T,A594C,E631G,F664Y,W703R SW124SW100 Q486H,F595V,D622A SW125SW101-SW126SW102-SW127 R533I,K537I,Q579A,E739R SW103 Q486H,D575T,N580S SW128* The dashes (-) represent polymerases with no amino acid mutations relative to the coTaq sequence.

PAGE 102

102 Table 3-6. Breakdown of types of mutations present after selection. Silent Nonsilent Total SW79X------000 SW80--E517G,A597S, A605G,D622A E517G,A597S, A605G,D622A -088 SW81E468G -----011 SW82L11P -----011 SW83X------000 SW84---A594C,F664Y --033 SW85---Q579A --134 SW86X------000 SW87F44S -----011 SW88X------101 SW89T31M -----011 SW90X------000 SW91V61L -----011 SW92X------000 SW93V646I -----011 SW94--D575T,F664H, F721L,E742V, R743A,E817G ---01616 SW95I60T -----112 SW96X------000 SW97X------000 SW98---R743A --033 SW99--I543T,A594C, E631G,F664Y, W703R I543T,A594C, E631G,F664Y, W703R --066 SW100---Q486H,F595V, D622A --044 SW101X------000 SW102X------000 SW103-Q486H,D575T, N580S ----077 SW104--P336L,M371T, N580S,L613A P336L,M371T, N580S,L613A --077 SW105X------000 SW106--E504G,R533I, R584V,F664L, F697S ---189 SW107--G195D,L230P, P552Q,D575T, F664L G195D,L230P, P552Q,D575T, F664L G195D,L230P, P552Q,D575T, F664L -189 SW108-L606P,I611E, E739R,R743A ----11011 SW109X------000 SW110X------000 SW111--K257E,S510I, D575F,L606C K257E,S510I, D575F,L606C K257E,S510I, D575F,L606C -1910SW112X------000 SW113X------000 SW114M314I -----011 SW115A201E -----011 SW116---N480R,T511A --044 SW117X------000 SW118-Q486H,F595V, D622A,F664Y ----055 SW119---S510I --033 SW120X------000 SW121---G327A,H330Y, N480R,R533I --077 SW122-------011 SW123-S510I,A605E, E612I ----077 SW124X------000 SW125X------000 SW126X------000 SW127-----R533I,K537I, Q579A,E739R 01010 SW128X------000 Cell Line CodonOptimized Taq No RD Mutations (Random Taq Mutations) RD Library Variants # of DNA Mutations RD Variants + Add'l Mutations RD Variants with Reversions RD Recombinants with 1 Crossover RD Recombinants with 2 Crossovers An X indicates polymerases with no amino acid mutations relative to the coTaq sequence.

PAGE 103

103 CHAPTER 4 DISTRIBUTION OF THERMOSTABILITY IN POLYMERASE MUTATION SPACE Introduction Recent years have seen a dramatic increase in the number of experiments being performed to optimize protein function utiliz ing directed evolution. With th e rise of directed evolution, there is a proportional escalation in the number and type of approaches used to create the libraries for these selections. Many different theories exist on the best met hods to create the best library, one that contains a large number of diverse, yet ac tive clones (Hibbert and Dalby, 2005, Arnold and Georgiou, 2003b). These theories contradict ea ch other on fundamental levels; for example, some say it is best to use random mutagenesi s throughout the entire gene (Drummond et al., 2005), and others think that it is better to perform random mutagenesis only within the region containing the active site of the protein (P ark et al., 2005, Dalby, 2003) Conversely, some researchers believe that site-saturation mutagenesis at carefully selected sites generates the best results (Parikh and Matsumura, 2005), while a few consider that mutagenesis at specific sites with specific amino acids will a llow for the creation of an optimal library (Crameri et al., 1998, Crameri et al., 1996, Castle et al., 2004). Our laboratory is interested in pursuing the directed e volution of polymerases to incorporate non-standard nucleotides (NSBs), specifically those exhi biting a C-glycosidic linkage (Fig. 2-3), such as 2-deoxypseudouridine (d U) and 2-deoxypseudothymidine (d T). To determine what type of mutagenic library would best suit our needs, we compared two libraries for their ability to perform at high temperatures, a prerequisite for selection in emulsions under the Ghadessy et al. conditions, as well as being desire d for a synthetic biology(Ghadessy et al., 2004, Ghadessy et al., 2001).

PAGE 104

104 The first was the rationally designed (RD) polymerase library, designed by Dr. Eric Gaucher using the REAP approach as discussed in the previous chapter, wh ere carefully selected residues were changed into other specific amino acids, and a random library (L4) with mutations spread across the whole polymerase sequence for their ability to function at various temperatures. The second was a randomly genera ted library (L4) with mutations spread across the entire polymerase sequence. The L4 lib rary was created using error-prone PCR with Taq polymerase and manganese chloride serving as the mutators (Arnold and Georgiou, 2003b). The starting gene was derived from the cotaq polymerase gene, which is the His(6)wt taq polymerase gene whose sequence had been optimized for codon usage in E. coli cells (Gustafsson et al., 2004). The 74 Taq polymerase mutants in each library we re first tested for their ability to incorporate dNTPs at various temperatures in a PCR reaction to determine the optimal temperature at which individual polymerases pe rformed, judging by the generation of full-length PCR products. In this case, it appeared that random mutagenesis was better able to yield thermostable variants than rational design met hod, but our RD library was specifically modified for identifying polymerases with altered catalytic activities, and therefore targeted sites where changes would be more likely to decrease thermostability. Then, variants from the RD library were tested for their ability to incorporate C-glycosides using mixtures of d UTP and TTP in different ratios, both at 94.0 C and at their optimal temperature. We identified only one muta nt with enhanced abilities over the coTaq polymerase to incorporate d UTP. Finally, the ability of d UTP to epimerize at high temperatures was of concern. Epimerization would lower the concentration of the -anomer, which is the desired substrate for

PAGE 105

105 the polymerase (Fig. 4-1). Therefore, para llel experiments were performed with d TTP, which is known not to epimerize (Wellington and Benner, 2006, Cohn, 1960, Chambers et al., 1963). These results suggested that d U is epimerizing to generate the -epimer, suggesting that d U is not as suitable as a C-glycosid e substrate in PCR experiments generally, as well as in directed evolution studies to develop thermostable polymerases having new catalytic activities. Materials and Methods DNA Sequencing and Analysis DNA sequencing was carried out by the University of Florida Interdis ciplinary Center for Biotechnology Research Sequencing DNA Core Facility using an ABI 3130 xl Genetic Analyzer (Applied Biosystems, Foster City, California) using primers P-6 through P-9 (Table 3-1). BLAST 2 Sequences software was used for se quence similarity sear ching (Tatusova and Madden, 1999); Dertis Reverse and/or compleme nt DNA sequences website was used to find the reverse complement of various DNA strands (Derti, 2003); and ExPASys translate tool was used to translate DNA sequences into their amino acid counterparts (Swiss Institute of Bioinformatics, 1999). Bacterial Growth Conditions and Strains The bacterial strains used in this study ar e listed in Table 3-3 (SW1, SW4 SW78, and SW134) and those in Table 4-4. The rich media used in these studies was Luria-Bertani (LB) medium (Difco Laboratories, Detroit, Michigan) (Miller, 1972). Ampici llin was provided in liquid or solid medium at a final concentration of 100 g/mL. Plasmids were transformed into the E. coli TG-1 cell line according to manufacturer s protocol (Zymo Research, Orange, California). Cell growth was determined by m easuring optical density at 550 nm using a SmartSpec Plus Spectrophotometer (Bio-Rad, Herc ules, California). Anhydrotetracycline (2

PAGE 106

106 mg/mL stock in N,N -dimethylformamide) was used at a final concentration of 0.2 ng/ L to induce expression. Synthesis of Triphosphates and Oligonucleotides Dr. Shuichi Hoshika, from the Foundation for Applied Molecular Evolution (FfAME, Gainesville, Florida), synthesized the pseudothymid ine precursor as described in Appendix A. Dr. Daniel Hutter (also of FfAME) synthesized 2-deoxyps eudothymidine-5-triphosphate (d TTP) as described in Appendix A. 2-deoxypseudouridine5-triphosphate (d UTP) was purchased from TriLink BioTechnol ogies (San Diego, California) Standard deoxynucleotide triphosphates (dNTPs) of 2-deoxyadenosine5-triphosphate (dATP), 2-deoxycytidine-5triphosphate (dCTP), 2-deoxyganos ine-5-triphosphate (dGTP), and thymidine-5-triphosphate (TTP) were purchased from Promega Co rporation (Madison, Wisconsin). d TNTP solutions were comprised of dATP, dCTP, dGTP, and d TTP, while d UNTPs were comprised of dATP, dCTP, d GTP, and d UTP. Random Mutagenic Library (L4 Library) Creation DNA 2.0 (Menlo Park, California) s ynthesized a form of the His(6)wt taq polymerase gene (cotaq ) that was optimized for the codon-usage of E. coli which was then used to construct the pSW2 plasmid (Fig. 3-3). Mutage nic PCR was performed on the cotaq gene to generate a library containing three to four amino acid changes per polymerase in a fashion similar to that described by Arnold and Geor giou (Arnold and Georgiou, 2003b). The PCRs contained the following: 1 X Mutagenic Taq Buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 15 mM MgCl2), 0.1 ng/ L pSW2, 200 M dNTPs, 300 nM P-4, 300 nM P-5, 5 U Taq polymerase (New England BioLabs, Beverly, Massachusetts), and MnCl2 (115 M). PCR reaction conditions were as follows: 5 min, 94 C; (30 s, 94.0 C; 20 s, 55.0 C; 3 min, 72.0 C)x15 cycles; 7 min, 72.0 C.

PAGE 107

107 Products were purified with the QIAquick PCR Purification Kit (Q iagen, Valencia, CA), eluted with Qiagen Buffer EB (50 L), and quantitated at an absorb ance of 260 nm using a SmartSpec Plus Spectrophotometer (Bio-Rad). The mutagenic PCR products were used in an adaptation of the Miyazaki and Takenouchi megaprimer PCR protocol (Miyazaki and Takenouchi, 2002). Samples (10 ng/ L final concentration) were added to a PCR mixture (1X Native Pfu Buffer, 100 ng pSW2, 500 M dNTPs, 6% DMSO). Mixture was heated to 96 C for 30 s prior to the addition of 0.05 U/ L Native Pfu Polymerase (Stratagene, La Jolla, California). Samples were then subjected to PCR [2 min, 96 C; (30 s, 96.0 C; 10 min, 68.0 C)x25 cycles; 30 min, 72.0 C]. The host strands of DNA (the pSW2 plasmi d in the PCR) were digested with 2 U Dpn I (New England BioLabs) at 37 C for 2.5 hrs. Reactions cooled to room temperature, purified using a QIAquick PCR Purificat ion Kit (Qiagen), and eluted with Qiagen Buffer EB (30 L). Purified products were transformed into the E. coli DH5 cell line according to manufacturers protocol (Invitrogen, Carlsbad, Ca lifornia). Seventy-nine isolated colonies were selected after the transformation (cell lines SW135 through SW 211). Each colony was grown in a separate overnight 5 mL LB-Amp culture (250 rpm, 37 C) in 14 mL 2059 Falcon Tubes (BD Biosciences, San Jose, California). Their plas mids were isolated using the QIAprep Spin Miniprep Kit (Qiagen). Plasmid constructs were verified both by restri ction digest analysis, using the enzymes Bam HI and Nco I according to the manufacturers protocol (New England BioLabs), and mutations were determined by sequencing. The 74 L4 Library plasmids containing mutations were transformed into the E. coli TG-1 cell line (ce ll lines SW212 through SW285) according to manufacturers protocol (Zymo Research, Orange, California).

PAGE 108

108 Incorporation of dNTPs by RD and L4 Libraries at Various Temperatures A single isolated colony from each of th e SW5 through SW78 (RD Library) and SW212 through SW285 (L4 Library) cell line s were used to inoculate a 148 individual cultures (5 mL LB-Amp), and were grown for 14.25 hrs at 250 rpm and 37 C in 14 mL 2059 Falcon Tubes (BD Biosciences). Approximately 2 x 108 colony-forming units (cfu), roughly equal to 500 L of a culture with an OD550nm of 4.0, were used to inoculate indi vidual 100 mL cultures of LB-Amp in 500 mL baffled flasks. These cultures were grown at 37 C and 250 rpm for 3.75 hrs to an approximate OD550nm of 1.8, and were then induced with an hydrotetracycline. The cells were allowed to grow for 1 hr longer to an approximate OD550nm of 3.0. Approximately 1 x 106 cfu (~2 L cells) were used as the sole source of polymerase and template in separate PCR reactions containing fi nal concentrations of these constituents: 1X Modified ThermoPol Buffer (2 mM Tr is-HCl, pH 9, 10 mM KCl, 1 mM (NH4)2SO4, 2.5 mM MgCl2, 0.2% Tween 20), 500 M dNTPs, 1.4 M P-4, 1.4 M P-5, 1.1 ng/ L RNaseA, and 6% DMSO. The PCRs (50 L) were run under the following conditions: 5 min, X C; (1 min, X C; 1 min, 55.0 C; 3 min, 72.0 C)x15 cycles; 7 min, 72.0 C, where X was a denaturing temperature of 75.0 C, 75.5 C, 76.6 C, 78.1 C, 80.4 C, 83.1 C, 86.3 C, 89.0 C, 91.1 C, 92.6 C, 93.7 C, or 94.0 C. Products were analyzed by agarose gel electrophoresis and quantitated using the GeneT ools Software, version 3.07 (SynGene, Cambridge, England). Incorporation of d UNTPs by RD Library at Optimal Temperatures A single isolated colony from each of the cell lines (SW4 through SW78) that were active at one of the temperatures tested, were used to inoculate a 33 indivi dual cultures (5 mL LBAmp). These were grown for 14.25 hrs at 250 rpm and 37 C in 14 mL 2059 Falcon Tubes (BD Biosciences). Approximately 2 x 108 cfu, roughly equal to 500 L of a culture with an OD550nm

PAGE 109

109 of 4.0, were used to inoculate individual 100 mL cultures of LB-Amp in 500 mL baffled flasks. These cultures were grown at 37 C and 250 rpm for 3.75 hrs to an approximate OD550nm of 1.8, and were then induced with anhydrotetracycline. The cells were allowed to grow for 1 hr longer to an approximate OD550nm of 3.0. Approximately 1 x 106 cfu (~2 L cells) were used as the source of polymerase and template in separate PCR reactions containing fi nal concentrations of these constituents: 1X Modified ThermoPol Buffer, 1.4 M P-4, 1.4 M P-5, 1.1 ng/ L RNaseA, and 6% DMSO. One of the following sets of nucleotide triphospha tes were added to th e reactions (final concentrations): 500 M dNTPs; 500 M dATP/dGTP/dCTP; 500 M dATP/dGTP/dCTP + 450 M TTP + 50 M d UTP; 10 M dATP/dGTP/dCTP + 400 M TTP + 100 M d UTP; 10 M dATP/dGTP/dCTP + 350 M TTP + 150 M d UTP; 10 M dATP/dGTP/dCTP + 300 M TTP + 200 M d UTP; 10 M dATP/dGTP/dCTP + 250 M TTP + 250 M d UTP; 10 M dATP/dGTP/dCTP + 200 M TTP + 300 M d UTP; 10 M dATP/dGTP/dCTP + 150 M TTP + 350 M d UTP; 10 M dATP/dGTP/dCTP + 100 M TTP + 400 M d UTP; 10 M dATP/dGTP/dCTP + 50 M TTP + 450 M d UTP; 500 M d UTPs. The PCRs (50 L) were run under the following conditions: 5 min, X C; (1 min, X C; 1 min, 55.0 C; 3 min, 72.0 C)x15 cycles; 7 min, 72.0 C, where X was each polymerases optimal denaturing temperature of 86.3 C, 89.0 C, 91.1 C, 92.6 C, 93.7 C, or 94.0 C. Products were analyzed by agarose gel electrophoresis and quantitated using th e GeneTools Software, version 3.07 (SynGene, Cambridge, England).

PAGE 110

110 Incorporation of d UTP and d TTP by coTaq Polymerase at Various Melting Temperatures The SW4 cell line was used to inoculate an LB-Amp culture (5 mL in a 14 mL 2059 Falcon Tube: BD Biosciences). The culture was grown for 14.25 hrs (250 rpm shaking at 37 C). Approximately 2 x 108 cfu of the resulting cell suspension (ca. 500 L of a culture with an OD550nm of 4.0) was used to inoculate a secondary culture of LB-Amp (100 mL in a 500 mL baffled flask). The secondary culture was grown (37 C, 250 rpm) for 3.75 hrs to an approximate OD550nm of 1.8. Expression of the poly merase was then induced with anhydrotetracycline. The cells were allowed to grow for 1 hr longer to an approximate OD550nm of 3.0. The cells themselves (approximately 1 x 106 cfu or ~2 L cells) were used as the sole source of polymerase and template in separate PCRs. These reactions contained a final concentrations of the following: 1X Modified ThermoPol Buffer, 1.4 M P-4, 1.4 M P-5, 1.1 ng/ L RNaseA, and 6% DMSO. The final concentr ation of nucleotide triphosphates added to the reactions can be found in Table 4-6. The PCRs (50 L) were run under the following conditions: 5 min, X C; (1 min, X C; 1 min, 55.0 C; 3 min, 72.0 C)x15 cycles; 7 min, 72.0 C, where X was a denaturing temperature of 86.3 C, 89.0 C, 91.1 C, 92.6 C, 93.7 C, or 94.0 C. Products were analyzed by agarose ge l electrophoresis and qua ntitated using the GeneTools Software, version 3.07 (SynGene, Cambridge, England). Results Random Mutagenic Library (L4 Library) Creation The L4 mutagenic library was created using the cotaq gene as the template sequence, and MnCl2 and Taq polymerase as the mutagens (Arnold and Georgiou, 2003b). Conditions were manipulated to create a library with approxima tely three amino acid changes per gene. After

PAGE 111

111 purification of the mutagenic PCR products, they were used in a variation of the Miyazaki and Takenouchi megaprimer PCR protocol (Miy azaki and Takenouchi, 2002), creating the fulllength plasmid (pASK-IBA43plus w ith insert). Purified produc ts were transformed into the E. coli DH5 cell line; 79 clones were isolated, sequenced, and compared to the coTaq amino acid sequence (Table 4-2). Of those 79 clones, only five retained the coTaq polymerase sequence; the remaining 74 contained at least one mutation. The plasmids containing the 74 mutant L4 genes were then transformed into the E. coli TG-1 expression cell line. Incorporation of dNTPs by RD and L4 Libraries at Various Temperatures To determine the optimal temperature for each of the cell lines in the RD and L4 Libraries, each of these mutant Taq polymerases were tested for their ability to incorporate dNTPs in PCR reactions at various temp eratures ranging from 75.0 C to 94.0 C. Reactions contained induced cells (1 x 106 cfu) as the sole source of polymerase and template plasmid, so active polymerases were forced to replicate th eir own encoding gene (2603 bp). Figure 4-2[A-C] shows the difference be tween the PCR products from the coTaq polymerase screen and representatives of the RD Library (SW17) and th e L4 Library (SW251). In these, and all of the other reactions screen ing various temperatures no full-length product (FLP) was observed at a temperature lower than 86.3 C. Based on the product band densities, the optimal temperature for each polymerase in these two libraries was determined (Table 4-3 and Table 4-4). Of the 74 L4 mutants, 39 were active at one of the temperatures tested, as compared to only 33 of the 74 RD mutants that were active. Figure 4-3[A-B] shows the distribution of the active polymerases in each library at the various temperatures. The average temperature for the RD mutants was 87.5 C and for the L4 mutants it was 89.0 C. It is interesting to note that

PAGE 112

112 within the RD Library, if the mutant was active with a 94.0 C temperature, it was active at the other five temperatures as well. This was not the case, however, w ith the L4 mutants; two of the mutants that were active at 94.0 C were not active at th e lower end of the spectrum. In addition, more mutants were stable at higher temperatur es in the L4 variants as opposed to the RD variants. Incorporation of d UNTPs by RD Library at Optimal Temperatures To find a polymerase that can incorporate and extend beyond d Us with higher efficiency than the co -Taq polymerase, each of the active mutant Taq polymerases in the RD Library were tested for their ability to incor porate varying concentrations of d UTP across from template dA in PCR reactions at their optimal temperature. Those polymerases that were not able to incorporate dNTPs at any temperature were not a ssayed in this experiment Reactions contained induced cells (1 x 106 cfu) as the sole source of polymeras e and template plasmid, again forcing active polymerases to replicate their own en coding gene (2603 bp). Control reactions, containing cells (SW4) expressing the coTaq polymerase, were performe d with the temperatures of 86.3 C and 89.0 C for comparative purposes. The raw densities of FLP bands, as measured by GeneTools Software (ver. 3.07, SynGene) of the RD mutants at their optim al temperatures were compared to those generated by the coTaq at that same temperature (Table 4-5). None of these polymerases showed an optimal temperature of 94.0 C; they all had optimal temperatures of 86.3 C or 89.0 C. In addition, all but one of the RD mutants failed to incorporate d UTP as efficiently as coTaq polymerase at their optimal temperature. The remaining mutant (SW29), howev er, showed an ability to generate FLP in all dNTP/d UNTP ratios up to 72% more efficiently, on average, than coTaq polymerase at 86.3 C (Fig. 4-4[A-C]).

PAGE 113

113 Although the RD mutants were unabl e to perform as well as coTaq polymerase at their optimal temperatures, as compared to the coTaq FLP at that same temperat ure, they were able to generate, on average, 40% more FL P at their optimal temperature th an when they were tested at 94.0 C (Table 4-5 versus Table 3-4). Figure 4-5[A-C] shows repr esentative (SW8) PCR products from the polymerase screen at 94.0 C (Fig. 4-5A) and at the SW8 polymerases optimal temperature of 86.3 C (Fig. 4-5B). In the first set of reactions the polymerase produced FLP with concentrations of 350 M d UTP; however, in the second set, the polymerase was able to generate FLP with concentrations of 400 M d UTP. This is graphically represen ted in Figure 4-5C. Incorporation of d UTP and d TTP by coTaq Polymerase at Various Melting Temperatures We performed a comparative analysis between the ability of coTaq polymerase to incorporate d UTP or d TTP in various concentrations at different temperatures. Table 4-6 shows the raw densities, as determined by the Ge neTools Software (ver. 3.07, SynGene), of the FLP bands generated by these experiments. Once the d TTP final concentration reached 300 M, all of the FLPs generated averaged a 23% higher density than those produced when using d UTPs at identical concentrations. Ho wever, at the temperatures of 91.1 C and 92.6 C, all concentrations of d TTP supported the synthesis of more FLP than d UTP. In addition, at every temperature tested, FLP was present when the d TTP concentration was in a 9:1 ratio with TTP, whereas the FLP was only obser ved at the highest ratio of d UTP to TTP of 8:2 when the temperature was 86.3 C or 89.0 C. Representative gels of these expe riments, at a temperature of 86.3 C, can be seen in Figure 4-6[A-C]. In the first gel (Fig. 4-6A), we see that FLP was generated up to an 8:2 ratio of

PAGE 114

114 d UTP to TTP; but was produced at a 9:1 ratio of d TTP to TTP (Fig. 4-6B). Easily visualized in the chart (Fig. 4-6C), the amount of FLP formed was higher when d UTP was present, until the final concentration reached 300 M; then the level of FLP was elevated when in the presence of d TTP. Graphical representations of the densities of the remaining five temperatures can be found in Figure 4-7[A-E]. Discussion In previous studies (Chapter 3), we showed that only 24% of the RD Library mutants generated PCR products in the presence of dNTP s when the highest cy cle temperature was 94.0 C (Table 3-4). This was, perhaps, consistent with expectation. A highe r percentage of active mutants, if randomly produced, might be expected to yield very few variants (Guo et al., 2004), but this might be balanced through the selection of sites less likely to cause unfolding, according to the REAP hypothesis (Gaucher, 2006). Unexp ectedly, we found that at least one of the 35 sites identified by the REAP approach had been pr eviously shown to modify the thermostability of Taq polymerase (Ghadessy et al., 2001). It is possible that other mutations affected thermostability as well. Therefore, polymerases in the RD Library were tested for their ability to incorporate dNTPs at temp eratures ranging from 75.0 C to 94.0 C. Of the 74 clones tested, none were able to generate FLP below a temperature of 86.3 C. This is most likely not due to any property of th e polymerase, but rather to the inability of the duplex DNA strands to melt at these lower temper atures, considering the temperature of the taq gene is ca. 88 C. Thirty-three of the clones (45%), in cluding the 18 previously shown to have activity (Table 3-4), were now able to incorporate dNTPs at lower temperatures down to 86.3 C (Table 4-3). This leads us to believe that so me of these replacements, located in and around the active site, indeed lowered the thermostability of the coTaq polymerase.

PAGE 115

115 It is also interesting to point out th at the SW36 and SW74 cell lines, containing polymerases with the F664H or F664L mutations re spectively, refutes Suzuki s theory that this phenylalanine residue can only be mutated to a tyrosi ne and retain activity (Suzuki et al., 1996). It may also be that the mutation to tyrosine allows the polymerase to retain the thermostability at 94.0 C; Suzuki et al. however, did not test the thermostability of the mutants in their experiments, and the activity seen with the F 664H and F664L mutations in this research was only at lower temperatures. We wanted to determine if a randomly create d mutagenic library would be as likely to create clones that display a d ecrease in the thermostability of the protein. A random, mutagenic library (L4) was created using MnCl2 and Taq polymerase (New England BioLabs) as the mutators (Table 4-2). We iden tified 74 clones, with unique sequen ces, that were also tested for their ability to incorporate dNTP s at various temperatures. Thirty-nine (53%) of the clones were able to generate FLP at one of the temperatures tested, and twenty-eight (38%) of these were able to function at 94.0 C (Table 4-4). Comparison of the thermostab ility of polymerases from the two libraries found more thermostability in the L4 random lib rary than in the RD library (F ig. 4-3[A-B]). Thirty-nine of the L4 mutants were active, with an average optimal temperature of 89.0 C, while only thirtythree of the RD clones retain ed activity, with an averag e optimal temperature of 87.5 C. It remains open whether this difference reflects the slightly greater number of replacements in the RD library members (3.5 residues/ protein) over the L4 library members (3 residues/protein). The ability to function at lower temperatures can be advantageous to the incorporation of non-standard bases. The litera ture reports examples where NSBs are incorporated more efficiently at lower temperatures (Rappaport, 2004, Horlacher et al., 1995 ). We, therefore,

PAGE 116

116 decided to test the incorporation of d UTP at each of the RD mutants optimal temperatures, comparing the results to those seen at 94.0 C (Table 3-4). Table 4-5 shows the ability of the active RD mu tants to incorporate various concentrations of d UTP in a PCR reaction with an optimal temperature of either 86.3 C or 89.0 C. Using this assay system, we identified a mutant (SW29) that was able to ge nerate, on average, 72% more product than coTaq polymerase with a temperature of 86.3 C (Fig. 4-4[A-C]). This mutant, containing the A597S, A740R, and E742V residue changes, produced FLP up to a final concentration of 400 M d UTP. The remaining 32 active polymerases that were tested were unable to incorporate the d UTP as well as coTaq polymerase; they were, however, able to generate, on average, 40% more FL P at their optimal temperature th an when they were tested at 94.0 C (Table 4-5 versus Table 3-4). This lends st rength to the theory that NSBs are easier to incorporate at lower temperatures. Another reason d UTP is more readily incorporated at lower temperatures could be due to its ability to epimerize (Fig. 4-1) (Wellin gton and Benner, 2006, Cohn, 1960, Chambers et al., 1963). Epimerization can occur at two stages. First, the d UTP might epimerize, converting d UTP (the substrate) to -d UTP (not a substrate). At worst, the -d UTP might be an inhibitor, but this possibility is not considered. The consequence of this conversion is to lower the concentration of -epimer, as well as its amount. If th e concentration of the triphosphate is less than its Michaelis constant this would slow the rate of primer extension. Here, the concentrations are 500 M, not dramatically higher than Taq polymerases reported Kms for dNTPs (16 M) (Kong et al., 1993), but gi ven the long extension time (3 min), it is doubtful that this is the origin of any effect seen. Alternativ ely, if the PCR is run to the point of exhaustion of the triphosphates, then a lower yield of PCR product is expected simply because the conversion

PAGE 117

117 of -d UTP to -d UTP leads to earlier exhaustion. Sin ce the amplicon is about 56% GC, this is considered not to be likel y, as the triphosphates are present in equal concentrations, implying that dGTP and/or dCTP would be the first to be exhausted. The alternative possibility is that the -d UTP is epimerizing at high temperatures after it is incorporated into the amplicon. Unlike with epimerization as the triphosphate, epimerized amplicon cannot simply be ignored. Rather, it creates serious problems with read-through by the polymerase; the amplicon may be lost to further PCR even with a single -d UTP. In all experiments, the total concentration (T+d UTP or T+d TTP) were kept constant (500 M). The total primer concentration (1.4 M each, 7 x 10-11 molecules per 50 L assay) is approximately five orders of magnitude greater th an the number of copies of the plasmids (about 300 copies per cell, and about 106 cells per assay). With 2603 nucleotides per amplicon, the triphosphates are nominally consumed after 15.5 P CR cycles; the primer is nominally consumed after 17 PCR cycles. This PCR was carried out for 15 rounds, but it is doubtful that nominal perfection (true doubling each round) was observe d here, or anywhere in PCR literature. The results with d UTP and d TTP in carious concentrations and at different temperatures are shown in Table 4-6. While errors in the first column, where the pseudo component concentration is zero, are large, trends across the series ar e consistent. It appears that d TTP supports the formation of PCR produc t at higher concentrations than d UTP. There is no obvious way to explain this if the limitation on product formation involves the exhaustion of either the triphosphate or the primer. Rather, it is consistent with a slow rate of epimerization of d UTP once it is incorporated into the amplicon. With d TTP, eventually at high concentrations, PCR product formation subsides. Th is is attributed to th e accumulative effects of too many unnatural nucleotides present in the amplicon (approaching 25%).

PAGE 118

118 Numerous approaches for library design exist to support directed evolut ion; this study has compared two of them: A) a rationally designed library based on comparative sequence analysis of the active sites of Family A polymerases, and B) a randomly mutagenized library with no preference to the location of mutations. Our st udies have shown that we are more likely to generate active, thermostable mutants with a ra ndomly mutated library than with our rationally designed library. This supports the conclusions drawn by Arnol d and colleagues (Drummond et al., 2005), who determined that libraries with high error-rates distributed throughout the entirety of the gene, result in a higher than exp ected number of active and unique variants. Our RD Library was designed to identify pol ymerases with an increased ability to incorporate NSBs, not for thermostability, so th e you get what you select for theory may be applicable in this situation. We were able to identify one mutant with an increased ability over coTaq polymerase to incorporate d UTP; this, however, is only one example of a C-glycoside. Since it is possible that d UTP is epimerizing at the temperatures tested, perhaps the testing of other C-glycosides, such as d TTP, can assist in the identifica tion of more mutants with the capability of incorporating these, or other NS Bs. Additionally, useful information could be gained by testing the ability all of our RD and L4 mutants for their ab ility to incorporate d TTP, or other NSBs, not only about inco rporation of NSBs, but also in regards to favorable library design.

PAGE 119

119 O H O R H H H H RO NNH O O H H OH H O R H H H H RO NNH O O H Figure 4-1. Epimerization of 2-deoxypseudour idine. 2-deoxypseudouridine can epimerize under acidic, basic, or even neutral conditions over time, in either the nucleoside or the oligonucleotide forms. Polymerases will not incorporate the epimerized form of this nucleotide, therefore use of the non-epimerizing 2-deoxypseudothymidine is recommended.

PAGE 120

120 Table 4-1. Additional bacteria l strains used in this study. NameStrainGenotypeNameStrainGenotypeSW135 E. coli DH5 SW1/pL4Mut1 (pASK-IBA43+ with co taq L4 mut 1, Apr) SW210 E. coli DH5 SW1/pL4Mut78 (pASK-IBA43+ with co taq L4 mut 78, Apr) SW136 E. coli DH5 SW1/pL4Mut2 (pASK-IBA43+ with co taq L4 mut 2, Apr) SW211 E. coli DH5 SW1/pL4Mut79 (pASK-IBA43+ with co taq L4 mut 79, Apr) SW137 E. coli DH5 SW1/pL4Mut3 (pASK-IBA43+ with co taq L4 mut 3, Apr) SW212 E. coli TG-1 SW1/pL4Mut1 (pASK-IBA43+ with co taq L4 mut 1, Apr) SW138 E. coli DH5 SW1/pL4Mut4 (pASK-IBA43+ with co taq L4 mut 4, Apr) SW213 E. coli TG-1 SW1/pL4Mut2 (pASK-IBA43+ with co taq L4 mut 2, Apr) SW139 E. coli DH5 SW1/pL4Mut5 (pASK-IBA43+ with co taq L4 mut 5, Apr) SW214 E. coli TG-1 SW1/pL4Mut3 (pASK-IBA43+ with co taq L4 mut 3, Apr) SW140 E. coli DH5 SW1/pL4Mut6 (pASK-IBA43+ with co taq L4 mut 6, Apr) SW215 E. coli TG-1 SW1/pL4Mut4 (pASK-IBA43+ with co taq L4 mut 4, Apr) SW141 E. coli DH5 SW1/pL4Mut7 (pASK-IBA43+ with co taq L4 mut 7, Apr) SW216 E. coli TG-1 SW1/pL4Mut5 (pASK-IBA43+ with co taq L4 mut 5, Apr) SW142 E. coli DH5 SW1/pL4Mut8 (pASK-IBA43+ with co taq L4 mut 8, Apr) SW217 E. coli TG-1 SW1/pL4Mut6 (pASK-IBA43+ with co taq L4 mut 6, Apr) SW143 E. coli DH5 SW1/pL4Mut9 (pASK-IBA43+ with co taq L4 mut 9, Apr) SW218 E. coli TG-1 SW1/pL4Mut7 (pASK-IBA43+ with co taq L4 mut 7, Apr) SW144 E. coli DH5 SW1/pL4Mut10 (pASK-IBA43+ with co taq L4 mut 10, Apr) SW219 E. coli TG-1 SW1/pL4Mut8 (pASK-IBA43+ with co taq L4 mut 8, Apr) SW145 E. coli DH5 SW1/pL4Mut11 (pASK-IBA43+ with co taq L4 mut 11, Apr) SW220 E. coli TG-1 SW1/pL4Mut9 (pASK-IBA43+ with co taq L4 mut 9, Apr) SW146 E. coli DH5 SW1/pL4Mut12 (pASK-IBA43+ with co taq L4 mut 12, Apr) SW221 E. coli TG-1 SW1/pL4Mut10 (pASK-IBA43+ with co taq L4 mut 10, Apr) SW147 E. coli DH5 SW1/pL4Mut13 (pASK-IBA43+ with co taq L4 mut 13, Apr) SW222 E. coli TG-1 SW1/pL4Mut11 (pASK-IBA43+ with co taq L4 mut 11, Apr) SW148 E. coli DH5 SW1/pL4Mut14 (pASK-IBA43+ with co taq L4 mut 14, Apr) SW223 E. coli TG-1 SW1/pL4Mut12 (pASK-IBA43+ with co taq L4 mut 12, Apr) SW149 E. coli DH5 SW1/pL4Mut15 (pASK-IBA43+ with co taq L4 mut 15, Apr) SW224 E. coli TG-1 SW1/pL4Mut13 (pASK-IBA43+ with co taq L4 mut 13, Apr) SW150 E. coli DH5 SW1/pL4Mut16 (pASK-IBA43+ with co taq L4 mut 16, Apr) SW225 E. coli TG-1 SW1/pL4Mut14 (pASK-IBA43+ with co taq L4 mut 14, Apr) SW151 E. coli DH5 SW1/pL4Mut17 (pASK-IBA43+ with co taq L4 mut 17, Apr) SW226 E. coli TG-1 SW1/pL4Mut15 (pASK-IBA43+ with co taq L4 mut 15, Apr) SW152 E. coli DH5 SW1/pL4Mut18 (pASK-IBA43+ with co taq L4 mut 18, Apr) SW227 E. coli TG-1 SW1/pL4Mut16 (pASK-IBA43+ with co taq L4 mut 16, Apr) SW153 E. coli DH5 SW1/pL4Mut19 (pASK-IBA43+ with co taq L4 mut 19, Apr) SW228 E. coli TG-1 SW1/pL4Mut17 (pASK-IBA43+ with co taq L4 mut 17, Apr) SW154 E. coli DH5 SW1/pL4Mut20 (pASK-IBA43+ with co taq L4 mut 20, Apr) SW229 E. coli TG-1 SW1/pL4Mut18 (pASK-IBA43+ with co taq L4 mut 18, Apr) SW155 E. coli DH5 SW1/pL4Mut21 (pASK-IBA43+ with co taq L4 mut 21, Apr) SW230 E. coli TG-1 SW1/pL4Mut20 (pASK-IBA43+ with co taq L4 mut 20, Apr) SW156 E. coli DH5 SW1/pL4Mut22 (pASK-IBA43+ with co taq L4 mut 22, Apr) SW231 E. coli TG-1 SW1/pL4Mut21 (pASK-IBA43+ with co taq L4 mut 21, Apr) SW157 E. coli DH5 SW1/pL4Mut23 (pASK-IBA43+ with co taq L4 mut 23, Apr) SW232 E. coli TG-1 SW1/pL4Mut22 (pASK-IBA43+ with co taq L4 mut 22, Apr) SW158 E. coli DH5 SW1/pL4Mut24 (pASK-IBA43+ with co taq L4 mut 24, Apr) SW233 E. coli TG-1 SW1/pL4Mut23 (pASK-IBA43+ with co taq L4 mut 23, Apr) SW159 E. coli DH5 SW1/pL4Mut25 (pASK-IBA43+ with co taq L4 mut 25, Apr) SW234 E. coli TG-1 SW1/pL4Mut26 (pASK-IBA43+ with co taq L4 mut 26, Apr) SW160 E. coli DH5 SW1/pL4Mut26 (pASK-IBA43+ with co taq L4 mut 26, Apr) SW235 E. coli TG-1 SW1/pL4Mut27 (pASK-IBA43+ with co taq L4 mut 27, Apr) SW161 E. coli DH5 SW1/pL4Mut27 (pASK-IBA43+ with co taq L4 mut 27, Apr) SW236 E. coli TG-1 SW1/pL4Mut28 (pASK-IBA43+ with co taq L4 mut 28, Apr) SW162 E. coli DH5 SW1/pL4Mut28 (pASK-IBA43+ with co taq L4 mut 28, Apr) SW237 E. coli TG-1 SW1/pL4Mut29 (pASK-IBA43+ with co taq L4 mut 29, Apr) SW163 E. coli DH5 SW1/pL4Mut29 (pASK-IBA43+ with co taq L4 mut 29, Apr) SW238 E. coli TG-1 SW1/pL4Mut30 (pASK-IBA43+ with co taq L4 mut 30, Apr) SW164 E. coli DH5 SW1/pL4Mut30 (pASK-IBA43+ with co taq L4 mut 30, Apr) SW239 E. coli TG-1 SW1/pL4Mut31 (pASK-IBA43+ with co taq L4 mut 31, Apr) SW165 E. coli DH5 SW1/pL4Mut31 (pASK-IBA43+ with co taq L4 mut 31, Apr) SW240 E. coli TG-1 SW1/pL4Mut32 (pASK-IBA43+ with co taq L4 mut 32, Apr) SW166 E. coli DH5 SW1/pL4Mut32 (pASK-IBA43+ with co taq L4 mut 32, Apr) SW241 E. coli TG-1 SW1/pL4Mut33 (pASK-IBA43+ with co taq L4 mut 33, Apr) SW167 E. coli DH5 SW1/pL4Mut33 (pASK-IBA43+ with co taq L4 mut 33, Apr) SW242 E. coli TG-1 SW1/pL4Mut34 (pASK-IBA43+ with co taq L4 mut 34, Apr) SW168 E. coli DH5 SW1/pL4Mut34 (pASK-IBA43+ with co taq L4 mut 34, Apr) SW243 E. coli TG-1 SW1/pL4Mut35 (pASK-IBA43+ with co taq L4 mut 35, Apr) SW169 E. coli DH5 SW1/pL4Mut35 (pASK-IBA43+ with co taq L4 mut 35, Apr) SW244 E. coli TG-1 SW1/pL4Mut36 (pASK-IBA43+ with co taq L4 mut 36, Apr) SW170 E. coli DH5 SW1/pL4Mut36 (pASK-IBA43+ with co taq L4 mut 36, Apr) SW245 E. coli TG-1 SW1/pL4Mut37 (pASK-IBA43+ with co taq L4 mut 37, Apr) SW171 E. coli DH5 SW1/pL4Mut37 (pASK-IBA43+ with co taq L4 mut 37, Apr) SW246 E. coli TG-1 SW1/pL4Mut38 (pASK-IBA43+ with co taq L4 mut 38, Apr) SW172 E. coli DH5 SW1/pL4Mut38 (pASK-IBA43+ with co taq L4 mut 38, Apr) SW247 E. coli TG-1 SW1/pL4Mut39 (pASK-IBA43+ with co taq L4 mut 39, Apr) SW173 E. coli DH5 SW1/pL4Mut39 (pASK-IBA43+ with co taq L4 mut 39, Apr) SW248 E. coli TG-1 SW1/pL4Mut40 (pASK-IBA43+ with co taq L4 mut 40, Apr) SW174 E. coli DH5 SW1/pL4Mut40 (pASK-IBA43+ with co taq L4 mut 40, Apr) SW249 E. coli TG-1 SW1/pL4Mut41 (pASK-IBA43+ with co taq L4 mut 41, Apr) SW175 E. coli DH5 SW1/pL4Mut41 (pASK-IBA43+ with co taq L4 mut 41, Apr) SW250 E. coli TG-1 SW1/pL4Mut42 (pASK-IBA43+ with co taq L4 mut 42, Apr) SW176 E. coli DH5 SW1/pL4Mut42 (pASK-IBA43+ with co taq L4 mut 42, Apr) SW251 E. coli TG-1 SW1/pL4Mut43 (pASK-IBA43+ with co taq L4 mut 43, Apr) SW177 E. coli DH5 SW1/pL4Mut43 (pASK-IBA43+ with co taq L4 mut 43, Apr) SW252 E. coli TG-1 SW1/pL4Mut44 (pASK-IBA43+ with co taq L4 mut 44, Apr) SW178 E. coli DH5 SW1/pL4Mut44 (pASK-IBA43+ with co taq L4 mut 44, Apr) SW253 E. coli TG-1 SW1/pL4Mut45 (pASK-IBA43+ with co taq L4 mut 45, Apr) SW179 E. coli DH5 SW1/pL4Mut45 (pASK-IBA43+ with co taq L4 mut 45, Apr) SW254 E. coli TG-1 SW1/pL4Mut46 (pASK-IBA43+ with co taq L4 mut 46, Apr) SW180 E. coli DH5 SW1/pL4Mut46 (pASK-IBA43+ with co taq L4 mut 46, Apr) SW255 E. coli TG-1 SW1/pL4Mut48 (pASK-IBA43+ with co taq L4 mut 48, Apr) SW181 E. coli DH5 SW1/pL4Mut47 (pASK-IBA43+ with co taq L4 mut 47, Apr) SW256 E. coli TG-1 SW1/pL4Mut50 (pASK-IBA43+ with co taq L4 mut 50, Apr) SW182 E. coli DH5 SW1/pL4Mut48 (pASK-IBA43+ with co taq L4 mut 48, Apr) SW257 E. coli TG-1 SW1/pL4Mut51 (pASK-IBA43+ with co taq L4 mut 51, Apr) SW183 E. coli DH5 SW1/pL4Mut49 (pASK-IBA43+ with co taq L4 mut 49, Apr) SW258 E. coli TG-1 SW1/pL4Mut52 (pASK-IBA43+ with co taq L4 mut 52, Apr) SW184 E. coli DH5 SW1/pL4Mut50 (pASK-IBA43+ with co taq L4 mut 50, Apr) SW259 E. coli TG-1 SW1/pL4Mut53 (pASK-IBA43+ with co taq L4 mut 53, Apr) SW185 E. coli DH5 SW1/pL4Mut51 (pASK-IBA43+ with co taq L4 mut 51, Apr) SW260 E. coli TG-1 SW1/pL4Mut54 (pASK-IBA43+ with co taq L4 mut 54, Apr) SW186 E. coli DH5 SW1/pL4Mut52 (pASK-IBA43+ with co taq L4 mut 52, Apr) SW261 E. coli TG-1 SW1/pL4Mut55 (pASK-IBA43+ with co taq L4 mut 55, Apr) SW187 E. coli DH5 SW1/pL4Mut53 (pASK-IBA43+ with co taq L4 mut 53, Apr) SW262 E. coli TG-1 SW1/pL4Mut56 (pASK-IBA43+ with co taq L4 mut 56, Apr) SW188 E. coli DH5 SW1/pL4Mut54 (pASK-IBA43+ with co taq L4 mut 54, Apr) SW263 E. coli TG-1 SW1/pL4Mut57 (pASK-IBA43+ with co taq L4 mut 57, Apr) SW189 E. coli DH5 SW1/pL4Mut55 (pASK-IBA43+ with co taq L4 mut 55, Apr) SW264 E. coli TG-1 SW1/pL4Mut58 (pASK-IBA43+ with co taq L4 mut 58, Apr) SW190 E. coli DH5 SW1/pL4Mut56 (pASK-IBA43+ with co taq L4 mut 56, Apr) SW265 E. coli TG-1 SW1/pL4Mut59 (pASK-IBA43+ with co taq L4 mut 59, Apr) SW191 E. coli DH5 SW1/pL4Mut57 (pASK-IBA43+ with co taq L4 mut 57, Apr) SW266 E. coli TG-1 SW1/pL4Mut60 (pASK-IBA43+ with co taq L4 mut 60, Apr) SW192 E. coli DH5 SW1/pL4Mut58 (pASK-IBA43+ with co taq L4 mut 58, Apr) SW267 E. coli TG-1 SW1/pL4Mut61 (pASK-IBA43+ with co taq L4 mut 61, Apr) SW193 E. coli DH5 SW1/pL4Mut59 (pASK-IBA43+ with co taq L4 mut 59, Apr) SW268 E. coli TG-1 SW1/pL4Mut62 (pASK-IBA43+ with co taq L4 mut 62, Apr) SW194 E. coli DH5 SW1/pL4Mut60 (pASK-IBA43+ with co taq L4 mut 60, Apr) SW269 E. coli TG-1 SW1/pL4Mut63 (pASK-IBA43+ with co taq L4 mut 63, Apr) SW195 E. coli DH5 SW1/pL4Mut61 (pASK-IBA43+ with co taq L4 mut 61, Apr) SW270 E. coli TG-1 SW1/pL4Mut64 (pASK-IBA43+ with co taq L4 mut 64, Apr) SW196 E. coli DH5 SW1/pL4Mut62 (pASK-IBA43+ with co taq L4 mut 62, Apr) SW271 E. coli TG-1 SW1/pL4Mut65 (pASK-IBA43+ with co taq L4 mut 65, Apr) SW197 E. coli DH5 SW1/pL4Mut63 (pASK-IBA43+ with co taq L4 mut 63, Apr) SW272 E. coli TG-1 SW1/pL4Mut66 (pASK-IBA43+ with co taq L4 mut 66, Apr) SW198 E. coli DH5 SW1/pL4Mut64 (pASK-IBA43+ with co taq L4 mut 64, Apr) SW273 E. coli TG-1 SW1/pL4Mut67 (pASK-IBA43+ with co taq L4 mut 67, Apr) SW199 E. coli DH5 SW1/pL4Mut65 (pASK-IBA43+ with co taq L4 mut 65, Apr) SW274 E. coli TG-1 SW1/pL4Mut68 (pASK-IBA43+ with co taq L4 mut 68, Apr) SW200 E. coli DH5 SW1/pL4Mut66 (pASK-IBA43+ with co taq L4 mut 66, Apr) SW275 E. coli TG-1 SW1/pL4Mut69 (pASK-IBA43+ with co taq L4 mut 69, Apr) SW201 E. coli DH5 SW1/pL4Mut67 (pASK-IBA43+ with co taq L4 mut 67, Apr) SW276 E. coli TG-1 SW1/pL4Mut70 (pASK-IBA43+ with co taq L4 mut 70, Apr) SW202 E. coli DH5 SW1/pL4Mut68 (pASK-IBA43+ with co taq L4 mut 68, Apr) SW277 E. coli TG-1 SW1/pL4Mut71 (pASK-IBA43+ with co taq L4 mut 71, Apr) SW203 E. coli DH5 SW1/pL4Mut69 (pASK-IBA43+ with co taq L4 mut 69, Apr) SW278 E. coli TG-1 SW1/pL4Mut72 (pASK-IBA43+ with co taq L4 mut 72, Apr) SW202 E. coli DH5 SW1/pL4Mut70 (pASK-IBA43+ with co taq L4 mut 70, Apr) SW279 E. coli TG-1 SW1/pL4Mut73 (pASK-IBA43+ with co taq L4 mut 73, Apr) SW203 E. coli DH5 SW1/pL4Mut71 (pASK-IBA43+ with co taq L4 mut 71, Apr) SW280 E. coli TG-1 SW1/pL4Mut74 (pASK-IBA43+ with co taq L4 mut 74, Apr) SW204 E. coli DH5 SW1/pL4Mut72 (pASK-IBA43+ with co taq L4 mut 72, Apr) SW281 E. coli TG-1 SW1/pL4Mut75 (pASK-IBA43+ with co taq L4 mut 75, Apr) SW205 E. coli DH5 SW1/pL4Mut73 (pASK-IBA43+ with co taq L4 mut 73, Apr) SW282 E. coli TG-1 SW1/pL4Mut76 (pASK-IBA43+ with co taq L4 mut 76, Apr) SW206 E. coli DH5 SW1/pL4Mut74 (pASK-IBA43+ with co taq L4 mut 74, Apr) SW283 E. coli TG-1 SW1/pL4Mut77 (pASK-IBA43+ with co taq L4 mut 77, Apr) SW207 E. coli DH5 SW1/pL4Mut75 (pASK-IBA43+ with co taq L4 mut 75, Apr) SW284 E. coli TG-1 SW1/pL4Mut78 (pASK-IBA43+ with co taq L4 mut 78, Apr) SW208 E. coli DH5 SW1/pL4Mut76 (pASK-IBA43+ with co taq L4 mut 76, Apr) SW285 E. coli TG-1 SW1/pL4Mut79 (pASK-IBA43+ with co taq L4 mut 79, Apr) SW209 E. coli DH5 SW1/pL4Mut77 (pASK-IBA43+ with co taq L4 mut 77, Apr)

PAGE 121

121 Table 4-2. L4 Mutant Library. Plasmid Name Mutations Present in L4 Taq Library Plasmid Name Mutations Present in L4 Taq Library pL4Mut1 L4Q,G16S,R91H,E292G,D575N,S620P pL4Mut41 E794G,M804V pL4Mut2 V110A pL4Mut42 L530P,K539N,L654P pL4Mut3 G197C,F269S,K790R pL4Mut43 F44I,E167G pL4Mut4 L409P,V615I,K828R pL4Mut44 Y392F,N412D,N562D,E649G pL4Mut5 L27Q,L30Q,R263S,L273R,L409P pL4Mut45 P809T,E227K pL4Mut6 F89S,I160T,P261L, GAP pL4Mut46 GAP pL4Mut7 V38G,K222E,F255I,E407A,E691A pL4Mut47 NONE pL4Mut8 P552L,L765P pL4Mut48 GAP pL4Mut9 N482I pL4Mut49 NONE pL4Mut10 A83G,I135N,L285Q,Y336H, GAP pL4Mut50 K216I,A455D,V651E pL4Mut11 G393S,T444P,M670T,E710G pL4Mut51 L108P,G197S,L377P,R390C,T503A,GAP pL4Mut12 L789P pL4Mut52 K125M pL4Mut13 E6D,K351R,A797D,G821D pL4Mut53 E167G,S309P,T719A,L814Q pL4Mut14 L122Q,D341G,A411V pL4Mut54 W315C,T506P pL4Mut15 I596M,M643V pL4Mut55 GAP pL4Mut16 A115P,L458R,H558P,H617R,L654P,K801E pL4Mut56 Y158C,S309T,A404T,S540G,M758T pL4Mut17 Y113H,G276V,L409P pL4Mut57 V38A,K337E,V796D pL4Mut18 R693C,E731G,V812A pL4Mut58 D101G pL4Mut19 NONE pL4Mut59 H330P, GAP pL4Mut20 F44L,T183A,K194E,L291P pL4Mut60 R91H,F561S pL4Mut21 K337E pL4Mut61 D493G pL4Mut22 L362P,M441I,E517V pL4Mut62 S121T,E599V,Y808HpL4Mut23 G81D,K203E,V446A,D634G pL4Mut63 A80V,R220C,G367C,D378N,R389L,S574G,G752D,M776L pL4Mut24 NONE pL4Mut64 L4R,I150N,K203R,K337E,Q563Stop,P647L,A774V pL4Mut25 NONE pL4Mut65 W425R,E771G,V796D pL4Mut26 GAP pL4Mut66 L13P,A565V pL4Mut27 L279P,S287T pL4Mut67 L93P,E156G,A213T,P299S,N580S,E771G,V796D pL4Mut28 L12P,V133M,N217D,L266P,E300G,L546P,W703R,L825P pL4Mut68 E109K,G209C,W240R,L373Q pL4Mut29 A231V, GAP pL4Mut69 K46E,V118A,L218P,I529T,Q579H pL4Mut30 K203I,A268G,D544N,R633L,T753A,V763A pL4Mut70 G184C,L491P,R556H pL4Mut31 L285P, GAP pL4Mut71 S309Y,D369G,F479S,I581V,A605T pL4Mut32 R91L,E534G pL4Mut72 E420D,E678G,K828E pL4Mut33 L221P,E264V,K528R,GAP pL4Mut73 K216I,T503A,T511M,Q589R,K759E pL4Mut34 L777P,Stop830 pL4Mut74 V780A pL4Mut35 N624Y pL4Mut75 K759R pL4Mut36 K337E pL4Mut76 E109K,G209C,W240R,L373Q pL4Mut37 A126P, GAP pL4Mut77 F721L pL4Mut38 D185V,L491P,M643V pL4Mut78 Y42H,R220C,W425Stop,R590W pL4Mut39 Y75C,K216E,S377T,A565V,M758T,E770D pL4Mut79 Y169C,T247A,D248G,E638G pL4Mut40 GAP,GAP *All are derivatives of the cotaq gene, and all are inserted into the pASK-IBA43plus vector. NONE means no mutations were found relative to the coTaq sequence, GAP denotes the presence of a frameshift mutation within the protein; and Stop indicates the presence of a Stop codon in the sequence.

PAGE 122

122 Figure 4-2. Representative images of ethidium-b romide stained agarose gels resolving products arising from PCR amplification using standard dNTPs and three different polymerases. Cells expressing the in dicated polymerase provided both the polymerase and the template plasmid for the reaction. Polymerases were therefore forced to replicate their own encoding ge ne (2603 bp) using primers P-4 and P-5. Optimal temperatures for each polymerase were determined by identifying the FLP band having the highest density. A) The coTaq polymerase having an optimal temperature of 89.0 C. B) The polymerase expressed in SW17 cells having an optimal temperature of 89.0 C. C) The polymerase expressed in SW251 cells having an optimal temperature of 94.0 C. A) B) C)

PAGE 123

123 Table 4-3. Generation of full le ngth PCR products from dNTPs by individual polymerases from the rationally designed (RD) Librar y at the indicated temperatures. Optimal Temp 86.389.091.192.693.794.0SW4Codon-Optimized (co) wt Taq89.0268194229250662705135257010124747212364200 SW5S573E,Y668F,A740S86.3316793560300000 SW6Q486H,K537I,M670G-000000 SW7A605G,L613A,E739P86.3260207325100251400114000 SW8D575F,L606C,A740S86.3351275533451223215369315530130026912914907 SW9T511V,R584V,I611E-000000 SW10N480R,F595V,E742H86.3178875974183210372981071869605564354588 SW11E517I,V583K,A597S89.0675098995847868690798423724526781743 SW12D575F,V583K,M670A86.3332982705726676270172780330258 SW13E517I,D607W,D622S-000000 SW14A594C,F664Y,A774H86.3168797715292991178437873870516390284889 SW15F595W,L606P,D622S86.33107200000 SW16S573E,D575F,F595V89.087879997897443058511256200 SW17S510I,A605K,L606S89.0206480722506151780581144747626625939332 SW18S573E,D622L,E742H-000000 SW19N480R,T511V,Y542E-000000 SW20A594C,F664H,M670G-000000 SW21Q486H,D575T,N580S86.3251691423776422415212200199520986861867511 SW22S510I,A605E,E612I-000000 SW23A594C,E612I,M670A-000000 SW24S510I,Q579A,I611Q-000000 SW25A594T,L606C,R657D89.0234369424146862041814189763314898401095528 SW26T541A,A605G,L606S86.31957603184683710809389678200 SW27E517G,K537I,L613A86.3172882514688641031245801755537510465426 SW28K537I,Q579A,E742V-000000 SW29A597S,A740R,E742V266993926726522649972230814422939932126919 SW30N580Q,A605E,L613I89.01055603173173514224634610047284828402 SW31N580S,F595V,A605G86.315212291380438119473546163010528243184 SW32N580S,D622L,A774H-000000 SW33R533I,R584V,F664L-000000 SW34Q486H,E517G,A605K89.0258296326735152412059229583523197942054398 SW35S573H,F664Y,R743A-000000 SW36D575T,N580Q,R584V89.011630319172316710714857210195277632 SW37T541A,F664L,R743A-000000 SW38T511V,R533I,D622A-000000 SW39A597S,I611E,Y668F89.0415640955460000 SW40Y542E,F595W,L606C-000000 SW41L606S,R657D,E739R89.072735927889782565988238419618471721518298 SW42S573H,D575T,L613I-000000 SW43T541A,L606P,L613D-000000 SW44Y542E,V583K,A605E-000000 SW45E517I,F595W,A605E,I611E-000000 SW46T541A,D575F,L613A,D622A89.0514801861910000 SW47T511V,A594C,L606S,A740R89.0164626023878012248001216287118239571588298 SW48Q486H,R533I,L606C,L613A-000000 SW49Q486H,F595V,D622A,F664Y89.018892220666128711000 SW50E517I,S573H,A605G,E612I-000000 SW51Y542E,R584V,A605K,E612I-000000 SW52D575T,A605E,L606C,D622A86.37086000000 SW53A594T,L613A,F664Y,E742H86.321252962020864148773622525400 SW54D575F,N580Q,W601G,D622S86.319848531070720000 SW55K537I,L606P,A740S,E742H86.329671022375974566103000 SW56A597S,W601G,L606S,F664H86.36042231618500000 SW57S510I,E517G,D607W,I611E-000000 SW58S510I,V583K,R584V,L606P-000000 SW59N480R,R533I,A597S,M670G-000000 SW60E612I,D622L,F664L,E739P-000000 SW61I611Q,M670G,E739P,E742H-000000 SW62F595W,F664H,Y668F,E739P-000000 SW63A597S,A605G,D622A,F664L-000000 SW64L606P,I611E,E739R,R743A-000000 SW65D607W,I611Q,R657D,E742V-000000 SW66T541A,I611Q,L613I,D622L-000000 SW67K537I,S573H,N580S,D622S-000000 SW68N480R,S573E,D607W,A740R-000000 SW69D575T,L613D,E739R,A774H86.33541500000 SW70Q579A,R657D,F664Y,A740R-000000 SW71R533I,K537I,A605K,L613I-000000 SW72T511V,E517G,L606C,F664Y89607861899097748202686452668056716970 SW73D575T,F664H,E742V,R743A-000000 SW74A594C,I611E,F664L,A740S86.314672399162620000 SW75N580S,L613A,A740S,R743A-000000 SW76S510I,T511V,L613I,E739R8910118151256969984215820803736008655700 SW77V583K,E612I,L613D,Y668F-000000 SW78S573E,R584V,A594C,D622S-000000Cell Line Substitutions RawDensities(CNT/mm2) *The pink rows indicate the sequences of polymer ases that generated full-length PCR product at a temperature at 94.0 C and below. The green rows indicate th e sequences of polymerases that generated full-length PCR product at a temperature between 86.3 C and 93.7 C, but not at 94.0 C, suggesting thermal instability. The blue rows indicate the seque nces of polymerases that lack evidence of activity at any temperature.

PAGE 124

124 Table 4-4. Generation of full le ngth PCR products from dNTPs by individual polymerases from the randomly generated (L4) Librar y at the indicated temperatures. Optimal Temp 86.389.091.192.693.794.0SW489.0268194229250662705135257010124747212364200 SW212pL4Q,G16S,R91H,E292G,D575N,S620P-000000 SW213V110A94.000629162696297776068789298 SW214G197C,F269S,K790R-000000 SW215pL409P,V615I,K828R-000000 SW216L27Q,L30Q,R263S,L273R,pL409P-000000 SW217F89S,I160T,P261L, GAP-000000 SW218V38G,K222E,F255I,E407A,E691A-000000 SW219P552L,L765P86.37549500000 SW220N482I91.1294562308802352467341925313543303106 SW221A83G,I135N,L285Q,Y336H, GAP-000000 SW222G393S,T444P,M670T,E710G-000000 SW223L789P89.019670623508036241000 SW224E6D,K351R,A797D,G821D-000000 SW225L122Q,D341G,A411V94.0276216318539401690406474409339439202 SW226I596M,M643V89.0197651122668752145291202764219638161765708 SW227A115P,pL458R,H558P,H617R,L654P,K801E-000000 SW228Y113H,G276V,pL409P-000000 SW229R693C,E731G,V812A89.022959072574544192912428735100 SW230F44L,T183A,K194E,L291P91.10152790181860171210136258109580 SW231K337E89.015565028302562594346246507024534822217280 SW232L362P,M441I,E517V-000000 SW233G81D,K203E,V446A,D634G-000000 SW234GAP-000000 SW235L279P,S287T92.6191930220367302023186214182719915951881015 SW236L12P,V133M,N217D,L266P,E300G,L546P,W703R,L825P-000000 SW237A231V, GAP-000000 SW238K203I,A268G,D544N,R633L,T753A,V763A86.3125304455440000 SW239L285P, GAP-000000 SW240R91L,E534G91.158916522023622313418208963620264481996181 SW241L221P,E264V,K528R,GAP-000000 SW242L777P,Stop830,Stop83189.0751487110538483247650416118801497316 SW243N624Y89.0337177805380736375693938672573595106 SW244K337E89.030555324944042346465218061820739382098704 SW245A126P, GAP-000000 SW246D185V,pL491P,M643V-000000 SW247Y75C,K216E,S377T,A565V,M758T,E770D89.01586157228832819739941367562456778125527 SW248GAP,GAP-000000 SW249E794G,M804V86.3956992887580701751485211214733116760 SW250L530P,K539N,L654P86.3544325413000000 SW251F44I,E167G94.045033510722661113110117097211655281283853 SW252Y392F,N412D,N562D,E649G89.057813546638440068335711216585156511 SW253P809T,E227K89.08289921840442140841200550618722901813403 SW254GAP-000000 SW255GAP-000000 SW256K216I,A455D,V651E86.3385109294115251941130359478790 SW257L108P,G197S,L377P,R390C,T503A,GAP-000000 SW258K125M89.0495313796179723534627670527588704237 SW259E167G,S309P,T719A,L814Q-000000 SW260W315C,T506P86.32319736328423784000 SW261GAP-000000 SW262Y158C,S309T,A404T,S540G,M758T86.31952541187997717164861218814828628460152 SW263V38A,K337E,V796D86.312471459353354286515095800 SW264D101G89.0257503026354502621795256949025963922453437 SW265H330P, GAP-000000 SW266R91H,F561S86.3213187018214857736515127300 SW267D493G86.3286101728455382527108231985724127472320855 SW268S121T,E599V,Y808H86.31142750111471110423421019279945295915580 SW269A80V,R220C,G367C,D378N,R389L,S574G,G752D,M776L-000000 SW270pL4R,I150N,K203R,K337E,Q563Stop,P647L,A774V86.3410343261521904000 SW271W425R,E771G,V796D-000000 SW272L13P,A565V-000000 SW273L93P,E156G,A213T,P299S,N580S,E771G,V796D86.333845221210000 SW274E109K,G209C,W240R,L373Q91.16621178915921018264961926605490705172 SW275K46E,V118A,L218P,I529T,Q579H-000000 SW276G184C,pL491P,R556H-000000 SW277S309Y,D369G,F479S,I581V,A605T-000000 SW278E420D,E678G,K828E86.31392467122746198092254000612969440950 SW279K216I,T503A,T511M,Q589R,K759E-000000 SW280V780A89.0146360415253261383685130521012396261132704 SW281K759R89.0171381417365441434711149336013785101262243 SW282E109K,G209C,W240R,L373Q92.6163899229883278944333824180119193040 SW283F721L93.7104251415106171497583150548215585791448625 SW284Y42H,R220C,W425Stop,R590W-000000 SW285Y169C,T247A,D248G,E638G91.1552304643967674392593949395651340181Raw Densities(CNT/mm2) Cell Line Substitutions *The pink rows indicate the sequences of polymer ases that generated full-length PCR product at a temperature at 94.0 C and below. The green rows indicate th e sequences of polymerases that generated full-length PCR product at a temperature between 86.3 C and 93.7 C, but not at 94.0 C, suggesting thermal instability. The blue rows indicate the seque nces of polymerases that lack evidence of activity at any temperature.

PAGE 125

125 Figure 4-3. Number of active RD and L4 mutant s at various temperatur es. A) The number of polymerases from the RD Library (a total of 74) that show a FLP band after a PCR run at the indicated temperature. B) The number of polymerases from the L4 Library (a total of 74) th at show a FLP band after a PCR run at the indicated temperature. 33 30 24 21 18 17 0 5 10 15 20 25 30 35Number of Active RD Mutants 86.3 89.0 91.1 92.6 93.7 94.0 Optimal Temperatures (oC) 37 37 35 32 29 28 0 5 10 15 20 25 30 35 40Number of Active L4 Mutants 86.3 89.0 91.1 92.6 93.7 94.0 Optimal Temperatures (oC)A) B)

PAGE 126

126 Table 4-5. Incorporation of d UTP by RD Library at optimal temperatures. Optimal Temp (oC) All dNTPs 9 mM dT/ 1 mM d U 8 mM dT/ 2 mM d U 7 mM dT/ 3 mM d U 6 mM dT/ 4 mM d U 5 mM dT/ 5 mM d U 4 mM dT/ 6 mM d U 3 mM dT/ 7 mM d U 2 mM dT/ 8 mM d U 1 mM dT/ 9 mM d U All d UNTPsSW4Codon-Optimized (co) wt Taq86.32312987224579021186821882497198660714670549047812780053747400 SW4Codon-Optimized (co) wt Taq89.02897387275582726183082278423195625612299737194591657563293700 SW5S573E,Y668F,A740S86.347790255425013124617572513672025594245420000 SW7A605G,L613A,E739P86.31602269164626414012681060751775918415561175702567003200300 SW8D575F,L606C,A740S86.3165171811603441175086125610514901729358595952031570664928700 SW10N480R,F595V,E742H86.32447053188390901937412211411113316459245212238214720100 SW11E517I,V583K,A597S89.0152258190882289567580425210199947326453907501197772040200 SW12D575F,V583K,M670A86.3244500000000000 SW14A594C,F664Y,A774H86.320425191602378142509812586171222901625304221786591123336700 SW15F595W,L606P,D622S86.300000000000 SW16S573E,D575F,F595V89.04991013011112295931484614725355941 00000 SW17S510I,A605K,L606S89.02533093208144322813081758469134736945164719613145945000 SW21Q486H,D575T,N580S86.31962394162200216466171759955180613910223595461301619263478400 SW25A594T,L606C,R657D89.0844336562078403590220913125825545140000 SW26T541A,A605G,L606S86.32092635220753821145001849280165317911636365713551662054149100 SW27E517G,K537I,L613A86.310965915530515473874342275430882353269467349521000 SW29A597S,A740R,E742V86.327937532548317271680923546232534067197610513381444711157912200 SW30N580Q,A605E,L613I89.07227114541554309933462762199601368387143435934000 SW31N580S,F595V,A605G86.31423097148359513104191238333162965812267287135711933365845700 SW34Q486H,E517G,A605K89.02258399219216316951771831735173280411284605227431496573929500 SW36D575T,N580Q,R584V89.01553454950046602523935406929149147700000 SW39A597S,I611E,Y668F86.375644657530434816321083913937747287281600000 SW41L606S,R657D,E739R89.0423788423788423788423788423788423788423788423788000 SW46T541A,D575F,L613A,D622A89.0541353801726102432803675129931 00000 SW47T511V,A594C,L606S,A740R89.05434104473103963383829123215331396615505120913000 SW49Q486H,F595V,D622A,F664Y89.04535643213053092882059219984638405381840000 SW52D575T,A605E,L606C,D622A86.33382123322862953452557352052851153094462118348000 SW53A594T,L613A,F664Y,E742H86.3178242012894521101912571901372153113447368700000 SW54D575F,N580Q,W601G,D622S86.315830121815276194069816510941410232846848395770804723643900 SW55K537I,L606P,A740S,E742H86.317545981788506163284412788791102959424994166452452632165200 SW56A597S,W601G,L606S,F664H86.31519620 000000000 SW69D575T,L613D,E739R,A774H86.3373410000000000 SW72T511V,E517G,L606C,F664Y89.024472118650016105113593096028478522417712878000 SW74A594C,I611E,F664L,A740S86.3130697610750337387814738352737451047174454029520000 SW76S510I,T511V,L613I,E739R89.0437213262515229710991671689081019173743222388000Cell Line Substitutions Raw Densities (CNT/mm2) *The pink rows indicate the polymerases that showed activity with a temperature of 94.0 C and lower; these data can be compared to those in Table 3-4. The green rows indicate the polymerases that showed activity with a temperature between 86.3 C and 93.7 C; these had no activity at 94.0 C, suggesting thermal instability.

PAGE 127

127 Figure 4-4. Generation of fu ll length PCR product at 86.3 C using d UTP by the coTaq polymerase and the RD polymerase in the SW29 cell line. Concentrations of dNTPs/d UNTPs listed are the starting concentr ations (see Materials and Methods for listing of final concentrations). A) Incorporation of various dNTP/d UNTP ratios by coTaq polymerase. FLP is not generated beyond the ratio of 2 mM TTP/8 mM d UTP. B) Incorporation of various dNTP/d UNTP ratios by SW29 cells. FLP is not generated beyond th e ratio of 2 mM TTP/8 mM d UTP. C) A graphical comparison of the band densities in each of these gels. The red columns correlate to the bands in gel A; the blue columns represen t those in gel B. Densities can also be found in Table 4-5. A ) B ) C ) All dNTPs 9 mM/1 mM 8 mM/2 mM 7 mM/3 mM 6 mM/4 mM 5 mM/5 mM 4 mM/6 mM 3 mM/7 mM 2 mM/8 mM 1 mM/9 mM All d UNTPs SW4 SW29 0 500000 1000000 1500000 2000000 2500000 3000000Raw Density (CNT/mm2)Ratios of dT/d U

PAGE 128

128 Figure 4-5. Generation of fu ll length PCR product at 94.0 C and 86.3 C using d UTP by the RD polymerase in the SW8 cell line. Concentrations of dNTPs/d UNTPs listed are the starting concentrations (see Materials and Methods for listing of final concentrations). A) Inco rporation of various dNTP/d UNTP ratios by SW8 cells at 94.0 C. FLP is not generated beyo nd the ratio of 3 mM TTP/7 mM d UTP. B) Incorporation of various dNTP/d UNTP ratios by SW8 cells at their optimal temperature of 86.3 C. FLP is not genera ted beyond the ratio of 2 mM TTP/8 mM d UTP. C) A graphical comparison of the ba nd densities in each of these gels. The red columns correlate to the bands in gel A; the blue columns represent those in gel B. Densities can also be found in Table 3-4 and Table 4-3. All dNTPs 9 mM/1 mM 8 mM/2 mM 7 mM/3 mM 6 mM/4 mM 5 mM/5 mM 4 mM/6 mM 3 mM/7 mM 2 mM/8 mM 1 mM/9 mM All d UNTPs 94.0 86.3 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000Raw Density (CNT/mm2)Ratio of dT/d UoCA) B) C)

PAGE 129

129Table 4-6. Incorporation of d UTP and d TTP by coTaq Polymerase at various temperatures. 500 mM dNTPs 450 mM dT/ 50 mM d U 400 mM dT/ 100 mM d U 350 mM dT/ 150 mM d U 300 mM dT/ 200 mM d U 250 mM dT/ 250 mM d U 200 mM dT/ 300 mM d U 150 mM dT/ 350 mM d U 100 mM dT/ 400 mM d U 50 mM dT/ 450 mM d U 500 mM d UNTPs SW486.32312987224579021186821882497198660714670549047812780053747400 SW489.02897387275582726183082278423195625612299737194591657563293700 SW491.16377503872434719694924142498951243695559027124000 SW492.6455238323427258032222532153441623793700824186000 SW493.7643793350500304969131233172586709103604825309000 SW494.02244256200537119956491535822125537958963718875264360000 Cell Line Melting Temp (oC) 500 mM dNTPs 450 mM dT/ 50 mM d T 400 mM dT/ 100 mM d T 350 mM dT/ 150 mM d T 300 mM dT/ 200 mM d T 250 mM dT/ 250 mM d T 200 mM dT/ 300 mM d T 150 mM dT/ 350 mM d T 100 mM dT/ 400 mM d T 50 mM dT/ 450 mM d T 500 mM d TNTPs SW486.3131505617050271783891183784616645751361746114312811600867936121592480 SW489.01383077149902116827361506228171960816567901374745915308543815729100 SW491.1805345860275820663804330826318729993593906425430278006588990 SW492.6714674107472711322787828011109007713425545528463738200326378580 SW493.7549273525350490532450728486603409646290744211733121997361270 SW494.0364939431868517729363169446363332797302432239203112759347630 Raw Densities (CNT/mm2) Cell Line Melting Temp (oC)

PAGE 130

130 Figure 4-6. Generation of fu ll length PCR product at 86.3 C by coTaq polymerase using various TTP:d UTP and TTP:d TTP ratios. Concentrations of dNTPs/d UNTPs/d TNTPs listed are the starting con centrations (see Materials and Methods for listing of final concentrati ons). A) Incorporation of various dNTP/d UNTP ratios by coTaq polymerase at 86.3 C. FLP is not generated beyond the ratio of 2 mM TTP/8 mM d UTP. B) Incorporation of various dNTP/d TNTP ratios by coTaq polymerase at 86.3 C. FLP is not generated beyond the ratio of 1 mM TTP/9 mM d TTP. C) A graphical comparison of the band densities in each of these gels. The red columns correlate to the bands in gel A; the blue columns represent those in gel B. Densities can also be found in Table 4-6. All dNTPs 9 mM/1 mM 8 mM/2 mM 7 mM/3 mM 6 mM/4 mM 5 mM/5 mM 4 mM/6 mM 3 mM/7 mM 2 mM/8 mM 1 mM/9 mM All d UNTPs/d TNTPs d U d T 0 500000 1000000 1500000 2000000 2500000Raw Density (CNT/mm2)Ratio of dT to d U/d TA) C) B)

PAGE 131

131 Figure 4-7. Graphical comp arisons of the band densities listed in Table 4-6. All of the reactions were identical except for the temperature of the PCR and ratios of dT:d U or dT:d T. The red columns corre late to the densities of full-length PCR product bands present on gels containing the d U studies. The blue columns correlate to the densities of the full-length PCR product bands present on gels containing the d T studies. Data for the 86.3 C PCR study is shown in Figur e 4-6C. A) A graphical comparison of the band densities at 89.0 C. B) A graphical comparison of the band densities at 91.1 C. C) A graphical comparis on of the band densities at 92.6 C. D) A graphical comparison of the band densities at 93.7 C. E) A graphical comparison of the band densities at 94.0 C. All dNTPs 9 mM/1 mM 8 mM/2 mM 7 mM/3 mM 6 mM/4 mM 5 mM/5 mM 4 mM/6 mM 3 mM/7 mM 2 mM/8 mM 1 mM/9 mM All d UNTPs/d TNTPs d U d T 0 200000 400000 600000 800000 1000000 1200000Density (CNT/mm2)Ratio of T to d U or d T 92.6 oC All dNTPs 9 mM/1 mM 8 mM/2 mM 7 mM/3 mM 6 mM/4 mM 5 mM/5 mM 4 mM/6 mM 3 mM/7 mM 2 mM/8 mM 1 mM/9 mM All d UNTPs/d TNTPs d U d T 0 500000 1000000 1500000 2000000 2500000 3000000Raw Density (CNT/mm2)Ratio of dT to d U/d T 89.0 oC All dNTPs 9 mM/1 mM 8 mM/2 mM 7 mM/3 mM 6 mM/4 mM 5 mM/5 mM 4 mM/6 mM 3 mM/7 mM 2 mM/8 mM 1 mM/9 mM All d UNTPs/d TNTPs d U d T 0 100000 200000 300000 400000 500000 600000 700000 800000 900000Raw Density (CNT/mm2)Ratio of dT to d U/d T 91.1 oC All dNTPs 9 mM/1 mM 8 mM/2 mM 7 mM/3 mM 6 mM/4 mM 5 mM/5 mM 4 mM/6 mM 3 mM/7 mM 2 mM/8 mM 1 mM/9 mM All d UNTPs/d TNTPs d U d T 0 100000 200000 300000 400000 500000 600000 700000Density (CNT/mm2)Ratio of T to d U or d T 93.7 oC All dNTPs 9 mM/1 mM 8 mM/2 mM 7 mM/3 mM 6 mM/4 mM 5 mM/5 mM 4 mM/6 mM 3 mM/7 mM 2 mM/8 mM 1 mM/9 mM All d UNTPs/d TNTPs d U d T 0 500000 1000000 1500000 2000000 2500000Density (CNT/mm2)Ratio of T to d U or d T 94.0 oCA) B) C) D) E)

PAGE 132

132 CHAPTER 5 CONCLUSIONS The notion of creating an arti ficially expanded genetic in formation system (AEGIS) by adding extra letters to the DNA al phabet has sparked interest in determining what features of these non-standard nucleotides (NSBs) could be a hindrance for incorporation by polymerases. Studies have been performed on the incorporation of a variety of different NSBs, such as those lacking minor-groove electrons (Hendrickson et al., 2004), those with a C-glycosidic linkage (Lutz et al., 1999), and those that do not allo w for the creation of hydrogen bonds between the nucleobases (Delaney et al., 2003) However, prior to this study, only a limited amount of research has studied the incorporation of multiple sequential NSBs. This dissertation focused on the stability of the duplex DNA containing multiple C-glycosides, the ability of polymerases to incorporate multiple, sequential nucleotides contai ning a C-glycosidic linkage, and the directed evolution of polymerases to incorporate thes e nucleotides more efficiently and faithfully. DNA Helical Structure in the Presence of C-Glycosides Using circular dichroism (CD), previous st udies of the helical structure of duplex DNA have shown that poly(U) poly(A) helices favor the Ahelical conformation while poly(dT) poly(dA) helices display B-DNA struct ure (Ivanov et al., 1973, Saenger, 1984, Chandrasekaran and Radha, 1992). It was necessar y to determine if the presence of multiple Cglycosides in double-stranded DNA (dsDNA) would alter the conf ormation of the helix to a point where there is a phase transition from B-DNA to A-DNA, possibl y making it difficult for polymerases evolved to handle B-DNA helices unable to replic ate the DNA containing multiple C-glycosidic nucleotides. CD measurements were used to test dsDNA containing from one to twelve consecutive d U dA or dT dA base pairs. Results indicated that at 25 C, the addition of more U did not

PAGE 133

133 generate a trend in the CD spectra that might i ndicate a change from a Bhelical conformation to an A-helical conformation. Relativ ely little difference was observed between the CD spectra of duplexes containing increasing numbers of 2-deoxypseudouridine (d U) nucleotides and those containing dT. These data suggest that gro ss conformational change should not present a problem for a polymerase to incorporate and replicate DNA containing these C-glycosides. Polymerase Screen for the Incorporation of C-glycosides Previous studies on the incorporation of Cglycosides required only that polymerases incorporate up to three consecu tive 2-deoxypseudothymdidine (d T) residues into a growing DNA strand (Lutz et al., 1999). To create an artificially expanded alphabet that freely incorporates C-glycosides, in cluding the AEGIS alphabet that has three species with a Cglycosidic linkage (Fig. 1-4), polymerases would be required to incorporat e more than three of these NSBs consecutively, efficiently, and faithfully. In the first part of this study, primer-extensi on assays were used to screen a number of Family A and Family B polymerases for their ab ility to incorporate and extend beyond four of the two representative C-glyc osides, 2-deoxypseudouridine (d U) and 2deoxypseudothymidine (d T). Studies described here showed that although the Klenow (exo-), Bst Large Fragment, and Therminator polymerases performed exceptionally well in their ability to incorporate and fully extend beyond four consecutive d T and d U nucleotides. Klenow (exo-) and Bst are not thermostable, and thus canno t support PCR. Further, according to its manufacturer, Therminator is not recommende d for any applications except DNA sequencing and primer-extension reactions. This means that none of these three polymerases were likely candidates for future studies. Taq polymerase, however, which was also able to incorporate and

PAGE 134

134 extend beyond the four NSBs, albe it with less efficiency, is ab le to support high temperature PCR. Taq was therefore selected as a candidate for further study. It is also interesting to note that base d on full-length product (FLP) band densities, it appears that the incorporation of d TTP by polymerases was more efficient than the incorporation of d UTP. Taq Polymerase Primer-Extension Assays If Taq was used for the starting point to obtain polymerases that accep t C-glycosides, it must replicate its own encoding polymerase gene, forcing it to incorporate four consecutive d T or d U across from template dA, as this is th e longest run of consecutive dAs in the taq polymerase gene. Since we have already shown that Taq can incorporate and extend beyond four consecutive C-glycosides, as seen in Chap ter 2 of this dissertati on, we next needed to demonstrate its ability to incorporate and extend beyond up to twelve consecutive d T-dA or d U-dA base pairs. Results showed that the production of FLP was terminated if it requi red the incorporation of more than five consecutive C-glycosides by Taq polymerase. The FLP band densities from this data showed that the d TTP was incorporated more efficiently than the d UTP. If this polymerase is to be used as a potential candida te for synthetic biology containing C-glycosides, it must first be modified by dire cted evolution experiments to allow it to incorporate more consecutive C-glycosides. Growth and Purification of Taq Polymerase A tightly regulated plasmid containing an N-terminal hexahistidine tagged wt taq gene (His(6)wt Taq ) was constructed and transformed into the E. coli TG-1 expression strain (Skerra, 1994). Growth and expression conditions were th en optimized prior to using the cells in

PAGE 135

135 selection experiments. Previous studies showed that the expression of polymerases in vivo is toxic to the cells (Moreno et al., 2005, Andraos et al., 2004); th is was also observed in these studies. Once the most favorable set of expressi on conditions was ascertained (a 1 hr expression following a late log phase induction), the His(6)wt Taq polymerase was purified via nickel chromatography and its activity was tested. Almo st identical amounts of FLP were found to be generated in a PCR reaction when identical concentrations (ng/ L) of the purified His(6)wt Taq polymerase and Taq polymerase purchased from New England BioLabs were used; this signifies that the purified protein isolated was indeed an active polymerase. It was noted that a low level of His(6)wt Taq polymerase was being produced after only 1 hr of induction, most likely best explained by polymerase toxicity. To rectify this situation, the gene encoding His(6)wt Taq polymerase was optimized for codon-usage in E. coli (cotaq gene) by our collaborators, DNA 2.0 Inc (Gustafsson et al., 2004). The codon-optimization does not affect the toxicity of the protein, but it does allow the E. coli cells to produce a greater amount of protein in the same amount of time. Once optim ized, after one hour of induction, at least three times as much polymerase was produced, as evidenced by the density of the bands on a Coomassie blue stained SDS-PA GE (7.5%) gel (Fig. 3-6C). The cotaq gene was cloned into the tightly re gulated plasmid with a histidine tag, transformed into E. coli cells, and its growth and expression conditions were compared to those of the His(6)wt Taq polymerase. The data revealed that under identical e xpression conditions, more polymerase was produced by cells expressing the coTaq polymerase than those expressing the His(6)wt Taq polymerase. To maximize the formati on of product in the directed evolution reactions, the cells containing coTaq polymerase were used in all further experiments. This is the first example of a polymerase that has been optimized for the codon usage of the expression

PAGE 136

136 cell strain. The success in the overproduc tion of large quantities of active coTaq polymerase in E. coli relative to the overproduction of His(6)wt Taq in cells, could be useful for other applications such as structural studies and commercial production, whic h require large amounts of protein. Creation of coTaq Polymerase Mutant Libraries Literature presents many different theories re garding the best methods to create a library most useful for directed evolut ion experiments. Such a libra ry contains a large number of diverse, yet active clones (Hibbert and Da lby, 2005, Arnold and Georgiou, 2003b, Drummond et al., 2005, Park et al., 2005, Dalby, 2003, Parikh and Matsumura, 2005, Crameri et al., 1998, Crameri et al., 1996, Castle et al., 2004). For this dissertation, two of these methods were selected for comparative analysis. The first wa s a rationally designed (RD) library, generated by Dr. Eric Gaucher (FfAME), th rough the selection of specific replacement amino acids based on a combination of evolutionary analysis and prev ious functional studies. In addition, a random library (termed L4) was generated with mutations randomly spread across the whole polymerase sequence. Creation of the Rationally Design ed Mutagenic Library (RD Library) The reconstructing evolutionary adaptive paths (REAP) approach, was used to create the RD Library, allowing for modification at residues where Type II functional divergence occurred within a family of polymerases. In this approach, sites were identified that, in the historical evolution of the polymerase, had a split conser ved but different pattern of evolutionary variation, and had previously been suggested to lead to a change in the function or behavior of the polymerase. Using this technique in comb ination with sequences discussed in a recent review on the evolution of novel polymerase activities (Henry and Romesberg, 2005), a total of 57 amino acid changes at 35 sites in the Taq polymerase sequence were chosen. The 57

PAGE 137

137 replacement amino acid residues were selected fro m those found at those sites within the Family A viral polymerase sequences, as li terature has revealed that vira l polymerases are more able to incorporate NSBs than other po lymerases (Sismour et al., 2004, Leal et al., 2006, Horlacher et al., 1995). The FfAME collaborators at DNA 2.0 th en created and synthesized the RD library containing 74 differe nt mutant sequences; the 57 amino acid changes we dictated were used in various combinations to yield three or four amin o acid mutations per sequence. This approach to creating mutagenic libraries restri cts the diversity based on evolu tionary data, but in doing so, was predicted to create a larg e number of active clones. Creation of the Random Mut agenic Library (L4 Library) The L4 random mutagenic library was created from the cotaq gene using error-prone PCR with the mutagens MnCl2 and Taq polymerase and primers flanking either end of the gene (Arnold and Georgiou, 2003b). This allowed mutations to be locat ed anywhere in the sequence of the gene. Rather than ri sk losing large quantities of the mutagenic PCR product during digestions, ligations, and purifica tion, a variation of the megaprim er PCR protocol was used to create the full length plasmids with inserts (Miyazak i and Takenouchi, 2002). This procedure was found to be extremely useful when creating lib raries, as it generates crossover mutations and reversions, introducing more diversity. The 74 unique clones generate d by these techniques contained approximately three am ino acid changes per sequence resu lting from an average of 4.3 base mutations per gene. These combined pr ocedures are recommende d for creating future mutagenic libraries because of their simplicity, th eir cost, and the ease with which they can be modified to increase or decrease the number of mutations per gene. Preliminary Studies of the Incorporation of d UTP by the RD Library Initially, members of the RD library were indivi dually tested for their ability to incorporate increasing concentrations of d UTP into PCR products. It was discovered that only 18 of the 74

PAGE 138

138 mutants tested were able to form FLP, even in the presence of only standard dNTPs. At this point, the design of the RD library was questioned, and it was noticed that at least one of the sites we had mutated had been previ ously shown to be involved in the thermostability of the Taq polymerase (Ghadessy et al., 2001). In addition, we designed our 57 replacement am ino acid residues based on the sequences of Family A viral polymerases, wh ich are only thermostable up to 37 C. Therefore, it is very likely that the mutations introduced in this appro ach caused a decrease in the thermostability of the RD polymerase variants The next step was to test the ability of the polymerase variants to function at a variet y of temperatures. Incorporation of dNTPs by RD and L4 Libraries at Various Temperatures The mutants from each library were individually tested for their ability to form FLP at various temperatures in PCRs containing only standard dNTPs. We found that by lowering the temperature from 94.0 C to 86.3 C, the number of mutant generating PCR products increased. In the RD Library, the number of active mutant polymerases increased from 18 to 33; in the L4 Library, 39 mutants were active when the temp erature was lowered, compared with only 27 active at a temperature of 94.0 C. These results suggest that it more likely to generate active, thermostable mutants with a randomly mutated libra ry than with a rationally designed library. This supports the conclusions drawn by Arnold and colleagues (Drummond et al., 2005), who determined that libraries with mutations distribu ted throughout the en tirety of the gene are more likely to result in active and unique variants than if the mutations were limited to the active site. Based on the generalization that approximately one-third of all random amino acid changes will result in the inactivation of a protein (G uo et al., 2004), and the design we employed to create the RD Library, it was perhaps reasonable to expect that more active mutants would be present in the RD Library than the randomly created L4 Librar y, when the same number of

PAGE 139

139 clones were tested. Since this was not the case, the design of the RD Library must be examined. It was also reasonable to concl ude that by focusing mutations in and around the active site, the risk of knocking out activity was increased, even though the s ites chosen were known to be variable, and the residues chosen were known to function in the evolutionary history of the polymerase. This library, however, was designed w ith the incorporation of NSBs in mind, so the possibility was investigated that the RD variants will have an incr eased ability to incorporate the C-glycosides when compared to coTaq polymerase. Incorporation of d UTP by the RD Library at Optimal Temperatures After the identification of the optimal te mperature for each of the 33 active RD polymerases, we challenged the polymerases to incorporate increasing concentrations of d UTP in PCR reactions at their optimal temperature. It was discovered that one RD mutant polymerase (pSW27: A597S, A740R, E742V) was able to incorporate d UTP more efficiently than the coTaq polymerase. The A597S mutation has previously been shown to assist in the incorporation of rNTPs (Xia et al., 2002), while the E742 mutation contributed to the incorporation of various NSBs (Ghadessy et al., 2004). Since d UTP is closely related to rUTP and is an NSB, it is arguable that these changes contri buted greatly to the activity of this mutant. The remaining 32 polymerases tested were unable to incorporate the d UTP as well as coTaq polymerase; they were, however, able to generate more FLP at th eir optimal temperature than when they were tested previously at 94.0 C. It is possible, that the increased ability to incorporate C-glycosides at lower temperatures can be attributed to the fact that NSBs are so metimes incorporated more efficiently at lower temperatures (Rappaport, 2004, Horlacher et al., 1995) Alternatively, it was considered that the d UTP is epimerizing at the higher temperatures, thereby making it difficult for the polymerase

PAGE 140

140 to incorporate the base into a growing DNA strand (Wellington and Benner, 2006, Cohn, 1960, Chambers et al., 1963). Therefore, a test was de signed to establish which of these theories was actually occurring. Incorporation of d UTP and d TTP by coTaq Polymerase at Various Temperatures The presence of the methyl group on d TTP inhibits the epimerization of the C-glycoside (Wellington and Benner, 2006), so it was possible to perform a comp arative analysis between coTaq polymerases ability to cope with d UTP and d TTP in various concentrations and at different temperatures. The coTaq polymerase was found able to incorporate final concentrations of d TTP greater than those with d UTP at all temperatures tested. This leads to the conclusion that the epimerization of the nuc leotide is hindering th e incorporation of d UTP, consequently this should not be used as a model C-glycoside in future studies. Selection of Thermostable RD Mutants Using Water-In-Oil Emulsions Selections require that some members of the library perform differently than the original protein of interest (Arnold and Georgiou, 2003a, Lutz and Patrick, 2004). One of the goals of this research is to evolve polymerases to incorporate various C-glycoside triphosphates efficiently and faithfully, thus it makes sense to perform an initial selection to identify mutants able into incorporate C-glycosides After noting that the d UTP was most likely epimerizing under our reaction conditions, and consid ering our availabl e quantities of d TTP were limited, we decided to select for the eighteen mutant polymerases in the RD Library that exhibited activity with dNTPs at a temperature of 94.0 C from the pool of the 74 RD mutants. In doing so, we were able to demonstrate our laboratorys ability to perform in vitro selections. A variation of the compartmen talized self-replica tion (CSR) method was used to create water-in-oil emulsions containing all 74 mutants, as a way to li nk genotype to phenotype (Fig. 1-

PAGE 141

141 13) (Miller et al., 2006, Tawfik and Griffiths, 1998, Ghadessy et al., 2001, Ghadessy et al., 2004). Products from the selection were recl oned into the expre ssion vector using the megaprimer PCR method previously discussed (Miyazaki and Takenouchi, 2002). Unfortunately, this protocol cause d numerous crossovers, reversi ons, and additions, so we were not able to determine the true sequences of th e all polymerases we isolated using the CSR. However, the megaprimer PCR reveals itself as an effective method for library rediversification between rounds of selection. We were able to identify one mutant from th e 50 clones we sequenced that coded for one of the eighteen variants previously shown to have activity under these reaction conditions. This demonstrates an ability to perform successful in vitro selections in our laboratory. If this selection was to be repeated, and products were cloned using the standard digestion, ligation, and purification techniques, it would most likely yield some to all of the eighteen sequences of active polymerases. Future Experimentation The results presented in this dissertation open the door for many future experiments. Further structural studies of the DNA contai ning multiple sequential C-glycosides can expound upon the knowledge gathered here. Given that we now know 2-deoxyp seudouridine is not a good representative of a C-glycoside, we can create duplex DNA containing 2deoxypseudothymidine and perform similar circular dichroism studies to di stinguish what helical form the DNA assumes. In addition, thermal dupl ex denaturation studies can be performed with these duplex struct ures containing d T to ascertain the st ability of the duplex DNA formed when multiple, sequential C-glycosides are present (Geyer et al., 2003).

PAGE 142

142 Additional study of rationally de signed library creation is no w possible. Since we now know that the use of viral re sidues to replace those of Taq polymerase at some sites may cause a decrease in protein thermostability, in the de sign of future librari es, we can avoid making mutations at these sites, and possibly increase th e percentage of active mu tants in the library. Furthermore, in future libraries, we can take into consideration the mu tation of sites throughout the polymerase sequence that display Type II func tional divergence, not just those in and around the active site. A distinct decrease in the amount of full-length PCR product was observed when the PCR based assays were performed using increasing co ncentrations of the C-glycoside and decreasing concentrations of dT. A contro l reaction using only decreasing levels of dT and no thymidine analogue should be performed to determine how much of the full-length PCR product generated in future reactions actually cont ains the C-glycosides versus ho w much is produced only using dT. Another reason we may be seeing this de crease in FLP formation with increasing Cglycoside concentration could be due to the ethidium bromide dye being used to aid in the visualization of the DNA. It is, perhaps, plausi ble that the ethidium br omide cannot intercalate as efficiently when multiple C-glycosides are present. Therefore, a comparative analysis between the amounts of FLP formed when using ethidium bromide versus another fluorescent dye, such as the SYBR Safe DNA Gel Stain, could be performed. It would also be interesting to test the 74 L4 mutants for their ability to incorporate Cglycosides more efficiently than the coTaq polymerase. This information will also help determine if the mutation of residues not in the ac tive site is beneficial to the incorporation of NSBs. Moreover, if the 74 RD mutants were rete sted for their ability to incorporate increasing

PAGE 143

143 levels of d TTP as opposed to d UTP, we may find more than one polymerase that incorporates the NSBs more efficiently than coTaq Once an acceptable library is created, in vitro evolution experiments can be performed to identify polymerases able to incorporate high levels of d TTP, rather than testing each mutant individually. These selections will begin with a moderate ratio of d T to dT, and increase with each round of selection. Between rounds, we now know that our libraries can be rediversified using the megaprimer PCR protocol, thereby redu cing the risk of large quantities of product being lost to the purification steps required fo r traditional recloning steps. After demonstrating our ability to perform a selection for polymeras es that can incorporate C-glycosides, we can begin to apply these techniques to develop pol ymerases that can incorporate more NSBs. Directed evolution is already being used in industry to improving the quality of and developing new industrial enzymes and therapeu tic treatments (Chirumamilla et al., 2001, Douthwaite and Jermutus, 2006). If the conjunct ion of the rationally de signed library with the modified CSR technique proves to be successful in the isolation of large numbers of active clones, there could be a commerci al impact for this system. Ri ght now it takes three to four rounds of selection to isolate cl ones with a desired trait, and e ach round takes at least one week; with our system, it is feasible that only one to two rounds of selection would be needed, thereby cutting the time in half. In addition, the use of synthetic gene libraries reduces the amount of time spent creating libraries de novo With an improved ability to produce more clones with desired activity from smaller st arting libraries, imagine how many products could be quickly isolated using these techniques.

PAGE 144

144 APPENDIX A SYNTHESIS OF PSEUDOTHYMIDINE AND PSEUDOTHYMIDINE-CONTAINING OLIGONUCLEOTIDES Synthesis of the 2-deoxypseudothymidine (d T) precursor was performed by Dr. Shuichi Hoshika according to the procedures previously set forth, with some modifications (actual scheme shown in Figure A-1) (Bhattacharya et al., 1995, Lutz et al., 1999, Zhang and Daves, 1992). The synthesis of 2-deoxypseudothymidine-5-triphosphate (d TTP) was performed by Dr. Daniel Hutter according to the standard Ludwig-Eckstein procedure for triphosphate synthesis (Ludwig and Eckstein, 1989). It was purified by HPLC on a Waters Delta 600 with Waters 2487 Dual wavelength absorbance detector controlled by Waters Millennium software. Initial purification was on i on-exchange column [GE Health care HiPrep 16/10 DEAE FF column, eluent A = 10 mM NH4CO3, eluent B = 1 M NH4CO3, gradient from 0 to 80% B in 40 min, flow rate = 3 mL/min, Rt = 22 min] followed by reverse pha se HPLC [Waters NovaPak HR C18 column, 19x300 mm, eluent A = 25 mM triet hylammonium acetate (TEAA) pH 7, eluent B = 10% CH3CN in 25 mM TEAA pH 7, gradient from 0 to 80% B in 32 min, flow rate = 5 mL/min, Rt = 16 min]. After l yophilization, it was twi ce re-dissolved in water and lyophilized again to remove excess TEAA. Analytical HP LC was performed to verify the purification [Waters Alliance 2695 with Waters 2996 PDA de tector, controlled by Waters Millennium software; Dionex DNAPac PA-100 colum n, 4x250 mm, eluent A = 10 mM NH4CO3, eluent B = 500 mM NH4CO3, gradient from 0 to 40% B in 20 min, flow rate = 0.5 mL/min: Rt = 17 min]. NMR (Varian Mercury 300 MHz spectrometer): 1H-NMR (D2O, 300 MHz): (ppm, rel to HDO = 4.65) = 1.97 (ddd, J = 5.9, 9.9, 13.3 Hz, 1H); 2.13 (ddd, J = 2.6, 5.9, 13.3 Hz, 1H); 3.27 (s, 3H); 3.94-4.00 (m, 3H); 4.39-4.41 (m, 1H); 4.97 (dd, J = 5.9, 9.6 Hz, 1H); 7.65 (d, J = 0.8 Hz,

PAGE 145

145 1H). 31P-NMR (D2O, 121 MHz): (ppm, rel to external standard H3PO4 = 0) = -10.7 (d, J = 20 Hz, 1P); -11.2 (d, J = 20 Hz, 1P); -23.3 ( t J = 20 Hz, 1P).

PAGE 146

146 S yn t hesis o f pseudo t hymidineO HO TBDPSO 8NH HN O I 9 a O HO TBDPSO NH HN O O HO O NH HN O O HO HO NH HN O b c 101112 Scheme 2. a) Pd(OAc)2, Ph3As, Bu3N, MeCN b) TBAF, AcOH, THF c) NaBH(OAc)3, AcOH, MeCN Scheme 3. a) Ac2O, DMAP, DMF, 30C b) MeI, N O -bis(trimethylsilyl)acetamide, CH2Cl2 ,reflux or MeI, ( i -Pr)2NEt, DMF c) K2CO3, MeOH d) DMTrCl, pyridine e) ClPN( i -Pr)2OCH2CH2CN, ( i -Pr)2NEt, CH2Cl2 f) aq. AcOH, THFO O O O O HO HO NH HN O 12O a b c O AcO AcO NH HN O 13O O AcO AcO NH N O 14O O HO HO NH N O 15O O HO AcO NH N O 17O O DMTrO HO NH N O 16O e O DMTrO O NH N O 18O fP O N CN d 2 steps 65% 2 steps 52% 96% 58% 82% 91% 73% Figure A-1. Synthesis of pseudothymidine precurs or. This scheme was designed by Dr. Shuichi Hoshika following protocol set forth previ ously (Bhattacharya et al., 1995, Lutz et al., 1999, Zhang and Daves, 1992).

PAGE 147

147 APPENDIX B PHYLOGENETIC TREES OF FAMILY A POLYMERASES The following are insets of the phylogenetic tr ee seen in Figure 3-1 and a seed alignment of twelve of the 719 Family A pol ymerases identified in this tr ee. These trees were generated using Pfam (Bateman, 2006, Finn et al., 2006), an d analyzed for sites that underwent Type II functional divergence. In this approach, Dr. Eric Gaucher identified sites that had a split conserved but different pattern of historical evolutionary variation, and had been previously suggested to lead to a change in the function or behavior of the polymerase (Henry and Romesberg, 2005). Using Pfam, 57 amino acid cha nges across 35 sites were identified within the 719 members of Family A polymerases that we re available to us (B ateman, 2006, Finn et al., 2006). Figure B-1. A seed alignment of the Family A pol ymerases. This tree was generated using Pfam (Bateman, 2006, Finn et al., 2006), and displays twelve representatives of the major genera found in the 719 Family A polymerase sequences.

PAGE 148

148 Figure B-2. Inset of the phylogene tic tree of Family A polymerases (from Fig. 3-1) showing the location of Taq polymerase. This tree was ge nerated using Pfam (Bateman, 2006, Finn et al., 2006).

PAGE 149

149 Figure B-3. Inset of the phylogene tic tree of Family A polymerases (from Fig. 3-1) showing the location of some viral polymerases. This tree was generated us ing Pfam (Bateman, 2006, Finn et al., 2006).

PAGE 150

150 APPENDIX C GENETIC CODE AND AMINO ACID ABBREVIATIONS Table C-1. The Genetic Code. UUUPheUCUSerUAUTyrUGUCysU UUCPheUCCSerUACTyrUGCCysC UUALeuUCASerUAAStopUGAStopA UUGLeuUCGSerUAGStopUGGTrpG CUULeuCCUProCAUHisCGUArgU CUCLeuCCCProCACHisCGCArgC CUALeuCCAProCAAGlnCGAArgA CUGLeuCCGProCAGGlnCGGArgG AUUIleACUThrAAUAsnAGUSerU AUCIleACCThrAACAsnAGCSerC AUAIleACAThrAAALysAGAArgA AUGMetACGThrAAGLysAGGArgG GUUValGCUAlaGAUAspGGUGlyU GUCValGCCAlaGACAspGGCGlyC GUAValGCAAlaGAAGluGGAGlyA GUGValGCGAlaGAGGluGGGGlyG GSecond LetterUCAFirst LetterThird LetterU C A G Table C-2. Amino acid abbreviations. Name3-Letter Code1-Letter Code AlanineAlaA Ar g inineAr g R AsparagineAsnN Aspartic acidAspD CysteineCysC GlutamineGlnQ Glutamic acidGluE GlycineGlyG HistidineHisH IsoleucineIleI LeucineLeuL MethionineMetM PhenylalaninePheF ProlineProP SerineSerS ThreonineThrT TryptophanTrpW TyrosineTyrY ValineValV

PAGE 151

151 LIST OF REFERENCES Allemann, R. K., Presnell, S. R. and Benner, S. A. (1991) Protein Engineering, 4, 831-835. Andraos, N., Tabor, S. and Richardson, C. C. (2004) Journal of Biological Chemistry, 279, 50609-50618. Argoudelis, A. D. and Mizsak, S. A. (1976) Journal of Antibiotics, 29, 818-823. Arnez, J. G. and Steitz, T. A. (1994) Biochemistry, 33, 7560-7567. Arnold, F. H. and Georgiou, G. (Eds.) (2003a) Directed Enzyme Evolution: Screening and Selection Methods, Humana Press, Totowa, N.J. Arnold, F. H. and Georgiou, G. (Eds.) (2003b) Directed Evolution Libr ary Creation: Methods and Protocols, Humana Press, Totowa, N.J. Bain, J. D., Switzer, C., Chamberlin A. R. and Benner, S. A. (1992) Nature, 356, 537-539. Bateman, A. (2006), Vol. 2006, Pfam, Sanger Institute, http://www.sanger.ac.uk/Software/Pfam/. Beard, W. A., Shock, D. D., Vande Be rg, B. J. and Wilson, S. H. (2002) Journal of Biological Chemistry, 277, 47393-47398. Beese, L. S., Derbyshire, V. and Steitz, T. A. (1993a) Science, 260, 352-355. Beese, L. S., Friedman, J. M. and Steitz, T. A. (1993b) Biochemistry, 32, 14095-14101. Benner, S. A. (2004) Accounts of Chemical Research, 37, 784-797. Bhattacharya, B. K., Devivar, R. V. and Revankar, G. R. (1995) Nucleosides & Nucleotides, 14, 1269-1287. Brakmann, S. (2005) Cellular and Molecular Life Sciences, 62, 2634-2646. Brock, T. D. and Freeze, H. (1969) Journal of Bacteriology, 98, 289-297. Castle, L. A., Siehl, D. L., Gorton, R., Patten, P. A., Chen, Y. H., Bertain, S., Cho, H. J., Duck, N., Wong, J., Liu, D. L. a nd Lassner, M. W. (2004) Science, 304, 1151-1154. Chambers, R. W., Kurkov, V. and Shapiro, R. (1963) Biochemistry, 2, 1192-1203. Chandrasekaran, R. and Radha, A. (1992) Journal of Biomolecular Structure & Dynamics, 10, 153-168. Charette, M. and Gray, M. W. (2000) International Union of Biochemistry and Molecular Biology Life, 49, 341-351. Chien, A., Edgar, D. B. and Trela, J. M. (1976) Journal of Bacteriology, 127, 1550-1557.

PAGE 152

152 Chirumamilla, R. R., Muralidhar, R., Marchant, R. and Nigam, P. (2001) Molecular and Cellular Biochemistry, 224, 159-168. Cline, J., Braman, J. C. and Hogrefe, H. H. (1996) Nucleic Acids Research, 24, 3546-3551. Cohn, W. E. (1960) Journal of Biological Chemistry, 235, 1488-1498. Collins, M. L., Irvine, B., Tyner, D., Fine, E ., Zayati, C., Chang, C. A., Horn, T., Ahle, D., Detmer, J., Shen, L. P., Kolberg, J., Bushnell, S., Urdea, M. S. and Ho, D. D. (1997) Nucleic Acids Research, 25, 2979-2984. Crameri, A., Raillard, S. A., Bermudez, E. and Stemmer, W. P. C. (1998) Nature, 391, 288-291. Crameri, A., Whitehorn, E. A., Tate, E. and Stemmer, W. P. C. (1996) Nature Biotechnology, 14, 315-319. Crick, F. (1970) Nature, 227, 561-563. Dalby, P. A. (2003) Current Opinion in Structural Biology, 13, 500-505. Davis, D. R. (1995) Nucleic Acids Research, 23, 5020-5026. Delaney, J. C., Henderson, P. T., He lquist, S. A., Morales, J. C., Essigmann, J. M. and Kool, E. T. (2003) Proceedings of the National Academy of Sciences of the United States of America, 100, 4469-4473. DeLano, W. L. (2002) PyMOL, DeLano Scientific, http://www.pymol.org. Derti, A. (2003), Vol. 2006, Reverse and/or complement DNA sequences, Harvard Medical School, http://arep.med.harvard.edu/labgc/a dnan/projects/Utilities/revcomp.html. Douthwaite, J. and Jermutus, L. (2006) Current Opinion in Drug Discovery & Development, 9, 269-275. Drummond, D. A., Iverson, B. L., Ge orgiou, G. and Arnold, F. H. (2005) Journal of Molecular Biology, 350, 806-816. Egli, M. (2004) Current Opinion in Chemical Biology, 8, 580-591. Emilsson, G. M. and Breaker, R. R. (2002) Cellular and Molecular Life Sciences, 59, 596-607. Eom, S. H., Wang, J. M. and Steitz, T. A. (1996) Nature, 382, 278-281. Fa, M., Radeghieri, A., Henry, A. A. and Romesberg, F. E. (2004) Journal of the American Chemical Society, 126, 1748-1754. Fairbanks, G., Steck, T. L. and Wallach, D. F. H. (1971) Biochemistry, 10, 2606-2617.

PAGE 153

153 Finn, R. D., Mistry, J., Schuster-Bockler, B., Griffiths-Jones, S., Hollich, V., Lassmann, T., Moxon, S., Marshall, M., Khanna, A., Durbin, R., Eddy, S. R., Sonnhammer, E. L. L. and Bateman, A. (2006) Nucleic Acids Research, 34, D247-D251. Forterre, P. (2006) Virus Research, 117, 5-16. Garrett, R. H. and Grisham, C. M. (1999) Biochemistry, Harcourt Brace College Publishers, Fort Worth, TX. Gaucher, E. A. (2006) In National Institute of Health STTR Phase 1 Grant Number 1 R41 GM074433-01, Foundation for Applied Molecula r Evolution, Gainesville, FL. Geyer, C. R., Battersby, T. R. and Benner, S. A. (2003) Structure, 11, 1485-1498. Ghadessy, F. J., Ong, J. L. and Holliger, P. (2001) Proceedings of the National Academy of Sciences of the United States of America, 98, 4552-4557. Ghadessy, F. J., Ramsay, N., Boudsocq, F., Loakes, D., Brown, A., Iwai, S., Vaisman, A., Woodgate, R. and Holliger, P. (2004) Nature Biotechnology, 22, 755-759. Ghosh, A. and Bansal, M. (2003) Acta Crystallographica Secti on D-Biological Crystallography, 59, 620-626. Goldman, M. and Marcy, D. (2001) In HIV-1 Reverse Transcriptase Tutorial pp. 1-3. Griffiths, A. D. and Tawfik, D. S. (2006) Trends in Biotechnology, 24, 395-402. Grosjean, H., Constantinesco, F., Fo iret, D. and Benachenhou, N. (1995) Nucleic Acids Research, 23, 4312-4319. Gu, X. (1999) Molecular Biology and Evolution, 16, 1664-1674. Gu, X. (2002), Vol. 2006, DIVERGE 2.0, Iowa State University, http://xgu.zool.iastate.edu/software.html. Guo, H. H., Choe, J. and Loeb, L. A. (2004) Proceedings of the National Academy of Sciences of the United States of America, 101, 9205-9210. Gustafsson, C., Govindarajan, S. and Minshull, J. (2004) Trends in Biotechnology, 22, 346-353. Hendrickson, C. L., Devine, K. G. and Benner, S. A. (2004) Nucleic Acids Research, 32, 22412250. Henry, A. A., Olsen, A. G., Matsuda, S., Yu, C. Z., Geierstanger, B. H. and Romesberg, F. E. (2004) Journal of the Americ an Chemical Society, 126, 6923-6931. Henry, A. A. and Romesberg, F. E. (2005) Current Opinion in Biotechnology, 16, 370-377. Hibbert, E. G. and Dalby, P. A. (2005) Microbial Cell Factories, 4

PAGE 154

154 Hirao, I., Kimoto, M., Mitsui, T., Fujiwara, T., Kawai, R., Sato, A., Harada, Y. and Yokoyama, S. (2006) Nature Methods, 3, 729-735. Hirao, I., Ohtsuki, T., Fujiwara, T., Mitsui, T ., Yokogawa, T., Okuni, T., Nakayama, H., Takio, K., Yabuki, T., Kigawa, T., Kodama, K., Nishikawa, K. and Yokoyama, S. (2002) Nature Biotechnology, 20, 177-182. Hogrefe, H. H., Cline, J., Lovejoy, A. E. and Nielson, K. B. (2001) In Hyperthermophilic Enzymes, Pt C Vol. 334, pp. 91-116. Horlacher, J., Hottiger, M., Podust, V. N., Hubscher, U. and Benner, S. A. (1995) Proceedings of the National Academy of Sciences of the United States of America, 92, 6329-33. Huisse, F. (2004) Journal of Clinical Virology, 30, S26-S28. Ivanov, V. I., Minchenk, L. E., Schyolki, A. K. and Poletaye, A. I. (1973) Biopolymers, 12, 89110. Johnson, S. C., Marshall, D. J., Harms, G., Miller, C. M., Sherrill, C. B., Beaty, E. L., Lederer, S. A., Roesch, E. B., Madsen, G., Hoffman, G. L ., Laessig, R. H., Kopish, G. J., Baker, M. W., Benner, S. A., Farrell, P. M. and Prudent, J. R. (2004) Clinical Chemistry, 50, 20192027. Joyce, C. M. and Benkovic, S. J. (2004) Biochemistry, 43, 14317-24. Kelman, Z., Hurwitz, J. and O'Donnell, M. (1998) Structure, 6, 121-125. Kim, Y., Eom, S. H., Wang, J. M., Lee, D. S., Suh, S. W. and Steitz, T. A. (1995) Nature, 376, 612-616. Kong, H. M., Kucera, R. B. and Jack, W. E. (1993) Journal of Biological Chemistry, 268, 19651975. Kornberg, A., Lehman, I. R., Bessma n, M. J. and Simms, E. S. (1956) Biochimica Et Biophysica Acta, 21, 197-198. Kunkel, T. A. and Bebenek, R. (2000) Annual Review of Biochemistry, 69, 497-529. Laemmli, U. K. (1970) Nature, 227, 680-685. Lane, B. G., Ofengand, J. and Gray, M. W. (1995) Biochimie, 77, 7-15. Leal, N. A., Sukeda, M. and Benner, S. A. (2006) Nucleic Acids Research, 34, 4702-4710. Lewin, B. (1997) Genes VI, Oxford University Press, New York. Li, J. S., Fan, Y. H., Zhang, Y., Marky, L. A. and Gold, B. (2003) Journal of the American Chemical Society, 125, 2084-2093.

PAGE 155

155 Li, J. S., Shikiya, R., Marky, L. A. and Gold, B. (2004) Biochemistry, 43, 1440-1448. Li, Y., Kong, Y., Korolev, S. and Waksman, G. (1998a) Protein Science, 7, 1116-1123. Li, Y., Korolev, S. and Waksman, G. (1998b) European Molecular Biology Organization Journal, 17, 7514-7525. Limbach, P. A., Crain, P. F. and McCloskey, J. A. (1994) Nucleic Acids Research, 22, 21832196. Lin-Goerke, J. L., Robbins, D. J. and Burczak, J. D. (1997) Biotechniques, 23, 409-12. Ludwig, J. and Eckstein, F. (1989) Journal of Organic Chemistry, 54, 631-635. Lutz, M. J., Horlacher, J. and Benner, S. A. (1998) Bioorganic & Medicinal Chemistry Letters, 8, 1149-1152. Lutz, S., Burgstaller, P. and Benner, S. A. (1999) Nucleic Acids Research, 27, 2792-8. Lutz, S. and Patrick, W. M. (2004) Current Opinion in Biotechnology, 15, 291-297. Michelet, W. and Genet, J. P. (2005) Current Organic Chemistry, 9, 405-418. Miller, J. H. (1972) Experiments in molecular genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Miller, O. J., Bernath, K., Agresti, J. J., Amitai, G., Kelly, B. T., Mastrobattista, E., Taly, V., Magdassi, S., Tawfik, D. S. and Griffiths, A. D. (2006) Nature Methods, 3, 561-570. Miyazaki, K. and Takenouchi, M. (2002) Biotechniques, 33, 1033-1038. Morales, J. C. and Kool, E. T. (2000) Journal of the Americ an Chemical Society, 122, 10011007. Moreno, R., Haro, A., Castellanos, A. and Berenguer, J. (2005) Applied and Environmental Microbiology, 71, 591-593. Muller, U. F. (2006) Cellular and Molecular Life Sciences, 63, 1278-1293. Najmudin, S., Cote, M. L., Sun, D. M., Yohannan, S ., Montano, S. P., Gu, J. and Georgiadis, M. M. (2000) Journal of Molecular Biology, 296, 613-632. Neumann, J. M., Bernassau, J. M., Gueron, M. and Trandinh, S. (1980) European Journal of Biochemistry, 108, 457-463. Ollis, D. L., Brick, P., Hamlin, R., X uong, N. G. and Steitz, T. A. (1985) Nature, 313, 762-766. Ong, J. L., Loakes, D., Jaroslawski, S., Too, K. and Holliger, P. (2006) Journal of Molecular Biology, 361, 537-550.

PAGE 156

156 Parikh, M. R. and Matsumura, I. (2005) Journal of Molecular Biology, 352, 621-628. Park, S., Morley, K. L., Horsman, G. P., Holmquis t, M., Hult, K. and Kazlauskas, R. J. (2005) Chemistry & Biology, 12, 45-54. Patel, P. H. and Loeb, L. A. (2001) Nature Structural Biology, 8, 656-659. Paul, N. and Joyce, G. F. (2004) Current Opinion in Chemical Biology, 8, 634-639. Pavlov, A. R., Pavlova, N. V., Kozyavki n, S. A. and Slesarev, A. I. (2004) Trends in Biotechnology, 22, 253-260. Perler, F. B., Kumar, S. and Kong, H. M. (1996) In Advances in Protein Chemistry Vol. 48, pp. 377-435. Piccirilli, J. A., Krauch, T., Morone y, S. E. and Benner, S. A. (1990) Nature, 343, 33-37. Piccirilli, J. A., Moroney, S. E. and Benner, S. A. (1991) Biochemistry, 30, 10350-6. Presnell, S. R. and Benner, S. A. (1988) Nucleic Acids Research, 16, 1693-702. Rappaport, H. P. (2004) Biochemical Journal, 381, 709-717. Raychaudhuri, S., Conrad, J., Hall, B. G. and Ofengand, J. (1998) RNA A Publication of the RNA Society, 4, 1407-1417. Rich, A. and Zhang, S. G. (2003) Nature Reviews Genetics, 4, 566-572. Rothwell, P. J. and Waksman, G. (2005) In Fibrous Proteins: Muscle and Molecular Motors Vol. 71, pp. 401-440. Roychowdhury, A., Illangkoon, H., Hendrickson C. L. and Benner, S. A. (2004) Organic Letters, 6, 489-492. Saenger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. and Erlich, H. A. (1988) Science, 239, 487-491. Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laborato ry, Cold Spring Harbor, NY. Sismour, A. M. and Benner, S. A. (2005) Nucleic Acids Research, 33, 5640-5646. Sismour, A. M., Lutz, S., Park, J. H., Lutz, M. J., Boyer, P. L., Hughes, S. H. and Benner, S. A. (2004) Nucleic Acids Research, 32, 728-735. Skerra, A. (1994) Gene, 151, 131-135.

PAGE 157

157 Steitz, T. A. (1999) Journal of Biological Chemistry, 274, 17395-17398. Suzuki, M., Baskin, D., Hood, L. and Loeb, L. A. (1996) Proceedings of the National Academy of Sciences of the Unit ed States of America, 93, 9670-9675. Swiss Institute of Bioinf ormatics. (1999) Vol. 2006, Translate, ExPASy, http://www.expasy.ch/tools/dna.html. Switzer, C., Moroney, S. E. and Benner, S. A. (1989) Journal of the American Chemical Society, 111, 8322-8323. Switzer, C. Y., Moroney, S. E. and Benner, S. A. (1993) Biochemistry, 32, 10489-96. Tatusova, T. A. and Madden, T. L. (1999) Fems Microbiology Letters, 177, 187-188. Tawfik, D. S. and Griffiths, A. D. (1998) Nature Biotechnology, 16, 652-656. Tindall, K. R. and Kunkel, T. A. (1988) Biochemistry, 27, 6008-6013. Vartanian, J. P., Henry, M. and WainHobson, S. (1996) Nucleic Acids Research, 24, 2627-2631. Watson, J. D. and Crick, F. H. C. (1953a) Nature, 171, 964-967. Watson, J. D. and Crick, F. H. C. (1953b) Nature, 171, 737-738. Wellington, K. W. and Benner, S. A. (2006) Nucleosides, Nucleotides, and Nucleic Acids, 25, 1309-1333. Williams, R., Peisajovich, S. G., Miller, O. J., Ma gdassi, S., Tawfik, D. S. and Griffiths, A. D. (2006) Nature Methods, 3, 545-550. Xia, G., Chen, L. J., Sera, T., Fa, M., Sc hultz, P. G. and Romesberg, F. E. (2002) Proceedings of the National Academy of Sciences of the United States of America, 99, 6597-6602. Yan, X. H. and Xu, Z. R. (2006) Drug Discovery Today, 11, 911-916. Zhang, H. C. and Daves, G. D. (1992) Journal of Organic Chemistry, 57, 4690-4696. Zhao, H. M., Giver, L., Shao, Z. X., Af fholter, J. A. and Arnold, F. H. (1998) Nature Biotechnology, 16, 258-261. Zhou, B. L., Pata, J. D. and Steitz, T. A. (2001) Molecular Cell, 8, 427-437. Zhou, J., Yang, M. M., Akdag, A. and Schneller, S. W. (2006) Tetrahedron, 62, 7009-7013.

PAGE 158

158 BIOGRAPHICAL SKETCH Stephanie Ann Havemann was born in Akron, Ohio and raised in Beaufort, South Carolina. She attended Beaufort Academy for primary school, where she began competing in cheerleading, softball, and golf. She particip ated in these sports throughout her high school career at Beaufort High School, where she graduated in the top 10 of her senior class. She also served on the Science Academic Challenge Team fo r 3 years, and led her team to one silver and two gold medals. She attended Mercer University in Macon, Ge orgia for her undergradu ate career, obtaining a Bachelor of Science in Biology and another Bachelor of Scien ce in Environmental Science in 2000. While there, she conducted a year of unde rgraduate research under Dr. Alan Smith characterizing the lipid transport proteins and pro-phenol oxidase of insects. Another semester of undergraduate research was performed, under the supervision of Dr. David Crowely, in attempts to identify an excision repair gene of the archaea, Haloferax volcanii that was homologous to that of the E. coli uvr A gene. She was also the fi rst non-engineering major at Mercer ever to participate in an engineering senior design project. Her three-person team designed and performed the initial construction of the Water Res ource Monitor for the City of Macon, allowing the city to monitor the depth, temperature, and pH of the Ocmulgee River. Her graduate career began in 2000, in the la boratory of Dr. Made line Rasche at the University of Floridas Department of Microbi ology & Cell Science. There, she devised and implemented an assay to detect the levels of methanopterin produced in various methanogenic and methylotrophic cells. She joined Dr. Steven Benners laboratories in 2002 in the University of Floridas Department of Chemistry where she studied the incorporation of non-standard bases into DNA. Her research focused on the directed evolution of polymerases to incorporate non-

PAGE 159

159 standard bases, exhibiting a C-glycosidic linkage, with efficiency and fidelity. She plans to continue her academic study as a post-doctoral research fellow.


Permanent Link: http://ufdc.ufl.edu/UFE0017563/00001

Material Information

Title: Directed Evolution of DNA Polymerases
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0017563:00001

Permanent Link: http://ufdc.ufl.edu/UFE0017563/00001

Material Information

Title: Directed Evolution of DNA Polymerases
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0017563:00001


This item has the following downloads:


Full Text





DIRECTED EVOLUTION OF DNA POLYMERASES


By

STEPHANIE ANN HAVEMANN













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007
































Copyright 2007

by

Stephanie Ann Havemann


































To my family and my husband. Without your constant and vigilant support, I would not be
where I am today. Thank you!









ACKNOWLEDGMENTS

I would like to begin by thanking my advisor, Dr. Steven Benner for all of his wisdom and

guidance; it has been an honor and a privilege to study under his tutelage. His passion for all

facets of science, and how they can be intertwined, should serve as inspiration to us all.

I would like to thank the rest of my committee: Dr. Tom Lyons, for always having an open

door and an receptive ear when I had questions; Dr. Nemat Keyhani, whose enthusiasm for

science was contagious and whose knowledge of microbial genetics was extremely valuable; Dr.

Nicole Horenstein, whose constant support and knowledge helped guide me throughout my

graduate career; and Dr. Rob Ferl, whose eagerness to learn and share information about various

aspects of astrobiology helped me determine the field of study I wish to pursue.

Special thanks go to Dr. Eric Gaucher, Dr. Ryan Shaw, and Dr. Nicole Leal, all of whom

have worked closely with me over the past few years and who have assisted me in various

experimental designs and implementations. Eric performed the rational design of the Taq

mutants and was my source of knowledge for all things dealing with evolutionary biology. Ryan

and I worked closely to discern the best method of creating and isolating DNA from oil-in-water

emulsions; his idea of changing the composition of the oil layer drastically improved our yields.

Nicole assisted me in performing some of my primer-extension assays and was a valuable source

of information and never-ending support.

I am extremely grateful to Dr. Daniel Hutter for the synthesis of the 2'-

deoxypseudothymidine-5 '-triphosphate, to Dr. Shuichi Hoshika for the synthesis of the

pseudothymidine precursor, and to Dr. Ajit Kamath for the synthesis and purification of the

pseudouridine-containing oligonucleotides. Special appreciation also goes to Dr. Michael

Thompson for providing the wt taq gene and his suggestions for the purification of the

polymerase, and to Gillian Robbins for assisting on the growth curve studies.









I am also thankful for the assistance of Dr. Art Edison and Omj oy Ganesh for their

assistance in the circular dichroism experiments. Finally, I would like to thank all the members

of the Benner group for their advice and discussions over the years, and Romaine Hughes,

without whom, our group would be in total chaos.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ............ ...... .__ ...............9....


LIST OF FIGURES .............. ...............10....


LI ST OF AB BREVIAT IONS ........._._ ...... .... ............... 12..


AB S TRAC T ............._. .......... ..............._ 15...


CHAPTER


1 INTRODUCTION ................. ...............17.......... ......


What are Nucleic Acids? .............. ...............17....
Rules of Complementarity ................. ...............17........... ....
DNA Helical Conformations ................. ...............18........... ....
Central Dogma of Molecular Biology ................. ...............19........... ...
W hat is AEGIS? ............ .. .. ...............20..
Use of AEGIS Components. ................ .......................... ......... ..........20
Problems with AEGIS Components ................. ...............22................
C-Glycosides .............. ...............23....
Pseudouridine ............ ..... .._ ...............23...
Pseudothymidine .............. ...............24....
DNA Polymerases ............... ...............25....
General Structure of Polymerases .............. ...............25....
Polymerase Families............... ...............27
Taq Polymerase .............. ...............28....
Directed Evolution............... ...............2

Mutagenic Libraries................ ...............3
Systems of Directed Evolution ............ ...... .... ...............32..
Phage display................ .... .. ...........3
Compartmentalized self-replication .............. ...............33....
Research Overview............... ...............34


2 POLYMERASE INCORPORATION OF MULTIPLE C-GLYCOSIDES INTO DNA:
PSEUDOTHYMIDINE AS A COMPONENT OF AN ALTERNATIVE GENETIC
SY STEM ................. ...............50................


Introducti on .................. ...............50._ ___......
Materials and Methods .............. .. ....... ...... ..........5

Synthesis of Triphosphates and Oligonucleotides............... ............5
Circular Dichroism .............. .... ...............53
Standing Start Primer-Extension As says............... ...............53.












Polymerase screen primer-extension assays............... ...............54.
Taq polymerase primer-extension assays............... ...............55.
Re sults ................. ...............56.................
Circular Dichroism .................. ..... .............5

Polymerase Screen Primer-Extension As says ................ ...............56................
Taq Polymerase Primer-Extension Assays............... ...............57.
Discussion ............._. ...._... ...............58....


3 CREATION OF A RATIONALLY DESIGNED MUTAGENIC LIBRARY AND
SELECTION OF THERMOSTABLE POLYMERASES USING WATER-IN-OIL
E MUL SIONS .............. ...............70....


Introducti on ............ ..... ._ ...............70....
M materials and M ethods .............. ...............74...
DNA Sequencing and Analysis............... ...............74
Construction of Plasmids............... ...............7
Construction of pSW 1 .................. .......... ..... ........7
Rationally designed mutagenic library (RD Library) creation. .............. .... ........._..75
Growth Curves and Cell Counts ........._._ ...... .... ...............75..
Purifieation of His(6)-wt Taq Polymerase ..........._..__.....__ ....___ ...........7
Incorporation of dyUTP by RD Library ........................ ..... ............7
Selection of Thermostable Mutants Using Water-In-Oil Emulsions .............. ................80
Water-in-oil emulsions ................. ...............80.................
Re-cloning of selected mutants .............. ...............81....
R e sults............... .. .... ...... ..... ......... .............8
Growth Curves and Cell Counts ................. ...............82........... ...
Purifieation of His(6)-wt Taq Polymerase ................. ...............83........... ...
Incorporation of dyUTP by RD Library ..................... ... .......... ...... .......8
Selection and Identification of Thermostable Mutants Using Water-In-Oil
Emul sions ................. ...............84........... ....
Discussion ................. ...............85.................


4 DISTRIBUTION OF THERMOSTABILITY IN POLYMERASE MUTATION SPACE.103


Introducti on ................. ...............103................
M materials and M ethods .............. ...............105...
DNA Sequencing and Analysis............... ...............10
Bacterial Growth Conditions and Strains ................. ...............105........... ...

Synthesis of Triphosphates and Oligonucleotides................... ........106
Random Mutagenic Library (L4 Library) Creation.................. ......... ........0
Incorporation of dNTPs by RD and L4 Libraries at Various Temperatures .................1 08
Incorporation of dyUNTPs by RD Library at Optimal Temperatures ................... .......108
Incorporation of dyUTP and dyTTP by co-Taq Polymerase at Various Melting
T emp erature s ................ ...............110....._._. ....
Re sults ................. ...... ....... ..... ..... .... ..... ..........11
Random Mutagenic Library (L4 Library) Creation. ...............__.. ......_._ ................110
Incorporation of dNTPs by RD and L4 Libraries at Various Temperatures .................11 1











Incorporation of dyUNTPs by RD Library at Optimal Temperatures ................... .......112
Incorporation of dyUTP and dyTTP by co-Taq Polymerase at Various Melting
T emp erature s ................ ...............113....._._. ....
Discussion ................. ...............114...............

5 CONCLUSIONS .............. ...............132....

DNA Helical Structure in the Presence of C-Glycosides ......... ................ ...............132
Polymerase Screen for the Incorporation of C-glycosides .............. ......................133
Taq Polymerase Primer-Extension Assays ...._ ........__.... ....._..............3
Growth and Purification of Taq Polymerase ........._._.... ...___....... ..........13
Creation of co-Taxq Polymerase Mutant Libraries .............. ........ ........ ... ........136
Creation of the Rationally Designed Mutagenic Library (RD Library) ................... ..... 136
Creation of the Random Mutagenic Library (L4 Library) ................. .....................137
Preliminary Studies of the Incorporation of dyUTP by the RD Library ................... ...........137
Incorporation of dNTPs by RD and L4 Libraries at Various Temperatures ........................ 138
Incorporation of dyUTP by the RD Library at Optimal Temperatures .............. ................139
Incorporati on of dyUTP and dyTTP by co- Taq Polymerase at Vari ous Temperature s...... 140
Selection of Thermostable RD Mutants Using Water-In-Oil Emulsions ................... ..........140
Future Experimentation .............. ...............141....

APPENDIX

A SYNTHESIS OF PSEUDOTHYMIDINE AND PSEUDOTHYMIDINE-
CONTAINING OLIGONUCLEOTIDES .............. ...............144....

B PHYLOGENETIC TREES OF FAMILY A POLYMERASES .................... ...............14

C GENETIC CODE AND AMINO ACID ABBREVIATIONS .............. ....................15

LIST OF REFERENCES ................. ...............151................

BIOGRAPHICAL SKETCH ................. ...............158......... ......










LIST OF TABLES


Table page

1-1 Comparison of the structural geometries of A, B, and Z-DNA forms. .............. ..... ..........3 8

1-2 Characteristics of the various polymerase families. ................ ................ ......... .46

2-1 Oligonucleotides used in this study. ............. ...............64.....

3-1 Oligonucleotides used in this study. ............. ...............91.....

3-2 Rationally Designed (RD) Mutant Library. ............. ...............95.....

3-3 Bacterial strains used in this study ................. ...............96........... ..

3-4 Incorporation of dyUTP at 94.0 oC by RD Library. ............. ...............100....

3-5 Mutations present after selection for active polymerases. ................... ...............10

3-6 Breakdown of types of mutations present after selection. ................ ............ .........102

4-1 Additional bacterial strains used in this study. ............. ...............120....

4-2 L4 Mutant Library ................. ...............121...............

4-3 Generation of full length PCR products from dNTPs by individual polymerases from
the rationally designed (RD) Library at the indicated temperatures. .............. ..... ........._.123

4-4 Generation of full length PCR products from dNTPs by individual polymerases from
the randomly generated (L4) Library at the indicated temperatures. .............. ..... ..........124

4-5 Incorporation of dyUTP by RD Library at optimal temperatures ................. ...............126

4-6 Incorporation of dyUTP and dyTTP by co-Taq Polymerase at various temperatures. ..129

C-1 The Genetic Code. ............. ...............150....

C-2 Amino acid abbreviations. ............ .............150......











LIST OF FIGURES


Figure page

1-1 The standard deoxyribonucleotides. ............. ...............37.....

1-2 Puckering of the furanose ring of nucleosides into various envelope forms. ................... .39

1-3 The central dogma of molecular biology. ................ .......................... ..........39

1-4 The six hydrogen bond patterns in an artificially expanded genetic information
sy stem (AEGI S) .............. ...............40....

1-5 The VersantTM branched DNA assay. ............. ...............41.....

1-6 An example of non-standard nucleobases coding for a non-standard amino acid.............42

1-7 Pseudouridine and pseudothymidine. ............. ...............43.....

1-8 The polymerization reaction of deoxyribonucleotides triphosphates catalyzed by
DNA polymerases ................. ...............44.................

1-9 Kinetic steps involved in the nucleotide incorporation pathway. .................. ...............44

1-10 Locations of active site residues in Taq polymerase. ............. ...............45.....

1-11 The staggered extension process (StEP) for rediversification of mutant libraries.............47

1-12 Phage display selection scheme. .............. ...............48....

1-13 General scheme for CSR ................. ...............49........... ...

2-1 A schematic representation of the CD spectra of A- and B-DNA forms. ................... .......62

2-2 The base pairing interactions between a standard A-T base pair and the non-standard
yT-A and yU-A base pairs............... ...............63.

2-3 Representative CD Spectra. ............. ...............65.....

2-4 Depiction of primer-extension assays used in the polymerase screen. .............. ..... ..........66

2-5 Family A polymerase screen. ..........._ ..... ..__ ...............67..

2-6 Family B polymerase screen. .............. ...............68....

2-7 Incorporation of one to twelve consecutive dT, dyT, or dyU residues by Taq
poly m erase. ............. ...............69.....

3-1 A phylogenetic tree of the Family A polymerases ................. ...............89........... .










3-2 Locations of the 3 5 rationally designed (RD) sites in the Taq polymerase structure.......90

3-3 View of the pASK-IBA43plus plasmid. ................ ...............92...............

3-4 View of the pSW1 plasmid. .............. ...............93....

3-5 View of the pSW2 plasmid ................. ...............94...............

3-6 Growth curves, cell counts, and expression of various E. coli TG-1 cell lines. ................97

3-7 Purification and activity of His(6)-wt Taq polymerase. ........._..._.. ...._.._ ........._......98

3-8 Representative gels showing the amount of full-length PCR products generated with
different dNTP/dyUNTP ratios and the indicated polymerases............... ...............9

4-1 Epimerization of 2' -deoxypseudouridine. .........._..._ ......... ...._.._ ..........19

4-2 Representative images of ethidium-bromide stained agarose gels resolving products
arising from PCR amplification using standard dNTPs and three different
polym erases............... ..............12

4-3 Number of active RD and L4 mutants at various temperatures ................. ................. 125

4-4 Generation of full length PCR product at 86.3 oC using dyUTP by the co-Taq
polymerase and the RD polymerase in the SW29 cell line ................. ............. .......127

4-5 Generation of full length PCR product at 94.0 oC and 86.3 oC using dyUTP by the
RD polymerase in the SW8 cell line ................. ...............128........... ..

4-6 Generation of full length PCR product at 86.3 oC by co-Taq polymerase using
various TTP:dyUTP and TTP:dyTTP ratios. ............. ...............130....

4-7 Graphical comparisons of the band densities listed in Table 4-6. ..........._.._ ..........._...13 1

A-1 Synthesis of pseudothymidine precursor. ............. ...............146....

B-1 A seed alignment of the Family A polymerases. ................ ...............147........... .

B-2 Inset of the phylogenetic tree of Family A polymerases (from Fig. 3-1) showing the
location of Taxq polymerase. ........._...._ ...._._. ...............148...

B-3 Inset of the phylogenetic tree of Family A polymerases (from Fig. 3-1) showing the
location of some viral polymerases............... ..............14









LIST OF ABBREVIATIONS

adenosine

artificially expanded genetic information system

ampicillin

ammonium persulfate

adenosine triphosphate

base pair

Bacillus stearothermophilus

cytosine

Curie (1 Ci = 3.7 x 107 Bequerel)

circular dichroism

cell-free extract

colony forming unit

counts per minute

counts

compartmentalized self-replication

dimethyl sulfoxide

deoxyribonucleoside (dA, dG, dC, T, yT, yU, etc.)

deoxyribonucleic acid

deoxyribonucleic acid specific endonuclease

double-stranded nucleic acid chain

1,4-dithio-DL-threitol

Escherichia coli


A

AEGIS

Amp

APS

ATP

bp

Bst

C

Ci

CD

Cfe

efu

CPM

CNT

CSR

DMSO

dN

DNA

DNase I

ds

DTT

E. coli









EDTA ethylendiamino tetraacetate

exo- lacking 3' 5' exonuclease activity

FLP full-length product

G guanosine

HIV human immunodeficiency virus type-1

hr hours

HPLC high performance liquid chromatography

isoC deoxyisocytidine

isoG deoxyisoguanosine

LB Luria-Bertani medium

mmn minutes

M-MuLV moloney murine leukemia virus

mRNA messenger ribonucleic acid

MWCO molecular weight cut-off

NMR nuclear magnetic resonance

NSB non-standard nucleobase

OD optical density

PAGE polyacrylamide gel electrophoresis

PCR polymerase chain reaction

Pfu Pyrococcus furious

PMSF phenylmethylsulfonyl fluoride

PNK polynucleotide kinase

REAP reconstructing evolutionary adaptive paths










ribonucleic acid

ribonucleic acid specific endonuclease

ribosomal ribonucleic acid

reverse transcriptase

sodium dodecylsulfate

seconds

staggered extension processes

thymidine

pseudothymidine

Thermus aquaticus DNA Polymerase I

Tris / borate / EDTA buffer

N,N,N,N-tetramethylethylenediamine

tetracycline

tri s(hy droxymethyl)ami nom ethane

octyl phenol ethoxylate

transfer ribonucleic acid

Thermus thermophilus

uracil

pseudouridine

ultraviolet

wild tyipe


RNA

RNase A

rRNA

RT

SDS

s

StEP

T




Taq

TBE

TEMED

Tet

Tris

Triton X-100

tRNA

Tth

U




UV

wt









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

DIRECTED EVOLUTION OF DNA POLYMERASES

By

Stephanie Ann Havemann

May 2007

Chair: Steven A. Benner
Major Department: Chemistry

To achieve the long-term goal of the Benner research group to create a synthetic biology

based on an Artificially Expanded Genetic Information System (AEGIS), polymerases that are

able to incorporate non-standard bases (NSBs) into DNA must be identified. In this dissertation,

a polymerase from Thermus aquaticus (Taq Polymerase) was identified that was able to

incorporate non-standard nucleotide analogs that contain a C-glycosidic linkage. This activity

was limited, meaning that the polymerase needed modification to support this goal. Further, we

asked whether sequential C-glycosides destabilized the duplex and altered its structure, to better

understand whether a synthetic biology based on C-glycoside nucleotides was possible.

To this end, two libraries of polymerases were created to identify mutations necessary to

alter the polymerases' ability to withstand high temperatures. One library was created by the

random mutagenesis of the taq gene, the other was rationally designed based on previous studies.

Seventy-four mutants from each library were screened for their ability to generate a full-length

polymerase chain reaction (PCR) product using standard nucleoside triphosphates at various

temperatures; the library of random mutants contained more thermostable polymerases than the

library obtained by rational design. Water-in-oil emulsions were then tested to determine

whether these, as artificial cells, might deliver thermostable polymerase variants from those used









in the screen. This identified difficulties in tools used to analyze the output of the library,

suggesting solutions that will guide future work. We also tested the individual components of

the rationally designed library for their ability to incorporate C-glycoside triphosphates in a PCR.

Structural studies with synthetic DNA containing multiple, consecutive C-glycosides showed no

change in conformation, at least not one that is detectible by circular dichroism.

These results represent a step towards the goal of creating an AEGIS-based synthetic

biology, an artificial chemical system that mimics emergent biological behaviors such as

replication, evolution, and adaptation. In addition, the mutant polymerases created in these

experiments are an inventory of polymerases useful in biotechnology, possibly allowing the

development of new, as well as improving on existing, clinical diagnostic techniques and helping

to facilitate a better understanding of polymerase-DNA interactions.









CHAPTER 1
INTTRODUCTION

What are Nucleic Acids?

Deoxyribonucleic acid (DNA), one of the fundamental constituents of life, serves as a key

component for the storage and transfer of genetic information. It is built from four building

blocks, adenosine, guanosine, cytidine, and thymidine, all of which are comprised of a

nucleobase attached to a 2'-deoxyribose molecule (Fig. 1-1). Similarly, ribonucleic acid (RNA)

is also built from four building blocks, except that thymidine is replaced by uridine and the sugar

moiety is a ribose. When a phosphate group replaces the 5'-hydroxyl group of these molecules,

they become acids that can be linked by their phosphate groups, resulting in the formation of the

backbone of a nucleic acid strand. Genetic information is commonly stored in a double stranded

(ds) helix, which is formed when the nucleobases are paired by hydrogen bonds. These helical

duplex strands are aligned so that the chains are anti-parallel to one another; in other words, one

strand lies in the 5' 3' direction and the complement is in the 3' 5' orientation.

Rules of Complementarity

Watson and Crick proposed that the interactions between nucleobases are governed by two

rules of complementarity: size complementarity and hydrogen-bonding complementarity

(Watson and Crick, 1953a, Watson and Crick, 1953b). Size complementarity means that a large

purine, such as adenosine or guanosine, pairs with a small pyrimidine, like cytosine, thymidine,

or uridine. Hydrogen-bonding complementarity means that hydrogen bond donors from one

nucleobase pair with the hydrogen bond acceptors from another. With these rules, it is expected

that in the formation of nucleic acid duplexes, guanosine must pair with cytosine and adenosine

must pair with either thymidine or uridine.









DNA Helical Conformations

The conformation of a DNA duplex is often assumed to be described using one of three

abstract models: A-DNA, B-DNA, or Z-DNA (Saenger, 1984). The most common form of

DNA found in living organisms is presumed to be the B-DNA helix. A-DNA is the common

helical structure whose geometries are described in Table 1-1. It is also interesting to note that

many other minor helical conformations of dsRNA or dehydrated DNA, but it can also be found

when certain DNA sequences repeat (Ghosh and Bansal, 2003). The only left-handed helix

known is the Z-DNA conformation, which appears to be a characteristic of alternating GC-rich

sequences that may help stabilize DNA during transcription (Rich and Zhang, 2003). Many

other helical conformations of DNA are possible, of course. Indeed, over twenty-six different

forms have been described in the literature to date (Egli, 2004, Ghosh and Bansal, 2003, Saenger,

1984). Nevertheless, for this work, we will reference the A-, B-, and Z-DNA models.

In actuality, the conformation of a DNA molecule must be described by examining the

structure atom by atom. Terms used to abstract the results of such an examination are described

in Table 1-1. Thus, the different types of helices are characterized by different geometries, such

as the number of base pairs per turn, the height of a turn, the rotation per base pair, the size and

depth of the maj or and minor grooves, and the type of sugar pucker. The sugar pucker refers to

the conformation of the sugar, which can exist in one of four envelope forms: C2'-endo, C2'-eXO,

C3'-endo, and C3'-eXO (Fig. 1-2) (Saenger, 1984).

In some cases, helical structures can be transformed from one conformation into another

simply by the modification of the humidity of the environment (for fibers) and/or the

concentrations of salt in the solution (Saenger, 1984). Helical structures can also be changed by

altering the chemical structure of the constituents. The conformation of the sugar pucker can

alter the helical form of the DNA by increasing or decreasing the distances between the










phosphate groups, thereby changing the number of base pairs per turn and the size of the

grooves. The C2'-endo conformation is usually found in B-DNA, while the A-DNA prefers the

C3'-endo pucker. The maj or and minor grooves found in B-DNA can act as binding pockets for

polymerases, since they allow for the presentation of nucleobase hydrogen bond donors and

acceptors (Garrett and Grisham, 1999). The grooves presented by A-DNA are more

symmetrical, making it difficult for polymerases to gain access to these potential hydrogen-

bonding sites (Garrett and Grisham, 1999).

The conformation of a DNA helix can be assessed in several ways. X-ray crystallography

is, of course, the best way to identify the position of individual atoms, with nuclear magnetic

resonance (NMR) emerging as a preferred choice in solution. The general overall conformation

can be estimated, however, by circular dichroism (CD) (Ghosh and Bansal, 2003).

Central Dogma of Molecular Biology

Nucleic acids maintain genetic information inside a cell by means of replication and

transcription; translation uses this genetic information to create proteins. This sequence has been

called the central dogma of molecular biology by Crick (Fig. 1-3) (Crick, 1970). DNA is

transcribed into messenger RNA (mRNA) using RNA polymerases, which is then translated into

proteins. The translation of the mRNA uses a combination of ribosomes, which are composed of

ribosomal RNA (rRNA) and proteins, and transfer RNA (tRNA), which carry amino acids to the

ribosomes. In situations where the genetic material is stored as RNA, such as in viruses, the

information is first converted back into DNA by enzymes known as reverse transcriptases prior

to being translated. DNA can replicate itself by employing enzymes known as DNA

polymerases, and RNA replicates itself using RNA polymerases.

This feature of life raises an obvious question: Which came first, nucleic acids or proteins?

At first glance, the answer appears to be nucleic acids, since proteins cannot store genetic









information. Upon further study, one realizes that without proteins, the genetic material could

not be replicated. One possible answer to this question is that the nucleic acids were once able to

act as both storage molecules and as proteins that could catalyze their own replication.

The discovery of ribozymes and deoxyribozymes lends support to this theory by showing

that nucleic acid molecules are not limited to the ability to store genetic information, they can

catalyze reactions both within their own structure or upon other structures (Muller, 2006,

Emilsson and Breaker, 2002, Paul and Joyce, 2004). Many of these nucleic acid catalysts have

been created using non-standard nucleobases (NSBs) to add additional functionality to the

nucleic acid molecules (Muller, 2006).

What is AEGIS?

Using Watson and Crick' s rules of complementarity and the requirement that the

nucleobases be joined with three hydrogen bonds, it is feasible to create an artificially expanded

genetic information system (AEGIS) containing eight additional base pairs (Fig. 1-4), thereby

expanding the genetic alphabet from four to twelve letters (Switzer et al., 1989, Piccirilli et al.,

1990, Geyer et al., 2003). Since these bases retain the Watson and Crick geometry, they can be

incorporated into growing DNA strands via synthesis, primer-extension experiments, or by the

polymerase chain reaction (PCR), which can subsequently be used in a variety of different

techniques.

Use of AEGIS Components

The importance of AEGIS components has already been illustrated in many ways. It has

been used in clinical diagnostics, to expand the genetic code, to understand DNA and polymerase

interactions, and has even been implicated as a factor for evolution of life on Earth. These

components have also been used in the first successful six-letter PCR reaction, lending support to

the development of a synthetic biology.










The powerful VersantTM branched-DNA assay, used to monitor the viral load of patients

infected with HIV, Hepatitis B, or Hepatitis C viruses, requires the use of at least two non-

standard nucleobases (NSBs) (Collins et al., 1997). This assay uses 5-methyl-2'-

deoxyisocytidine (isoC) and 5-methyl-2'-deoxyi soguanosine (isoG) to decrease the non-specific

binding of a nucleic acid probe (Fig. 1-4), thereby increasing signal amplification relative to

noise by eight-fold over previous systems used (Fig. 1-5) (Huisse, 2004, Collins et al., 1997).

EraGen Biosciences (Madison, WI) is now using these AEGIS components in a similar

multiplexed system to identify newborns with cystic fibrosis (Johnson et al., 2004). These

assays have barely begun to scratch the surface of the potential clinical diagnostic uses of this

expanded genetic alphabet.

The current genetic code uses 64 three-letter codons to encode for the incorporation of 20

canonical amino acids (Appendix C); use of all twelve AEGIS nucleotides would allow for 1728

three-letter codes, and if the AEGIS components were functionalized, the possibilities are

seemingly nearly endless. AEGIS components have been already been used to encode for the

incorporation of non-standard amino acids in ribosome-mediated translation. For example, in

1992 Bain et al. used isoC and isoG in a codon-anti-codon pair to generate peptides containing

the non-standard amino acid L-iodotyrosine (Bain et al., 1992). More recently, Hirao et al. used

the 2-amino-(2-thienyl)purine and pyridine-2-one in a codon-anti-codon pair in an in vitro

transcription study to generate peptides containing 3-chlorotyrosine (Fig. 1-6) (Hirao et al., 2002,

Hirao et al., 2006).

Some of these AEGIS components have also been used in the characterization of the

kinetic parameters of polymerases (Joyce and Benkovic, 2004, Sismour and Benner, 2005), and

in the first six-letter PCR, which was catalyzed by a mutant of the HIV-reverse transcriptase










(Sismour et al., 2004). AEGIS components have also been used to better understand the

interactions between polymerases and DNA (Lutz et al., 1998, Joyce and Benkovic, 2004,

Hendrickson et al., 2004, Delaney et al., 2003). For example, studies have been performed using

variety of different NSBs, such as those lacking minor-groove electrons (Hendrickson et al.,

2004) and those with a C-glycosidic linkage (Lutz et al., 1999), in order to identify

characteristics of nucleobases that are essential for correct incorporation by polymerases.

Problems with AEGIS Components

Although the AEGIS components retain Watson and Crick geometry, it is possible that

some of the features present on the NSBs, such as the absence of minor groove electrons or the

presence of C-glycosidic linkages, may present a challenge to polymerases. The ability of

polymerases to function in the absence of an unshared pair of electrons in the minor grove of

dsDNA, as seen in the pyDAD-puADA base pair (Fig. 1-4), was previously examined by

Hendrickson et al (Hendrickson et al., 2004). In those studies, Hendrickson discovered that the

presence of electrons in the minor grove may only be necessary for exonuclease activity of

polymerases, and not for incorporation (Hendrickson et al., 2004). This, however, presents a

problem when trying to incorporate NSBs with efficiency and fidelity, since the polymerase has

no proofreading ability. Lutz et al. examined the ability of polymerases to function in the

presence of nucleosides exhibiting a C-glycosidic linkage, a carbon-carbon bond between the

nucleobase and sugar as seen in the pyDAD, pyAAD, and pyADD nucleosides (Fig. 1-4) (Lutz et

al., 1999). He also reported that polymerases with exonuclease activity were less likely to accept

the C-glycoside than were those lacking the proofreading ability, making replication with fidelity

difficult.










C-Glycosides

An N-glycoside is a nucleoside with a carbon-nitrogen bond linking the nucleobase to the

sugar; all standard nucleosides are therefore N-glycosides. However, three of the AEGIS

nucleosides use a carbon-carbon bond to join the nucleobase to the sugar, making these

nucleosides C-glycosides by definition (Fig. 1-4). This carbon-carbon linkage can cause a

structural change in the sugar pucker of the nucleoside, making it a C3-endo pucker instead of a

C2--endo pucker, possibly changing the form of the DNA from B-DNA to A-DNA (Davis, 1995).

Wellington and Benner detailed strategies by which these molecules can be chemically

synthesized in a current review article (Wellington and Benner, 2006). C-glycosides have also

been found in vivo in various types of RNA, however (Charette and Gray, 2000). These C-

glycosides are of great interest, not only because of their presence in the AEGIS nucleosides, but

also for their clinical uses; many naturally occurring C-glycosides are antibiotics or antiviral

agents (Michelet and Genet, 2005, Zhou et al., 2006). More generally, C-glycosides can be used

in gene therapy (Li et al., 2003, Li et al., 2004).

Pseudouridine

Pseudouridine (yU), the 5-ribosyl isomer of uridine (Fig. 1-7A), is present in both tRNA

and rRNA and is vital to the fitness of organisms (Raychaudhuri et al., 1998, Charette and Gray,

2000). This modified nucleoside, found in all three domains of life, was the first naturally

occurring NSB discovered (Charette and Gray, 2000), and is introduced into the RNA sequences

by the posttranscriptional modification of uridine (Argoudelis and Mizsak, 1976, Grosjean et al.,

1995). Pseudouridine has been reported to have a propensity to adopt a syn conformation around

the glycosyl bond when in solution, although the data supporting this are questionable; it is,

however, found only in the anti conformation when in a nucleic acid strand (Fig. 1-7B) (Lane et

al., 1995, Neumann et al., 1980). The anti conformation allows the coordination of a water









molecule between the 5' phosphate group of the yU residue, the 5' phosphate group of the

preceding residue, and the N1-H of the yU residue (Fig. 1-7C) (Arnez and Steitz, 1994). The

coordination of this water molecule results in an enhanced base stacking ability and a reduced

conformational flexibility of the RNA molecule, thus increasing the local rigidity of the RNA

(Charette and Gray, 2000, Davis, 1995).

Pseudouridine is thought to play several roles in Nature, as described in the review by

Charette and Gray (Charette and Gray, 2000). In tRNA, it is thought to play a critical role in the

binding of the tRNA to the ribosome during translation because it stabilizes the tRNA structure,

allowing tighter binding to occur, thereby increasing translational accuracy. Pseudouridine also

has been implicated in alternative codon usage in tRNA, and as a player in the folding of rRNA

and ribosome assembly by its contributions to RNA stability.

Pseudothymidine

Pseudothymidine (qT), or 1 -methylpseudouridine (Fig. 1-7D), was originally isolated from

Streptomyces platensis in 1976 by Argoudelis and Mizsak (Argoudelis and Mizsak, 1976). This

naturally occurring C-glycoside, found in RNA, is also thought to be created by a

posttranscriptional modification ofuridine (Limbach et al., 1994). The first successful in vitro

transcription of yT was performed by Piccirilli et al. using T7 RNA polymerases with a template

containing yT and standard ribonucleosides (Piccirilli et al., 1991). Further studies, conducted

by Stefan Lutz, observed the ability of DNA polymerases to not only incorporate this NSB into a

growing DNA strand in primer-extension assays, but also challenged a polymerase to use yT in

a PCR reaction that required the successful incorporation of up to three consecutive dyT

residues. (Lutz et al., 1999). Since then, no further studies requiring the incorporation of this C-

glycoside into nucleic acids have been performed.









DNA Polymerases

DNA polymerases are the enzymes that perform template directed DNA synthesis from

deoxyribonucleotides and an existing DNA template. These enzymes, essential for the

replication of the genetic information carried in all living organisms, were originally discovered

in 1956 by Arthur Kornberg (Kornberg et al., 1956), for which he was awarded a Nobel Prize in

1959. The synthesis of the complementary DNA strand always occurs in the 5' 3' direction

through the addition of incoming nucleotide's triphosphate group onto the 3'-OH group of the

preceding nucleotide, releasing a pyrophosphate group in the process (Fig. 1-8) (Garrett and

Grisham, 1999, Lewin, 1997). After the successful replication of a DNA strand, the new strand

is complementary to the template (leading) strand, and identical to the lagging strand. Since all

DNA polymerases function in this manner, it is easy to comprehend that their structures are also

generally conserved.

General Structure of Polymerases

All DNA polymerases share a common structural framework that is commonly referred to

as a right hand comprised of three subdomains: the fingers, the palm, and the thumb. The fingers

domain is responsible for nucleotide recognition and binding, the thumb domain binds the DNA

substrate, and the palm domain is the catalytic center of the protein. It appears that this

framework is the same in all DNA polymerase families. It is not clear whether this represents

convergent or divergent evolution; there is no sequence similarity between, for example, Family

A and Family B polymerases that makes a case for their distant homology (Rothwell and

Waksman, 2005). In 1985, the laboratory of Thomas Steitz first solved the crystal structure of

the Klenow fragment, the C-terminal domain of the Escherichia coli DNA Polymerase I (Ollis et

al., 1985). Since then, the crystal structure of many different polymerases have been solved, not

only in their nascent states, but some with DNA or dNTPs and pyrophosphate bound to the









catalytic site (Rothwell and Waksman, 2005, Beese et al., 1993b, Beese et al., 1993a). It has also

been determined that during polymerization, divalent metal cations, such as Mg2+, r

coordinated in polymerase active sites to help activate the 3'-OH group for attack on the

incoming nucleotide (Steitz, 1999).

Features of polymerases that are not conserved throughout the families include both the 5'

-3' and 3' 5' exonuclease subdomains that allow for proofreading, and other subunits used for

different types of repair. The exonuclease subdomains, when present, are the proofreading

centers of the polymerase. The 5' 3' exonuclease activity is usually involved in nick

translation, or the synthesis of DNA at a location where there is a break in the phosphodiester

bond of one strand (Perler et al., 1996). The 3' 5' exonuclease activity is the true

"proofreading" activity of the polymerase, responsible for the excision of a newly synthesized

mismatch (Perler et al., 1996).

The process by which a DNA polymerase adds an incoming nucleotide onto the 3'-

hydroxyl group of the preceding nucleoside involves many steps, which are only now being fully

understood. Figure 1-9 details the kinetic steps involved in this addition (Patel and Loeb, 2001,

Rothwell and Waksman, 2005). In Step 1, the polymerase (E) binds to the DNA primer:template

complex (TP); the polymerase then binds the incoming nucleotide triphosphate (dNTP) in Step 2.

The polymerase then undergoes a conformational change (E') in Step 3 that brings the various

components into positions that can support the chemistry of this reaction; this is the rate-limiting

step of polymerization. The polymerase performs the addition of the nucleotide, remains

completed with the pyrophosphate, and undergoes another conformational change in Step 4.

The pyrophosphate group is released in Step 5; in Step 6, the polymerase can dissociate from the

DNA or translocate the substrate for another round of synthesis.










Polymerase Families

Based on sequence similarity, seven major families of homologous polymerases have been

classified (Patel and Loeb, 2001, Rothwell and Waksman, 2005): A, B, C, D, X, Y, and RT. The

most extensively studied are those of the Family A and Family B polymerases, but Table 1-2

identifies characteristics and representative polymerases of all seven families. Polymerases

behave differently not only between the families, but also within the families themselves, based

on their ability to repair, their processivity, and their fidelity. Processivity is defined as the

ability of the polymerase to continue catalysis without dissociating from the DNA (Kelman et al.,

1998); this is important when dealing with AEGIS components since it has been previously

shown that polymerases tend to "pause," or fall off the DNA, after the incorporation of a NSB

(Lutz et al., 1999, Sismour and Benner, 2005). Fidelity is the ability of the polymerase to select

and incorporate the correct complementary nucleoside opposite the template from a pool of

similar structures (Beard et al., 2002, Cline et al., 1996); this is important to AEGIS components

to guarantee that the newly replicated DNA contains the correct sequence.

Family A polymerases, which contain some of the prokaryotic, eukaryotic, and viral

polymerases, are best known for the E. coli DNA Pol I, Thermus aquaticus (Taq) Pol I, and the

T7 DNA polymerases (Perler et al., 1996). The E. coli DNA Pol I and Taq polymerases are

known as repair polymerases since they contain the 5' 3' exonuclease domains, while the T7 is

known as a replicative polymerase since it has a strong 3' 5' exonuclease activity (Rothwell

and Waksman, 2005, Kunkel and Bebenek, 2000).

Family B polymerases contain representatives from prokaryotic, eukaryotic, archaeal, and

viral polymerases, this is the only family of polymerases with members from all four of these

populations (Patel and Loeb, 2001). This family of polymerases is predominately involved with

DNA replication, as opposed to repair, and exhibit extremely strong 3' 5' exonuclease









activities. In eukaryotes, these polymerases carry out the replication of chromosomal targets

during cell division. The most well known of the archaeal polymerases from this family,

Pyrococcus furious (Pfu) DNA Polymerase, has the lowest known error rate of all thermophilic

DNA polymerases that can be used for PCR amplification mutationall frequency/bp/duplication

is 1.3 x 10-6 ) (Hogrefe et al., 2001, Cline et al., 1996).

Family C polymerases contain the bacterial chromosomal replicative polymerases, and

Family D polymerases are suggested to act as archaeal replicative polymerases (Patel and Loeb,

2001, Rothwell and Waksman, 2005). Family X polymerases are found in eukaryotes, and are

believed to play a role in the base-excision repair pathway that is important for correcting abasic

sites in DNA (Patel and Loeb, 2001, Rothwell and Waksman, 2005). Family Y polymerases,

found in prokaryotes, eukaryotes, and archaea, are part of a replicative complex, and function by

recognizing and bypassing lesions created by UV damage so that replication of the DNA is not

stalled (Zhou et al., 2001, Rothwell and Waksman, 2005). The last characterized family of

polymerases, the reverse transcriptases (RT), found in eukaryotes and viruses, catalyze the

conversion of RNA into DNA, but they can also replicate DNA templates as well (Najmudin et

al., 2000, Goldman and Marcy, 2001, Rothwell and Waksman, 2005).

Taq Polymerase

Thermus aquaticus, an organism found in thermal springs, hydrothermal vents, and even

hot tap water, was first isolated by Brock and Freeze in 1969 (Brock and Freeze, 1969). Taq

polymerase, a 94 kDa protein, was isolated from this organism by Chien et al. in 1976 (Chien et

al., 1976), and belongs to the Family A polymerases. This thermophilic polymerase has 5' 3'

exonuclease activity, but lacks the 3' 5' exonuclease activity required for the proofreading

ability, therefore giving this polymerase a low replication fidelity of about 8 x 10-6 mutationall

frequency/bp/duplication) (Cline et al., 1996). However, Taxq is fairly processive with an









average incorporation of 40 nucleotides before dissociating from the DNA, and it has a quick

extension rate of about 100 nucleotides per second (Pavlov et al., 2004, Perler et al., 1996).

Taq polymerase, one of the most extensively studied polymerases, was the first

thermostable polymerase to be used in PCR; thereby eliminating the need to add additional

polymerase after every round of PCR as was necessary when E. coli DNA Pol I was used for

thermocycling experiments (Saiki et al., 1988). In 1995, the Steitz laboratory was the first to

crystallize nascent Taxq polymerase (Kim et al., 1995), and have since crystallized the polymerase

with DNA at the active site (Eom et al., 1996). These, and other studies, have allowed

researchers to identify the active site of the polymerase and the specific residues which contact

the DNA, the incoming nucleotides, or are involved in metal ion chelation (Eom et al., 1996, Fa

et al., 2004, Li et al., 1998b, Li et al., 1998a, Kim et al., 1995, Suzuki et al., 1996).

Due to Taq polymerase's lack of proofreading ability, it has been identified previously as a

candidate for replication of DNA containing non-standard nucleosides (Lutz et al., 1999). Taq

has been used to incorporate and/or replicate NSBs exhibiting C-glycosidic linkages (Lutz et al.,

1999), NSBs lacking an unshared pair of electrons in the minor groove (Hendrickson et al.,

2004), and nonpolar nucleoside isoteres (Morales and Kool, 2000). Directed evolution has

created Taq polymerase mutants that have been used to incorporate an even larger repertoire of

NSBs (Henry and Romesberg, 2005).

Directed Evolution

A recent review by Griffiths and Tawfik discussed the application of techniques developed

for the in vitro evolution of various proteins to increase their rate of catalysis, perform different

functions, and accept new substrates (Griffiths and Tawflk, 2006). These procedures all select

for desired enzyme characteristics from pools of millions of genes with schemes designed to link

genotype to phenotype. This provides a great advantage over the older methods of screening









mutant library members individually, because these approaches use a "one-pot" technique that

allows for the testing of a large number of variants (2 x 10s or more) at once (Griffiths and

Tawfik, 2006)

Other common features of these directed evolution systems include the development of a

mutagenic library, expression of this library, a high-throughput assay designed to identify

individuals with the desired characteristics, and a means for reshuffling mutants between rounds

of selection (Brakmann, 2005, Lutz and Patrick, 2004, Arnold and Georgiou, 2003a). The most

challenging part of any selection experiment is the design of the technique that will be used to

isolate variants with the desired characteristics (Brakmann, 2005), because "you get what you

select for." In other words, scientists may want to select for a specific characteristic of an

enzyme, but if the technique is not designed correctly, they may end up selecting for an enzyme

with a different characteristic.

Mutagenic Libraries

The first step in any directed evolution experiment is to create a large library of mutant

enzymes. There are many ways to accomplish this task, varying from the rational design of

mutations at selected sites to the random mutagenesis of residues along the length of the

sequence. Francis Arnold co-authored a book with George Georgiou that gave detailed

instructions on how to perform nineteen different techniques to generate libraries for directed

evolution (Arnold and Georgiou, 2003b). This book gave attention to standard error-prone PCR

techniques that use MnCl2 inStead of MgCl2 in PCR reactions catalyzed by a polymerase with

low fidelity, such as Taq, and to methods that could be used for the rediversification of libraries

between rounds of selection, such as the staggered extension process (Fig. 1-11).

An important consideration when creating a true random library of mutants is the bias of

some techniques to create certain transitional or transversional mutations preferentially.









Transitional mutations occur when one purine-pyrimidine pair is replaced with another purine-

pyrimidine pair; this creates four possible transition mutations with the standard nucleotides.

Transversional mutations occur when a purine-pyrimidine pair is replaced by a pyrimidine-

purine pair, creating eight possible transition mutations when using standard dNTPs. When

creating an unbiased library, sometimes it is necessary to use two or more methods in order to

allow for the same approximate percentage of transitional and transversional mutations to occur.

The use of the MnCl2 and Taxq polymerase in an error-prone PCR allows for all four

transitions and all eight transversions to occur, however the A-T to T-A transition and A-T to G-

C transversion tend to be more prevalent when using this technique (Vartanian et al., 1996, Lin-

Goerke et al., 1997, Arnold and Georgiou, 2003b). Biases such as this can be altered by

increasing or decreasing the concentrations of some of the nucleotides in the reaction. This

technique can be performed on a low budget, and can be easily modified to increase or decrease

the frequency of mutagenesis by altering the concentration of dNTPs or the number of PCR

cycles (Arnold and Georgiou, 2003b).

Another method of creating mutagenic libraries is by rational design. The random library

approach generates a large, diverse repertoire of polymerases, but a low number of active clones.

Guo et al. has shown that at least one-third of all random amino acid changes will result in the

inactivation of a protein (Guo et al., 2004), so it is likely that a protein with more than a few

random amino acid changes will be inactive. Furthermore, Guo et al. also calculated that

approximately 70% of random mutations in the active sites of polymerases will result in an

inactive polymerase variant (Guo et al., 2004). A desirable library for directed evolution

experiments would optimally have a large, diverse number of proteins with a high number of

active clones (Hibbert and Dalby, 2005). To generate a library such as this, the reconstructing









evolutionary adaptive paths (REAP) approach can be used (Gaucher, 2006); this approach allows

researches to modify only the sites where functional divergence occurred within a family of

polymerases. In other words, sites that, in the historical evolution of the polymerase, had a split

"conserved but different" pattern of evolutionary variation, are chosen for modification. In

theory, this technique has a high probability to generate new activities and functions (Gaucher,

2006).

Systems of Directed Evolution

Some of the more common methods used in directed evolution experiments include phage

display (Fa et al., 2004), ribosome display (Yan and Xu, 2006), complementation (Arnold and

Georgiou, 2003a), and compartmentalized self-replication (CSR) (Ghadessy et al., 2001, Tawfik

and Griffiths, 1998). Two of these techniques, phage display and CSR (Henry et al., 2004), were

applied to the evolution of polymerases to increase thermostability (Ghadessy et al., 2001),

permit activity in the presence of an inhibitor (Ghadessy et al., 2001), and allow incorporation of

non-standard bases (Ghadessy et al., 2004, Fa et al., 2004, Xia et al., 2002). Both phage display

and CSR systems have been successfully used to evolve Taq polymerase in vitro (Ghadessy et

al., 2001, Ghadessy et al., 2004, Fa et al., 2004).

Phage display

The phage display directed evolution system was developed by attaching a fragment of

Taq polymerase and an oligonucleotide primer substrate to the exterior of a phage particle via its

minor phage coat protein pIII (Fa et al., 2004). Since there are approximately five of these coat

proteins per phage, all localized to one area on the phage coat, researchers were able to

successfully link phenotype to genotype (Fig. 1-12). The mutant polymerases were challenged

to add non-standard nucleosides and one biotinylated nucleoside onto the oligonucleotide primer

by template directed synthesis; those polymerases with the ability to do so were immobilized on










streptavidin beads, and were recovered. The genes encoding the active polymerases were

identified by sequencing, or rediversified and shuttled into another round of selection. This

technique, while excellent for identifying polymerase mutants able to incorporate a small number

of non-standard bases, does not require the polymerase to perform a PCR; this would not be

conducive to the design of an AEGIS based synthetic biology that requires the polymerase to

replicate its own gene.

Compartmentalized self-replication

Compartmentalized self-replication makes use of water-in-oil emulsions as a way to link

genotype to phenotype, and requires polymerase mutants to replicate their encoding gene in a

PCR reaction (Tawflk and Griffiths, 1998, Ghadessy et al., 2004, Ghadessy et al., 2001,

Williams et al., 2006), theoretically an excellent technique for developing polymerases for a

synthetic biology. A library of polymerase gene variants is cloned and expressed in cells (Fig. 1-

13A); the bacterial cells containing the polymerases and their encoding genes are then suspended

in aqueous droplets in an oil emulsion. Each of these droplets, on average, contains one cell as

well as the primers and dNTPs/NSBs required for PCR (Fig. 1-13B). The thermostable

polymerase is released from the cell during the first denaturing cycle of PCR, allowing

replication of its encoding gene to proceed. Poorly adapted polymerases fail to replicate their

encoding gene, while better-adapted polymerases succeed in replication (Fig. 1-13C). The

resulting polymerase genes are then released from emulsions by extraction with ether; those

encoding the most active polymerases dominate these clones. A run-off PCR using standard

nucleotides prepares the DNA for recloning, which can then be subj ected to another cycle of

selection (Fig. 1-13E).

CSR has been previously used to generate Taq polymerase variants that are more

thermostable (Ghadessy et al., 2001), have an increased resistance to inhibitors (Ghadessy et al.,










2001), and are able to incorporate various non-standard bases (Ghadessy et al., 2004). More

recently, Philipp Holliger and co-workers, who originally performed the aforementioned

selections, have modified this technique to change a selected region of the polymerase sequence,

and replicate that region in CSR reactions (Ong et al., 2006). This short-patch

compartmentalized self-replication reaction (spCSR) has already been used to develop Taq

polymerase variants able to function with both NTPs and dNTPs, and variants that are able to

incorporate NSBs with 2'-substitutions. This technique allows the researcher to mutate only the

active site of the polymerase, and then challenges the polymerase to amplify the region encoding

the active site; this makes it easier for polymerases with the ability to incorporate NSBs, but who

lack the catalytic efficiency and processivity, to be isolated from a pool of mutants. By reducing

the stringency of the initial selections, more clones can be isolated with the desired traits;

catalytic efficiency and processivity of the polymerase can be selected for later using the

polymerase sequence of the desired variant under normal CSR conditions.

Research Overview

To create an AEGIS, the first step should be to create or identify polymerases with the

ability to incorporate multiple, consecutive NSBs into a growing strand of dsDNA, efficiently

and faithfully. Rather than challenging a polymerase with a gamut of NSBs containing different

unique features, we decided to focus on one unique characteristic of AEGIS nucleosides, the C-

glycosidic linkage. Previous studies have shown that polymerases have a difficult time

incorporating the non-standard base pairs containing a C-glycosidic linkage (Switzer et al., 1993,

Sismour et al., 2004), therefore representative C-glycosides, 2' -deoxypseudouridine (dyU) and

2' -deoxypseudothymidine (dyT), that could base pair with a canonical nucleotide, in order to

decrease the strain on the polymerase, were selected for study (Lutz et al., 1999).









The research presented here began with the determination of the effect of multiple,

sequential C-glycosides on duplex DNA structure, to better understand the obstacles a

polymerase would have to overcome in order to incorporate bases exhibiting C-glycosides.

Next, a screening of a variety of Family A and Family B polymerases, identified Taxq as a

polymerase that exhibited a limited ability to incorporate non-standard bases that contain a C-

glycosidic linkage. However, further modification of the protein sequence of this enzyme was

needed to identify a mutant Taq polymerase with an increased ability to incorporate multiple,

sequential C-glycosides NSBs more efficiently.

To achieve this, the second part of this dissertation focused on the creation of a rationally

designed (RD) library of 74 mutant Taq polymerases. Variants were screened for the ability to

incorporate dyU in a PCR amplification of their encoding gene. None of these variants were

shown to produce more full-length PCR product than the wild tyipe Taq polymerase. Only 18

variants showed any activity at all in this first test, even with standard dNTPs, under these

reaction conditions. A rationally designed library was then used to perform an initial selection,

by using water-in-oil emulsions to select for the active mutant polymerases we identified in our

initial screen.

It was postulated that the low number of active variants in our RD library was due to a

decrease in the thermostability of the enzyme. After altering the PCR reaction conditions to test

this hypothesis, we were able to identify 33 active mutant polymerases in this library. Since this

library was rationally designed, it was interesting to speculate as to whether a randomly created

library of polymerase clones would tend to have increased or decreased thermostability when

compared to the number of active clones in our RD library. A random library (L4) was created

for this purpose, and was screened for activity at various temperatures in PCR reactions; 39









clones were found to be active. This comparison of the thermostability of the two libraries

shows that the randomly created library has an enhanced ability to retain polymerase

thermostability when compared to our rationally designed library.

The RD library was designed to identify mutants able to incorporate non-standard bases,

and not to have a high degree of thermostability. Optimal temperatures for function in a PCR

were determined for each of the RD variants, and the mutants were then screened for their ability

to incorporate various concentrations of dyU at that optimal temperature. One mutant in the

pSW27 plasmid, containing the A597S, A740R, and E742V residue changes, was identified with

the ability to generate, on average, 72% more product at all dyU concentrations tested, than wt

Taq polymerase at a temperature of 86.3 oC.

While dyU is a C-glycoside with the ability to pair with 2'-deoxyadenosine, it has been

shown to epimerize (Wellington and Benner, 2006, Cohn, 1960, Chambers et al., 1963). Since

dyT cannot epimerize, due to the presence of the extra methyl group, we performed a

comparative analysis between wt Taq polymerases' ability to cope with dyU and dyT in various

concentrations and at different temperatures in a PCR. Results indicated that it may be the

epimerization of the nucleotide hindering the incorporation of dyU, and therefore it should not

be used as a model C-glycoside for directed evolution studies.

These results presented in this work represent a significant step towards the long-term goal

of creating an AEGIS-based synthetic biology. In addition, the repertoire of mutant polymerases

designed and created in these experiments will assist in creating an inventory of polymerases

useful in biotechnology, possibly allowing the development of new, as well as improving on

existing diagnostic techniques and helping to facilitate a better understanding of polymerase-

DNA interactions.






















OH H OH H
2'-deoxyadenosine 2'-deoxyguanosine


OH H OH H
2'-deoxyvthymidine 2'-deoxyvcytosine


Figure 1-1.


The standard deoxyribonucleotides. The nucleobases pair based on the two rules of
complementarity: hydrogen-bonding complementarity, when the hydrogen bond
donor from one nucleobase pairs with the hydrogen bond acceptor from another, and
size complementarity, when a large purine (top row) pairs with small pyrimidine
(bottom row) (Watson and Crick, 1953a, Watson and Crick, 1953b). Therefore, 2'-
deoxyadenosine j oins with 2'-deoxythymidine and 2'-deoxyguanosine j oins with 2'-
deoxycytosine. When a phosphate group replaces the 5'-hydroxyl group of these
molecules, they become acids and can be linked by their phosphate groups to create
a DNA strand.










Table 1-1. Comparison of the structural geometries of A, B, and Z-DNA forms.

Geometry A-DNA B-DNA Z-DNA

Helical Sense Right-handed Right-handed Left-handed


Helix diameter 2.6 nm 2.0 nm 1.8 nm


Repeating unit 1 base pair 1 base pair 2 base pairs


Rotation per base pair 340 360 600/2


Rise per base pair 0.256 nm 0.338 nm 0.38 nm


Base pairs per turn 11 10 12


Pitch per turn of helix 2.82 nm 3.38 nm 4.56 nm

Very narrow and Very wide and
Major Groove Flat
very deep deep

Very broad and Very narrow and
Mmnor Groove Narrow and deep
very shallow deep

C: C2'-endo &
Sugar Pucker C3'-endo C2'-endo
G: C2'-eXO
*Data adapted from Saenger and Garrett & Grisham (Saenger, 1984, Garrett and Grisham, 1999).











B) c's

3'


Figure 1-2.


Puckering of the furanose ring of nucleosides into various envelope forms. In the
envelope form, four of the five atoms are coplanar, the remaining atom departs this
plane: A) a C2--eXO sugar pucker, B) a C2'-endo sugar pucker, C) a C3--eXO sugar
pucker, and D) a C3-endo sugar pucker. B-DNA has a C2 -endo pucker, while A-
DNA exhibits a C3 -endo pucker (Saenger, 1984).


T rans cr iption
DNA~ dep enders
RNA Polym erases






Rever se T rans cr iption
RNA depndernDNA
Polymerases or Reverse
Tranisciptases


Replication,
RNAL dependent
RNAPolyrm rases
or eplicans





RNA


mRNA tRNA anid
riba somes






Pr otein


Replic~atin ',(., .
DNAdpedn.
DNA1Polymerases~ --- NA


Figure 1-3. The central dogma of molecular biology (Lewin, 1997, Crick, 1970). Genetic
material, in the form of DNA, is first transcribed into RNA and then is translated
into proteins. On the occasion that genetic material is stored as RNA, it first
undergoes reverse transcription to create DNA before it is shuttled back into the
system.















V aminoA





donor
H\ acceptor
donor



pu-DAD




N~ isoG


donor
acceptor
acceptor


CR

py-DAA pu-ADD


py-ADA


acceptor
acceptor
donor









isoC R

py-AAD




acceptor
acceptor
donor


donor
acceptor
donor


donor

ace or



pu-DDA


py-DAD pu-ADA


acceptor
donor
donor


py-AAD pu-DDA


py-ADD pu-DAA


Figure 1-4.


The six hydrogen bond patterns in an artificially expanded genetic information
system (AEGIS). These patterns are constrained by Watson and Crick's rules of
complementarity and by the requirement that the nucleobases be joined by three
hydrogen bonds (Switzer et al., 1989, Piccirilli et al., 1990, Geyer et al., 2003,
Benner, 2004, Watson and Crick, 1953a, Watson and Crick, 1953b). Purines are
denoted by "pu," pyrimidines by "py," hydrogen-bond acceptors by "A," hydrogen
bond donors by "D," and R indicates the point of attachment of the backbone. Note
the presence of a C-glycosidic linkage in the pyDAD, pyADD, and pyDDA
nucleotides.




















ratio


Solid
Support


SAnalyte DNA
- Capture Strand
-Branched DNA
NSB-containing Duplex


Figure 1-5. The VersantTM branched DNA assay. This assay exploits the pairing of non-
standard bases (NSBs) to reduce the signal to noise ratio 8-fold over a previous
version of the assay that did not use NSBs (Huisse, 2004, Collins et al., 1997). The
branched DNA assay is used to monitor the viral load counts of patients with the
HIV, Hepatitis B, or Hepatitis C viruses (Collins et al., 1997).











A) -pyridin-2-one (y) B) 3' 5
H ~DNA -sTC-
Transcription
S 5' 3'
mRNA yACP~I-
/N rasato
,H Ribose
N ~N tRNA


QN H ,

Ribose
2-amino-6-(2-thienyl)purine~s)







o = -O



3-chlorotyrosine


Figure 1-6. An example of non-standard nucleobases coding for a non-standard amino acid.
This shows the transcription and translation (seen in B) of the non-standard base pair
(seen in A and denoted as s and y) to generate a protein containing the non-standard
amino acid 3-chlorotyrosine. This picture is adapted from Hirao et al (Hirao et al.,
2002, Hirao et al., 2006).
























B) K K D) .
HN NH HN NH N NH

O O'r -O



HO H HO H HO H
OH OH OH OH OH OH

Figure 1-7. Pseudouridine and pseudothymidine. A) This naturally occurring C-glycoside,
found in RNA, is thought to be created by a posttranscriptional isomerization of
uridine (Argoudelis and Mizsak, 1976, Grosjean et al., 1995). B) Pseudouridine has
a propensity to adopt a syn conformation around the glycosyl bond when in solution,
but it is only found in the anti conformation when in a nucleic acid strand (Lane et
al., 1995, Neumann et al., 1980). C) The anti conformation allows for the
coordination of a water molecule between the 5' phosphate group of the yU residue,
the 5' phosphate group of the preceding residue, and the N1-H of the yU residue
(Arnez and Steitz, 1994). The coordination of this water molecule results in an
enhanced base stacking ability and a reduced conformational flexibility of the RNA
molecule, thus increasing the local rigidity of the RNA (Charette and Gray, 2000,
Davis, 1995). D) The structure of pseudothymidine (1 -methylpseudouridine). This
naturally occurring C-glycoside, found in RNA, is also thought to be created by a
posttranscriptional modification ofuridine (Limbach et al., 1994).















o.,
0-
HO O\


O


Figure 1-8.


The polymerization reaction of deoxyribonucleotides triphosphates catalyzed by
DNA polymerases. The triphosphate of the incoming group is linked to the 3'-
hydroxyl group of the preceding nucleoside, releasing a pyrophosphate in the
process; therefore DNA synthesis requires synthesis of new molecules in the 5' 3'
direction (Garrett and Grisham, 1999).


1 2 3 4 5 6
E + TP E -TP E -TP-dNTP E' -TP-dNTP E -TP z-PP, E -TP z+PPi E -TP,


Figure 1-9.


Kinetic steps involved in the nucleotide incorporation pathway. The kinetic steps
involved in the addition of a nucleotide onto a growing DNA strand (Patel and Loeb,
2001, Rothwell and Waksman, 2005). In Step 1, the polymerase (E) binds to the
DNA primer:template complex (TP); the polymerase then binds the incoming
nucleotide triphosphate (dNTP) in Step 2. The polymerase then undergoes a
conformational change (E') in Step 3 that brings the various components into
positions that can support the chemistry of this reaction; this is the rate-limiting step
of polymerization. The polymerase performs the addition of the nucleotide, remains
completed with the pyrophosphate, and undergoes another conformational change in
Step 4. The pyrophosphate group is released in Step 5; in Step 6, the polymerase can
dissociate from the DNA or translocate the substrate for another round of synthesis.









1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801


MRGMLPLFEP
LLKALKEDGD
ELVDLLGLAR
DRIHALHPEG
EKTARKLLEE
DLPLEVDFAK
PPEGAFVGFV
LLAKDLSVLA
EAGERAALSE
LDVAYLRALS
PAIG G
DLIHPRTGRL
EEGWLLVALla
REAV PLMRig
PKVRAWIEKT
MPV GTAADL
RLAKEVMEGV


KGRVLLVDGH
AVIVVFDAKA
LEVPGYEADD
YLITPAWLWE
WGSLEALLKN
RREPDRERLR
LSRKEPMWAD
LREGLGLPPG
RLFANJLWGRL
LEVAEEIARL
K ~AAVLE
HTRFNJQT
YSQIELRVLA
AAK3IN VL
LEEGRRRGYV
MKLAMVKLFP
YPLAVPLEVE


HLAYRTFHAL
PSFRHEAYGG
VLASLAKKAE
KYGLRPDQWA
LDRLKPAIRE
AFLERLEFGS
LLALAA~ARGG
DDPMLLAYLL
EGEERLLWLY
EAEVFRLAGH
ALREAHPIVE
TG L Pg
HLSGDENJLIR
~GMAHPLSQ
ETLFGRYV
RLEEMGARML
VGIGEDWLSA


KGLTTSRGEP
YKAGRAPTPE
KEGYEVRILT
DYRALTGDES
KILAHMDDLK
LLHEFGLLES
RVHRAPEPYK
DPSNJTTPEGV
REVERPLSAV

KILQ CELT
LQ TPL
VFQEGRDIHT
ELAIPYEEAQ
PDLEARV SV
Le~I LVLE
KE


VQAVYGFAKS
DFPRQLALIK
ADKDLYQLLS
DNJLPGVKGIG
LSWDLAKVRT
PKALEEAPWP
ALRDLKEARG
ARRYGGEWTE
LAHMEATGVR
ERVLFDELGL
L IDDPLP
GQRIRRAFIA
ETASWMFGVP
AFIERYFQSF
rAAg FAF
APKERAEARA


Figure 1-10. Locations of active site residues in Taxq polymerase. Residues shown in blue are
involved in contacting the DNA during polymerization; those shown in red indicate
residues involved in metal ion coordination (Eom et al., 1996, Fa et al., 2004, Li et
al., 1998b, Li et al., 1998a, Kim et al., 1995, Suzuki et al., 1996).















Feature A B C D X Y RT
Prokarvotes,
Domin Cntinng roarotsEukaryotes, Archaea, Prokaryotes Archaea Eukaryotes PrkroeEukaryotes, Viruses
Polymerase Eukaryotes, Viruses Eukaryotes, Archaea
Viruses

E colz DNA Pol I; HIV-RT;
Representative Pfu DNA Pol I; E. colz DNA Pol IV;
Taq Pol I; E. colz Pol III(a) Pfu DNA Pol II Eukaryotic DNA Pol b M-MuLV-RT;
Polymerases Eukaryotic DNA Pol a E colz DNA Pol V
T7 DNA Pol Eukaryotic telomerases

General Use Repair Replicative Replicative Replicative Repair Replicative/Repair Replicative
Fidelity Good Excellent Excellent Excellent N/A Poor Good


Table 1-2. Characteristics of the various polymerase families.











A





Bm


C


mm


mm mmmm mm m


Figure 1-11.


The staggered extension process (StEP) for rediversification of mutant libraries.
This process has already been successfully used to rediversify libraries between
rounds of selection in CSR reactions (Arnold and Georgiou, 2003b, Zhao et al.,
1998, Ghadessy et al., 2001). A) Denatured template genes are primed with the
same primer. B) Short fragments are produced by brief primer-extension. C) In
the next cycle, fragments randomly prime the templates and extend further. D)
This process is repeated until full-length genes are produced. E) Full-length genes
are then purified, amplified, and recloned into a vector for another round of
selection.











A) ,
pol.Pept de
pilll
(T28)ATCCCA~n)GGCTC
~(P60)TAGGG
Basic peptide-DNA duplex
(T28)ATCC
B) (P660) TAG




dATP, dGTP,
Blotin-16-dCTP
(T28)ATCC
C) (P660) TAG




Streptavidin Coated Beads

(T28)ATCC
D) _L-~(P60) TAGG


Blotin









BSt etvdin


Figure 1-12.


Phage display selection scheme. This details the scheme used in the directed
evolution of a Taq polymerase fragment to incorporate non-standard nucleosides
into a growing DNA strand (Fa et al., 2004). A) A phage particle is displaying an
acidic peptide and a mutant polymerase on the pIII minor coat protein of the phage.
These coat proteins are localized to one area on the phage molecule, allowing
genotype to be linked to phenotype. B) The primer-template complex is attached to
the phage particle via a basic peptide, which links with the acidic peptide displayed
on the coat protein. C) The polymerase incorporates modified nucleotides in a
primer-extension assay, which terminates with the addition of a biotinylated
standard nucleotide. D) The biotin tag is captured by streptavidin and the entire
complex is immobilized on magnetic beads, allowing those phage particles
displaying inactive polymerases to be washed away. E) DNase I is used to
dissociate the phage complex from the DNA strands, allowing the phage displaying
the active polymerase to be captured in an elution. The genes encoding the active
polymerases can then be identified by sequencing and/or rediversified and shuttled
into another round of selection.


D seI clevag


















Heating In first PCR
cycle lyses cell



C)
water drop In oil

O on
plasmid OO
dATP,
dCTP,
dGTP, TTP,
ad/or
PCR primers




temperature
cycle


Suspend In
water-oll
emulsion )


A)




E coli cell


Run-off with standard
nucleotides & re cone
for next round of
selection


Extac
oilawa



Maycolsofgn
(l o s cie


Figure 1-13.


General scheme for CSR. CSR allows for the selection of polymerases with an
ability to incorporate an unnatural nucleotide using water-in-oil emulsions. ) A
library of polymerase gene variants is cloned and expressed in E. coli. Spheres
represent active polymerase molecules inside of a bacterial cell. B) The bacterial
cells containing the polymerases and their encoding genes are suspended in
aqueous droplets in an oil emulsion. C) The thermostable polymerase enzyme and
encoding gene are released from the cell during the first denaturing cycle of PCR,
allowing self-replication to proceed. D) The resulting mixture of polymerase genes
is released by extraction with ether. E) A single run-off PCR with standard
nucleotides prepares the DNA for recloning and another cycle of selection.









CHAPTER 2
POLYMERASE INCORPORATION OF MULTIPLE C-GLYCOSIDES INTTO DNA:
PSEUDOTHYMIDINE AS A COMPONENT OF AN ALTERNATIVE GENETIC SYSTEM

Introduction

Each of the four standard nucleobases found in natural DNA (adenine, guanine, cytosine,

and thymine) is joined to their sugar via a carbon-nitrogen bond. This, by definition, makes

standard nucleotides N-glycosides. The nature of the glycosidic linkage is believed to have

consequences on the detailed conformation of the nucleoside, including through the operation of

the anomeric effect. In particular, the nature of the glycosidic bond may influence the puckering

of the sugar.

Unlike the standard nucleotides, the nucleotides that allow artificially expanded genetic

information systems (AEGIS) to be created are frequently C-glycosides, which have a carbon-

carbon bond between the nucleobase and the sugar. This is exemplified in the case of non-

standard pyrimidines that present Donor- Donor-Acceptor, Donor-Acceptor-Donor and

Acceptor-Donor-Donor hydrogen bonding patterns seen in Figure 1-4. If replacing the N-

glycosidic linkage by a C-glycosidic linkage changes features of the nucleoside that are

important specificity determinants for polymerases, problems are created for those seeking to

expand the genetic alphabet artificially and develop a synthetic biology from an expanded

genetic alphabet.

Reverse transcriptases have an ability to process both DNA and RNA, whose sugars have

different conformations. Reverse transcriptases, therefore, should be able to accept components

of an artificially expanded genetic information system that incorporate C-glycosides. Perhaps it

is not surprising that the first reported example of PCR amplification of a six letter genetic

alphabet, where one the extra two letters was a C-glycoside, exploited HIV-RT (Sismour et al.,

2004).









When attempting to develop a synthetic biology using C-glycosides, the physical structure

of the DNA must be considered, especially since the presence of multiple, sequential C-

glycosides can possibly alter the structure and stability of duplex DNA. Previous studies have

shown that poly(U)*poly(A) helices favor the A-DNA form while poly(T)*poly(A) helices

display perfect B-DNA structure (Ivanov et al., 1973, Saenger, 1984, Chandrasekaran and

Radha, 1992). Circular dichroism was employed to infer the secondary structure of our DNA,

since the spectra generated by A-DNA and B-DNA are quite different (Fig. 2-1) (Ivanov et al.,

1973). Duplex DNA containing one to twelve consecutive dA-dyU base pairs was studied and it

was determined that all remained in the B-DNA form.

To take the next step towards a synthetic biology with an expanded genetic alphabet, it

would be desirable to have DNA polymerases that accept multiple C-glycoside nucleotides. To

determine whether natural DNA polymerases have this capability and the extent to which this

capability is conserved, four Family A DNA polymerases and four Family B DNA polymerases

were screened for their ability to incorporate multiple 2' -deoxypseudothymidine-5 '-triphosphate

(dyTTP) and 2' -deoxypseudouridine-5 '-triphosphate (dyUTP) across from template dA. These

C-glycosides are steric analogs of thymidine-5' -triphosphate (TTP) and present the same

hydrogen bonding pattern to a complementary strand as TTP (Fig. 2-2). Consequently, they

should serve as a relatively specific probe for this non-standard structural feature.

In these experiments, all of the polymerases tested were able to incorporate both C-

glycosides to an extent; but there was room for improvement in some, such as Taxq. To

determine the extent of Taxq polymerase's ability to incorporate the C-glycosides, it was screened

for its ability to incorporate anywhere from one to twelve consecutive dyTTP or dyUTP across

from template dA.









Materials and Methods

Synthesis of Triphosphates and Oligonucleotides

Dr. Shuichi Hoshika, from the Foundation for Applied Molecular Evolution (FfAME,

Gainesville, Florida), synthesized the pseudothymidine precursor as described in Appendix A.

Dr. Daniel Hutter (FfAME) synthesized 2' -deoxypseudothymidine-5 '-triphosphate (dyTTP) as

described in Appendix A. 2'-Deoxypseudouridine-5 '-triphosphate (dyUTP) was purchased

from TriLink BioTechnologies (San Diego, California). Standard deoxynucleotide triphosphates

(dNTPs) of 2'-deoxyadenosine-5 '-triphosphate (dATP), 2'-deoxycytidine-5' -triphosphate

(dCTP), 2'-deoxyganosine-5' -triphosphate (dGTP), and thymidine-5' -triphosphate (TTP) and

were purchased from Promega Corporation (Madison, Wisconsin). Triphosphate solutions

identified as dyTNTPs were comprised of dATP, dCTP, dGTP, and dyTTP, while those

acknowledged as dyUNTPs were contained dATP, dCTP, d GTP, and dyUTP.

The oligonucleotides used for these experiments are listed in Table 2-1. Those sequences

containing only standard nucleotides were commercially obtained from Integrated DNA

Technologies (Coralville, Iowa) as desalted or PAGE (Polyacrylamide Gel Electrophoresis)

purified oligonucleotides. Those oligonucleotides containing dyU were synthesized by Dr. Ajit

Kamath (University of Florida, Gainesville, Florida) and were prepared using standard

monomers and reagents (Glen Research, Sterling, Virginia) on an Expedite 8909 DNA

Synthesizer (PerSeptive Biosystems, Inc., Framingham, Massachusetts). The crude products

were digested, with agitation, in 1 mL of concentrated ammonium hydroxide at 55 oC for 16 hrs

to release and deprotect the oligonucleotide (Sambrook et al., 1989). The mixtures were briefly

centrifuged and the supernatants were passed through 2 Clm cellulose acetate syringe filters. The

residual products were washed three times with 1 mL portions of sterile water. The combined









fi1trates were lyophilized to dryness and were purified by polyacrylamide gel electrophoresis

(PAGE) and isolated by reversed-phase chromatography on a silica gel as described previously

(Sambrook et al., 1989).

Circular Dichroism

Each template, containing one through twelve consecutive dA or dyU residues (T-13

through T-22 or T-23 through T-34, respectively), was annealed to its complement template,

containing consecutive dT or dA residues (T-3 5 through T-46 or T-47 through T-58,

respectively). Reactions contained 5 nmol of each template and 290 CIL of CD buffer (1 M

NaC1, 10 mM Na2HPO4, 1 mM Na2EDTA at pH 7.0) for a total volume of 300 CIL. The mixtures

were incubated for 5 min at 96 oC and allowed to cool to room temperature over the course of 1

hr.

The CD spectra from 200 to 320 nm, using a wavelength step of 1 nm, were measured in a

nitrogen atmosphere at 25 OC in a 0.1 cm pathlength cuvette, using an Aviv Model 215 Circular

Dichroism Spectrometer (Proterion Corporation, Inc., Piscataway, NJ). Scans were performed in

triplicate for each sample mixture and the data was averaged.

Standing Start Primer-Extension Assays

Radiolabeled primer was prepared by incubating 0.5 nmol P-1, 100 CICi y-32P-ATP, lX T4

Polynucleotide Kinase (PNK) Buffer, 50 U T4 PNK (New England BioLabs, Beverly,

Massachusetts), and sterile dH20 in a Einal volume of 100 C1L, for 1 hr at 37 oC. The

radiolabeled primer was purified using the QIAquick Nucleotide Removal Kit (Qiagen,

Valencia, California) and eluted from the column in 100 CIL Buffer EB (10 mM Tris-HC1, pH

8.5).









Radiolabeled template, to depict the location of full-length product (FLP), was prepared by

incubating 50 pmol T-4, 10 CICi y-32P-ATP, lX T4 PNK Buffer, 25 U T4 PNK, and sterile dH20

in a final volume of 50 CIL, for 1 hr at 37 oC. The radiolabeled T-4 was purified using the

QIAquick Nucleotide Removal Kit, and eluted from the column in 50 CIL Buffer EB. 200 CIL

DNA PAGE Loading Dye (98% formamide, 10 mM EDTA, 1 mg/mL xylene cyanol, and 1

mg/mL bromophenol blue) was added to the 1 C1M radiolabeled T-4 for a final concentration of

0.2 CIM radiolabeled T-4.

Radiolabeled 10 base-pair (bp) ladder was prepared by first incubating 1.95 Clg 10 bp DNA

Step Ladder (Promega Corporation), 30 CICi y-32P-ATP, lX T4 PNK Buffer, and sterile dH20 in

a final volume of 27 CIL, for 1 min at 90 oC. Immediately following, 30 U T4 PNK was added

and the mixture was incubated for 30 min at 37 oC. The radiolabeled 10 bp ladder was purified

using the QIAquick Nucleotide Removal Kit, and eluted from the column in 30 CIL Buffer EB.

120 CIL DNA PAGE Loading Dye was added to the 65 ng/CIL radiolabeled 10 bp DNA Ladder

for a final concentration of 13 ng/CIL radiolabeled 10 bp DNA Ladder.

Polymerase screen primer-extension assays

Klenow Fragment (3' 5' exo-), Bst DNA Polymerase (Large Fragment), Taxq DNA

Polymerase, VentR~ (eXO-) DNA Polymerase, Deep VentR~ (eXO-) DNA Polymerase, and

TherminatorTM DNA Polymerase were purchased from New England BioLabs. Tth DNA

Polymerase was purchased from Promega Corporation. Pfu (exo-) DNA Polymerase was

purchased from Stratagene (La Jolla, California). Buffers used in these experiments were

supplied by the manufacturer as follows: reactions using Bst, Taq, Tth, Vent (exo-), Deep Vent

(exo-), and Therminator were performed in lX ThermoPol Buffer (20 mM Tris-HCI (pH 8.8), 10

mM (NH4)2SO4, 10 mM KC1, 2 mM MgSO4, 0. 1% Triton X-100); Klenow (exo-) reactions were










performed in lX NEBuffer 2 (10 mM Tris-HCI (pH 7.9), 50 mM NaC1, 10 mM MgCl2, 1 mM

dithiothreitol); and reactions using Pfu (exo-) were performed in lX Cloned Pfu Buffer (20 mM

Tris-HCI (pH 8.8), 2 mM MgSO4, 10 mM KC1, 10 mM (NH4)2SO4, 0.1% Triton X-100, 0.1

mg/mL nuclease-free Bovine Serum Albumin). Optimal temperatures for polymerase function

were 37 oC for Klenow (exo-), 65 oC for Bst, and 72 oC for Taq, Tth, Vent (exo-), Deep Vent

(exo-), Pfu (exo-), and Therminator.

T-4 Primer-Template complex was prepared by mixing 25 pmol radiolabeled P-1, 200

pmol non-radiolabeled P-1, and 300 pmol non-radiolabeled T-4, in a final volume of 15 CIL. The

mixture was incubated for 5 min at 96 oC and allowed to cool to room temperature over the

course of 1 hr.

For primer-extension assays, 1.5 CIL of the primer-template complex, lX of the appropriate

manufacturer' s supplied buffer, 1 U/CIL of the appropriate polymerase, and sterile dH20 were

used in a final volume of 9 C1L. Reactions were then incubated at the appropriate temperature for

30 s. Each reaction was initiated by adding 1 C1L of one of the following: 1 mM dTTP, 1 mM

dyTTP, 1 mM dyUTP, 1 mM dNTPs, 1 mM dyTNTPs, or 1 mM dyUNTPs, and incubated for

two more minutes at the appropriate temperature. Reactions were immediately quenched with 5

CIL of DNA PAGE Loading Dye. Samples (1 CIL) were resolved on denaturing PAGE gels (7 M

Urea and 20% 40:1 acrylamide: bisacrylamide) and analyzed on a Molecular Imager FX System

(Bio-Rad, Hercules, California).

Taq polymerase primer-extension assays

Primer-Template complexes were prepared by mixing 25 pmol radiolabeled P-1, 200 pmol

non-radiolabeled P-1, and 300 pmol of non-radiolabeled template (T-1 through T-12), in a final










volume of 15 CIL. The mixtures were incubated for 5 min at 96 oC and allowed to cool to room

temperature over the course of 1 hr.

For primer-extension assays, 1.5 CIL of the appropriate primer-template complex, lX

ThermoPol buffer, 1 U/CIL Taq Polymerase, and sterile dH20 were used in a final volume of 9

CIL. Reactions were then incubated at 72 oC for 30 s. Each reaction was initiated by adding 1 CIL

of one of the following: 1 mM dNTPs, 1 mM dyTNTPs, or 1 mM dyUNTPs, and incubated for

two more minutes at 72 oC. Reactions were immediately quenched with 5 CIL of DNA PAGE

Loading Dye. Samples (1 CIL) were resolved on denaturing PAGE gels (7 M Urea and 20% 40:1

acrylamide: bisacrylamide) and analyzed on a Molecular Imager FX System (Bio-Rad).

Results

Circular Dichroism

Duplexes were formed by annealing each template (T-13 through T-34) to its complement

sequence (T-3 5 through T-58) creating twelve control helices containing only thymidine and

twelve helices containing pseudouridine. Figure 2-3 [A-E] shows a representative set of these

spectra, specifically the spectra of duplexes containing 1, 3, 6, 9, or 12 A-yU base pairs. When

compared to the spectra seen in Figure 2-1, all spectra are consistent with B-DNA being the

overall conformation of all duplexes. In addition, the spectra representing the oligonucleotides

containing the dA-dyU base pairs are similar to the patterns of the spectra containing the dA-dT

base pairs.

Polymerase Screen Primer-Extension Assays

Four Family A and four Family B polymerases were screened for their ability to

incorporate non-standard bases exhibiting a C-glycosidic linkage with efficiency. Polymerases

were tested in both 4-base and 13-base extension assays, and were challenged to incorporate (4-










bases) or incorporate and extend beyond (13-bases) four consecutive dT, dyT, or dyU residues

across from template dA under the polymerases' optimal conditions (Fig. 2-4). Reactions used

TTP, dyTTP, or dyUTP in the 4-base extensions and either dNTPs, dyTNTPs, or dyUNTPs for

the 13-base extension reactions. Family A polymerases (Fig. 2-5[A-B]) were represented by

Klenow (exo-), Bst, Taq, and Tth; Family B polymerases (Fig. 2-6[A-B]) were represented by

Vent (exo-), Deep Vent Exo-, Pfu (exo-), and Therminator.

Pfu (exo-) was the only polymerase that was not able to generate FLP when challenged to

incorporate and extend beyond both of the non-standard bases. All other Family A and Family B

polymerases were able to incorporate the four consecutive non-standard bases (NSBs) and

extend beyond them, to some measure, to generate FLP. Bst and Therminator polymerases

appeared to have consumed almost all of the primer in the course of their reactions, generating

large amounts of FLP, with all of the different NTPs tested. Klenow (exo-) and Vent (exo-) also

did an exceptional j ob at incorporating the NSBs, but the remainder of the polymerases did

appear to have difficulty given the intensity of the pause sites relative to the intensity of the FLP

bands.

Taq Polymerase Primer-Extension Assays

To replicate its own encoding polymerase gene, Taq polymerase would be required to

incorporate and extend beyond four consecutive dT, dyT, or dyU residues. In these

experiments, Taq polymerase was challenged to incorporate and extend beyond twelve

consecutive dT/dyT/dyU residues opposite template dA. From these results (Fig. 2-7[A-B]), it

was determined that Taq appears to have some difficulty incorporating twelve consecutive dT

residues, as evidenced by the pausing in those lanes, but it is still able to generate FLP (N+13).

It is also apparent that Taq has difficulty incorporating multiple consecutive residues of C-










glycosides, since it was not able to generate FLP when forced to incorporate five or more dyT or

dyU residues. However, it does, generate a small amount of FLP when challenged to insert four

consecutive dT, dyT, or dyU residues, and therefore should be able to replicate its own gene

using a C-glycoside substitute for TTP.

Discussion

It was first necessary to determine if the presence of multiple dyU residues in double-

stranded DNA would perturb the helical structure to a point where there is a phase transition

from B-DNA to A-DNA, perhaps making it difficult for polymerases to replicate the DNA. It is

well known that poly(U)*poly(A) favors the A-helicies, while poly(T)*poly(A) favors B-DNA

helicies (Ivanov et al., 1973, Saenger, 1984, Chandrasekaran and Radha, 1992).

The distinctive differences in the CD between the canonical A-duplex and the canonical B-

duplex structures involves a shift of the positive potion of the spectrum to shorter wavelengths,

to 267 nm for the A-form compared to 275 nm for the B-form (Ivanov et al., 1973). A similar

shift with a similar magnitude is seen in the negative portion. Further, the Q-DNA shows a

stronger Cotton effect than the B-DNA. Therefore, to determine whether the addition of C-

glycosidic units tends to drive the conformation of the duplex from B towards A, we look for an

increase in the Cotton effect and a shift towards shorter wavelengths.

Circular dichroism was performed on 24 duplex DNA molecules containing anywhere

from one to twelve consecutive dyU~dA or dTedA base pairs. The observed spectra (Fig. 2-

3 [A-E]) were compared to those in Figure 2-1, the reference spectra for canonical A and B

duplexes. In all spectra containing dyU, the wavelength shifted marginally (ca. 4 nm) towards

longer wavelengths. This shift does not display a trend, however. The shift is the same no

matter how many dyU units are incorporated into the strand.









The only possible trend is a change in the relative intensity of the positive (at 275 nm) and

negative (at 264 nm) band intensities (Ivanov et al., 1973). Here the intensity of the 246 nm

band and the 275 nm band both decrease. As concentrations were carefully controlled, we do not

believe that this reflects a change in the concentration of the oligonucleotides. This is also

suggested by the intensity of signals at lower wavelengths, although these are notoriously

compromised by any trace of impurity. Disregarding this detail, the trend is the opposite of what

one expects for the conversion of the duplex structure from canonical B to canonical A.

These results provide no evidence that addition of dyU units causes the duplex structure to

change from a B-DNA to an A-DNA conformation. Thus, there was no evidence to suggest that

there would be a conformational problem with the duplex structure when incorporating multiple,

sequential C-glycosides. It should be mentioned, however, that CD is indicative only of the

gross properties of the system; it does not provide information about detailed structure. It is

conceivable that the conformation is changed in a different way, or some subtly.

Nevertheless, these results encouraged us to test polymerases for their ability to work with

C-glycosides. Polymerases that already display some of the desired catalytic activity, in this case

the incorporation of the C-glycosides, should facilitate in the evolution and/or creation of an

AEGIS. Previous studies have shown that polymerases are able to incorporate up to three C-

glycosides, but have not tested their ability to incorporate more than three multiple, sequential C-

glycosides that would be required for an AEGIS (Lutz et al., 1999, Sismour et al., 2004, Piccirilli

et al., 1991). Accordingly, four Family A polymerases, Klenow (exo-), Bst, Taq, and Tth, and

four Family B polymerases, Deep Vent (exo-), Vent (exo-), Pfu (exo-), and Therminator, were

screened for the ability to incorporate TTP, d yTTP, and dyUTP across from template dA in

both 4-base and 13-base primer extension assays (Fig. 2-5[A-B] and Fig. 2-6[A-B]). In the 4-









base extension assay, polymerases were challenged to incorporate four consecutive TTP,

dyTTP, or dyUTP across from template dA during two-minute incubations at the optimal

temperature for each enzyme. The 13-base assay, incubated as described above, took place in

the presence of dCTP, dGTP, dATP, and TTP, dyTTP, or dyUTP, and required incorporation

and extension beyond the four consecutive TTP, dyTTP, or dyUTP.

The Bst and Therminator polymerases appeared to have worked extremely well and

consumed almost all of the primer in the course of all of their reactions, while Pfu (exo-) did not

appear to generate any 13-base FLP when presented with either of the two NSBs. All other

polymerases generated varying amounts of FLP with both of the NSBs, suggesting that any of

the aforementioned polymerases could be potential candidates for adaptation to an AEGIS, based

on the qualification that the polymerase must already be able to incorporate C-glycosides.

However, two of these polymerases, Klenow (exo-) and Bst are not thermostable, and thus could

not undergo PCR and, according to the manufacturer, Therminator is not recommended for any

applications except DNA sequencing and primer-extension reactions, thereby making these three

polymerases unlikely candidates for future studies. As in previous studies, Taq was selected as

the best polymerase candidate to undergo further testing since it so readily accepted the

consecutive non-standard bases (Lutz et al., 1999).

In an AEGIS system, a polymerase would be required to replicate its own encoding gene

with efficiency and fidelity. In order for Taq to replicate its encoding polymerase gene, it would

be required to incorporate four consecutive dyT or dyU across from template dA. Since we

have already shown that Taq can in fact incorporate and extend beyond four consecutive C-

glycosides, we next tested its ability to incorporate and extend beyond up to twelve consecutive

dyT-dA or dyU-dA base pairs. Primer extension experiments were performed under optimal










polymerase conditions using templates T-1 through T-12. Based on the results of the study (Fig.

2-7[A-B]), Taq polymerase will not readily incorporate and extend beyond more than five

consecutive C-glycosides to generate FLP. If this polymerase is to be used as a potential

candidate for an AEGIS system, it must be modified, possibly by directed evolution experiments,

so that it can incorporate more of these non-standard bases.


































220 260 300
Wavelength (nm)


Figure 2-1. A schematic representation of the CD spectra of A- and B-DNA forms. The dotted
line indicates the position of the absorption maxima (adapted from Ivanov et al.,
1973 (Ivanov et al., 1973)).




















Ro minor groove o-s

T A

mqjor groove n






ROO OR
o ~minor groove


RO O
yT A


mqjor groove n






ROO OR
o_ minor groove

RO O
VTU A

Figure 2-2. The base pairing interactions between a standard A-T base pair and the non-standard
yT-A and yU-A base pairs. Note the C-glycosidic bond (shown in blue) between
the base and the sugar in both yT and yU.





Oligo Sequence (5'-3' Direction)


Purification


:AG AGA CGlly CIA 1AG
:GG ACG Allpy CTA TAG
:GG CGA Ilnly CTA TAG
;GC GAlly lnly CTA TAG
;CG Allpy Ilnly CTA TAG
:GA AAI nliqAJI CTA TA(


~The yr represent the incorporation of a pseudouridine residue.


Table 2-1. Oligonucleotides used in this study.


























20* 12 A+1 T6 -*-1 A+1 psedo







Wavelength (nm)
*- 3A+3 T -*- 3A+ pseudoU














Wavelength (nm)
~-*-A+6T-*-6A+6 pseudoU


Waveleneth (nm)
t9A+9 T -*-9A+9pseudoU















--12A+12T--12A+12 pseudoU


Figure 2-3.


Representative CD Spectra. Circular dichroism spectra of select double stranded
templates with their complements containing varying amounts of dA-dT or dA-dyU
base pairs at 25 oC. All of the spectra above are indicative of B-DNA (Ivanov et al.,
1973). Note that the conformation does not dramatically change as the amount of
yU is increased. (A) The spectra of duplexes containing 1 dA-dT base pair vs. 1
dA-dyU base pair. (B) The spectra of duplexes containing 3 dA-dT base pairs vs. 3
dA-dyU base pairs. (C) The spectra of duplexes containing 6 dA-dT base pairs vs. 6
dA-dyU base pairs. (D) The spectra of duplexes containing 9 dA-dT base pairs vs.
9 dA-dyU base pairs. (E) The spectra of duplexes containing 12 dA-dT base pairs
vs. 12 dA-dyU base pairs.











TTrP, dyCITTP
or dyrUTP



7P .Primer P-1l


STo plateT-4 A AA AC CT GT GT C G-


13-base exten sion
FLP IdNTPs,
d~yTNTPs, or
dyU]NTPs are
present)
4-base extension FLP
(only TTP, dqiTTP, or
dyrUTP is present)


Figure 2-4. Depiction of primer-extension assays used in the polymerase screen. In the 4-base
extension assays, polymerases were challenged to incorporate up to four consecutive
dT, dyT, or dyU residues across from template dA. In the 13-base extension
assays, the polymerases were forced to incorporate and extend beyond those first
four residues.




























IC
N br

20 bp I
~6;~ PP~ d ~ C t B P P a p~ a a b~
8T 2 CC
CCC ~CCCCC
P~LCCI. C~~ C~
05
2 r 322 2
-E ~05 P+ .t
"~' 3 3
na o
o-,
IS
a
i:


Klenow exo- Bot Taq Tth
Polymerase Polymerase Polymerase Polymerase


B) 3 Klenow exo- But Taq Tth
B) Polymerase Polymerase; Polymerase Polymerase


30 bp *


N+13


30 bp
a
)1 rr
N+a CI r r


srarlr


lZ
N+*


~L~LIC

11441
III~~~~


ZI
II


Figure 2-5.


Family A polymerase screen. Unextended primer is at position N; N+4 is the full-

length product (FLP) for the 4-base extension assays; N+13 is the FLP for the 13-
base extension assays. Final concentrations: TTPs/dyTTPs/dyUTPs/dNTPs/

dyTNTPs/dyUNTPs (100 CIM), radiolabeled P-1 (2.5 pmol), non-radiolabeled P-1
(20 pmol), non-radiolabeled template T-4 (30 pmol), and appropriate polymerase (1
U). The mixtures were prewarmed to the polymerase's optimal temperature for 30 s
and initiated with the appropriate NTP mixture. The mixtures were incubated at the

polymerase's optimal temperature for 2 min and immediately terminated with DNA
PAGE Loading Dye (formamide, EDTA, and dyes). An aliquot (1 CIL) was loaded
onto denaturing polyacrylamide gels (20%, 7 M urea) and resolved. A) The

incorporation and extension of dT and dyT by various Family A polymerases. All

polymerases were able to incorporate and extend beyond the four consecutive A-T
or A-yT base pairs to generate some FLP in both the 4-base and 13-base extension

assays. Klenow (exo-) and Bst most likely generated higher amounts of yT
containing FLP since their optimal temperatures are lower than that of Taq and Tth.

B) The incorporation and extension of dT and dyU by various Family A
polymerases. All polymerases were able to incorporate and extend beyond the four
consecutive A-T or A-yU base pairs to generate some FLP in both the 4-base and
13-base extension assays. Klenow (exo-) and Bst most likely generated higher

amounts of yU containing FLP since their optimal temperatures are lower than that
of Taq and Tth.


~~- ~ *L
1 "+ I ~i*e ~,
it
*Q,.. 4
9*r1~6(1 i 4L
r
*
r


'"^"pggap~~~Dg$~~
PP
IX
P+ C3~~C3 3~~ 3~
PiC 5 ~5:5: piC
E a ~o 3 a B 3 a ~ 3
o,
'E
g
a
ii:












Whnt exo- Deep Vent exo Pfu exo- Therrninator
Polymerase Polymerase Polymerase Polymerase B)


Wnt exo- Deep Vnt exo- Pfu exo- Therminator
Polymeras Polymerase Polymerase Polymerase


A)
N+18 ""


N+~18


i


1
IIC


N+19


It
90 bp Ir.


8
3Pbp 11


ic

rlr


N~ ~~cs~~rr~-

rIIiel)iclCIII

#
2U ~p 1L


N*4 Ill*"~LCLllnr*~ 1
eL Icr *C ~r, cll
PI ." CC I* ; *L ~r, lli bjlr
cc ~ ~r*lrrl~ra~
N ~r~ rrr II *P 1 4 1* *L ~ ~ ~~


2~ bp ~
1
EfiZ~~p~
2; bt
Zi','=~

op
~1
d


Figure 2-6.


Family B polymerase screen. Unextended primer is at position N; N+4 is the full-
length product (FLP) for the 4-base extension assays; N+13 is the FLP for the 13-
base extension assays. Final concentrations: TTPs/dyTTPs/dyUTPs/dNTPs/

dyTNTPs/dyUNTPs (100 CIM), radiolabeled P-1 (2.5 pmol), non-radiolabeled P-1
(20 pmol), non-radiolabeled template T-4 (30 pmol), and appropriate polymerase (1
U). The mixtures were prewarmed to the polymerase's optimal temperature for 30 s
and initiated with the appropriate triphosphate mixture. The mixtures were
incubated at the polymerase's optimal temperature for 2 min and immediately
terminated with DNA PAGE Loading Dye (formamide, EDTA, and dyes). An

aliquot (1 CL) was loaded onto denaturing polyacrylamide gels (20%, 7 M urea) and
resolved. A) The incorporation and extension of dT and dyT by various Family B
polymerases. All polymerases, except Pfu (exo-), were able to incorporate and
extend beyond the four consecutive A-T or A-yT base pairs to generate some FLP
in both the 4-base and 13-base extension assays. Pfu (exo-) was able to generate
FLP in the 4-base assay, but not the 13-base assay. Therminator was extremely

adept at incorporating the dyT residues, as depicted by the low levels of unextended
primer remaining in those lanes. B) The incorporation and extension of dT and dyU
by various Family B polymerases. All polymerases, except Pfu (exo-), were able to
incorporate and extend beyond the four consecutive A-T or A-yT base pairs to
generate some FLP in both the 4-base and 13-base extension assays. Pfu (exo-) was
able to generate FLP in the 4-base assay, but not the 13-base assay. Therminator
was extremely adept at incorporating the dyU residues, as depicted by the low levels
of unextended primer remaining in those lanes.


~~npp~g~~gg~~pp~$a
eZ..z t ~te E;;
+ I--Cc cce
E rr B -d ~Z'E'' c
~t"
,~ B B rr B
a

p


'LCDPB~~~LL
9~, cc21 c ~ c c
2 3 22 ,2r +22
3 TI 'D 3 3 3 3
3 +
rr











A) 40 bp





+1 12 I 12~LO~
Inoprtino t 2Icrprro a o1
CosctveTParos Cnectv pTPare
frm eplt AfrmTepat


B) 40 bp
N+14
N+13

30 op

N+4


e


* .


Figure 2-7.


Incorporation of one to twelve consecutive dT, dyT, or dyU residues by Taq
polymerase. Unextended primer is at position N; FLP is denoted by N+13 in all of
these assays (see Table 2-1 for oligonucleotides used). Final concentrations:
dNTPs/dyTNTPs/dyUNTPs (100 CIM), radiolabeled P-1 (2.5 pmol), non-
radiolabeled P-1 (20 pmol), non-radiolabeled templates T-1 through T-12 (30 pmol),
and Taq polymerase (1 U). The mixtures were prewarmed to 72 OC for 30 s and
initiated with the appropriate NTP mixture. The mixtures were incubated at 72 OC
for 2 min and immediately terminated with DNA PAGE Loading Dye (formamide,
EDTA, and dyes). An aliquot (1 CIL) was loaded onto denaturing polyacrylamide
gels (20%, 7 M urea) and resolved. A) The incorporation and extension of 1 to 12
dT or dyT residues across from template A by Taq polymerase. It appears that very
little to no FLP is generated after the incorporation of five or more consecutive
dyTs. B) The incorporation and extension of 1 to 12 dT or dyU residues across
from template A by Taq polymerase. It appears that very little to no FLP is
generated after the incorporation of five or more consecutive dyUs.


bp

r Incorporation of 1 to 12 Incorporati~on of 1 to 12
-1 4 Consecutive TTP across consecutive dyUTP
I rom Template A across from Template A









CHAPTER 3
CREATION OF A RATIONALLY DESIGNED MUTAGENIC LIBRARY AND SELECTION
OF THERMOSTABLE POLYMERASES USING WATER-IN-OIL EMULSIONS

Introduction

To create synthetic biology using an artificially expanded genetic information system

(AEGIS), a polymerase that is capable of incorporating non-standard nucleotides (NSBs) is

needed. Unfortunately, studies have not found an extant thermostable polymerase able to

incorporate a variety of NSBs with efficiency and fidelity. Polymerases usually perform more

efficiently with one type ofNSB, than they do with another (Hendrickson et al., 2004, Leal et al.,

2006, Roychowdhury et al., 2004).

Directed evolution may help to rectify this situation and allow us to mutate an existing

polymerase to generate one with an increased ability to incorporate a variety of NSBs (Ghadessy

et al., 2001, Ghadessy et al., 2004). Therefore, we became interested in directed evolution as a

way to modify Taq polymerase to better incorporate NSBs, specifically ones exhibiting a C-

glycosidic linkage.

Taq polymerase, a member of the Family A polymerases, has already been successfully

evolved under direction to incorporate various other NSBs using directed evolution (Ghadessy et

al., 2001, Ghadessy et al., 2004, Fa et al., 2004). Ghadessy et at. provided a procedure for doing

so using water droplets in oil (Ghadessy et al., 2004, Ghadessy et al., 2001); these served as

artificial cells. They began with large, diverse random libraries of the Taxq polymerase, with

approximately 7 amino acid residue replacements. Ghadessy et at. found that three to four

rounds of selection was sufficient to identify a polymerase able to incorporate various NSBs

using these random libraries.

This result was initially surprising, as Guo et at. has shown that approximately one-third of

all random multiple amino acid changes will result in the inactivation of a protein, and that 70%









of random changes in the active site of a polymerase will also result in inactivation (Guo et al.,

2004). This implies that a protein having more than a few random amino acid changes has a

high likelihood of being inactive. One might have expected that a very large fraction of the

variants created by Ghadessy et al. would have been inactive, especially at high temperatures,

and this expectation is consistent with results reported below.

This raises a general question: What is the likelihood that a library contains a protein

having a novel but desirable property? A desirable library for directed would optimally have a

large, diverse number of proteins with a high number of active clones (Hibbert and Dalby, 2005).

One approach to achieving this goal involves the selection of sites to introduce replacements.

For example, if replacements throughout the protein are equally likely to lower thermal stability,

while replacements in sites near the active site are more likely to change catalytic behavior, it

makes sense to focus randomization in residues near the active site (Arnold and Georgiou,

2003b, Arnold and Georgiou, 2003a, Fa et al., 2004, Miller et al., 2006, Ghadessy et al., 2004,

Ghadessy et al., 2001).

An alternative approach recognizes that natural history has already explored polymerase

"sequence space." Much of this natural history is available to us in genomic sequence databases.

This permits an approach, originally called "evolutionary guidance," that extracts information

from that history to identify sites that are more likely to influence behavior in a way that is

desired, and less likely to damage the enzyme (Allemann et al., 1991, Presnell and Benner,

1988).

Eric Gaucher, at the Foundation for Applied Molecular Evolution (FfAME), recently

developed this approach a step further under the reconstructing evolutionary adaptive paths

(REAP) rubric (Gaucher, 2006). He identified sites where functional divergence occurred within









a family of polymerases, but where natural history suggested that the site was under strong

selective pressure. In theory, this has the highest probability to generate new activities and

functions.

Using the sites identified by the REAP approach, the Type II sequence divergence of the

Family A polymerases was studied (Gu, 2002, Gu, 1999). In this approach, sites were identified

that had a split "conserved but different" pattern of historical evolutionary variation, and had

been previously suggested to lead to a change in the function or behavior of the polymerase.

Using Pfam (Fig. 3-2), a total of 57 amino acid changes across 35 sites within the 719 members

of Family A polymerases that were available were identified (Bateman, 2006, Finn et al., 2006).

The 35 sites for mutational studies, distributed as seen in Figure 3-2, were derived from these

analyses, and from sequences discussed in a recent review by Henry and Romesberg on the

evolution of novel polymerase activities (Henry and Romesberg, 2005). The 57 replacement

amino acid residues were selected based on the Family A viral polymerase sequences at the 35

mutational sites. The viral sequences were exploited since literature has told us that viral

polymerases are more adept at incorporating NSBs than other polymerases (Sismour et al., 2004,

Leal et al., 2006, Horlacher et al., 1995), and ancient viruses have also been implicated in the

origins of cellular DNA replication machinery (Forterre, 2006).

The company DNA 2.0 created and synthesized the rationally designed (RD) library

containing 74 different mutants using the 57 amino acid changes identified by REAP, in various

combinations to yield three or four amino acid mutations per sequence. In addition to creating

the library, DNA 2.0 also designed and generated a version of the wt taq polymerase gene that

was optimized for codon usage in E. coli cells (co-Taq polymerase). The optimization of codon

usage results in higher expression levels of the protein within the cell (Gustafsson et al., 2004).









Each of these 75 polymerases (co-Taq and the 74 mutants) were tested for their ability to

incorporate increasing concentrations of a representative C-glycoside (Fig. 2-3), 2'-

deoxypesuouridine-5'triphosphate (dyUTP). None were able to incorporate dyUTP more

efficiently than the co-Taq polymerase, and only eighteen of the 74 mutants of the RD Library

showed activity with the canonical dNTPs under the conditions with which they were presented.

Selections require that some members of the library perform differently than the original

protein of interest (Arnold and Georgiou, 2003a, Lutz and Patrick, 2004). We did not perform a

selection to identify a polymerase with an increased ability to incorporate dyUTP, since we

determined there were no clones in the RD Library that functioned with the NSB better than co-

Taq polymerase. In order to demonstrate our laboratories ability to perform in vitro selections,

we decided select for the eighteen mutant polymerases that exhibited activity with dNTPs from

the pool of 74 mutants.

To perform our selection experiments, we used a variation of the compartmentalized self-

replication (CSR) method developed in the laboratories of Griffiths and Holliger to create water-

in-oil emulsions as a way to link genotype to phenotype (Miller et al., 2006, Tawfik and

Griffiths, 1998, Ghadessy et al., 2001, Ghadessy et al., 2004). This method (Fig. 1-13) uses cells

expressing the polymerase as the sole source of polymerase and plasmid template in a PCR

reaction, which takes place inside the aqueous phase of the emulsion. Inactive polymerases fail

to replicate their encoding gene, so they are effectively selected against after the extraction of

products from the emulsion.

After our selection, products were recloned into the expression vector using a version of

the megaprimer PCR method (Miyazaki and Takenouchi, 2002). As this protocol generated

products that were crossover mutations, sequencing of the products provided a list of the









mutations that survived the selection, without providing information about which mutations were

associated with each other. The megaprimer PCR is, nevertheless, an effective method for

library rediversification between rounds of selection.

Materials and Methods

DNA Sequencing and Analysis

DNA sequencing was carried out by the University of Florida Interdisciplinary Center for

Biotechnology Research, DNA Sequencing Core Facility using an ABI 3 130Oxl Genetic Analyzer

(Applied Biosystems, Foster City, California) and primers P-6 through P-9 (Table 3-1). BLAST

2 software was used for sequence similarity searching (Tatusova and Madden, 1999); Derti's

Reverse and/or complement DNA sequences website was used to find the reverse complement of

various DNA strands (Derti, 2003); and ExPASy's translate tool was used to translate DNA

sequences into their amino acid counterparts (Swiss Institute of Bioinformatics, 1999).

Construction of Plasmids

Construction of pSW1

The gene (wt taq) encoding wt Taq polymerase was cloned from a vector generously

donated by Dr. Michael Thompson (UNC, Chapel Hill, North Carolina) using primers P-2 and P-

3. The product was digested with the SacII and Ncol restriction enzymes (New England

BioLabs, Beverly, Massachusetts) according to manufacturer' s protocol. The restricted wt taq

was then ligated into the identically digested pASK-IBA43plus vector (IBA GmbH, St. Louis,

Missouri)(Fig. 3-3), using T4 DNA ligase (New England BioLabs) according to manufacturer' s

protocol (16 oC overnight with a 4: 1 insert:vector ratio) to make the new plasmid pSW1 (Fig. 3-

4), and adding an N-terminal hexahistidine tag onto the wt taq gene (His(6)-wt Taq). Plasmid

constructs were verified by restriction digest analysis, using the enzymes BamnHI and Ncol

according to the manufacturer' s protocol (New England BioLabs), as well as sequencing.









Rationally designed mutagenic library (RD Library) creation

DNA 2.0 (Menlo Park, California) synthesized a variant of the wt taq polymerase gene

(co-taq) that was optimized for the codon-usage ofE. coli, which was then used to construct the

pSW2 plasmid (Fig. 3-5). Plasmids pSW3 pSW76 (Table 3-2) were designed by Dr. Eric

Gaucher (Foundation for Applied Molecular Evolution, Gainesville, Florida) and DNA 2.0 using

the REAP approach. Sequence alignments and phylogenetic tree construction of 719 Family A

polymerase protein sequences were generated using the Pfam website (Bateman, 2006, Finn et

al., 2006). Type II functional divergence between the bacterial/eukaryotic Family A

polymerases and the viral Family A polymerases was estimated with DIVERGE 2.0 software

(Gu, 2002, Gu, 1999). The 35 sites for mutational studies were derived from these analyses, as

well as sequences discussed in Henry and Romesberg (Henry and Romesberg, 2005); the

replacement amino acid residues were selected based on the viral sequences at those sites. The

sites chosen are all located in or near the active site of the polymerase.

DNA 2.0 randomized the mutations throughout the 74 sequences so they were equally

distributed (3 to 4 amino acid changes per gene). In addition to the synthesis of the genes, DNA

2.0 cloned all 75 of these plasmids (co-taq and 74 mutants) into the pASK-IBA43plus vector

using the SacII and Ncol restriction sites. Plasmid constructs were verified both by restriction

digest analysis, using the enzymes BamHI and Ncol according to the manufacturer's protocol

(New England BioLabs), and by sequencing.

Growth Curves and Cell Counts

The bacterial strains used in this study are listed in Table 3-3. The rich media used in these

studies was Luria-Bertani (LB) medium (Difco Laboratories, Detroit, Michigan) (Miller, 1972).

Ampicillin was provided in liquid or solid medium at a final concentration of 100 Clg/mL.

Plasmids were transformed into the E. coli TG-1 cell line according to manufacturer' s protocol









(Zymo Research, Orange, California). Cell growth was determined by measuring optical density

at 550 nm using a SmartSpec Plus Spectrophotometer (Bio-Rad, Hercules, Califomnia).

Anhydrotetracycline (2 mg/mL stock in N,N-dimethylformamide) was used at a final

concentration of final concentration of 0.2 ng/C1L to induce expression.

Inocula for the growth experiments were prepared as follows: bacterial strains were grown

overnight (14.25 hrs) at 37 oC and 250 rpm in LB medium (supplemented with ampicillin, if

applicable) in 14 mL 2059 Falcon Tubes (BD Biosciences, San Jose, Califomnia). Cells (1 mL)

from the 5 mL overnight culture were used to inoculate 100 mL LB or LB-Amp cultures in 500

mL baffled flasks. Cultures were grown at 37 oC and 250 rpm for 8.75 hrs. Cell counts were

measured by performing a dilution series using 10-fold dilutions of the cells in 0.85% NaC1.

Dilutions were plated onto LB plates (supplemented with ampicillin, if applicable), grown

overnight at 37 oC, and colonies were counted the next morning to determine the number of

colony-forming units per milliliter of culture (cfu/mL).

Samples of cells were taken at various time points to determine the levels of protein

expression, before and after induction. 2X SDS-PAGE (62.5 mM, pH 6.8, 25% glycerol, 2%

SDS, 0.01% bromophenol blue, 5% P-mercaptoethanol (Laemmli, 1970))loading dye was added

to the samples, and to 50 U Taq Polymerase (New England BioLabs). Samples were boiled for 8

minutes, then loaded onto a Tris-HCI Ready Gel (7.5%, Bio-Rad) and resolved for 45 min at 200

V. Gels were stained via the Fairbanks Method (Fairbanks et al., 1971).

Purification of His(6-wt Taq Polymerase

The SW3 cell line was grown overnight in 5 mL of LB-Amp broth for 14.25 hr at 37 oC

and 250 rpm in 14 mL 2059 Falcon Tubes (BD Biosciences). Approximately 2 x 10s colony-

forming units (cfu), roughly equal to 500 CIL of a culture with an OD5sonm Of 4.0, were used to









inoculate two 100 mL cultures of LB-Amp in 500 mL baffled flasks. These cultures were grown

at 37 oC and 250 rpm for 3.75 hrs to an approximate OD5sonm Of 1.8, and were then induced by

addition of anhydrotetracycline (0.2 ng/CIL final concentration). The cells were allowed to grow

for an additional 5 hrs to an approximate OD5sonm Of 3.5. Samples of the undinduced and

induced cells were taken and stored at -20 oC for further analysis.

Cultures were then combined and the cells harvested by centrifugation (9000 rpm, 10 min,

4 oC). The SW3 cells were washed in 40 mL of Cell Harvest Buffer (50 mM Tris-HC1, pH 7.9,

50 mM dextrose, 1 mM EDTA, 4 oC) and centrifuged again (8000 rpm, 10 min, 4 oC). The cell

pellet was then resuspended in Cell Lysis Buffer (20 mM Tris-HC1, pH 7.9, 50 mM NaC1, 5 mM

imidazole, 1 mg/mL lysozyme, 5 Clg/mL DNasel, and 10 Clg/mL RNasel) at a concentration of 2

mL/gram of cells.

The cells were gently lysed by rocking (GyroMini Nutating Mixer) at ambient temperature

for 15 min, the proteins were then denatured by heating to 75 oC for 20 min. The lysed cells

were centrifuged (39,000 x g, 10 min, 4 oC) and the cell-free extract (cfe) removed and placed

into a clean tube. The efe was then sonicated with six 10 s bursts at 71% output with a 10 s

cooling periods at 4 oC between each burst (Model 500 Sonic Dismembrator with a 1/2 inch

tapped horn with flat tip, Fisher Scientific, Suwannee, Georgia). The efe was centrifuged

(39,000 x g, 10 min, 4 oC) and the supernatant (cleared efe) was removed.

The cleared efe was added to 1 mL of a 50% Ni-NTA slurry (Qiagen, Valencia, California)

and incubated at 4 oC for 60 min with gentle mixing (GyroMini Nutating Mixer). The lysate-Ni-

NTA mixture was loaded onto a Poly-Prep Column (Bio-Rad, Hercules, California) and allowed

to settle for 10 min at 4 oC. A portion of the flow-through (10 CIL) was then collected and saved

for analysis. The column was washed twice with 4 mL of Ni-NTA Wash Buffer (20 mM Tris-










HC1, pH 7.9), 50 mM NaC1, 60 mM imidazole) and a portion of the flow-through (10 CIL) was

saved for future analysis. The protein was eluted four times (0.5 mL each) with Ni-NTA Elution

Buffer (10 mM Tris-HCL, pH 7.9, 250 mM NaC1, 500 mM imidazole) and portions of each (10

CIL) were saved for future analysis at -20 oC. 2X SDS-PAGE loading dye was added to each of

the samples mentioned above. Samples were prepared, resolved, stained, as described in the

previous section, and the elutions containing the majority of the purified Hi ssc-wlf Taq

polymerase were identified.

Elution fractions 2 4 were combined and loaded into a Slide-A-Lyzer 10K MWCO 0.5 -

3 mL Dialysis Cassette (Pierce, Rockford, Illinois) that was pre-hydrated in Taq Dialysis Buffer

A (50 mM Tris-HC1, pH 8.0, 50 mM KC1, 0.1 mM EDTA, 0.5 mM PMSF, 0.5% Nonidet-P40,

0.5% Triton X-100). The sample was dialyzed at 4 oC for 4 hrs against 500 mL of Dialysis

Buffer A. It was then dialyzed for another 4 hrs at 4 oC against 500 mL of Taq Dialysis Buffer B

(50 mM Tris-HC1, pH 8.0, 50 mM KC1, 0.1 mM EDTA, 0.5 mM PMSF, 0.5% Nonidet-P40,

0.5% Triton X-100, 1 mM DTT). Finally, it was dialyzed for 8 hrs at 4 oC against 1 L of Taq

Storage Buffer (50 mM Tris-HC1, pH 8.0, 50 mM KC1, 1 mM DTT, 0.1 mM EDTA, 0.5 mM

PMSF, 0.5% Nonidet-P40, 0.5% Triton X-100, 1 mM DTT, 50% glycerol). The sample was

removed, quantitated, and the protein concentration determined using the Bio-Rad Protein Assay

Dye Reagent according to manufacturer' s instructions.

The purified His(6)-wt Taq polymerase and Taxq polymerase (New England BioLabs) were

used in separate PCR reactions. The same concentration of each polymerase (enough protein to

equate to 3 U of Taq polymerase from New England BioLabs) were added to PCR reactions

containing: lX Modified ThermoPol Buffer (2 mM Tris-HC1, pH 9, 10 mM KC1, 1 mM

(NH4)2SO4, 2.5 mM MgCl2, 0.2% Tween 20), 250 CIM dNTPs, 1.0 CIM P-4, 1.0 CIM P-5, and 1









ng/CIL pSW1. The PCRs (50 CIL) were run under the following conditions: 5 min, 94 oC; (1 min,

94.0 oC; 1 min, 55.0 oC; 3 min, 72.0 oC)x15 cycles; 7 min, 72.0 oC. Products were analyzed by

agarose gel electrophoresis and quantitated using the Molecular Imager Software (Bio-Rad).

Incorporation of dyUTP by RD Library

2' -deoxypseudouridine-5 '-triphosphate (dyUTP) was purchased from TriLink

BioTechnologies (San Diego, California). Standard deoxynucleotide triphosphates (dNTPs)

were comprised of 2'-deoxyadenosine-5 '-triphosphate (dATP), 2'-deoxycytidine-5' -triphosphate

(dCTP), 2'-deoxyganosine-5' -triphosphate (dGTP), and thymidine-5' -triphosphate (TTP) and

were purchased from Promega Corporation (Madison, Wisconsin). dyUNTPs were comprised

of dATP, dCTP, d GTP, and dyUTP.

Individual cultures (5 mL LB-Amp ) of the SW5 SW78 cell lines were grown for 14.25

hrs at 250 rpm and 37 oC in 14 mL 2059 Falcon Tubes (BD Biosciences). Approximately 2 x

10s colony-forming units (cfu), roughly equal to 500 CIL of a culture with an OD5sonm Of 4.0,

were used to inoculate individual 100 mL cultures of LB-Amp in 500 mL baffled flasks. These

cultures were grown at 37 oC and 250 rpm for 3.75 hrs to an approximate OD5sonm Of 1.8, and

were then induced with anhydrotetracycline. The cells were allowed to grow for 1 hr longer to

an approximate OD5sonm Of 3.0.

Approximately 1 x 106 ofu (~2 CIL cells) were used as the sole source of polymerase and

template in separate PCR reactions containing final concentrations of these constituents: lX

Modified ThermoPol Buffer, 1.4 CIM P-4, 1.4 CIM P-5, 1.1 ng/CIL RNaseA, and 6% DMSO. The

final concentration of nucleotide triphosphates added to the reactions were one of the following:

500 C1M dNTPs; 500 CIM dATP/dGTP/dCTP; 500 CIM dATP/dGTP/dCTP + 450 CIM TTP + 50

CIM dyUTP; 10 C1M dATP/dGTP/dCTP + 400 C1M TTP + 100 CIM dyUTP; 10 CIM









dATP/dGTP/dCTP + 350 CIM TTP + 150 CIM dyUTP; 10 CIM dATP/dGTP/dCTP + 300 CIM

TTP + 200 C1M dyUTP; 10 CIM dATP/dGTP/dCTP + 250 CIM TTP + 250 CIM dyUTP; 10 CIM

dATP/dGTP/dCTP + 200 CIM TTP + 300 CIM dyUTP; 10 CIM dATP/dGTP/dCTP + 150 CIM

TTP + 350 C1M dyUTP; 10 CIM dATP/dGTP/dCTP + 100 CIM TTP + 400 CIM dyUTP; 10 CIM

dATP/dGTP/dCTP + 50 CIM TTP + 450 CIM dyUTP; 500 CIM dyUTPs. The PCRs (50 CIL) were

run under the following conditions: 5 min, 94 oC; (1 min, 94.0 oC; 1 min, 55.0 oC; 3 min, 72.0

oC)x15 cycles; 7 min, 72.0 oC. Products were analyzed by agarose gel electrophoresis and

quantitated using the GeneTools Software, version 3.07 (SynGene, Cambridge, England).

Selection of Thermostable Mutants Using Water-In-Oil Emulsions

Water-in-oil emulsions

The appropriate cell line was grown overnight in LB-Amp broth (5 mL) for 14.25 hr at 37

oC and 250 rpm in 14 mL 2059 Falcon Tubes (BD Biosciences). Approximately 2 x 10s colony-

forming units (cfu), roughly equal to 500 CIL of a culture with an OD5sonm Of 4.0, were used to

inoculate a 100 mL culture of LB-Amp in 500 mL baffled flasks. These cultures were grown at

37 oC and 250 rpm for 3.75 hrs to an approximate OD5sonm Of 1.8, induced with

anhydrotetracycline, and allowed to grow for 1 hr longer to an approximate OD5sonm Of 3.0. The

amount of culture containing 2 x 10s ofu was determined; that amount was centrifuged (13,000

rpm, 2 min), the supernatant removed, and the remaining pellet was stored on ice.

The aqueous phase of the emulsions was prepared by resuspending the cell pellet in a 200

CIL solution containing: lX Modified ThermoPol Buffer, 500 CIM dNTPs, 1.4 C1M P-4, 1.4 CIM P-

5, 1.1 ng/CIL RNaseA, and 6% DMSO. For control reactions, without cells, 1 ng/CIL of pSW2

and 10 U Taxq Polymerase were added to the aqueous phase. Reactions were stored on ice until

further use.










To prepare the oil-phase of the emulsions, Arlacel Pl3 5 (Uniqema, New Castle, Delaware)

was heated to 75 oC, as was mineral oil (Sigma-Aldrich, St. Louis, Missouri). The mineral oil

was mixed with the Arlacel Pl35 (1.5% v/v) in a 5 mL Corning Externally Threaded Cryogenic

Vial (Corning, Acton, Massachusetts) containing an 8 x 3 mm stir bar with pivot ring. The oil-

phase was stirred at 1000 rpm on ice while the 200 CIL aqueous phase was added drop-wise over

a period of 2 minutes. The emulsion was stirred for 5 min longer, then subj ected to PCR [5 min,

94 oC; (1 min, 94.0 oC; 1 min, 55.0 oC; 3 min, 72.0 oC)x15 cycles; 7 min, 72.0 oC].

Products were extracted from the emulsions with the addition of two volumes of water-

saturated ether. The ether and emulsions were mixed by vortexing, centrifuged (5 min, 8000

rpm), and the aqueous phases extracted. To rid the aqueous phases of contaminating enzyme, the

products were subj ected to a QIAquick PCR Purification Kit (Qiagen), and products were eluted

from the column in Qiagen Buffer EB (50 C1L). Products were separated using agarose gel

electrophoresis; the product band was extracted and then purified using a QIAquick Gel

Extraction Kit (Qiagen). Samples were eluted in Qiagen Buffer EB (50 CIL), and product

concentration was determined by measuring absorption at 260 nm.

Re-cloning of selected mutants

The final products of the emulsions were used in an adaptation of the Miyazaki and

Takenouchi megaprimer PCR protocol (Miyazaki and Takenouchi, 2002). CSR products were

digested with Ncol and SacII according to manufacturer' s protocol (New England BioLabs).

Digested samples (10 ng in 1 C1L) were added to a 49 CIL PCR mixture (lX Native Pfu Buffer,

100 ng pSW2, 500 C1M dNTPs, 6% DMSO). Mixture was heated to 96 oC for 30 s prior to the

addition of 0.05 U/C1L Native Pfu Polymerase (Stratagene, La Jolla, California). Samples were









then subjected to PCR [2 min, 96 oC; (30 s, 96.0 oC; 10 min, 68.0 oC)x25 cycles; 30 min, 72.0

oC].

The template strands of DNA (pSW2 plasmid in the PCR) were digested with 2 U DpnI

(New England BioLabs) at 37 oC for 2.5 hrs. Reactions were cooled to room temperature,

purified using a Qiagen PCR Purifieation Kit, and eluted with Qiagen Buffer EB (30 CL).

Purified products were transformed into the E. coli DH500 cell line according to manufacturer's

protocol (Invitrogen, Carlsbad, California). Fifty isolated colonies were selected after the

transformation (cell lines SW79 through SW128). Overnight 5 mL LB-Amp cultures (250 rpm,

37 oC) were grown for each colony, and their plasmids isolated using the QIAprep Spin

Miniprep Kit (Qiagen). Plasmid constructs were verified by restriction digest analysis, using the

enzymes BamnHI and Ncol according to the manufacturer' s protocol (New England BioLabs),

and mutations were determined by sequencing.

Results

Growth Curves and Cell Counts

Growth curves, cfu counts, and protein expression studies were performed on the SW1 -

SW4 cell lines to determine the optimal times for induction (Fig. 3-6[A-C]). The optimal time (1

hr) for induction for both the SW3 and SW4 cell lines was found to be during late log phase at an

optical density of approximately 1.8 at 550 nm. The optimal length of induction was 1 hr, due to

the rapid death of the cells after the induction of the taq gene, as is evidenced by a drop in the

efu/mL counts (Fig. 3-6B). Inductions longer than 1 hr, or induction at early to mid-log phases

caused the cells to perish due to toxicity because of the over-expression of a polymerase in vivo

(data not shown) (Moreno et al., 2005, Andraos et al., 2004). When the migration of the

recombinant Taq polymerases (His(6)-wt Taq and co-Taq) are compared to that of the Taq









Polymerase purchased from New England BioLabs, they all appear to have the same observed

molecular weight of 94 kDa on a Coomassie Blue stained SDS-PAGE (7.5%) gel (Fig. 3-6C).

Purification of His(g)-wt Taq Polymerase

The His(6)-wt Taq polymerase was purified from SW3 cells that were over-expressing the

His(6)-wt taq gene using nickel affinity chromatography. The polymerase was purified to a single

band on a Coomassie Blue stained SDS-PAGE (7.5%) gel (Fig. 3-7A), and elution fractions 2 -

4 were combined and concentrated via dialysis to generate a working stock of His(6)-wt Taq

polymerase. The protein concentration was determined to be 0.744 Clg/CIL, using the Bio-Rad

Protein Assay Dye Reagent. To verify the ability of the purified Hi sgc-wlf Taq polymerase to

amplify DNA in a PCR reaction, similar to that of Taq polymerase (New England BioLabs),

each of these polymerases were used in separate, identical PCRs. The final concentration of

polymerase (5.5 Clg/mL) in each reaction was kept constant. Figure 3-7B shows the products of

these PCRs, and after analysis it was determined that the densities of these two bands were

almost identical.

Incorporation of du/UTP by RD Library

In efforts to find a polymerase that can incorporate and extend beyond dyUs with higher

efficiency than the co-Taq polymerase, each of the mutant Taq polymerases in the RD Library

were tested for their ability to incorporate dyUTP across from template dA in PCR reactions

containing varying ratios of TTP to dyUTP. Reactions contained induced cells as the sole

source of polymerase and template plasmid, so active polymerases were forced to replicate their

own encoding gene (2603 bp).

Figure 3-8[A-B] shows the difference between the PCR products from the co-Taq

polymerase screen (Fig. 3-8A) and a representative (SW21) of the RD Library (Fig. 3-8B). In









both of these reactions, the polymerase could not produce full-length product (FLP) with

concentrations of dyUTP higher than 400 C1M (final concentration). Based on the product band

densities, it was found that none of the active RD Library polymerases displayed a higher

propensity for the incorporation of dyUTP than the co-Taq polymerase (Table 3-4). It was also

noted that only 18 of the 74 mutant polymerases tested showed activity with only dNTPs under

these assay conditions (Table 3-2 and Table 3-4).

Selection and Identification of Thermostable Mutants Using Water-In-Oil Emulsions

We pooled all 74 RD Library strains to perform a selection in water-in-oil emulsions to

isolate those 18 mutants that showed activity. After the products were isolated, they were used

in a modified version of the Miyazaki and Takenouchi megaprimer PCR protocol (Miyazaki and

Takenouchi, 2002), creating the full-length plasmid (pASK-IBA43plus with insert). Purified

products were transformed into the E. coli DH500 cell line; fifty clones were isolated, sequenced,

and compared to the co-Taq amino acid sequence (Table 3-5). Of these fifty clones, 22 showed

no changes relative to the co-Taq sequence, and the remaining 28 had at least one residue

modified. Table 3-6 shows a breakdown of these mutations, and states whether they are random

mutations or RD Library mutations. In the case of the RD Library mutations, it is indicated if

they are true RD Library sequences, RD Library sequences with additional mutations, RD

Library sequences with reversions to the co-Taq sequence, and/or crossovers between two or

more RD Library sequences. In addition, only 5% of the mutations found in these sequences

encode silent mutations (Table 3-6).

As a control, the selection was also performed using only cells expressing the co-Taq

polymerase. Five clones were submitted for sequencing following the megaprimer PCR

protocol. Of these five, four were the correct co-Taq polymerase sequence found in SW4, and









the fifth contained only two amino acid mutations in relation to the co-Taq sequence (data not

shown).

Discussion

Previously, directed evolution experiments have defined mutations that allow Taxq

polymerase, and other Family A polymerases, to be used in different situations; for example, a

few allow for the incorporation of non-standard bases, others are more thermostable, and some

are resistant to inhibitors (Ghadessy et al., 2001, Ghadessy et al., 2004, Henry and Romesberg,

2005). The design of our RD Library was based off mutations discussed in the review by Henry

and Romesberg (Henry and Romesberg, 2005), and were carried out by using the REAP

approach with the Family A polymerases. A library of 74 polymerases was designed, which

contained three to four amino acid mutations per polymerase out of a pool of thirty-five possible

mutations, in an attempt to identify a polymerase with the ability to incorporate non-standard

bases, exhibiting a C-glycosidic linkage, with efficiency and fidelity.

It has been demonstrated previously that the over-expression of a polymerase in a cell can

cause toxicity problems and cause premature cell death (Moreno et al., 2005, Andraos et al.,

2004). To circumvent this problem, the gene encoding His(6)-wt Taxq polymerase was optimized

for codon-usage in E. coli, and cloned into a tightly-regulated plasmid (Skerra, 1994) in an

attempt to express the polymerase at higher levels only after induction. After appropriate

expression conditions were found, the members of the RD Library were individually tested for

their ability to incorporate dyUTP, a representative non-standard nucleotide exhibiting a C-

glycosidic linkage. The polymerases were challenged with increasing concentrations of the

dyUTP as the concentration of TTP presented was decreased. None of the RD Library

polymerases were able to incorporate dyUTP more efficiently than the codon-optimized Taq










sequence. In the future, other possible mutation sites and combinations of mutations may need

to be made and tested to find a polymerase that can accomplish this task. Interestingly, only

eighteen of the 74 mutant polymerases tested showed activity with standard dNTPs under these

assay conditions.

Ideally, a selection would have been performed using the RD Library to identify

polymerases able to incorporate dyUTP with efficiency. Since none were able to incorporate the

NSB more efficiently than the co-Taq polymerase, as evidenced by the densities of the FLP

bands, a selection was performed to identify those polymerases that showed activity with the

dNTPs under these assay conditions. A water-in-oil emulsion system, similar to that Ghadessy et

al. described (Ghadessy et al., 2001), was used as a means to link geneotype to phenotype,

forcing active polymerases to replicate their own genes in a PCR reaction. All 74 cell lines

containing the RD-Library were used in equal proportions to perform such a selection. After

products were extracted from the emulsion system, they were recloned into a plasmid using a

version of the megaprimer PCR (Miyazaki and Takenouchi, 2002).

The megaprimer PCR method was chosen as the method for recombining the polymerase

genes with the plasmid based on its "one pot" approach. After extracting the final products from

the emulsions, all further recloning can take place in one reaction vessel, and undergoes only one

purification step prior to transformation into a cell line. Other methods, using digestions and

ligations, require several purification steps between the various procedures, resulting in low

yields of final product.

After sequencing, it was noted that 22 out of the 50 clones sequenced contained the

original co-Taxq polymerase sequence; 15 carried partial forms of the original RD Library

sequences, and only four were true RD library sequences. The remaining nine sequences were









random mutations most likely created during the PCR in the emulsions. This could be due to the

fact that Taq polymerase has an error rate of approximately 8 x 10-6 mutationall

frequency/bp/duplication) (Cline et al., 1996). It is also noteworthy that two of 50 sequences

(SW119 and SW122) contained frameshift mutations, which tend to occur once every 2.4 x 10-5

base pairs when using Taq polymerase (Tindall and Kunkel, 1988).

Since the plasmid carrying the co-taq gene was only introduced during the megaprimer

PCR, and the plasmid used as template was digested with DpnI, it was determined that during the

course of the megaprimer PCR reaction, recombinations and reversions of the various sequences

most likely occurred during this procedure. This would explain the high number of co-Taq

sequences and the large number that contain various additions, reversions, and crossovers

relative to the original RD Library mutations. This also accounts for the presence of the

numerous co-Taxq polymerase clones identified after sequencing.

Out of the four exact RD library sequences that were recovered, only one coded for a

mutant that was previously shown to have activity in the assay using dyUTP. This could

indicate that the emulsions are breaking, allowing active polymerases to replicate the genes of

inactive polymerases. Further tests could be performed to confirm or deny this conclusion; an

example would be using two different cell lines in an emulsion, one expressing active

polymerase and one expressing inactive polymerase. Identification of the Einal product would

allow us to determine if indeed these emulsions are rupturing. If this is the case, modifications

could be made to the oil phase of the emulsions, such as increasing the percentage of Arlacel

Pl35, to prevent this from occurring.

We have determined that the megaprimer PCR method would be an efficient way of

introducing diversity into a library between rounds of selection, but it is not an effective means









for recloning if trying to identify specific products. Once the stability of the emulsion system is

verified, and the recloning of the CSR products is performed using the standard

digestion/ligation/transformation protocol (Sambrook et al., 1989), it is likely that we will be

able to identify thermostable polymerases using this technique. The next step would be using

this method with a random library, instead of a rationally designed library, to identify

thermostable polymerases and/or polymerases that can incorporate C-glycosides with efficiency

and fidelity. After several rounds of evolution, we may be able to identify a polymerase capable

of functioning with an AEGIS.



























Non-viral Fblymerases






















Viral Polymerases











Figure 3-1. A phylogenetic tree of the Family A polymerases. This tree was generated using
Pfam (Bateman, 2006, Finn et al., 2006), and analyzed for sites that underwent Type
II functional divergence. Appendix B has parts of this tree expanded so that it is
readable.










































Figure 3-2.


Locations of the 3 5 rationally designed (RD) sites in the Taq polymerase structure.
These held the mutations in the RD Library. There were 57 mutations made at these
sites: sites in red were sites where the natural amino acid was replaced by one
different amino acid. Amino acids in blue indicate sites that were replaced by two
different amino acids. Sites in green represent sites where three residues were
substituted for the original amino acid. Image created by Dr. Eric Gaucher using the
PyMOL Molecular Graphic System (DeLano, 2002).















Oligo Sequence (5'-3' Direction) Purification
P-2 GAT GAC CGC GGT ATG CTG CCC CTC Desalted
P-3 CAT TAC AGA CCA TGG TCA CTC CTT GGC GGA G Desalted
P-4 CAA ATG GCT AGC AGA GGA TCG CAT CAC CAT CAC Desalted
P-5 CAG GTC AAG CTT ATT ATT TTT CGA ACT GCG GGT GGC Desalted
P-6 GAG TTA TTT TAC CAC TCC CT Desalted
P-7 CGC AGT AGC GGT AAA CG Desalted
P-8 GAA AAC CGC GCG TAA ACT GC Desalted
P-9 CCT GGA ACA CGC GAA TCA GG Desalted

*All oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, Iowa).


Table 3-1. Oligonucleotides used in this study.













Sac II
Eco RI
Ec113611
Sacl
Acc651


B HI

Accl
Sally
BpIMI
Pstl
NcolI
Eco 4711l


aN in Ill
Prom %Fis Tag
Strep-Tag ivae l
Ngo""MIV


3000
Sfl Crigin


pASK-IBA43plus
2500
3286 bps ,000


2000
Tet-Repressor


1500


Ppu 11


Amp R


Xmn I


Eco NI/
Bpu 101
Nsp


Scal


Fspl
Mun I


Figure 3-3. View of the pASK-IBA43plus plasmid. This plasmid was purchased from IBA
GmbH (St. Louis, Missouri) and it can generate an N-terminal hexahistidine and a
C-terminal Strep-tag". This high copy number plasmid is a tightly controlled
tetracycline expression system conferring ampicillin resistance.














Hincll


Promoter


Spel~
Ndel
Ppul01
Nsil
Bsml
Nrul
SnaBI


sooo
Tet-Repressor


1000




2000


wt taq


4000 5723 bps


Ahdl,
Bmrl

MunI


AmpR


Pvul
Scal


BamHI


fl ori


PshAl


Figure 3-4. View of the pSW1 plasmid. This is a ligation of the pASK-IBA43plus plasmid with
the His(6)-wt taq polymerase gene using the SacII and Ncol restriction sites. This
plasmid generates an N-terminal hexahistidine translated with the His(6)-wt taq gene.
This high copy number plasmid is a tightly controlled tetracycline expression system
conferring ampicillin resistance.


pSW1





















Promoter


Ppul0\
Nsil


.Eco521


sooo
Tet-Repressor


1000




2000


co taq


4000 5723 bps


Ahdl


AmpR


,Ecl1361l
Sacl


fl Origin


Figure 3-5. View of the pSW2 plasmid. This is a ligation of the pASK-IBA43plus plasmid with
the codon-optimized taq polymerase gene using the SacII and Ncol restriction sites.
This plasmid generates an N-terminal hexahistidine translated with the co-taq gene.
This high copy number plasmid is a tightly controlled tetracycline expression system
conferring ampicillin resistance.


pSW2
















Plasmid DNA 2.0 Mutations Present Plasmid DNA 2.0 Mutations Present
Name Gene ID # in RD Taq Library Name Gene ID # in RD Taq Library


pSW14 5351 S573E,D575F,F595V

pSW16 533 S7E,D6 L,E JHH
pSW17 55 404 ,T 11 J4E
pSW18 55 94 ,F 64H,H60

pSW20 359 S1 I, 60E,E 1 I
pSW21 360 A54 ,E 1 I,H60
pSW22 361 S1 I, 59,I 11

pSW24 5364 T 41 65,L6S


pSW31 5371 R5 I,R5 4 ,F 4L pSW68 5421 Q579A,R657D,F664Y,A740R
pSW69 5422 R533I,K537I,A605K,L613I
pS33 5375 S7H,F 64 ,R 43
pSW71 5425 D575T,F664H,E742V,R743A
pSW355377 T541A,F664L,R743A pSW72 5426 A594C,I611E,F664L,A740S
pSW 6 5378 T511V,R533I,D622A pSW7 3 5427 N5 80OS, L613A, A7 40S, R7 43A
pSW375379 A597S,I611E,Y668F
pSW38 5381 Y542E,F595W,L606C pSW75 52 V583K,E612I,L613D,Y668F
pSW76 53 S573E,R584V,A594C,D622S

*The pink cells denote the sequences of polymerases showing activity. The blue cells signify the
sequences of polymerases that lack evidence of activity under these assay conditions. All are derivatives
of the co-taq gene and inserted into the pASK-IBA43plus vector. Mutations were designed by Dr. Eric
Gaucher (Foundation for Applied Molecular Evolution) and were synthesized and assembled by DNA
2.0.


41A,L606P,L61
42E,V583K,A60
,F595W,A605E,
.D575F.T,613A.


Table 3-2. Rationally Designed (RD) Mutant Library.




















Name Strain Genotype IName Strain Gntp


SW1 cobTG-1F'traD36lacl"A~lacZ) MI5 proA B' /supE A~hsdM-morB)5 (rk
mi MorB) thl A(lac-proAB)


E cobr TG-1 SW1/pSW66 (pASK-IBA43+ with co taq mutant 5419, Ap')


E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1


SW1/pASK-IBA43+
SW1/pSW1 (ASK-IBA43+
SW1/pSW2 (pASK-IBA43+
SW1/pSW3 (pASK-IBA43+
SW1/pSW4 (pASK-IBA43+
SW1/pSW5 (pASK-IBA43+
SW1/pSW6 (pASK-IBA43+
SW1/pSW7 (pASK-IBA43+
SW1/pSW8 (pASK-IBA43+
SW1/pSW9 (pASK-IBA43+
SW1/pSW10 (pASK-IBA43
SW1/pSW11(pASK-IBA43
SW1/pSW12 (pASK-IBA43
SW1/pSW13 (pASK-IBA43
SW1/pSW14 (pASK-IBA43
SW1/pSW15 (pASK-IBA43
SW1/pSW16 (pASK-IBA43
SW1/pSW17 (pASK-IBA43
SW1/pSW18 (pASK-IBA43
SW1/pSW19 (pASK-IBA43
SW1/pSW20 (pASK-IBA43
SW1/pSW21(pASK-IBA43
SW1/pSW22 (pASK-IBA43I
SW1/pSW23 (pASK-IBA43I
SW1/pSW24 (pASK-IBA43I
SW1/pSW25 (pASK-IBA43
SW1/pSW26 (pASK-IBA43I
SW1/pSW27 (pASK-IBA43I
SW1/pSW28 (pASK-IBA43I
SW1/pSW29 (pASK-IBA43
SW1/pSW30 (pASK-IBA43
SW1/pSW31 (pASK-IBA43
SW1/pSW32 (pASK-IBA43I
SW1/pSW33 (pASK-IBA43I
SW1/pSW34 (pASK-IBA43
SW1/pSW35 (pASK-IBA43
SW1/pSW36 (pASK-IBA43
SW1/pSW37 (pASK-IBA43I
SW1/pSW38 (pASK-IBA43
SW1/pSW39 (pASK-IBA43
SW1/pSW40 (pASK-IBA43
SW1/pSW41 (pASK-IBA43I
SW1/pSW42 (pASK-IBA43I
SW1/pSW43 (pASK-IBA43I
SW1/pSW44 (pASK-IBA43
SW1/pSW45 (pASK-IBA43I
SW1/pSW46 (pASK-IBA43I
SW1/pSW47 (pASK-IBA43I
SW1/pSW48 (pASK-IBA43
SW1/pSW49 (pASK-IBA43
SW1/pSW50 (pASK-IBA43
SW1/pSW51 (pASK-IBA43I
SW1/pSW52 (pASK-IBA43I
SW1/pSW53 (pASK-IBA43
SW1/pSW54 (pASK-IBA43
SW1/pSW55 (pASK-IBA43I
SW1/pSW56 (pASK-IBA43I
SW1/pSW57 (pASK-IBA43
SW1/pSW58 (pASK-IBA43
SW1/pSW59 (pASK-IBA43
SW1/pSW60 (pASK-IBA43I
SW1/pSW61 (pASK-IBA43I
SW1/pSW62 (pASK-IBA43I
SW1/pSW63 (pASK-IBA43
SW1/pSW64 (pASK-IBA43


E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1
E cobr TG-1


SW1/pSW67 (pASK-IBA43
SW1/pSW68 (pASK-IBA43
SW1/pSW69 (pASK-IBA43
SW1/pSW70 (pASK-IBA434
SW1/pSW71 (pASK-IBA434
SW1/pSW72 (pASK-IBA43
SW1/pSW73 (pASK-IBA43
SW1/pSW74 (pASK-IBA434
SW1/pSW75 (pASK-IBA434
SW1/pSW76 (pASK-IBA434


with co taq mutant 5420, Ap')
with co taq mutant 5421, Ap')
with co taq mutant 5422, Ap')
with co taq mutant 5423, Ap')
with co taq mutant 5425, Ap')
with co taq mutant 5426, Ap')
with co taq mutant 5427, Ap')
with co taq mutant 5428, Ap?
with co taq mutant 5429, Ap')
with co taq mutant 5430, Ap')


with wt taq nsert, Ap')
wi~th co taq Insert, Ap')
with co taq mutant 5339, Ap')
with co taq mutant 5340, Ap')
wi~th co taq mutant 5342, Ap')
wi~th co taq mutant 5343, Ap')
with co taq mutant 5344, Ap)
with co taq mutant 5345, Ap')
with co taq mutant 5346, Ap')
+ wth co taq mutant 5347, Ap')
+ wth co taq mutant 5348, Ap')
+ wth co taq mutant 5349, Ap')
+ with co taq mutant 5350, Ap')
+ with co taq mutant 5351, Ap')
+ wth co taq mutant 5352, Ap')
+ wth co taq mutant 5353, Ap')
+ wth co taq mutant 5356, Ap')
+ with co taq mutant 5357, Ap')
+ wth co taq mutant 5358, Ap')
+ wth co taq mutant 5359, Ap')
+ wth co taq mutant 5360, Ap')
+ with co taq mutant 5361, Ap')
+ with co taq mutant 5363, Ap')
+ with co taq mutant 5364, Ap')
+ wth co taq mutant 5365, Ap')
+ with co taq mutant 5366, Ap')
+ with co taq mutant 5367, Ap')
+ with co taq mutant 5368, Ap')
+ wth co taq mutant 5369, Ap')
+ wth co taq mutant 5370, Ap')
+ wth co taq mutant 5371, Ap')
+ with co taq mutant 5372, Ap')
+ with co taq mutant 5375, Ap')
+ wth co taq mutant 5376, Ap')
+ wth co taq mutant 5377, Ap')
+ wth co taq mutant 5378, Ap')
+ with co taq mutant 5379, Ap')
+ wth co taq mutant 5381, Ap')
+ wth co taq mutant 5382, Ap')
+ wth co taq mutant 5383, Ap')
+ with co taq mutant 5384, Ap')
+ with co taq mutant 5385, Ap')
+ with co taq mutant 5387, Ap')
+ wth co taq mutant 5388, Ap')
+ with co taq mutant 5389, Ap')
+ with co taq mutant 5390, Ap')
+ with co taq mutant 5391, Ap')
+ wth co taq mutant 5393, Ap')
+ wth co taq mutant 5395, Ap')
+ wth co taq mutant 5396, Ap')
+ with co taq mutant 5397, Ap')
+ with co taq mutant 5398, Ap')
+ wth co taq mutant 5399, Ap')
+ wth co taq mutant 5400, Ap')
+ with co taq mutant 5401, Ap')
+ with co taq mutant 5402, Ap')
+ wth co taq mutant 5405, Ap')
+ wth co taq mutant 5408, Ap')
+ wth co taq mutant 5409, Ap')
+ with co taq mutant 5410, Ap')
+ with co taq mutant 5411, Ap')
+ with co taq mutant 5413, Ap')
+ wth co taq mutant 5414, Ap')
+ wth co taq mutant 5417, Ap')


E cobr DH5a SW1/pCSRMut1 (pASK-IBA43+ with co taq CSR mut 1, Apr)
E cobr DH5a SW1/pCSRMu2 (pASK-IBA43+ with co taq CSR mut 2, Ap,)
E cobr DH5a SW1/pCSRMut (pASK-IBA43+ with co taq CSR mut 3, Ap,)
E cobr DH5a SW1/pCSRMut4 (pASK-IBA43+ with co taq CSR mut 4, Ap,)
E cobr DH5a SW1/pCSRMut5 (pASK-IBA43+ with co taq CSR mut 5, Ap,)
E cobr DH5a SW1/pCSRMut6 (pASK-IBA43+ with co taq CSR mut 6, Ap,)
E cobr DH5a SW1/pCSRMut7 (pASK-IBA43+ with co taq CSR mut 7, Ap,)
E cobr DH5a SW1/pCSRMut8 (pASK-IBA43+ with co taq CSR mut 8, Ap,)
E cobr DH5a SW1/pCSRMul9 (pASK-IBA43+ with co taq CSR mut 9, Ap,)
E cobr DH5a SW1/pCSRMutl0 (pASK-IBA43+ with co taq CSR mut 10, Ap,)
E cobr DH5a SW1/pCSRMutl1 (pASK-IBA43+ with co taq CSR mut 11, Ap,)
E cobr DH5a SW1/pCSRMutl2 (pASK-IBA43+ with co taq CSR mut 12, Ap,)
E cobr DH5a SW1/pCSRMutl3 (pASK-IBA43+ with co taq CSR mut 13, Ap,)
E cobr DH5a SW1/pCSRMutl4 (pASK-IBA43+ with co taq CSR mut 14, Ap,)
E cobr DH5a SW1/pCSRMutl5 (pASK-IBA43+ with co taq CSR mut 15, Ap,)
E cobr DH5a SW1/pCSRMutl6 (pASK-IBA43+ with co taq CSR mut 16, Ap,)
E cobr DH5a SW1/pCSRMutl7 (pASK-IBA43+ with co taq CSR mut 17, Ap,)
E cobr DH5a SW1/pCSRMutl8 (pASK-IBA43+ with co taq CSR mut 18, Ap,)
E cobr DH5a SW1/pCSRMutl9 (pASK-IBA43+ with co taq CSR mut 19, Ap,)
E cobr DH5a SW1/pCSRMut20 (pASK-IBA43+ with co taq CSR mut 20, Ap,)
E cobr DH5a SW1/pCSRMut2 1 (pASK-IBA43+ with co taq CSR mut 21, Ap,)
E cobr DH5a SW1/pCSRMut22 (pASK-IBA43+ with co taq CSR mut 22, Ap,)
E cobr DH5a SW1/pCSRMut23 (pASK-IBA43+ with co taq CSR mut 23, Ap,)
E cobr DH5a SW1/pCSRMut24 (pASK-IBA43+ with co taq CSR mut 24, Ap,)
E cobr DH5a SW1/pCSRMut25 (pASK-IBA43+ with co taq CSR mut 25, Ap,)
E cobr DH5a SW1/pCSRMut26 (pASK-IBA43+ with co taq CSR mut 26, Ap,)
E cobr DH5a SW1/pCSRMut27 (pASK-IBA43+ with co taq CSR mut 27, Ap,)
E cobr DH5a SW1/pCSRMut28 (pASK-IBA43+ with co taq CSR mut 28, Ap,)
E cobr DH5a SW1/pCSRMut29 (pASK-IBA43+ with co taq CSR mut 29, Ap,)
E cobr DH5a SW1/pCSRMut30 (pASK-IBA43+ with co taq CSR mut 30, Ap,)
E cobr DH5a SW1/pCSRMut31 (pASK-IBA43+ with co taq CSR mut 31, Ap,)
E cobr DH5a SW1/pCSRMut32 (pASK-IBA43+ with co taq CSR mut 32, Ap,)
E cobr DH5a SW1/pCSRMut33 (pASK-IBA43+ with co taq CSR mut 33, Ap,)
E cobr DH5a SW1/pCSRMut34 (pASK-IBA43+ with co taq CSR mut 34, Ap,)
E cobr DH5a SW1/pCSRMut35 (pASK-IBA43+ with co taq CSR mut 35, Ap,)
E cobr DH5a SW1/pCSRMut36 (pASK-IBA43+ with co taq CSR mut 36, Ap,)
E cobr DH5a SW1/pCSRMut37 (pASK-IBA43+ with co taq CSR mut 37, Ap,)
E cobr DH5a SW1/pCSRMut38 (pASK-IBA43+ with co taq CSR mut 38, Ap,)
E cobr DH5a SW1/pCSRMut39 (pASK-IBA43+ with co taq CSR mut 39, Ap,)
E cobr DH5a SW1/pCSRMut40 (pASK-IBA43+ with co taq CSR mut 40, Ap,)
E cobr DH5a SW1/pCSRMut41 (pASK-IBA43+ with co taq CSR mut 41, Ap,)
E cobr DH5a SW1/pCSRMut42 (pASK-IBA43+ with co taq CSR mut 42, Ap,)
E cobr DH5a SW1/pCSRMut43 (pASK-IBA43+ with co taq CSR mut 43, Ap,)
E cobr DH5a SW1/pCSRMut44 (pASK-IBA43+ with co taq CSR mut 44, Ap,)
E cobr DH5a SW1/pCSRMut45 (pASK-IBA43+ with co taq CSR mut 45, Ap,)
E cobr DH5a SW1/pCSRMut46 (pASK-IBA43+ with co taq CSR mut 46, Ap,)
E cobr DH5a SW1/pCSRMut47 (pASK-IBA43+ with co taq CSR mut 47, Ap,)
E cobr DH5a SW1/pCSRMut48 (pASK-IBA43+ with co taq CSR mut 48, Ap,)
E cobr DH5a SW1/pCSRMut49 (pASK-IBA43+ with co taq CSR mut 49, Ap,)
E cobr DH5a SW1/pCSRMut50 (pASK-IBA43+ with co taq CSR mut 50, Ap,)
E cobr DH5a SW1/pCSRwt1 (pASK-IBA43+ with co taq wt mut 1, Ap,)
E cobr DH5a SW1/pCSRwt2 (pASK-IBA43+ with co taq wt mut 2, Ap,)
E cobr DH5a SW1/pCSRwt3 (pASK-IBA43+ with co taq wt mut 3, Ap,)
E cobr DH5a SW1/pCSRwt4 (pASK-IBA43+ with co taq wt mut 4, Ap,)
E cobr DH5a SW1/pCSRwt5 (pASK-IBA43+ with co taq wt mut 5, Ap,)
F-80dlac ZAM15 A(lac ZYA-argF) Ul69 rec Al endAl
E o HuhsdR17(rk-, mk+ gal phoA sup E44 hE thf 1 gvr A96 relAl


E cobr TG-1 SW1/pSW65 (pASK-IBA43+ with co taq mutant 5418, Ap')


Table 3-3. Bacterial strains used in this study.

























Time (hr)
-*SW1 SW2 -SW3-U -tnSW3- -eSW4-U -*-SW4-1


Ti me (hr)
SW1 SW2 SW3-U SW3-I SW4-U SW4-I
0 1.36E+07 2.01E+07 6.10E+06 7.95E+06 6.00E+06 7.22E+06
1 5 .40E+07 1.31E+07 1.22E+07 8.73E+06 9.53E+06 1.04E+07
2 1.80E+08 3.04E+07 8.14E+07 5.21E+07 2.65E+07 3.09E+07
3 6.48E+08 8.63E+07 2.87E+08 2.79E+08 3.88E+08 1.90E+08
3.75 1.47E+09 3.22E+08 6.83E+08 4.93E+08 4.09E+08 4.36E+08
4.75 2.73E+09 7.95E+08 1.08E+09 2.36E+08 8.26E+08 5.66E+08
5.75 2.94E+09 1.01E+09 1.08E+09 5.07E+07 1.10E+09 6.29E+08
6.75 6.06E+09 1.39E+09 1.88E+09 1.22E+07 1.53E+09 5.01E+08
7.75 3.08E+09 1.11E+09 1.77E+09 7.39E+06 2.23E+09 7.63E+08
8.75 3.14E+09 1.14E+09 1.55E+09 3.90E+06 2.15E+09 5.69E+08


MWU U 13 14 U E



Rorrair psW RentpS


B)


Colony Counts (cfulmL)


Figure 3-6.


Growth curves, cell counts, and expression of various E. coli TG-1 cell lines. The
SW3 (denoted SW3-I) and SW4 (denoted SW4-I) cell lines were induced after 3.75
hrs with a final concentration of 0.2 ng/CIL anhydrotetracycline. A) Growth curves
of various cell lines. Samples were grown in LB media, cultures SW2 SW4 were
supplemented with ampicillin (100 Clg/mL final concentration), at 250 rpm and 37
OC for 8.75 hrs. B) Colony counts (cfu/mL) of each of the cell lines in part A at the
various time points. Cells were grown on LB or LB-Amp agar overnight at 37 oC.
C) Coomassie Blue stained SDS-PAGE (7.5%) gel showing protein expression of
induced cells at various time points. U stands for uninduced cells, I-1 through I-4
indicate time-points at hours one through four after induction (t = 4.75 through t =
7.75 hrs), and NEB Taq depicts the migration of the 94 kDa Taq polymerase
purchased from New England BioLabs. Since their genetic code has been optimized
for use in E. coli cells, the SW4 strain, containing the co-taq gene, appear to grow to
a higher OD550nm than the SW3 strain containing the His(6)-wt taq gene.











A)
250 kDa
150 kDa

100 kDa .
94 kDa-
75 kDa ag


B)

4000 bp
3000 bp

S.2000 bp
1550 bp
1400 I:-p


50 kDa

37 kDa


Polymerase Polymerase


MW U 1-5 L W-1 W-2 E-1 E-2 E-3 E-4


Figure 3-7.


Purification and activity of His e,,-in't Taq polymerase. A) The purification ofHis(6)-
wt Taq polymerase from SW3 cells after five hours of induction. U uninduced
cells, I-5 cells after 5 hrs of induction, L load from the Ni2+ COlumn, W-1 and W-
2 wash fractions from the column, E-1 through E-4 elution fractions from the
column. Elution fractions 2 4 were combined and subjected to dialysis. B)
Products of PCRs comparing identical concentrations of Taq polymerase (New
England BioLabs) and Hi s e,,-~in' Taq polymerase. The amount of product generated
with each polymerase was almost identical considering the density of the product
band using Taq polymerase was 1980 CNT/mm2 and the density of the product band
using His(6)-wt Taq polymerase was 1925 CNT/mm2













































E E E E E E E E E EU

E2E2E E2E2E E2E2E
D E ,_, ,_,E _,E ,,E ,_ E E ,,E ,_ E


Representative gels showing the amount of full-length PCR products generated with
different dNTP/dyUNTP ratios and the indicated polymerases. Concentrations of
dNTPs/dyUNTPs listed are the starting concentrations (see Materials and Methods
for listing of final concentrations). All PCRs used 1 x 106 CfU Of cells expressing
polymerase as the sole source of polymerase and template plasmid for the reaction.
Polymerases were forced to replicate their own encoding gene (2603 bp). A)
Incorporation of various dNTP/dyUNTP ratios by co-Taq polymerase. FLP is not
generated beyond the ratio of 3 mM TTP/7 mM dyUTP. B) Incorporation of
various dNTP/dyUNTP ratios by a representative of the RD Library (SW21). FLP
is not generated beyond the ratio of 3 mM TTP/7 mM dyUTP.


A)
4000 bp
3000 bp


2000 bp

1550 bp
1400 bp










B)
4000 bp

3000 bp


2000 bp
1550 bp
1400 bp












Figure 3-8.


a F F F F F F F F Fa
z O ~z
UU
Ci U O U C)U 0'3 O U U U O ~ O U O i~J
E E E E r E E E E
U
n~E~E~E~E~E~E~E~ESE
o r00 O
U U ~i U E~ ~3 P U r U ~ ;3 t U ~ ;3 ~ E
E~~~ahahahaha~aha
o TJ ~ TJ ~ rJ ~ rJ ~ rJ ~ TJ ~ n ~ rJ ~ n ~ O
EEEEETEEE
ETETETETETETETETET
EEEEEEEEE
ooooooooo














Raw Densities (CNT/nd2)
Cell
LieSubstitutions Al 9 mM dT/ 8 mM dT/ 7 mMdT/ 6 mM dT/ 5 mM dT/ 4 mM dT/ 3 mM dT/ 2 mM dT/ 1 mM dT/ AHl
dNTPs 1 mM dyU 2 mM dyU 3 mM dyU 4 mM dyU 5 mM dyU 6 mM dyU 7 mM dyU 8 mM dyU 9 mM dyU dyrUNTPs


Table 3-4. Incorporation of dryUTP at 94.0 oC by RD Library.