<%BANNER%>

Molecular Analysis of Two Putative Mediator Subunits in Arabidopsis thaliana

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 E20101113_AAAAQU INGEST_TIME 2010-11-14T00:35:42Z PACKAGE UFE0018461_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 34154 DFID F20101113_AADXYO ORIGIN DEPOSITOR PATH pan_w_Page_30.QC.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
52c6262db74f62725586ccd2d8292dbf
SHA-1
201913ddf49fdb364e020cf8a7953329dbfe5700
25271604 F20101113_AADXOU pan_w_Page_47.tif
9b1844178d6627b45b3303dcda5e5f95
8d78e183e03a9c6ab1c7b0c69ba96d02b160a066
119493 F20101113_AADXJX pan_w_Page_46.jpg
266e40edbb53c925cf615060c55f04c4
5a45afa898257592886133ad62d2bfe1519fb650
375 F20101113_AADXTR pan_w_Page_51.txt
3b5ff714a704785122f8723aa67294be
8269c9ef060373c60c967385c2b8ad225a52e138
7062 F20101113_AADXYP pan_w_Page_28thm.jpg
45c951059c4f009f0b991fd0353fca15
abd28daedaeddccd0e3aa834dfa8951c5c5906cd
F20101113_AADXOV pan_w_Page_48.tif
6a970b159ed548e19d3ab6e67602f450
473f04f97daa68f27d0e7e0471e0cd1f91c416f8
82433 F20101113_AADXJY pan_w_Page_47.jpg
24258a3198e29acd6912ce99c4101d83
fdd105e9a65dd98d4a8e4196aa20b0a7564ce494
351 F20101113_AADXTS pan_w_Page_53.txt
0862fb1e78d3242dfa27cf3c81eeba45
f852ccc3a7933bcb1318f796d9c94460af783f9c
36668 F20101113_AADXYQ pan_w_Page_68.QC.jpg
e649234f8d3d963e178affd275b54024
10cdea05aa3326c4a972fa33667e2b92433fca56
F20101113_AADXOW pan_w_Page_49.tif
68c22889c82c0d3e8e5de11ecf9d46dd
e3794ff43553d26f83ed2f1787e81cc9dce0d87d
10114 F20101113_AADXHA pan_w_Page_50.pro
65134af2bf6c343bd165259e53e0cf8c
0c9cb4946671dbe0d5a002781f2139424ab3af3a
99987 F20101113_AADXJZ pan_w_Page_48.jpg
33c7cfb8cbf75e928a52bc732744a204
4b46cd5c762f29d67993f16185dabba7f7c008cc
519 F20101113_AADXTT pan_w_Page_54.txt
09ac883c850cd46ffa031b7d3e481ecc
ba7656470c688245e6a7de292e2492bf14bde8fd
8260 F20101113_AADXYR pan_w_Page_30thm.jpg
dee89c6e15816024ecb9f2dceb2b3203
1a4a478b1a7dd47c137dcbc39499a0e8ab76a358
F20101113_AADXOX pan_w_Page_50.tif
b710dff585a2e0679994f7cf24930e5a
16c688028ff6b5c751f6d1a0c24df41ba9a10d15
1951 F20101113_AADXHB pan_w_Page_30.txt
63f98e0068d81e272ceedd38c6aa6568
64ef57158f04708cb4ce36923121cbe01fd30bba
4828 F20101113_AADXTU pan_w_Page_55.txt
9545adb90eb32dd134747c35a50d1c89
c6ec9c7ee21af73bca47d4ed438cad45348423e2
109429 F20101113_AADXYS UFE0018461_00001.xml FULL
4814105ce28eb7180d1b0a224755c2fb
cb82edbc7b064acf40df4721e3b70576ab531c9a
F20101113_AADXOY pan_w_Page_51.tif
20af276d6105af314e2aecf0666e3b2f
fe2de0396fcebeda30b63922807eb012e098b940
13753 F20101113_AADXHC pan_w_Page_44.jpg
e3ab7ea0fee66f5519486a4d8149d826
5001a22d5afd7906708a0e6bb23ea2441c5e9247
1783 F20101113_AADXTV pan_w_Page_56.txt
9434656632024616e943901754eae3e7
c731a36223ff099acf15e022dae6d78a47203e6a
33861 F20101113_AADXYT pan_w_Page_08.QC.jpg
e17010d2f4dd345a1e5e7e7c58f8d979
80f747dab0483553a938a92caff16f55b8f4ea92
F20101113_AADXOZ pan_w_Page_52.tif
0d8b5947d9c6c1d75ee2720fded06a78
a28b56fa1b5039e88f630ec9fbf1431c7b6c6835
125029 F20101113_AADXHD pan_w_Page_40.jp2
6fc0089d7d3cfb3b7c6ef1484b65e166
09d2e21f44030b85ef34ebd2f21523823c34d44d
763 F20101113_AADXTW pan_w_Page_57.txt
09ff61a8a8e01222c3abc87eba54323a
edce95ccc1c85f7e68cb120a968c5d865dc36474
120695 F20101113_AADXMA pan_w_Page_37.jp2
4cecb9f5bccf5d72572a49741cc2d3b4
41a5ea62e0d1398511717377cf9279f240ed2904
4483 F20101113_AADXYU pan_w_Page_10thm.jpg
f016ba260f9fe3fa65f53d4793a590ff
0e73ca0e01538c7bba73a6117a4a50e178f1c703
7765 F20101113_AADXHE pan_w_Page_18thm.jpg
c21bf6af68a61c35dd29c427226b7013
9a90ef59389399a5ccdc8ee53d5a89d6119c7f74
2086 F20101113_AADXTX pan_w_Page_58.txt
55b8616db18a624c550054898809b450
4fab6c7088d0400f75aac2182175aef0c061a023
120621 F20101113_AADXMB pan_w_Page_38.jp2
5770e9c9655ca0b0cb19f52447fc2469
fb89d2fc3c24c28f08d1a70d31e6805f22ade3fe
34157 F20101113_AADXYV pan_w_Page_20.QC.jpg
a5ca4191d0bac1448b512de0b9e5f1f1
0b7a42ec6fc65657199c86a290a80bfb4c068679
1713 F20101113_AADXHF pan_w_Page_07thm.jpg
360c4cc46716170e30731ac1b2e4dc80
f8e5b3a3920b47d8d52dd624dc0ef3450f6ba09f
57439 F20101113_AADXRA pan_w_Page_39.pro
7d63a33063fbd40a16843e86cbb08610
01dae955c4e7337da07abd56576c436cd16057b2
171 F20101113_AADXTY pan_w_Page_60.txt
5c99c7dffa5196d6c73b62592a546297
f5940c362e8f29fcc9045c534755fbb29c01a87f
123709 F20101113_AADXMC pan_w_Page_39.jp2
0cf3a287ae49da6031e9541352691a8c
bf1b6ffe83088fe6cd3eec987777408bfb710ab7
36340 F20101113_AADXYW pan_w_Page_21.QC.jpg
c1cfe437dbeefa6297eaa0a784c2d317
966851d5bf0179c6d61d1884da41cb028994d81f
1053954 F20101113_AADXHG pan_w_Page_59.tif
bf773afd7769424cb20ead546e1a74a5
84e035149d7b0c431db7e0cf7fc1107c608f33f2
57693 F20101113_AADXRB pan_w_Page_40.pro
83f3fc14118d8861b482305823a50f55
4ad2fede405af592ea4335c00f0c77e85533ae05
2720 F20101113_AADXTZ pan_w_Page_61.txt
9263009b429fc9817c9a1f9db95bcf1b
b1ed6e151c4fa30a1401927c0264a855ebf7e78b
1051984 F20101113_AADXMD pan_w_Page_41.jp2
b70164c3a84fb9132bcae7a7aa25d410
5091efb6482fb36a2eba923e1f638184725a5e6d
27956 F20101113_AADXYX pan_w_Page_25.QC.jpg
a23a286633cc638dccb938a245ecfa33
90980ee27b806569fd54da7114e440e5bfa43530
118125 F20101113_AADXHH pan_w_Page_35.jp2
10116df9c3579e00b90664c46d1c2564
9c1b60d047af027f399158f31a6ef5e83e3321ce
55988 F20101113_AADXRC pan_w_Page_41.pro
4874b8dd21355aaf81387f082f5f6923
287989a50f1f8fbda116b44ee40fb5f0b52598f3
117119 F20101113_AADXME pan_w_Page_42.jp2
c766597414cac9d59c8f7603e0db01cd
829aa5e028a6c9894671a29704317de8b67fb6f4
8442 F20101113_AADXYY pan_w_Page_31thm.jpg
4c0a136547fd1d6141690079c4493d66
bc8f23a91c4a3d1df479e8f19d06fdb737081df2
5452 F20101113_AADXWA pan_w_Page_27thm.jpg
c0018fd42dc296a06c54da937c4527fb
6d83ae7d9be5fb19921014c1d124256a0fb450a2
23933 F20101113_AADXHI pan_w_Page_01.jpg
1b46088248398c7edbee3f8c75fddfd0
52e073675d4999404b9d487a3490b49321c5426b
117278 F20101113_AADXMF pan_w_Page_43.jp2
1a9a8fce95f8589e97558401fa89140c
0a7c6b4a78cb772e7ca220464dac870eb45c3c89
9386 F20101113_AADXYZ pan_w_Page_33thm.jpg
609b9e4363c6f73c2bb341b6f806ebe2
36462074078a6ae285435a104006352ba5e00fc7
9169 F20101113_AADXWB pan_w_Page_37thm.jpg
f17887d92501a5f2d84e3e9fe7fa8014
5c6338751347220e44711b504d12a4492a73230f
1051971 F20101113_AADXHJ pan_w_Page_46.jp2
4394f149dc97c24d46336b6760136242
9319b41762de411391f7f124cdbbd68e656c1630
51738 F20101113_AADXRD pan_w_Page_42.pro
b6aa986a8a2b677e8ac38fe37bfe63b9
b5a59f45cbcc3447c1aef221b51eb549c87b9e5f
14381 F20101113_AADXMG pan_w_Page_44.jp2
beedade5a3c60678ebbc86a8387f4e57
4a2863ad98257db1335ba48be8e077762e2de704
1872 F20101113_AADXWC pan_w_Page_73thm.jpg
f53f4eec490da90b78b483dba422f500
0e672f3c40f56cc6090433f33278a36ecc4df102
5026 F20101113_AADXHK pan_w_Page_44.pro
be522abb8b763525a6ab46fb33fc8ffb
f7b46df4f3e6810fd9b183b2dca8b52c906ad84b
53805 F20101113_AADXRE pan_w_Page_43.pro
ab652cfc5640fcc1fd29d12186ac3a23
88300f72f343826ecd7824e8f1420a9bf0ea8d21
1051931 F20101113_AADXMH pan_w_Page_47.jp2
c546e73f327ed0ea8f2255b6e0af86f5
21e0608e3e3b426e2df7bfffcd4238e20e891c06
18038 F20101113_AADXWD pan_w_Page_51.QC.jpg
b480632c9878349498230375ea4c6e82
a5defeb513f3ac16edd3a7c96842fa890b86e83a
1520 F20101113_AADXHL pan_w_Page_04.txt
cf8186c37d89b592626d68ed380cdc49
9977f60e52baada808764ce2c9e91b8552e0bfbe
48805 F20101113_AADXRF pan_w_Page_45.pro
0d5dcbeef215f85ba054f2c7f00a065c
5c819b67dedc48c6e62702b66673d5a3e9edf167
1051986 F20101113_AADXMI pan_w_Page_48.jp2
9f72d1dc449bcd5b9cc7f6e338340618
0dc413f1e32f7b912de7f293a5750cc506c0e387
8026 F20101113_AADXWE pan_w_Page_29thm.jpg
b0389c522e86282f2fc1bad332566252
834e97e8679da37e0623e45777774b8f4b162f5e
3508 F20101113_AADXHM pan_w_Page_52thm.jpg
35fbc9e03e831b90dada8f9afda829eb
f1ec4bca3b1ba7890b52d63635955ee8bd6cba19
90191 F20101113_AADXRG pan_w_Page_46.pro
e19f25a8eac100cc8aa243918fa61f4b
411f8a386d362595abec286cdfc1442ed2bf157b
1051981 F20101113_AADXMJ pan_w_Page_49.jp2
7b75d050a434a10affdb38465b35e25d
e46074342eea8de0d8404a864340607802b8a223
37858 F20101113_AADXWF pan_w_Page_33.QC.jpg
46e14b13ec7b8d4d305520fb39ea3060
56649b50d3c46bed528926e022e92cd32caf8c74
67014 F20101113_AADXHN pan_w_Page_66.pro
18f0b48f6a5286c598c27f9d91c5293a
4df148f8b300f3151d318cc124bb3f0c323b711f
15611 F20101113_AADXRH pan_w_Page_47.pro
1c65759b2252a4507fb195a128d33935
e904910a46c9d9c471795e43225bef6a01787a3a
760313 F20101113_AADXMK pan_w_Page_50.jp2
13f9afe1c10f8a1c2c0bf9189a3bc9fe
375355a9e1b55b2fc92be1997375084caae961e6
39934 F20101113_AADXWG pan_w_Page_65.QC.jpg
8f8eeb94ab6e087748a60af594707ea7
b95bfb11d99d8f1486335e3eafc9b6d539a80359
37349 F20101113_AADXHO pan_w_Page_63.QC.jpg
05c2fd95bf5114b1c4be40dde9db5369
6b725390e196d84ed0bd8696aa05ca38a6766534
14572 F20101113_AADXRI pan_w_Page_48.pro
58e5f5fc6a65c8e9f62ec53377eb3409
490dc7a11f41984a9873545833fa7760632db080
826844 F20101113_AADXML pan_w_Page_51.jp2
ce78c699308dd4ef11294554e67f5238
78aa89380599213720ccde99e61ae1a8eac70749
10618 F20101113_AADXWH pan_w_Page_13.QC.jpg
22c67a2f7a15fe0ad48649738ea4bb97
f557c91a558b1af9044b6ba75aad6c932c541b7c
F20101113_AADXHP pan_w_Page_19.tif
0e4932ba54337d08a54640f9e83f9f61
016a594f0ca0494d5889971288a533cc0c5bcdfe
6692 F20101113_AADXRJ pan_w_Page_49.pro
45f79307aa7f890f3e4a638ec4eb4421
ffa7ac3ef0b7d3f957f2d8bee74245a68c622c69
1011072 F20101113_AADXMM pan_w_Page_53.jp2
f6cf3e128a0798ea7b52e6308547bc38
4139f22d2add0fe451d97334200d935e05bbfc7c
79490 F20101113_AADXHQ pan_w_Page_26.jp2
a672a414bd6bdd42732210ca8404ad09
9976d7b235dd49d2b6210b12a8d45cd1f075542f
7556 F20101113_AADXRK pan_w_Page_51.pro
3bbd0e8f81588faef1f8c0c545900966
429b313b68d93e1dd5b2f2367139ae002a9c52e4
1015413 F20101113_AADXMN pan_w_Page_54.jp2
b169c447f30ebeef757e176d44dc70bd
01fc6887bdc0822484b6e1b392fff8d02029dfff
15747 F20101113_AADXWI pan_w_Page_45.QC.jpg
4b26e8178d99e81405848d34d95849ea
25b7ae67ae660515ce9b46956b7f3c9a71db55d5
F20101113_AADXHR pan_w_Page_07.tif
05e72549319f917e25f225b1191707a4
e33500782f8281e43556933e0ba5f445985e9801
7412 F20101113_AADXRL pan_w_Page_52.pro
e66fb79ef110356f60acee55b066e19d
a81832c4499c3a83884dc63f314204f22a4a253a
1051979 F20101113_AADXMO pan_w_Page_55.jp2
48f6d678feb8bd4e0bec24546d4c8c9d
f1e5ff52b5eecd53cd5a8c2025004c2b69bc7038
8355 F20101113_AADXWJ pan_w_Page_14thm.jpg
cac9312f6bd2f753e80b13cb4c88b7c5
8bd1d2161739e4491971889930b6da48393fb044
48599 F20101113_AADXHS pan_w_Page_10.jpg
65550d08a3c691ac984068dbd25d0e1e
2ad740d4910ee83dc2f094c47cc52bee0ae12152
6965 F20101113_AADXRM pan_w_Page_53.pro
786c3b024f01ac845c83f13e16bcd2bc
1500bd4514271f87d9fb9faa414109950a789b72
1033922 F20101113_AADXMP pan_w_Page_56.jp2
8f664b8220d1b20d39d868ca8807123f
160692fe26f7b495fd43b84e350b89b29f1d7a37
35272 F20101113_AADXWK pan_w_Page_42.QC.jpg
7e3aa87ac4ce691d14da6e159e33208a
6fc71807c54356a192132d4561d8d71fc47820f1
10492 F20101113_AADXRN pan_w_Page_54.pro
be4ec7070cdf9630de2a5cad95e5b9c1
cf5e7c640bcf7c02c51fad19dd46320125a7d974
411494 F20101113_AADXMQ pan_w_Page_57.jp2
c93be1e64a4e473eedf5b7c968a40170
c572a66f82a6cefea824b5a6dfb15d13ea8b2522
135014 F20101113_AADXHT pan_w_Page_65.jpg
3da9610abb6291ba0bc60d017ca382f9
70b3639e91a2563348aa9bde00988228c0d5473f
8674 F20101113_AADXWL pan_w_Page_23thm.jpg
ca611ee2273c261685f230c040510478
d5a8cd471ca9dd6b05791066d9c70d480f8de1f4
113103 F20101113_AADXRO pan_w_Page_55.pro
602de9ab9955be737fdb462a644394c5
2ddbaf49fb0d881721f749bd87a40852243eb541
108462 F20101113_AADXMR pan_w_Page_58.jp2
8d82949e03a036b7e293ffb0687ffb42
44c1df9322fedca9247ce312342a7e2f0a2c09f5
43352 F20101113_AADXHU pan_w_Page_18.pro
b35f5b3e9e189e69cf171d414aae6d56
462467e945be2d989cd01467703ef8d29fce3e39
9563 F20101113_AADXWM pan_w_Page_69thm.jpg
270afb5f713c2ce719fbb4a3d42b2877
00dc4b03d9ff2d9440733dff5dfd6acb3eb41714
39676 F20101113_AADXRP pan_w_Page_56.pro
4e8bb91864e8d221b03cf0f341bf636b
383b90c3f4e4384cb7546b23f38da0db97c0fbb8
120539 F20101113_AADXMS pan_w_Page_59.jp2
415ee29fee30ede068aee0ced2c46db3
5f7fb2b3bcbf693d48399d18da468f21c9e79480
68331 F20101113_AADXHV pan_w_Page_67.pro
e32cae450cfa167cdb4ce0ec8630acf9
ddb5688e4671df89705a089537ad678436035c00
4248 F20101113_AADXWN pan_w_Page_06thm.jpg
3ef86e3b04a00afe4df9ef56ed8e420f
f12cb99ad42fb15ea1bf468e25056aa769430baa
16820 F20101113_AADXRQ pan_w_Page_57.pro
4fa64e4ea7ab056abc0c22406d1c1b6c
5230e7e8163b70dc7ef353eee7602982de394738
12726 F20101113_AADXMT pan_w_Page_60.jp2
7e1856bc64da803f2b7ef7661fb8a23a
fccaf70db50d4447f660bfcc79649dde380699de
96873 F20101113_AADXHW pan_w_Page_18.jp2
9ae13e44aebebb1c72e7c36989a9077f
27ed8206c45b55d5435ff862fa899c8aca9aad6a
9993 F20101113_AADXWO pan_w_Page_67thm.jpg
93845bfe852def288a2d9d78b6e87ea2
2918feb0d9c5a6fd5d912bc74d1ee639261d6700
50659 F20101113_AADXRR pan_w_Page_58.pro
d2c94813f7fec47871e002017a6c19c3
bfa80ad1e6b0f517adbef3663c4be77adee8dd87
137915 F20101113_AADXMU pan_w_Page_61.jp2
a7de429e62d5c3c08b9dd34613d69f1e
821356a052aba447fa454d3fbe84fc7c79bfb838
118813 F20101113_AADXHX pan_w_Page_40.jpg
66689181eed3971d13305f7ff61ea657
4fa29577e98d18f2131089f8e474676ae613b32b
614 F20101113_AADXWP pan_w_Page_02thm.jpg
cab0a39572b4f212a62220e4a49f4d29
e6398787040e19a25ee696bee3293b7210b6b55d
54656 F20101113_AADXRS pan_w_Page_59.pro
437483f365198909be0df15c2317086e
9ac0fff227c54fd63b1c539b36387d8c3ef5f5b8
144598 F20101113_AADXMV pan_w_Page_62.jp2
f427eef846ce444985e6eeca371e4e78
e0ad85831c240cde2c531b42fcf74b870028fb7d
38264 F20101113_AADXHY pan_w_Page_37.QC.jpg
2d38d3d933818a1e197367133dc41147
bb92a8b6c766d0eedb3b92049e28593eba3ce269
8324 F20101113_AADXWQ pan_w_Page_17thm.jpg
16f84153e60f947b107d62e4d1fd730c
7c02323d9715c71ea8d5aa6b3584dde969e2f2f5
4220 F20101113_AADXRT pan_w_Page_60.pro
424dbe71078c95fbb2ac09976ff4872c
20f62fa49df4e423634d70280af9efacd6adf990
135770 F20101113_AADXMW pan_w_Page_63.jp2
512009036a94cd7d42ef5091a28eefd8
d6da73075c2742e2107e8e17c1e7c9e6f59aecd8
8621 F20101113_AADXWR pan_w_Page_16thm.jpg
7d464d5e0333aac88940a61465ede268
1ff3becb75e69752ee0a8327d11e650903af45fb
67145 F20101113_AADXRU pan_w_Page_61.pro
c6902f4783d8b839bab5fe60f86efa6a
4012efaa1ccb451620f6468bee9fb38d1820546f
136804 F20101113_AADXMX pan_w_Page_64.jp2
772e180405e4fd1ebd6833679d21de46
4d2d7f056e8ac72682c9e468370b2a4316b39056
35098 F20101113_AADXHZ pan_w_Page_14.QC.jpg
49436d331d6e742bb6848059716516d7
2328423bb27ba75c28b2a03b9f56989bf83696b6
34587 F20101113_AADXWS pan_w_Page_58.QC.jpg
e6518368c6819acf1858a125fc427ddf
5dabbde184bb2a6f081130bcb9cd3525ed1d8935
69780 F20101113_AADXRV pan_w_Page_62.pro
a995ad65c9d8722398795c8183aa55ed
1c1d3088721af64a337a39e7353381f2d0773c57
140822 F20101113_AADXMY pan_w_Page_66.jp2
9200f97c3cee3365c4e6c6f88304cb89
056c63a105c60082469c872cda2abd12cdd2313e
33134 F20101113_AADXWT pan_w_Page_35.QC.jpg
62fb95a84b8d94f6009d501ab9653895
a71f86f5d3484c21441084b06341250f699f9045
64134 F20101113_AADXRW pan_w_Page_63.pro
b4122186aeb1d7d6515c2908a0d6cf6b
f015ab5833bb82f547d0764f093052a5b15a2876
54027 F20101113_AADXKA pan_w_Page_49.jpg
42797b896597cd88a1c4ed095e46ca8b
f21130e76f0f2e76fb0154849e6384d9bed27e01
141701 F20101113_AADXMZ pan_w_Page_67.jp2
8543ccd37768ed416556dcbe55f049ab
da160060a71119528e201bf1df56f42054e16745
8593 F20101113_AADXWU pan_w_Page_36thm.jpg
24dd42d07a91499dd266e2c40ecbfa31
d052a0a879b8f1ced1ab61fee5036a8dfdca5537
65665 F20101113_AADXRX pan_w_Page_64.pro
980595c2622baf2417ad3abe5cb06951
e15d7fbf6039480b44b3aefb694c3e292261d34e
55892 F20101113_AADXKB pan_w_Page_50.jpg
9cb35bfc2856d0dce8be3c3a098f1db5
9779bbf7d0bef26e4db0d50d550c9f10e2523252
39413 F20101113_AADXWV pan_w_Page_69.QC.jpg
638f17bae69f53b7ee519682860bc656
e474ec844f6a4503b1064fe553c9dbacecfe6dc5
67531 F20101113_AADXRY pan_w_Page_65.pro
a981a0ff4f517279732569629de11f22
9f2d2176bededfe5719556fdc6cad8bb67fcacb1
53639 F20101113_AADXKC pan_w_Page_51.jpg
722bae57c8f1e9d51c393df15ba783c5
52052127c6e060c7e75b9878aa7cee8dc0c5a18f
F20101113_AADXPA pan_w_Page_53.tif
907d4acac5be895d2270cb28038d4c9e
f7081b184b349177d496476f9aa54238ca9f4b3c
9949 F20101113_AADXWW pan_w_Page_65thm.jpg
b5a5eda74fbec78979ac758d8c9088cf
1a9b0813e4962228c5173b42522e95707ac5e984
63248 F20101113_AADXRZ pan_w_Page_68.pro
e7bf3cd93da352eeeb71e9528c69811a
2a025336bd4cdd37d0820422e6b92dacbab09a48
59508 F20101113_AADXKD pan_w_Page_53.jpg
2c630aa6158a2bd3461f05bfbdd12756
6002985eecb2b3523d47f3eee46c8dc0a20ec076
7782 F20101113_AADXWX pan_w_Page_35thm.jpg
410d374dc5b2a5281c7e85f70364eb7e
0e3a356c2030ded6907a2f77867f7596d626f0a0
55624 F20101113_AADXKE pan_w_Page_54.jpg
bb059f75385071afed6c716dfeee3352
76f72a94b98dd3ed53e3a066dec93bf3077a0a34
F20101113_AADXPB pan_w_Page_54.tif
0c77c1f3fea4457b7984537777c5a6ab
80f927da113cf78d224346017bbd575ed19da221
1305 F20101113_AADXWY pan_w_Page_44thm.jpg
4c5d56ced726b7b17f3592618be3e7ef
475b38fc2729d116e77dec4979736974cdcb90f4
108120 F20101113_AADXKF pan_w_Page_55.jpg
e77998b44000c71f16ce458217fca64d
f78ccdfd73e3bb1ec8c0423b2efd47be8dd4f9a9
2599 F20101113_AADXUA pan_w_Page_63.txt
bbbb7f144860eca8f67a400e935fb375
d12927a34411429a9edd0df0f7ea56c501a3a21b
F20101113_AADXPC pan_w_Page_55.tif
8414ec87377c909238dd385351d8b088
6a0adfb56b8cd91e7df740e06d8780dd2e20bb5f
34779 F20101113_AADXWZ pan_w_Page_16.QC.jpg
7568c55c89b96bd76323224a9c61be93
b4b934bf5b605bc4674dedcc336d51609706bb0f
94880 F20101113_AADXKG pan_w_Page_56.jpg
1830fc666d9e3cab85805dfd2a6175a7
302411181df62eac33301825da2b9da115cd3fe9
2657 F20101113_AADXUB pan_w_Page_64.txt
7f91483f7824043ae4480281d39a2cd5
5140b4283d68b7d257861aad129b9ac53e7553c7
F20101113_AADXPD pan_w_Page_56.tif
b5a54d12ef911775c914702c80a49976
224020c93ef02ce759166ef94018180494370a03
40552 F20101113_AADXKH pan_w_Page_57.jpg
11d95187287fb3e8638c378c52cac1a1
c5f822d9bcdbc31d04d90657d3e9dcb010b804d4
2735 F20101113_AADXUC pan_w_Page_65.txt
79698581e491f8432d9b6d55471a9b0b
94f65ce61f44ef902c8315432cc3c34f10902db0
F20101113_AADXPE pan_w_Page_57.tif
8afbd58ec86a8df4eeda5f79c0075770
5ac9c0db50ae1f80ac35c8585a9efae5de169236
8613 F20101113_AADXZA pan_w_Page_34thm.jpg
0280c5cb179d63c90792389552745aec
6fe98a44e7d4e7fe9507468292a28e5c145051e7
105007 F20101113_AADXKI pan_w_Page_58.jpg
dd0f8c5b1437ca113e2564d81f8fa6d4
47b298a00771ce2ac55a020fb264b62c2fd7e83b
2706 F20101113_AADXUD pan_w_Page_66.txt
2fdbea8cad771feee07848ec13a2f886
a3f0688a045d0d014c4b6cc0d7c8b0191b6cce93
24219 F20101113_AADXFL pan_w_Page_47.QC.jpg
970f2ab7ddf08a522be8fcf056253709
b0a4ff7ea0d7bdce10f3c803d77c0c0215d75772
F20101113_AADXPF pan_w_Page_58.tif
3a1afb518758697ea0f8f660a0f2df02
0e0515cf0da5f7d7b9834f3d2b059984cfd0e716
35477 F20101113_AADXZB pan_w_Page_36.QC.jpg
1a02096ce265806bb4736589c7d5effa
08e485c81ba51456c3be57b780d960788c6c06a2
112687 F20101113_AADXKJ pan_w_Page_59.jpg
666c6484f2631a758508cee5f9910013
7f946bd196441914f2c623cc405275223debe2eb
2759 F20101113_AADXUE pan_w_Page_67.txt
7d25eb8ef9ed57c8455b7aa50e655475
ad8b9da3c28b1b875636f435a402c336b9b31267
9073 F20101113_AADXFM pan_w_Page_38thm.jpg
247cb62af6673657ca66afec9ff9ca80
7b5b539dd28594730ede69d3cb236b818389b9ae
F20101113_AADXPG pan_w_Page_60.tif
a70b5f54b78da768cebbd5b549243af8
2c7d74bd06761cae179d02c1004d61ea14a6b27a
9226 F20101113_AADXZC pan_w_Page_40thm.jpg
a001281df37006d32b5029899b8fb7bd
957c34ccc24961a11aa57567e9a3efc31c504f03
11601 F20101113_AADXKK pan_w_Page_60.jpg
46ad19ecd90a7b59a773baad19427deb
afc8ddc4ba855bad1841b37cca9ce6a3bc7894ae
2565 F20101113_AADXUF pan_w_Page_68.txt
71f3fa0ecfc953d350b3983273904b22
216cd0571686da9d4b3da06d4b0bd91a3b09d138
2826 F20101113_AADXFN pan_w_Page_62.txt
ab4a1998612c0a546b97d7b335a2bd07
a1bf050369c335e81f585a0e2e169505d97e518c
F20101113_AADXPH pan_w_Page_61.tif
763cf11e1387b3d667d16ccb003f5b18
fb4bf6fff42f79973925d0519d1e0ab3b9540a79
27370 F20101113_AADXZD pan_w_Page_46.QC.jpg
b98e402c1442514b5fdc829f834e0bcd
bc4a1b57233c022ced67c5f676e6eb787653cdc0
141298 F20101113_AADXKL pan_w_Page_61.jpg
7ed6cfea9bf526ad0bb2990e351be1cd
286ede2d723131910ce05a2579a664a157530399
52499 F20101113_AADXFO pan_w_Page_36.pro
6fc8bb0bbe4d27bb535c5e7d2411c74c
701a336acf1550b0d9beff7347c97dd7b901655d
F20101113_AADXPI pan_w_Page_62.tif
1461095523ea380decdf61b47999450e
73d0205ded86236e0baf3bf240cc0ed1253cc4e7
27884 F20101113_AADXZE pan_w_Page_56.QC.jpg
e8014a943d3ea2d055f6d6880868a492
280c1a0380b665dce5c1457119eba3d58d810bdf
139702 F20101113_AADXKM pan_w_Page_62.jpg
84faee7e6ab9021b37a313d135c5ac8a
07818abd4e7fa7ca74b370798dc78dabdc7f68a5
2734 F20101113_AADXUG pan_w_Page_69.txt
4de7b1f563ffa3f6fb3a91de097ef12e
a41ba98156872d0af4e4bd111c8fd6a63b451e5c
46763 F20101113_AADXFP pan_w_Page_30.pro
2baaaddad44edb5898152561442d06a4
863bdaf4774945979fe9ba0c2e8bc80f57c3da86
F20101113_AADXPJ pan_w_Page_63.tif
1959c8dcf6fc5afdc1a5aa01f760a262
6d05635a44e6ae0496144ec1d1b17d48199364f6
8389 F20101113_AADXZF pan_w_Page_58thm.jpg
7af84dc2ead54fae1002d3fbdc948568
2facf2c929134a4f9f0d4eaa2ea69835de3e72c0
127601 F20101113_AADXKN pan_w_Page_63.jpg
1795b4d6b0de2b35f21582649b4e8a64
dac6e161a4237d5a684208585f80a2936858b6f1
2683 F20101113_AADXUH pan_w_Page_70.txt
7d5223f2a2d2d9315c5e196a678159e4
0648f354ec7e98aeb53280cde616c6733999bfc4
347 F20101113_AADXFQ pan_w_Page_52.txt
9cde32e2685ea90458aed77bf87f8d36
decc1f7511e9ebfdb12bbea54617817aad7755db
F20101113_AADXPK pan_w_Page_64.tif
0b0440076d1fb0a8451611e43c4fa549
c371046a61adc184af6c35b0142879b15ae2dba2
9036 F20101113_AADXZG pan_w_Page_59thm.jpg
d4670648efab2f8273d0e60d21aef416
85a48d0ccd7521bad705969b67802f4b5ba466c2
132714 F20101113_AADXKO pan_w_Page_64.jpg
55a0764c9cbe43b52ed0967baf20c9e3
f436277eaea42af1d89a6cdc6c8ce2b52b5ac7df
2651 F20101113_AADXUI pan_w_Page_71.txt
0fac463a6b8c9e1097b3698acfde334e
0042929f880da273c6a8e9450acf65dc1b223fea
5143 F20101113_AADXFR pan_w_Page_44.QC.jpg
724d47051766f6651387b6ce26e3f238
870e7a6b11d4915872dcec5547a36304e04431a8
F20101113_AADXPL pan_w_Page_66.tif
9b744e11d38d501a48dc1267a6c2a9a9
8d1f99cee045534b9b10c76a0139b1e473c1eec7
2802 F20101113_AADXUJ pan_w_Page_72.txt
ee436b2e6caa819cab952fbc1b75d8fa
3f65835801197244c7448990fc312200789b834c
F20101113_AADXFS pan_w_Page_65.tif
876e04f35f0266969f49cd6bd17430d4
f714eb4ef342c7adc8520d07b120af29bd897e4e
F20101113_AADXPM pan_w_Page_67.tif
b8af83133431384faee248d68387b4a1
b3e891b377e8f419e3ce2c8f845d2ff4b7ff96e8
135146 F20101113_AADXKP pan_w_Page_67.jpg
8ebdd1f9978c03013a3734c7b3281740
166fc3e9176f8b771e8d28700f2ccb122a39281b
2313 F20101113_AADXUK pan_w_Page_01thm.jpg
4225a3962b33e380564554d42097a067
a07deddd917cab4a7ffd959bf757681686d95177
23690 F20101113_AADXFT pan_w_Page_73.jpg
1cc042c5711c9a0c51747ccecc9dccd6
32827c72ab17b573c1873d20fefefcc02888f7d2
F20101113_AADXPN pan_w_Page_68.tif
7aafdddcc08e0f3305c38be962571402
f59ab55c6f2ac00b1bc6f08004c1e610ff3aa604
122174 F20101113_AADXKQ pan_w_Page_68.jpg
1c7ac02c7400a703f95a34dcece70f61
2d40584da544494094d956defdc8731793e8e8b2
1972988 F20101113_AADXUL pan_w.pdf
80d2bea9a15d68553787bc7238023d38
40c626174243ff019a7cd4ef19afcb263d48e470
F20101113_AADXFU pan_w_Page_11.tif
8a4c86d9808f2154c4997e57ec511fd4
f3556aa1083e756b1b11384b2ad43744612cbeca
F20101113_AADXPO pan_w_Page_69.tif
f9999052f89f8245d95aed9ace35dde5
be8ba43b2c8b9ecbbd21b8e2bf649890246d0535
136788 F20101113_AADXKR pan_w_Page_69.jpg
a13cc8d42a928db1520dd3db6fef10c0
784fe60665dcc04352bff92dd2238ab1775900c0
3923 F20101113_AADXUM pan_w_Page_45thm.jpg
949e4d1c1d059adbc42060fdfc6aa78b
d53e01f2a554f11f41d8910a59150ec3e482ee26
108459 F20101113_AADXFV pan_w_Page_36.jpg
5777e3d7231425c170d132d95cc896a3
a064425a00d2a28792187b2b8d57706fc249b6ca
F20101113_AADXPP pan_w_Page_70.tif
630f7517df4285eeb67bd80ee3549656
3f50bd04c556d2ce3eab251d8d4fd1f8cb271fc8
140203 F20101113_AADXKS pan_w_Page_70.jpg
ae3c03ebad5331d50f3481b72e1edbe5
39432471af05acd0ca3504052cf38d9e29a9bc5b
5595 F20101113_AADXUN pan_w_Page_07.QC.jpg
64cd356526497da2edd51ae6a3ad37f6
9598064ce2d3f3a31829e2ab1a5d21cfc349773c
67457 F20101113_AADXFW pan_w_Page_69.pro
717317f97d70a42c1022bf2a6d26bcf8
29faded04b5b0186815c9fa0b3696266a9fee499
F20101113_AADXPQ pan_w_Page_71.tif
630724f282a595f10401a41d9076452f
7ebf83f0c60e28d15aebf29defca6232695a2c99
131928 F20101113_AADXKT pan_w_Page_71.jpg
1011f67277eea383786b1bfac440c8b0
f4fc88489fcc11af65c602996c5dbc8e5bbfaeca
39050 F20101113_AADXUO pan_w_Page_67.QC.jpg
eaf8c800e86f31ec9ef6162c4481ffd5
41ac5c30b227b53c410d27e19ce710e8124e8132
F20101113_AADXPR pan_w_Page_72.tif
58891e4faa67bff0a52638eb86268d91
39af56f88cb89f05beccca29af0c90296a9791fd
141886 F20101113_AADXKU pan_w_Page_72.jpg
0c9114f6bd99633bc46c140495b9d837
7a30ed2e7692902fe89acfa309802d8f729a9764
37632 F20101113_AADXUP pan_w_Page_59.QC.jpg
71455cd25008195d4e348faf7512e7a5
c5a7945fd4229130397ff6581db9056e77b5ba8b
33063 F20101113_AADXFX pan_w_Page_52.jpg
9b91e53359084b13e2de63f60c22ea07
167c3f6379f49f1b0837d1547fd1c4fefd7f839f
F20101113_AADXPS pan_w_Page_73.tif
b5d973d643361a0dec6053828971c76d
3098732df134159dec2b541107860e56b970ed4d
22561 F20101113_AADXKV pan_w_Page_01.jp2
be41fbb7301eb24c7b1d01beab1ffef4
b2abacadc10faff2e08bcb822128b11762448504
9410 F20101113_AADXUQ pan_w_Page_39thm.jpg
a0476b80e2072407044a7d44a861bec6
cbe130dc99e0147f60ac275e5a9daf4f61db8a1f
96993 F20101113_AADXFY pan_w_Page_12.jpg
866444faf67a36c556cb27987754aa0c
0386a15a7b88a7ca3b99c26a2207b592a1640e0f
7472 F20101113_AADXPT pan_w_Page_01.pro
789049b1b820ffd2d09ef51730720dc2
04e91ff29410a6bd19bb391b011560cad87133f1
5524 F20101113_AADXKW pan_w_Page_02.jp2
029d6445f47604d5212d0ca64d7b6824
f5c1554d355bad36e7a381288581e08b8971fcf5
39437 F20101113_AADXUR pan_w_Page_39.QC.jpg
8b53fd6ffce3d7802069dfbc758db3ed
2fc5dded4a0b37aae76a719ccdf2ef57dab82da6
2279 F20101113_AADXFZ pan_w_Page_40.txt
7032ae2b04df81f9d6b274a86bd62149
50bb10ad4c40f852505aa417d957b760e6b9f3a8
960 F20101113_AADXPU pan_w_Page_02.pro
6456b57762a450aec46741f87c8b54db
0a382722b95d2f1f5c7764397572a27222005d43
82262 F20101113_AADXKX pan_w_Page_04.jp2
258c33457f0d4379c59e23adf9f276ef
a4ee3ab98f94045d63c40070bfecbade25012b10
8859 F20101113_AADXUS pan_w_Page_42thm.jpg
a678f775a594fc1b05890157b7984ae1
a04ec87c01fde34c4afe2c22736f5937b28d7efd
592 F20101113_AADXPV pan_w_Page_03.pro
ac3ec192a0f599ab89792f9e7aabe60c
594a4dd9dc83bf41c003ccbc5481f07871ad01c2
1051975 F20101113_AADXKY pan_w_Page_05.jp2
bac836957c654ec143081fed512d8039
19ec433ec506b7707a06a0e6184476289f6ed529
34275 F20101113_AADXUT pan_w_Page_34.QC.jpg
d6f012f4db5ce945b37fb5eb87619907
fd33f56370cc45267de4dcb28b986b007eb42ba6
36801 F20101113_AADXPW pan_w_Page_04.pro
ae22d1391089aab97fe467c3763732a8
52ce0a20e5ccf2561f67e21057d470cd8a4ea623
376099 F20101113_AADXIA pan_w_Page_52.jp2
b065d0d350ff21bced842813ea0ad25b
3fca289b9aed397423e02576127dcf6e43cc5aa7
1051929 F20101113_AADXKZ pan_w_Page_06.jp2
248f1ce84d788d5b731eeaf4225f1d26
657ea6f376466aa4397dad9298df96e73dc9e467
39088 F20101113_AADXUU pan_w_Page_40.QC.jpg
ece735d2b5373e0bad6ce7894249eb5f
50d1ae0c2c02597c76e5c1a617a30820cd4e4ee6
77455 F20101113_AADXPX pan_w_Page_05.pro
5e9600d25eb6eacf38059969b50f8052
0082d8a35ab439fc4f5c97233db7f44c8821fb84
515 F20101113_AADXIB pan_w_Page_01.txt
8053d81f0e7ee058ad19e8c6b2de3348
fd980acc0384b9385f2ad0cfdd70cf6a17c1622e
38066 F20101113_AADXUV pan_w_Page_38.QC.jpg
be0668a1499ac8d46f5e1d6cb762342e
7e68594cd6a8076c484262eae945f7f1283fff2d
37850 F20101113_AADXPY pan_w_Page_06.pro
ad802bbb02a7bf572a20f051f03f5604
19f3c669b43ac67cd8c71e4a990b323a082e22a2
139753 F20101113_AADXIC pan_w_Page_65.jp2
d433def6b41e137b2e3a97696261e74f
49174872ac7631a59abb01859707189aa063d518
9369 F20101113_AADXUW pan_w_Page_71thm.jpg
5f707264a645f5fd636d9b92f2e20754
ccff8af408b7fbf5c1ee13b8c6f9e45533802dfd
134068 F20101113_AADXNA pan_w_Page_68.jp2
0b826c628dd5dd52bf811bd1622077fe
6dbb456c8902e08fad0321deddc9e3283dea3bed
6354 F20101113_AADXPZ pan_w_Page_07.pro
5e63311e4d6c4b9c60ee130d1bd3d6c4
4379b78b2c34fcce2d284a2dcc2e05b393df9e20
2092 F20101113_AADXID pan_w_Page_19.txt
ae27350125cf1ce5b4263ec5934236e2
475c2bb65350f9546a7d62353b7f9a1b2cd20564
18101 F20101113_AADXUX pan_w_Page_50.QC.jpg
b7cf19589e9836915ea6c5c15346ab4a
8a1883d36c9a69bc126557cd9587304ba36d288e
137651 F20101113_AADXNB pan_w_Page_69.jp2
a16a9f4810dfe2b8214a9d12eedac76c
97c8f43c6db025d257729b1c7f698c214a80cf88
53223 F20101113_AADXIE pan_w_Page_15.pro
cfa6f3e702c1cc9ead5dede0fa708c12
057bd99b47354def813040bacd87ed20991ae08c
9217 F20101113_AADXUY pan_w_Page_63thm.jpg
8358019c89dc0095323cbc56347915e8
1907ac3371a6cb84469dae5f836d213b75a4e743
140312 F20101113_AADXNC pan_w_Page_70.jp2
01037d42390d1cd3b10d335b847dcf2f
41f397b6484c08d111e9ebde0301db15425cc174
27568 F20101113_AADXIF pan_w_Page_05.QC.jpg
4db708591941375ac0524304f5b309f5
36a3908d542b0f08d063a60814ec4c369dc9b068
66288 F20101113_AADXSA pan_w_Page_70.pro
dfbfe85c3171b2fd6f34e0eb08ee4624
26869b4cbcb6221803602497c08f1b22fcb10cc7
37949 F20101113_AADXUZ pan_w_Page_71.QC.jpg
c7014e8c25a69a3726c65ccaf30f29dd
d72cb83296148e0ebefa2ebad87f73e087e6df79
137956 F20101113_AADXND pan_w_Page_71.jp2
3ee41522ccb4549dc30892fe9f83b372
1f58747cf63283458b7f4ebefc4175afe17d0210
40961 F20101113_AADXIG pan_w_Page_26.pro
f236ee8e783215f4b873ab2ae86dc53f
95ffaba25a9597c318fd0391f20bd3be82000807
65545 F20101113_AADXSB pan_w_Page_71.pro
e429e9ea494fc811a30a159bc9878295
a01aa7c5e4d3b44c800ddb3ab35d0047cd4a65de
144925 F20101113_AADXNE pan_w_Page_72.jp2
10affe3ededb3609c911a2d1778469aa
b784bb4419da13f4a384733e25f87750fcc2c76d
1051951 F20101113_AADXIH pan_w_Page_45.jp2
11faf561bc59fd0fd9b777441e2d7cf8
b241a7383eb05f1aa6ecb2ef7b1541ded5022a15
69451 F20101113_AADXSC pan_w_Page_72.pro
0f5b085b39bcbabd83d810e96986b7f5
6aeaf14cc0ce2524e5acad02c50d678e1438ddc6
25176 F20101113_AADXXA pan_w_Page_28.QC.jpg
c0435db59addbf62f9d2144c489a07c8
7d4b80a295177a49de5e81b8ccf1cd5cd2a0b126
F20101113_AADXNF pan_w_Page_01.tif
87180bac7e450533f054456e49ea96a8
a3d4dd1076dc3cedcfc5feb18308d06a92b2c712
7742 F20101113_AADXII pan_w_Page_56thm.jpg
a31efb316acabc7c899e29e520e398e7
429d0544e78065e229e9fcaa4e2a2c3d17c5f740
9258 F20101113_AADXSD pan_w_Page_73.pro
60157863341238c23f0f970ea042261f
40307432c3199138b3ec96ede87652d8225a6279
6414 F20101113_AADXXB pan_w_Page_04thm.jpg
ae168cd81f237792faa57d6493318112
0e931bc06e703e8f6b0391848b3940fdf1ed686c
F20101113_AADXNG pan_w_Page_02.tif
fafd87b54895b1ebc4970d4b01d2f15a
df66134ad667b74379def0a70d2789593fb1738b
84887 F20101113_AADXIJ UFE0018461_00001.mets
b27e305af402d4d398b18a662c9d9c90
59d159e75dfbd30c955b70f77b6549fada22fb89
37023 F20101113_AADXXC pan_w_Page_43.QC.jpg
3e8fa4d29d9a3a7d816ecf0547dc56d8
ada26da1bd6b9c0e4549888354e0e9fad0c8bd4c
F20101113_AADXNH pan_w_Page_03.tif
8d73d8990f06b63939f89a075b4f3086
6a6603d0b3eb7db64b89eb71e8de14bfbd031541
96 F20101113_AADXSE pan_w_Page_02.txt
c3441e1b21f60623c57c3d47cb19184c
6daf25bd3c0e46f17a43b34b74475d1eacd2c77a
19408 F20101113_AADXXD pan_w_Page_54.QC.jpg
c16d05e3bc4af22616aed88c6221286a
baf46d63f8bdb453cdc55ce7405e8c9ea1a6b60b
F20101113_AADXNI pan_w_Page_04.tif
8fc660f72913692a107011433c337304
4dabeed5e7c5c6081db0b6a659a9cc24178f321a
3416 F20101113_AADXSF pan_w_Page_05.txt
635e0cab8201340e32c51ba29c1c4b50
8a0dae1b99af930b141c64de59946fae7acc962b
4326 F20101113_AADXXE pan_w_Page_60.QC.jpg
08c29d3939c9c198c9ddfb8b8f328714
2a939bd186511e78e621c07ebb4f97fda4eca121
F20101113_AADXNJ pan_w_Page_05.tif
856a80bb1e663222c77bc7d98afc10da
7d23d8bea80e5d0b84f83199567ea1bbb1a068d0
4235 F20101113_AADXIM pan_w_Page_02.jpg
0f26c65c6010490ef6e30797159fda4f
71533df71356931b7d292a7e47f964c852ff103e
1633 F20101113_AADXSG pan_w_Page_06.txt
d81ca8721ee9e56d0d3de98cd5f5bd46
177c3694c985f53f9dbef186b0ed4692190c0e45
23253 F20101113_AADXXF pan_w_Page_26.QC.jpg
a43e1c0d4011d782d90e9e0a01265ad6
3746576e16d4bacb22958dd4fb27690d7340ba1d
F20101113_AADXNK pan_w_Page_06.tif
d9fdda0ad2c94618c8901d07cb338ccb
e0fe9c479a0ca156c5537e0467613acf44b33b67
3210 F20101113_AADXIN pan_w_Page_03.jpg
be29e572e73a264bfca5f83e3d3b9e56
13325b8a8924b23d490cb2beb01c5291010c1834
260 F20101113_AADXSH pan_w_Page_07.txt
4f267e039e94624fa7f68fcc7646d554
f3204267568341cb8931aacfe9e7d483543fc347
31501 F20101113_AADXXG pan_w_Page_48.QC.jpg
3afc6edc2040b60ee146887095fe2f99
3d8652d22a3f349ec2f44b53224719bcf42f8491
126673 F20101113_AADXIO pan_w_Page_05.jpg
69645afda42670921b918c7ed3016709
3dc103d8f21325ec447fe453129a7869ac4c3e48
2403 F20101113_AADXSI pan_w_Page_08.txt
a44fde67ed64d0d50ae383b855b50444
cc27a8b6690fd2c63a5d9ff5625051f29a1dbb27
F20101113_AADXNL pan_w_Page_08.tif
416c2c6cd8d998eefc7eb56e2019b469
d91a22f63022e305f7bbdecd9ee9c2917c8ddd19
37605 F20101113_AADXXH pan_w_Page_32.QC.jpg
3cb8fb982f800cfd4e0404fc8fd33f6d
589060da1f5ac2c912b2891c1ed10b0bb82d1546
67639 F20101113_AADXIP pan_w_Page_06.jpg
96dcf5d0662c5acf0331334a498717b9
98da9ab4945981815b343aa34f2eb01cd4186591
364 F20101113_AADXSJ pan_w_Page_09.txt
3783a486fed63d35a3ec6874a835ee6f
3ff97389f11a1f9613bd5b82564daf9c52c181b3
F20101113_AADXNM pan_w_Page_09.tif
c8a924d534741228602a4d2c696d22e5
7dcecab4b07258dc07f1d81e093e278147a4532b
19553 F20101113_AADXXI pan_w_Page_27.QC.jpg
666098e16832a47f139963b7e4a0c96c
65bef060a454dcb271dde5ffdf2da976e06db555
17703 F20101113_AADXIQ pan_w_Page_07.jpg
65adfb92176fb7af78a62b56c5dcae9e
8b7346ec37dff0cfc1718fa61935f2676bb41189
289 F20101113_AADXSK pan_w_Page_11.txt
2c6ebbce4dc9a8fd3918ad9741048fe3
b9cd91a8c0de1cd464a08df8a02a4ec13f35a03c
F20101113_AADXNN pan_w_Page_10.tif
e7d59df857263d424b6f8c46df816405
a1bb4b2e4319bb89527546faff12c2f7cee2bea3
118250 F20101113_AADXIR pan_w_Page_08.jpg
ab9a915a3551c068e7ff367def9fef93
4ddf9b41a03dc2095fd8cab059a2dd0e378f2e80
2021 F20101113_AADXSL pan_w_Page_12.txt
a3ad02d73515ffc94a1f49840f4ca1d1
766d16c656de8f48a0afbc5f8e08d28ba09cff8c
F20101113_AADXNO pan_w_Page_12.tif
cb3c8108b213bd4fd257228583e1d5ab
f9a63ba11b02bd2f239353819deaebd8ac2f618c
7315 F20101113_AADXXJ pan_w_Page_73.QC.jpg
420c5d02b5b25b15f85b26793e11615d
a84aab7876aa58ba232956a7a85147c6f0c78b56
22287 F20101113_AADXIS pan_w_Page_09.jpg
03f6496b53222ff28842557e60783ff4
43564c0794e5c27a3cb4da463b446464cebdcb51
537 F20101113_AADXSM pan_w_Page_13.txt
77ca3f77ff70e6e79c88e0014fc2bb71
74053ffb2a363b808c394a44daefd97b5b1bcb01
F20101113_AADXNP pan_w_Page_13.tif
545946809315c6b790ed21d41d977002
3fe9a22576d219344aeef9a7f23f0ab22570ebc1
27653 F20101113_AADXXK pan_w_Page_55.QC.jpg
808de3ed0e01670e5c4d439b730c9c5e
7f7e59e642b2c7ad12c051d11e581b7545a83454
17722 F20101113_AADXIT pan_w_Page_11.jpg
d5edd58ccacf31c1d8d3cce783f2901d
72d8c457535bb9b97bd421fff5e84f668a1fa0da
2139 F20101113_AADXSN pan_w_Page_14.txt
2443139bb167f27a5ea0b0c0cb771e26
48f60f63e7f778bcbd200a45d158e81bee1489f9
F20101113_AADXNQ pan_w_Page_14.tif
e1ca0e512ab3fa15ecb547db583d40ad
b612ee4f9c072a24aea50a8124282b019544fa71
16470 F20101113_AADXXL pan_w_Page_10.QC.jpg
6b78245e9cf6aa841773370d593fd3c4
1e9db6689a1f57d09e2ec1e7492ebfe22de33c41
30903 F20101113_AADXIU pan_w_Page_13.jpg
cc9fd5391afa8b00bf8211788b652fe7
de591a7983902221e6c9c42f29b27f782af68ccd
2091 F20101113_AADXSO pan_w_Page_15.txt
b6f3bf5767bfecea4ead821c950b37bf
6a6bb5a15ab00be711a6a8e11f166644f48beccb
F20101113_AADXNR pan_w_Page_16.tif
fe30a5fbaaafb0daca7914887fc337c0
cbb526c7e2f4c83e7e790c008b898b4352ffb9cf
2975 F20101113_AADXXM pan_w_Page_57thm.jpg
a531f0ea91aa9acf1707c79423e61617
7da2b1802cda327c6bfc499b0a9b80e96794e1bb
106872 F20101113_AADXIV pan_w_Page_14.jpg
ae4ebc135fb7e6ff271e9f845c116361
6458fb4d67d345f647dc84dce6d3bf22eb1203ae
2023 F20101113_AADXSP pan_w_Page_17.txt
361c054b224b6e6da42886a5e7a48bc5
4dfd74246769fd3f26f6f834de38d3211e7c34d8
F20101113_AADXNS pan_w_Page_17.tif
85cbd52d756fcd570478d458c5dbb41c
af58b9e6c6cce165fd92606f8e24a570ccc2d03c
8961 F20101113_AADXXN pan_w_Page_43thm.jpg
ef5391cf2a56186c0c864cd0cc3c9450
790ac7d27530f50e867f4b859e3d84d4c54ab513
109142 F20101113_AADXIW pan_w_Page_15.jpg
255c404f29445f0dd851633c87979342
f5f6bd4b9d2bf1de36d6b41e10e4219cf55c6c7b
1735 F20101113_AADXSQ pan_w_Page_18.txt
739a3713e6b77b1699545ba813fd9165
64a827b74e33ea79da1a4cc5cb0fd27782eb7fd3
F20101113_AADXNT pan_w_Page_18.tif
2b864136b391a37f5cc65a77c8aa3e55
20bcc2595c4403d0eaaafc4500ac2ef5f18ce95b
9012 F20101113_AADXXO pan_w_Page_19thm.jpg
2820dfbc024c9ca32f1c71d9a46af38a
46d9fd2a676051d6fc1997bc50c92cf54b5363df
106526 F20101113_AADXIX pan_w_Page_16.jpg
79db9d6d6f877703b591e1d4dabe9643
482ff03a7df540b1cde59779896410ac5ea5b548
1965 F20101113_AADXSR pan_w_Page_20.txt
328639232d83d3915b010649416d98c7
fcf6c1bc968fab3e4efb649465a299385a5095fc
F20101113_AADXNU pan_w_Page_20.tif
d1ee214e41d0efaf624bb44962dcc8b8
2342d546eef5d467218c83fed257010368d39a02
9249 F20101113_AADXXP pan_w_Page_48thm.jpg
a3fedc61d698b6f3b74265e8aad1db1a
90951924f8214acb9f61762660618d333f245b60
105444 F20101113_AADXIY pan_w_Page_17.jpg
3bc135843e5a58e8858fb12d991eb193
4a9d3a912940f77bca1d4a42b455ff167e4d49da
2119 F20101113_AADXSS pan_w_Page_21.txt
a74f712694870f156584f376e0222d17
725433415a163b3ff08912339c108b02085459d4
F20101113_AADXNV pan_w_Page_21.tif
a26a6e86637ad81e087fa4acb833b6fe
737d7bb2988c797cbd2b0297b0c2e4e7a8daccaa
11190 F20101113_AADXXQ pan_w_Page_57.QC.jpg
658c42f09d7a256a41c0ebefa1a1476b
7f3e6495eea996e00f1a08a505ebf0c4f88d8c58
93837 F20101113_AADXIZ pan_w_Page_18.jpg
99a3f92964b14207f1cab831fc78d7cc
e73abd8eb73534e8cbf02b2614f966dad886fe55
2149 F20101113_AADXST pan_w_Page_23.txt
efdbf6e5d91555d7a6d881d55e7a2c23
5f74657b4a50fb129ebe72e88b26aea0b9d2ee45
F20101113_AADXNW pan_w_Page_22.tif
5bccb8dab670ff306af6811c9b084c6b
6591f3c4c7a01ebd57de195148e1f898212adb48
F20101113_AADXGA pan_w_Page_36.tif
5a696ae392f9b67ea52188eb792c8080
6df545498a54331e57a35a7942eef7b9061b7187
1148 F20101113_AADXXR pan_w_Page_60thm.jpg
a429de5e92e80988a52c4f92abb109b3
f5f695c39627c010a36e97e3ca7896943b5deef3
2272 F20101113_AADXSU pan_w_Page_24.txt
8f9910bc7ce432c26ab9073ccc4c877a
b3ab16f883ef3e9b4b0815f701d780c7ce445490
F20101113_AADXNX pan_w_Page_23.tif
46e3c20da6b6622b1327ff9538316d4f
11d58e2e1f466be4110d6515ce68d1c175ef2349
112904 F20101113_AADXGB pan_w_Page_21.jp2
5627c587cb8470b467b7120d82bf0dee
719c65aac0d2d8313041e9d6cb04858434fa59ed
4779 F20101113_AADXXS pan_w_Page_53thm.jpg
eb79d4554605b8e672097c9f9377f06c
bb2ad4193b08d8f420cd48021ce0805faace03a8
1608 F20101113_AADXSV pan_w_Page_25.txt
88567292f208bc163de82ce4eec5ff0f
11cfc39efcb31526b43ad0bf8ac4410263148981
F20101113_AADXNY pan_w_Page_24.tif
18b9bc9ab477398a57c67eb95e2e0ed9
443b73b460f00e4788a7d4278eff1c9fc33756ac
39718 F20101113_AADXGC pan_w_Page_41.QC.jpg
97206edf3664278b7a00cbc0aeae05b6
6720aedf90dece06c257d65b65340bb2b7da64fb
9228 F20101113_AADXXT pan_w_Page_61thm.jpg
992ffa30869ca35c0ce03436ef7b184e
9c6fb1009b809e37a8ce677e6ebc65326115ac7b
1869 F20101113_AADXSW pan_w_Page_26.txt
11914045a31fba5446a765c589feddde
9e79dfa0359c796e403eb35e00639719142e4544
270612 F20101113_AADXLA pan_w_Page_07.jp2
9b6366275cca0698a276f38bd477c35d
79366ec0c6fe11f6899f8aa02ddfa9850d730f84
F20101113_AADXNZ pan_w_Page_25.tif
675e134594b8198f25709dfcc32c30e6
33def7c73508c81cace11565403b51ca417d1177
18974 F20101113_AADXGD pan_w_Page_53.QC.jpg
89538d85614d8112b4b163f0eb31e892
98ddbe47837bd5916d07b84120ed31c41353fa9e
451 F20101113_AADXXU pan_w_Page_03thm.jpg
585e6e457eb0ad2f583db9eb98b0fd55
fa83a260f54ca791dc40035c30e45dc807d69b84
1565 F20101113_AADXSX pan_w_Page_27.txt
c211aadf561ab5636acf85084676d5da
62fbbf4d8cb2eb8c630dc942210050fd334a6ae8
1051953 F20101113_AADXLB pan_w_Page_08.jp2
60e10d711229223c489ee78d634b3531
3b94cd0a3d5636012b9605f9a169032b6d7991f9
2163 F20101113_AADXGE pan_w_Page_59.txt
131992da239dae634e1349ca90ea3dfd
22d7b5d7e02ad43243199799e261459f89c9e681
1467 F20101113_AADXXV pan_w_Page_02.QC.jpg
2074257bee8554752892e079fda31e55
2fb513a507c88a705b2e595104a9d7cc61e0cfc6
58554 F20101113_AADXQA pan_w_Page_08.pro
59ba3f41e05a7906d0bfeb42b304b472
6b7dc613706cf5b13017c74805c251f7280d0384
1729 F20101113_AADXSY pan_w_Page_28.txt
8b68101b683056ca2e17779e7197ff37
c03cc27242b238454e234e41960af0928a032f60
390207 F20101113_AADXLC pan_w_Page_09.jp2
334ebec2e799044904a2119c19793443
897345e490d6a0982fd76b6a32248d00531c8408
113648 F20101113_AADXGF pan_w_Page_33.jpg
a1d0c8e66e766039d37c7d851d496079
24606f212e562ca908b319211c0e41ebaff6d3f3
40318 F20101113_AADXXW pan_w_Page_62.QC.jpg
85c6f4587cb7f6b3d73a0bcdf219b379
a00d00ee7b259e95a00acdef509d78fd1a60957f
8673 F20101113_AADXQB pan_w_Page_09.pro
0ec9667148d371f1459de494334c2fb4
d0494c3881a45200004ad2f78967f91b57e985ec
1896 F20101113_AADXSZ pan_w_Page_29.txt
f2fc17523725b3964eb0f19c414a23ba
e146f5eba37d4f5494acec0a3d3283b5059336b9
51199 F20101113_AADXLD pan_w_Page_10.jp2
bbbc9de947972bd06eae6f2736395f3d
0cf362f162c1383e9bd9e72882081b8fd2d24e27
1906 F20101113_AADXGG pan_w_Page_22.txt
7aa283fbc7356faf882a4102e875ec7c
9085a275b237b58f6b9e82b93d937eeebe11fedc
38741 F20101113_AADXXX pan_w_Page_24.QC.jpg
f35931ad5df8dcc2bfb51919ac8f09e0
1ed17356de7a125705d1a8e7c1d495d7a9f1a8c3
17873 F20101113_AADXLE pan_w_Page_11.jp2
1edcd375e1231a1c0d4ed24a625f7a6b
7d2d0d226314f8dd8b85560e62ae3f1810c71f73
31753 F20101113_AADXGH pan_w_Page_22.QC.jpg
a41d05c63891422178cf481eca1a3294
434421781a73b9d5675efba018150365b7ddf2f5
6493 F20101113_AADXXY pan_w_Page_09.QC.jpg
b6c09a4c3dd227d2933997932c215f6c
3dd97b247757ea66a38cb4156e26978baf451ca2
9889 F20101113_AADXVA pan_w_Page_72thm.jpg
c519066d02b10e25ae3f7e4672c40ae1
ff5a2e847fc1011d74835f93f0af4a2d07ba4307
20447 F20101113_AADXQC pan_w_Page_10.pro
3474be7d5074a06f3f6144b43ef370a5
1ba0e62bd986e457fc018eead33176837f2a330c
100349 F20101113_AADXLF pan_w_Page_12.jp2
b8a716aab21f1559b441073eff9a4516
bed8d3153507704342da4ebc1a93685a6167962a
319 F20101113_AADXGI pan_w_Page_49.txt
1778490d5822387c5d80f499b39d3b8b
6faa4c8f45a3387d530fbbc4430b3ba4da734a63
F20101113_AADXXZ pan_w_Page_21thm.jpg
218ca04e105b74095690a6c0493f7068
fb790bda0fec2fbf51397de1be0458b2aa67be66
35507 F20101113_AADXVB pan_w_Page_15.QC.jpg
ffdecf4174d90f15a16920ec88da789a
26c3d6d3248ca2e74dbed7626b3049a72088abc4
6910 F20101113_AADXQD pan_w_Page_11.pro
30f42091139632f16c2a2b25c82f546a
7f854e08e8832adbdae1e60ce86aa78d1fd943fa
33176 F20101113_AADXLG pan_w_Page_13.jp2
7c4245187cee56d06527dda63bed955e
28f7c6347a80e192db5bb697a6fcb00800f2439c
2097 F20101113_AADXGJ pan_w_Page_16.txt
bc1021343893447d6de95a0d785e75d9
e90f63a81f0580f9fe14a10dab76259738a0659f
6400 F20101113_AADXVC pan_w_Page_11.QC.jpg
bbe33802722833a604096080b9c9cbfd
8843c6b30ecc97ad08ad5dd6d40adacc85a082de
45682 F20101113_AADXQE pan_w_Page_12.pro
3c4a06a3a845a71949dbda29a1d29bac
a9860ad569fa2f12249aa9431ab5ebb9e9d2d605
110620 F20101113_AADXLH pan_w_Page_14.jp2
1bc41125335f160a8ab29e52615ff8ea
c361ec4d9aef7bae6413cc8cdcf9ff44527a6458
9611 F20101113_AADXGK pan_w_Page_70thm.jpg
745b66206dd93efeef7778df54a7c8c4
2f0a5a1727a3c3b558bac6fdbf5994ea9d98b8c0
40629 F20101113_AADXVD pan_w_Page_72.QC.jpg
0112b59bdfe8c0f48d74b3d6eb7c84d8
d48cc2bdf32edea3829d98982925f02a1e8f12fb
13482 F20101113_AADXQF pan_w_Page_13.pro
ef6afbafedd9e4f40dee2e836dea7bd2
66a93632e52cb1249e45c03202f75edae95c6f22
113538 F20101113_AADXLI pan_w_Page_15.jp2
9a98a04e32acfb8a5c1f924cee7d7729
1f7c2b9b7c61abc40fd758bbfd2b57473e26ea6b
5900 F20101113_AADXGL pan_w_Page_26thm.jpg
7118539d476cb3f9121c71c1fbbc1a5f
d518937c774e973c4120e18ca0d87e8f95680bd1
39572 F20101113_AADXVE pan_w_Page_70.QC.jpg
eb12bd91f6996e3f7986393fd2e05f94
62017ae87e79e3ba5c40dd591e42ae2f59f0084f
51889 F20101113_AADXQG pan_w_Page_14.pro
6bd9bcb90a1438c56c67affff595c03d
05ba9be4c5979e5b60550320bb0baf66b6a9a84b
113332 F20101113_AADXLJ pan_w_Page_16.jp2
2d416ff2ca27b292e8609da68b887656
a61a7f416f1339c681bd43a7f949a9bed050d541
24622 F20101113_AADXGM pan_w_Page_73.jp2
045a2c8c5d039acb0f469f7cb17115e4
719780dec4107f2d9498abc94abde69e07b3cb73
9027 F20101113_AADXVF pan_w_Page_32thm.jpg
37be5b37bcd1f8953fb5bacf14bd3525
95cd5348b2edf738b35dd800677bd8493661e08e
110406 F20101113_AADXLK pan_w_Page_17.jp2
814d19c9eff64484d3e5ec6a9975f53b
7f4869c93fc7d74b4bbbae2a003fed30dcb761e3
1985 F20101113_AADXGN pan_w_Page_09thm.jpg
56b1dd81ea952d37717aba90edaa077a
4c4c0a6cc6328e13f8d19468981ae3ba8a524d5e
52537 F20101113_AADXQH pan_w_Page_16.pro
5b13ce2b055e8dfb7205b8931576fe70
b2e49aeabb066580c35f60bc945b5a17873b2668
40385 F20101113_AADXVG pan_w_Page_61.QC.jpg
0d83b86024807db474b6a19d60991575
e526b8c7f98e7de6e1e1ebb892cf9a90ce38d60c
117227 F20101113_AADXLL pan_w_Page_19.jp2
69860a7949069291d8f5c2b2f0ea3139
74f857b090fea0d9423f624651f7478a2cd96b4d
871 F20101113_AADXGO pan_w_Page_10.txt
4220e44e4fb18b2d79f42a45c3870a12
5af51459d3fc91e5e6a515c7904bc908c6a769b6
50704 F20101113_AADXQI pan_w_Page_17.pro
6f786ba495a27919fab07609d9f95c79
dc68c5db7cdc0b5ecd799ad5429308330d838278
109334 F20101113_AADXLM pan_w_Page_20.jp2
f3e9358634c1b6603e838ef669c92868
955d9b35a9a39a69636eb3c931f0b52f14fa5df2
9417 F20101113_AADXGP pan_w_Page_64thm.jpg
20886359796f54895cba6ef208d818c6
71973c62621581e645f118022b2db9cc7ee69951
53042 F20101113_AADXQJ pan_w_Page_19.pro
92850f4cfc1f34160fc977fa36387ace
4be9a1c7f742d9ca0a7b077966a13a72439855dc
9215 F20101113_AADXVH pan_w_Page_24thm.jpg
015588b34961dbfe72ca02b8c22f12ac
f7dccd72e2c48acce1ec87fd6d0891a2c1e0f0b9
102198 F20101113_AADXLN pan_w_Page_22.jp2
1d4354e03fdb31fcf8268629700c7332
aa7409016d89c244f59d80af5ba82a29906b5a37
9701 F20101113_AADXGQ pan_w_Page_62thm.jpg
9e03304c5b35c4edcbf009dc3d9ce290
aaa49020de7fe368cd9e4708fbdeb3b65a91d057
49111 F20101113_AADXQK pan_w_Page_20.pro
434e61f2744bee3de1e1c48a40101552
7495df099840f4c6e0a743ca597ff6993046b17b
7872 F20101113_AADXVI pan_w_Page_22thm.jpg
2cae3380ea7ef0b7c554dc12af8cc13b
ffa99c9b2d3bdc47faf0d0aa2de6389cd81ed518
113787 F20101113_AADXLO pan_w_Page_23.jp2
bcdc86c14352b5933c036cad06f95805
44318e966a31491a13b84a2930c4e58aa9081364
65 F20101113_AADXGR pan_w_Page_03.txt
addd7b7426fd041582c158c4f1908570
73e27e09ce07d5250e5c97d788b89796599412a9
52545 F20101113_AADXQL pan_w_Page_21.pro
be3f38fc86238771434af23cfe49e61e
5280ca617baded48430612b4dd496b82c469802d
5350 F20101113_AADXVJ pan_w_Page_51thm.jpg
2c0ff1b1cf84625f6c5f36875b71538e
95cfe5ca122cc1f669b320c834c0a2f562405052
121374 F20101113_AADXLP pan_w_Page_24.jp2
06b402acd2c9ce02d14115b96d7e43a3
149074c8dc6c48e8f55a432fd1c8448db77fdb58
33646 F20101113_AADXGS pan_w_Page_31.QC.jpg
c6e105f13cc92f1002f6ba7c060d52fa
6cb5dd90e562c5c2827f32bf2c69c8d520b4fd30
47444 F20101113_AADXQM pan_w_Page_22.pro
57165d4a7d1ccd6257ab8a2f2a7c4db0
af084f800a3babaeab6ed0a9a9303226ef07a59a
1823 F20101113_AADXVK pan_w_Page_11thm.jpg
675bbb1c12898354a7387549ebac4b5a
12f3c7b42ac101eaacbcd833c486359ea6c5eee0
90075 F20101113_AADXLQ pan_w_Page_25.jp2
66c43210c3f77e9de98a6974d0fdfa96
70324a2c25a706d91ea66332a7dc46b4e4db71f7
135943 F20101113_AADXGT pan_w_Page_66.jpg
fcf13fc3e109bd0339de5f0bdb83528c
6945cc25b2e3e717fc3c564607a2cb854f5fd219
53195 F20101113_AADXQN pan_w_Page_23.pro
fa48461e0d468a317cd6b22d5e6ceb42
438572752d83f43f544ad7801695c32b0942b2c3
8497 F20101113_AADXVL pan_w_Page_08thm.jpg
f1faef4d5017a623948ba3d8e32ed36b
d484c7a2bad426c555ea1cf470a1aeb84d3c2938
65514 F20101113_AADXLR pan_w_Page_27.jp2
727b8a6701d5597a9dc9060eecd0727c
92bb9f26ba9218f0c3afad757b9134acba2228bf
4395 F20101113_AADXGU pan_w_Page_03.jp2
c22b50959cc4f0c199c36a7b3f9cb677
ffc6adb44647fce404027efbbb89ddbf076dbdcd
57059 F20101113_AADXQO pan_w_Page_24.pro
c5f70eeb6ec9754212506fe0cb744f60
35bf53b1dfb81b1baece868bded4c81f277bf236
34630 F20101113_AADXVM pan_w_Page_17.QC.jpg
63c5f618d63bf00d36ab170dcd28f680
4050f594bbcc8424668442ab2dac036a52b1e63c
90342 F20101113_AADXLS pan_w_Page_28.jp2
1f901f45911906e7aa4aa28752e8bf52
5adf228173f71d1a67a7e5de6026f67183faa8f9
30004 F20101113_AADXGV pan_w_Page_12.QC.jpg
ead41264e3da980d6e80e8b1ce92e4cb
be0afe9118a3693c0329d2143473ad5d31f21ef0
40480 F20101113_AADXQP pan_w_Page_25.pro
b11a414994b05498c2ed79001c207dad
edb9168f43f62d3d4a068c04bd63d58a2e0db656
38698 F20101113_AADXVN pan_w_Page_64.QC.jpg
78fc39b013fd5130f470f6605ec59e32
c4f36c2e4cfe3b61c361e28a76f3e4b777ed6b17
103591 F20101113_AADXLT pan_w_Page_29.jp2
ec95913c2358644abf83fdb90d293a11
fbc4a18c11923ae7021534dc4aedbecadf78416b
79083 F20101113_AADXGW pan_w_Page_04.jpg
658ea59424ccd9dd73381c1e6c5ec1a3
e56b9f1d62bf86a6295981ec1a3158c8ba1b80bb
32822 F20101113_AADXQQ pan_w_Page_27.pro
b1cdaa3e217bec7924e8acde2d594094
69f0ade9412f40ba3ea3ddb114a5f551fcb715a4
986 F20101113_AADXVO pan_w_Page_03.QC.jpg
87220f0dff0cacaf853317f5e218751c
22099b486ecbff57cf8987bddc02b23bc8f43e6e
106048 F20101113_AADXLU pan_w_Page_30.jp2
16bbe6544eba1fc1321cc0622428867d
c339f0dd5fdcfd4f1ff7a74e30b118651e3f990b
407 F20101113_AADXGX pan_w_Page_73.txt
0189f84c6daf1df211c382d20ac6578c
a4ff80d81379761832342abe0127a23d510b8125
39830 F20101113_AADXQR pan_w_Page_28.pro
79f14dbdf0fd42004e27e04def9ba89a
9648e99d5dc42052499cdba48b212055382418df
6268 F20101113_AADXVP pan_w_Page_46thm.jpg
6741160938b044280f0231e8f1ac8f65
07217926868039fed8132195462b10948611358b
109431 F20101113_AADXLV pan_w_Page_31.jp2
8a10364a93b98bc8eb6aae89270fc875
4ab06175b2597c48160faadab2718d8b9c628fa6
45077 F20101113_AADXQS pan_w_Page_29.pro
ed5e061abec5656c134ec3c201c92b60
94359d90a904bb33ce1db5b95e102c57416c9d86
6759 F20101113_AADXVQ pan_w_Page_25thm.jpg
f34acc847f83d2e9547c348f4f473944
b1ffc54c00a4d45e8695d8e0d3b78de211e4f862
117051 F20101113_AADXLW pan_w_Page_32.jp2
a94a77cb649148ac98af9f9f2c626933
ac5f3f05070b2d332b0d74fd8564ea3bcbd16b6e
F20101113_AADXGY pan_w_Page_15.tif
51cbe4f1b5cc1ed5e8c1d6ddba686711
f1e20a51c2a838a05660ad48ce9d151d643bb8e2
48514 F20101113_AADXQT pan_w_Page_31.pro
5a4b969b6c52391839c8186cea491ddf
a5374bc26fc4ad0bf344c4ae0e82980aad241265
30839 F20101113_AADXVR pan_w_Page_18.QC.jpg
58a8aed04048dc57c6f7fe45059b3056
37f7a9f0fc6c1d0877a02b1c8ab5013e665b4de2
118474 F20101113_AADXLX pan_w_Page_33.jp2
8c75ddd70353fe38684067b311316bc3
5c71cb487cd50f12714c2146763d5d4bc606367e
2148 F20101113_AADXGZ pan_w_Page_36.txt
a06e36716a0b455575d4c73c2bf93836
a3ef7ba713b10c98362efee3825d4fb28d0928f9
53827 F20101113_AADXQU pan_w_Page_32.pro
01726cb0250f7466a84803c2e981d78c
7f1457d1a947dce6b484e3c20d315d47e7200f40
11033 F20101113_AADXVS pan_w_Page_52.QC.jpg
4da0b0a4c9fdccc16409b4aa8097b3c9
5d5041c2ff1c29023dda0db7ea7d8257dce7e906
109100 F20101113_AADXLY pan_w_Page_34.jp2
4b0fcdb87b50f9b065ebdc6bd6629ca7
a9fb5b284da07e3cb3759fcbfc39e2383ce38f92
54273 F20101113_AADXQV pan_w_Page_33.pro
24885e2883aa2f8134a10656795a0e32
2238f008cdfc39639f56335e42ca675aa2c35f0e
7865 F20101113_AADXVT pan_w_Page_12thm.jpg
bfdd437db4f5bcaafc1b98594cd14ceb
7df656870749cd466282974f343e26f0a6bc17a0
117416 F20101113_AADXLZ pan_w_Page_36.jp2
fdd4a9321bfd5cfa37bdcae57f021a9e
3c76fb323c7884addfc28f2a78fe4cf6fe1ec00e
48924 F20101113_AADXQW pan_w_Page_34.pro
2d8750bc20b6f6baebbd33d3b53c268b
5e92afb0e8c2ffde88a70cdb55fdf39c76504cb6
111508 F20101113_AADXJA pan_w_Page_19.jpg
17467e4244b134a389d36d058d110706
b525705e5f7da58e83a2995faa1ec8b2b9ee092b
5230 F20101113_AADXVU pan_w_Page_50thm.jpg
1b9fd440c50f1cebd46930706bb81d23
77e4f84f8e1b3ec5b6ca68e91e2319b57b97dd31
46166 F20101113_AADXQX pan_w_Page_35.pro
890f51d644354e325ecf2dfecfc580be
119ea84196f6eab4fd1d16ac5fe7f06267d4073c
103201 F20101113_AADXJB pan_w_Page_20.jpg
44cc959edc66c44824c9338e01ac234c
fd5c69f98dae23a7d4fd0776c917ae9712d047fd
17501 F20101113_AADXVV pan_w_Page_49.QC.jpg
9aa915633a9c3ffa5b8e922da237aade
b0388c98b7614732c3fad7c4dd8e33e473d8ea76
55859 F20101113_AADXQY pan_w_Page_37.pro
fd73ebda415c75b5bfdd787f3e5f1ee2
4ccdc5e6ce6251ce916d303c6f17c742362a7be9
108698 F20101113_AADXJC pan_w_Page_21.jpg
96dd8516b6e5afb0ca75a2713ddd1356
b97b52a82a67b1f4cedec880cdf264c28852248b
8219 F20101113_AADXVW pan_w_Page_20thm.jpg
a9fe1f93f535cc2731e1b3669eeb6ce2
b6bf29d3e4c8974092d1ac87e3a83ad212009618
F20101113_AADXOA pan_w_Page_26.tif
eea8c3826c643512c60744ac45a5c75e
ffff9ef0b24d43d7886f08784c2551e4fe49f61a
55140 F20101113_AADXQZ pan_w_Page_38.pro
d9f6224462c2da004addcf5e392c9729
6b7f1200a5783afcb39d3b277bbabc4887c1ae95
97664 F20101113_AADXJD pan_w_Page_22.jpg
ddb320bf7a7c95a7db46b1ec0655baaf
0cca077f0b62237f0b8ba0410350fdd948fc4df1
7110 F20101113_AADXVX pan_w_Page_47thm.jpg
b8105024c753a0242813333a93e87c92
02790608619ff13118ad666290f141aaceec934d
F20101113_AADXOB pan_w_Page_27.tif
69dc76a6135c05e17c8cf6c4944ee5a2
d60d682d40dd659efd86c233500d0f2126c4b4be
109701 F20101113_AADXJE pan_w_Page_23.jpg
a097352ca12dcd7072b0b86c91c08c0f
983d59053fbae13afeb9c4386ea72764d3b19a8d
36009 F20101113_AADXVY pan_w_Page_19.QC.jpg
3adc2c384eac2e41019cc054cb9704aa
18a4f1af1370c2570b67cea9d62674179f5bbb4a
1970 F20101113_AADXTA pan_w_Page_31.txt
e59b5fd084c48f48ebba0a16262e54a3
b8ce27b68d7723489b48ccc4ee6f2d0ff13c62e5
F20101113_AADXOC pan_w_Page_28.tif
81ea2087748f9ef8cd0665ad18bfdbfb
9b947b0247947122690a1367ed32a429a4b70af6
117802 F20101113_AADXJF pan_w_Page_24.jpg
562986665423260ff390ddb8fda187bf
5eafa23a54b27a1509cdce97ee5875fa1afa9983
4784 F20101113_AADXVZ pan_w_Page_49thm.jpg
106b7c64c5c6a7b8869afe1b2bbddbe9
560d0519c13613e624eddb7e8fa09e73e3286377
2140 F20101113_AADXTB pan_w_Page_32.txt
046f71ae5d8b605d83b469509d81e57a
2601ed338b7ff6fb18bd3e2298b68d4bb34f32c8
F20101113_AADXOD pan_w_Page_29.tif
757551dae609d663111d03dfa5d62b19
493c026661c456d6869a55ddf7f6d5b4ac76e122
83838 F20101113_AADXJG pan_w_Page_25.jpg
e2c0152dc36593269da6a37fb92224b3
67fa2a65f0c72e90c31be990fa0976fc18bddbe3
2128 F20101113_AADXTC pan_w_Page_33.txt
7eeb23ca8e992d742ad727cd4d4099b0
9797b8666722df364c85a9c85a09b07702023022
F20101113_AADXOE pan_w_Page_30.tif
db815526c42c165c96de01e8fc528523
5dfa4a0e8846e2b21bce077fae9b1278954311c0
74600 F20101113_AADXJH pan_w_Page_26.jpg
1c6be347645ed68d296f584150d45d1d
4836ba20a03da6502cdec5616bf1e3fc6f717294
8713 F20101113_AADXYA pan_w_Page_15thm.jpg
dd6e3bd8372052c070d45aa9bc2815f9
33c2eb2f25025a1337572cc0e3622c3690373ea1
1950 F20101113_AADXTD pan_w_Page_34.txt
7a77cdfacd77211ae346564502a2717c
4bc69f953108bb0e7289b1b890f6cbddc497d915
F20101113_AADXOF pan_w_Page_31.tif
0cb1a08ecf2fe6dd932f42c6d975c29a
e501d00f07256945a4ae7767c0dc0af77754d43e
60192 F20101113_AADXJI pan_w_Page_27.jpg
aa617acfc76000d92f91d29613abddca
e4137ee974a8e59375eb9c06bbfe0fdefcfff5ea
33195 F20101113_AADXYB pan_w_Page_29.QC.jpg
79dcc12a2e4e9b90a83d6e8d53e68486
59fb14473804b166b5f5c225c39c649420d5dc9d
F20101113_AADXOG pan_w_Page_32.tif
3bf20adea02b280cebe6813acd1e4c43
8eeaa0d20f544eb353e256a543b3c70992b3bca7
79718 F20101113_AADXJJ pan_w_Page_28.jpg
ac3b7cd066b5176e59a4bd70cd1968c7
7cf81842695e91cdb726cea14053a12853d0049f
1866 F20101113_AADXTE pan_w_Page_35.txt
555af025ce57236a870febda2d2964a1
f72d18e77cd417319c3cbe82870d65eb4a497c4c
6597 F20101113_AADXYC pan_w_Page_05thm.jpg
2fbb55b1cff3464ddbb334f14419c503
1c7a448a75bdb500ba7a2e24547ef800bc29a8e2
F20101113_AADXOH pan_w_Page_33.tif
92a5495be1480fc8bf53481b1bbb6a7c
870cfb6b9073c2c1652497f5986592f4f6aee629
99845 F20101113_AADXJK pan_w_Page_29.jpg
c7bb1e0334c5581f614246ea835b4b95
33594628c4f8b42b2e0981bdc5eb758f622c609a
9291 F20101113_AADXYD pan_w_Page_41thm.jpg
847bca994cabcc52d2bb1cc00974b996
3d232d9519c17e1e1e5aa55a7a8d44ac55ad75b6
F20101113_AADXOI pan_w_Page_34.tif
a9a5b2755a20eb90da2bb28874c2fbde
026833b6776db6b8a06dbd229d7a391314295b5d
99768 F20101113_AADXJL pan_w_Page_30.jpg
c8f77e7747a63eda41c361560652453c
aea6af942dec9cbc8aa44b28a19facbf8b54050f
2212 F20101113_AADXTF pan_w_Page_37.txt
e61307a7885f7a9c11b6f43919123906
4e411c72eb1768c186a1e3732149779be77607e7
9541 F20101113_AADXYE pan_w_Page_68thm.jpg
4cf33b0334488bcdbe0f47e1923ab250
02ba13607436d75b71b75fd885019fb806236065
F20101113_AADXOJ pan_w_Page_35.tif
066bdc05716ce07bb9c3b122ece3329e
a174c352b37272cd4f0be075d2448a50cebcbcbb
102793 F20101113_AADXJM pan_w_Page_31.jpg
7539364185265a10ee24a582b8e8ff21
3e0fda1f82bc7ac9b06c9ac14222d4c47c290d3a
2195 F20101113_AADXTG pan_w_Page_38.txt
9fba710e70b19b6698442cc3448b25a6
baba18b9e44b7f68b0747961277a43027b404548
25440 F20101113_AADXYF pan_w_Page_04.QC.jpg
726d6d958964c30d6c27122b3ce36ed8
c2fb68fd696c102a2d013f0420a46b63c598001d
F20101113_AADXOK pan_w_Page_37.tif
f640a4ed5abdc119921641e20eb4c53c
5700f991365954cf7951035b3179e407385f751c
113220 F20101113_AADXJN pan_w_Page_32.jpg
06bc57e934faed21448d6c1732e661c5
d1be0085768992cca9fc81a31acb6cd0e3e3803b
2270 F20101113_AADXTH pan_w_Page_39.txt
5123902606e38b6a47d37ea9b4b8da55
7721d786b173127e5af945fd25b300b384bec193
7090 F20101113_AADXYG pan_w_Page_01.QC.jpg
b5116ff6c2ce194283f7a0750e88a285
ed4ecd7fb225ba07724f2e9b119c66de50ea463d
F20101113_AADXOL pan_w_Page_38.tif
825ddecc49f6fea1a07ce15075d8674d
73d405f93e2a30ee7b8589f66466785082eb2d37
104006 F20101113_AADXJO pan_w_Page_34.jpg
6ea9d16477897ffda8046f923d162bb8
edd9543caf5cedc7048c13dffcfe9c3f75e777b6
2194 F20101113_AADXTI pan_w_Page_41.txt
90827fb251096dc1e5e9278531af3c8e
53520b9fe305d0ab0eaa10689be26ea81d576002
15949 F20101113_AADXYH pan_w_Page_06.QC.jpg
08466efeb4e68a9a79011f3ab8589bc4
ca2500a00241068c997d7ccffbafd736335e2092
F20101113_AADXOM pan_w_Page_39.tif
512c2a9a54743473f234f874c13f840f
ed0ed56eb6c93589c5e57bd6289b968611773066
103421 F20101113_AADXJP pan_w_Page_35.jpg
e09569bdfcf55849a4cf137285359f42
dd1cfe80c47bac12620be26f5fa42f19aa8fa50a
2039 F20101113_AADXTJ pan_w_Page_42.txt
3a587f51e3b720c09c615e6ab09a10d9
d9526bbd9afd942e16d557af534a205d0e2552dd
39904 F20101113_AADXYI pan_w_Page_66.QC.jpg
544da80566b075fef210c065a98d17c9
1eeb8f0a27ee8eb59c5284e4a1982b9d9a1b6635
F20101113_AADXON pan_w_Page_40.tif
bf52ebcc33744d7cf2c0de6ac40e150c
76abae42dd65e94e88c46af9b17bc8d0dd9399ee
113947 F20101113_AADXJQ pan_w_Page_37.jpg
cd2d9c85b0993acdacbfdb3d364b782f
02e4a217ca5c6a6d06d7bf0deee287528f6180f9
2160 F20101113_AADXTK pan_w_Page_43.txt
18eaeb15eefdf4416683a155ab789526
d5df7107e0b23ab20b68fc691de7261549072768
7117 F20101113_AADXYJ pan_w_Page_55thm.jpg
9887aaa087ef0fdc4e53d22643262442
c82cf7bb30133ef012de9c52b830f45546759b67
F20101113_AADXOO pan_w_Page_41.tif
8915a6cad08f32c243c8f32f0196c110
6999e37fad049a92d0c434ca794907d7aebb3c5b
114386 F20101113_AADXJR pan_w_Page_38.jpg
3ddac53afdc562efa4157f9d1c2c26af
e49c3baaa65dbc52ca548b6fad6a2487b4ab7303
202 F20101113_AADXTL pan_w_Page_44.txt
3971436617e8a85cf4a2861e114d0850
9debe63e6ff5a5ffa9231762c3cc7bee6cca8823
F20101113_AADXOP pan_w_Page_42.tif
b9b21bd528825d5a4127df34a145ce34
648f8c6b9a50c4f24ea06e02a4dd78e7314f4ea3
117209 F20101113_AADXJS pan_w_Page_39.jpg
5123fc095e4a0ad3f8db8dc4c3bc161b
e04442674499dc4028a089dfe3621807c2af68b8
2156 F20101113_AADXTM pan_w_Page_45.txt
baca6b8b288d43d5ae680ef5318d3d3e
8687bf4b1a4a77fb25871774c3a1ce008f1be61e
6550 F20101113_AADXYK pan_w_Page_54thm.jpg
da16435d6ba02d3d22e0c5b5e7de924a
990c01e4d25ca622f863fa9bf0060d17144d7561
F20101113_AADXOQ pan_w_Page_43.tif
7be8d204913403edad4167cf46ea1044
7aec9730aa59cabd2e3e9575ba0524261ffabdd1
117014 F20101113_AADXJT pan_w_Page_41.jpg
0e54d591753213cd0df6c21c052ddd2c
a193eb6fcadb72bd40aec29a29bd88fde7bf06e0
4128 F20101113_AADXTN pan_w_Page_46.txt
58d058d146c813880315c473af10f17f
8427b70c8872e8ab0b6cb890e576f09a05ec20bd
2568 F20101113_AADXYL pan_w_Page_13thm.jpg
ebf8943b7da61b7b1804ffd2fc49b36a
184aaf6d067c8fa9ab06dc26ef178ef31e18fe41
F20101113_AADXOR pan_w_Page_44.tif
648a7740c06422f76fe1492aeb779fcc
74959e728092847ef5fa24d0cbca615f023fe1aa
110394 F20101113_AADXJU pan_w_Page_42.jpg
8f4829307d52e1b42f341ef5f235b79e
65befab0dfadaf550c44e902803abd3a6233a465
741 F20101113_AADXTO pan_w_Page_47.txt
a48e7827c3e25656cdc42139ce97e991
1bf7dcf649bed92ef6dfbf3d55a9a524f0301a50
9692 F20101113_AADXYM pan_w_Page_66thm.jpg
1b8cfcf72d17f10a0db3c16e82bf5f47
3f6547fcadafe6261b11fd343c5b9d1657d9b462
F20101113_AADXOS pan_w_Page_45.tif
0215d8344468549c58007819f0507904
c29d90b24f3c3784c478789e071e540a7a222396
111173 F20101113_AADXJV pan_w_Page_43.jpg
5f645585eedc051cefe588cb7f5d88de
4926ccb6e87e9693eaed2712defc1641ed96c69f
641 F20101113_AADXTP pan_w_Page_48.txt
85f4c836893d2fd844e3e7a98186adc7
5b3afe674b23f297692717be56db9e3b7cd8f666
36738 F20101113_AADXYN pan_w_Page_23.QC.jpg
9afdffde7cf101d3d4ac7b0b7823a300
1c5fea251157a3a23e4820e8bef70eb617d30350
F20101113_AADXOT pan_w_Page_46.tif
67b8d51947d17f76cd15cf5d7dfd3736
8331bc31aa0f9c094a962f9005a13bfd0ea00ba8
65965 F20101113_AADXJW pan_w_Page_45.jpg
8cfa515977207f1d283ecffdbd91de63
fe0b7db08ea7b77d201dcf17e041573365cf2630
566 F20101113_AADXTQ pan_w_Page_50.txt
ca4a8c8ee4de9b1a419b9174308a4496
a8826646ecb84651ff272068daa43393c6d27b5e



PAGE 1

1 MOLECULAR ANALYSIS OF TWO PUTATIVE MEDIATOR SUBUNITS IN Arabidopsis thaliana By WEI PAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 Copyright 2007 by Wei Pan

PAGE 3

3 To my family

PAGE 4

4 ACKNOWLEDGMENTS I thank Dr. Bill Gurley for his kindness, commitment and great mentoring. He is an excellent advisor because he always encouraged me to develop indepe ndent thinking and gave me the opportunity to pursue my research inte rests. His careful reading and editing greatly improved this thesis. I thank Dr. Robert Ferl fo r his suggestion on my wo rk and giving me the pBI101sGFP vector. I thank Dr. Kevin OGrady for his continuous help with my experiments and teaching me many molecular biology tec hniques. I extend thanks to Dr. Eva Czarnecka-Verner for her suggestions on my research. I thank Dr. Zhonglin Mou and Ms. Xudong Zhang fo r their valuable assistance with some techniques such as Northern blotting and GUS st aining, and generously letting me share some of their facilities. I thank Ms. Donna Williams fo r her assistance with the con-focal observation. I thank Dr. Masaharu Suzuki for helping me ge t started in research in the Plant Molecular and Cellular Biology Program (PMCB); Dr. Alice Ha rmon for the invaluable training I gained in her lab; and Dr. David Cl ark for his kindness and support for my study in PMCB. I also wish to express my gratitude to the entire PMCB faculty who taught me in classes and journal clubs. I benefited a lo t from the wonderful courses. I am extremely grateful for all my family and friends for their understanding and support over the years.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 LIST OF ABBREVIATIONS........................................................................................................10 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 INTRODUCTION..................................................................................................................14 Assembly of the Preinitiation Complex..................................................................................14 Identification of the Mediator Complex in Yeast...................................................................16 Identification of the Mediator Complex in Human Cells.......................................................17 TRAP Complex...............................................................................................................17 SMCC Complex..............................................................................................................18 DRIP Complex................................................................................................................18 ARC Complex.................................................................................................................18 CRSP Complex................................................................................................................18 PC2 Complex...................................................................................................................18 Mediator Interacts w ith Transactivators.................................................................................20 Mediator Interacts with RNA pol II........................................................................................21 Phosphorylation of RNA Pol II CTD.....................................................................................21 Mediator Interacts with Coactivators......................................................................................22 Mediator Promotes the Formation of a Stable PIC.................................................................23 Mediator is Required in the Reinitiation Scaffold..................................................................23 Mediator Stimulates both Basal and Activated Transcription................................................23 Model for Mediator Function in Activated Transcription......................................................24 Hypothesis for a Mediator Complex in Arabidopsis ..............................................................24 2 MATERIALS AND METHODS...........................................................................................29 Plant Growth Conditions........................................................................................................29 Genotyping of the T-DN A Insertion Lines.............................................................................29 RNA Analysis................................................................................................................... ......30 Microscopy..................................................................................................................... ........30 Plasmid Construction........................................................................................................... ...30 GUS Staining................................................................................................................... .......31 Agrobacterium Transformation Technique............................................................................31 Chromatin Immunoprecipitation............................................................................................32 PCR Analysis of Chroma tin Immunoprecipitation.................................................................34

PAGE 6

6 Bioinformatics................................................................................................................. .......35 3 RESULTS........................................................................................................................ .......36 Analysis of Arabidopsis Med31 Gene by Multiple Sequence Alignments............................36 Phenotype Characterization of med31 Mutants......................................................................37 Med31 Expression in the med31-2 Plants...............................................................................38 Subcellular Localization and Tissue Expression Pattern of Med31::GFP Fusion Proteins....39 Tissue Expression Pattern of Med31 Promoter::GUS Fusions...............................................39 Co-immunoprecipitation Maps Med6 and Med31 to Promoter DNA....................................40 ChIP Analysis for Med31................................................................................................41 ChIP Analysis for Med6..................................................................................................42 Conclusion..................................................................................................................... .........43 4 DISCUSSION..................................................................................................................... ....58 Phenotype Characterization of med31 Mutants......................................................................58 Evidence for a Mediator Complex in Arabidopsis .................................................................59 LIST OF REFERENCES............................................................................................................. ..61 BIOGRAPHICAL SKETCH.........................................................................................................73

PAGE 7

7 LIST OF TABLES Table page 1-1 Interaction of the transact ivators with the Mediator subu nits in different organisms..........26 1-2 Mediator subunits in yeast, Arabidopsis Drosophila and humans ....................................28

PAGE 8

8 LIST OF FIGURES Figure page 3-1 Multiple sequence alignments of Med31 homologs in different species.....................45 3-2 Multiple alignments of AtMed31 with the deduced amino acid sequences of its homologs in other plant species................................................................................ ...46 3-3 Diagrammatic representati on of the insertions of the T-DNA in med31-1 and med31-2........................................................................................................................47 3-4 Germination rate and root length of WT and med31-1 seedlings (9-day-old).............47 3-5 Nine-day-old WT and med31-2 seedlings grown under continuous light....................47 3-6 Nine-day-old WT and med31-2 seedlings grown under dark.......................................48 3-7 Ten-day-old WT and med31-2 seedlings......................................................................48 3-8 Comparison of adult WT plants and med31-2 plants...................................................48 3-9 Northern blot analysis of Med31 expression in WT and med31-2 plants....................49 3-10 Subcellular locali zation of Med31::GFP fusion prot eins in the root tip of a 35-day-old plant............................................................................................... ............49 3-11 Expression of Med31::GFP fusion proteins in lateral roots.........................................50 3-12 Expression of Med31::GFP fusion proteins in a root hair............................................50 3-13 Expression of Med31::G FP fusion proteins in a leaf...................................................51 3-14 Expression of Med31::GFP fusion proteins in a trichome...........................................52 3-15 Expression of Med31::GFP fusion proteins in a petiole...............................................53 3-16 Med31 promoter directed GUS tissue e xpression pattern in young plants (16-day-old)..................................................................................................................54 3-17 Med31 promoter directed GUS tissue expr ession pattern in adult plants (46-day-old)..................................................................................................................54 3-18 Multiple sequence alignments of Med6 homologs in different species.......................55 3-19 Med31 associates with the promoters of CCA1 Hsp18.2 and Adh1 but not with the intergenetic region........................................................................................ ..........56

PAGE 9

9 3-20 Med6 associates with the promoters of CCA1 Hsp18.2 and Adh1 but not with the intergenetic region........................................................................................ ..........56 3-21 Immunoglobulin G Sepharose and c-Myc antibody cannot immunoprecipitate the CCA1 promoter from WT Arabidopsis ........................................................................57

PAGE 10

10 LIST OF ABBREVIATIONS Adh ARC CCA1 ChIP c-Myc CRSP CTD DRIP EST GFP GUS HAT Hsp18.2 IgG PC2 PIC RNA pol Sep10 SMCC Soh1 SWI/SNF TAP TFIIA TFIIB alcohol dehydrogenase activator-recruited cofactor circadian clock associated 1 chromatin immunoprecipitation cellular myelocytomatosis oncogene cofactor required for Sp1 activation carboxy-terminal domain vitamin D receptor interacting protein expressed sequence tag green fluorescent protein -glucuronidase histone acetyltransferase heat shock protein 18.2 immunoglobulin G positive cofactor 2 preinitiation complex RNA polymerase Separation10 SRB/MED Cofactor Complex suppressor of hpr1 switching/sucrose non-fermenting tandem affinity purification Transcription Factor II A Transcription Factor II B

PAGE 11

11 TFIID TFIIE TFIIF TFIIH TRAP UTR VP16 WT Transcription Factor II D Transcription Factor II E Transcription Factor II F Transcription Factor II H thyroid hormone receptor-associated protein untranslated region herpes simplex virus protein 16 wild type

PAGE 12

12 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MOLECULAR ANALYSIS OF TWO PUTATIVE MEDIATOR SUBUNITS IN Arabidopsis thaliana By Wei Pan May 2007 Chair: William B. Gurley Major Department: Plant Molecular and Cellular Biology Mediator is a conserved coactivator comp lex that has been identified in yeast, Drosophila and humans. It plays a critical role in gene transcription me diated by RNA polymerase II (RNA pol II) by serving as a bridge between activators bound to the prom oter and other transcription machineries, including RNA pol II. Despite eviden ce suggesting such a vital role of Mediator in gene expression, the subunit composition and functi on of Mediator has not been determined in plants. Based on the conserved transcriptional machineries (RNA pol II, general transcription factors and some coactivators) in plants, metazo ans and yeast, we hypothesized the plant also has the Mediator coactivator. Identification of the homologs of most of the yeast and metazoan Mediator subunits in Arabidopsis supported this hypothesis. This study characterized the function of tw o putative Mediator subunits, Med6 and Med31. Two T-DNA insertion lines in the Med31 promoter or 5 untranslate d region were identified. The med31-1 mutant line had shorter root length and a reduced germination rate. The med31-2 plants had shorter root length, aberrant patterns of cotyledon development, and smaller size compared with wild type plants. We found the Med31::GFP (green fluorescent prot ein) fusion proteins were localized to the nucleus. The Med31::GFP signal was detected in the roots, leaves,

PAGE 13

13 trichomes and petioles. In addition, we found the Med31 promoter::GUS fusions were expressed in the shoot apexes and latera l roots of the young seedlings (1 6 days old), and in the young inflorescences, anthers, stigmas of adult plants (46 days old) and in developing seeds. Both Med6 and Med31 proteins were localized to the promoters of three unrelated genes ( CCA1, Hsp18.2 and Adh1 ). These results strongly support th e conclusion that Med6 and Med31 are members of the Mediator complex in Arabidopsis

PAGE 14

14 CHAPTER 1 INTRODUCTION Assembly of the Preinitiation Complex Transcription is one of the most significant st eps that occur during ge ne expression. It is carried out by RNA polymerases and additiona l factors. There are four kinds of RNA polymerases in plants, RNA polymerase (RNA pol) I, II, III and IV. RNA pol I is located in the nucleolus, and it transcribes rRNA genes, excep t 5S rRNA. RNA pol II is located in the nucleoplasm and transcribes hnRNA, the precursor of mRNA. RNA pol III is also located in the nucleoplasm and is responsible for the synthe sis of tRNA, 5S rRNA and other small RNAs (Thomas and Chiang, 2006). And last, an RNA pol ymerase unique to plants, RNA polymerase IV, is involved in the siRNA silencing pa thway, RNA-dependent DNA methylation and the formation of heterochroma tin (Onodera et al., 2005). Transcription by RNA pol II can be broadl y categorized as basal transcription (activator-independent) and activ ated transcription (activator-dependent). A simplified sequence of activated transcription initiation for RNA pol II has been postulated as follows. Activators (transactivators or transcripti on factors) bind the re gulatory motifs of DNA and then recruit a variety of additional factors that prepare the promoter for the arrival of RNA pol II and the formation of the preinitiation complex (PIC ) (Thomas and Chiang, 2006). One of the first components to arrive is a kinase which phosphor ylates histone H3 (Featherstone, 2002). Then coactivators that can modify chromatin structur es are recruited. For example, HAT (histone acetyltransferase) arrives at the promoter early in the activation proce ss and its role is to acetylate specific lysines in histone amino-termin i and other transcriptio n factors (Roth et al., 2001; Naar et al., 2001; Clayton et al., 2006). Another complex that is recruited early in the process of gene activation is SWI/SNF (switchi ng/sucrose non-fermenti ng), which remodels the

PAGE 15

15 chromatin structure and facilita tes the accessibility of other me mbers of the transcriptional apparatus to the DNA (Gavin et al., 2001, Havas et al., 2000). After the promoter is made accessible, the TFIID complex is recruited to the TATA box in the promoter (Pugh, 2000). Activators also recruit Mediator complex which facilitates the formation of pol II PIC, which consists of RNA pol II and genera l transcription factors (TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) (Conaway et al., 2005). Next, TFIIH facilitates promoter melting a nd phosphorylates the CTD (carboxy-terminal domain) of the largest subun it of RNAP II (Jiang et al., 1996; Kim et al., 1994). This phosphorylation event is thought to be require d for promoter clearance and the start of transcriptional elonga tion (Dvir et al., 1997; Kugel and Goodrich, 1998; Kumar et al., 1998). After the synthesis of the initial transcript, most members of the PI C (with the exception of TFIIB and TFIIF) remain at the promoter and form a structure know as the scaffold that facilitates the reentry of RNA pol II, TFIIB and TFIIF for subsequent rounds of synthesis (Yudkovsky et al., 2000). In the process of reinitiation, the CTD of RNA pol II is dephosphorylated by a CTD phosphatase that is stim ulated by TFIIF (Friedl et al., 2003). This cycle of phosphorylation and dephos phorylation of the CTD is essent ial to the entry of RNA pol II to the PIC (with a hypophosphorylated CTD) and subsequent promoter clearance (hyperphosphorylated CTD) (Oelgeschlager, 2002). One of the key regulatory complexes involved in the process of promot er activation is the Mediator. This large assemblage of proteins (~ 2MDa) is conserved from yeast to humans and is composed of 25-29 subunits (Boube et al., 2002). It is becoming incr easingly clear that Mediator plays a critical role in both ac tivated and basal transcription me diated by RNA pol II in yeast and metazoans (Baek et al., 2002, Nair et al., 2005), b ecause it serves as a br idge between activators

PAGE 16

16 bound to the promoter and other ge neral transcription factors, as well as RNA pol II (Kornberg, 2005). Identification of the Mediator Complex in Yeast The presence of Mediator was proposed becau se of the discovery of activator inhibition in yeast. The activator GAL4-VP16 was found to repress the activatio n effect of another activator (a factor binding to a thymidine-rich DNA element) both in vivo and in vitro This phenomenon led to the hypothesis that the two ac tivators competed for a common intermediate factor. Activator inte rference was relieved in vitro with the addition of the fraction containing this intermediate factor, which was na med Mediator (Kelleher et al., 1990). The Mediator fraction from column chroma tography was shown to be required for GAL4-VP16 and GCN4-dependent gene transcription in an in vitro transcription system (Flanagan et al., 1991). This was th e initial direct evidence that Mediator was involved in gene transcription. The In vitro transcription system was reconsti tuted with purified RNA pol II and general transcription factors from yeast and wa s widely used for checking the presence of Mediator, thereafter. Three experimental approaches were originally used to identify proteins as Mediator subunits: 1) Identify the suppre ssors of RNA pol II CTD trunca tion mutations; 2) Isolate the fraction (RNA pol II holoenzyme) that can stim ulate the activator-dep endent transcription. Separate the proteins by electr ophoresis in a gel, and then identify the proteins by peptide sequencing; and 3) Identify the proteins th at co-immunoprecipitate with known Mediator subunits. The presence in the RNA pol II holo enzyme and support of activator-dependent transcription were two criteria that were used to confirm the identities of Mediator subunits. In total, 25 Mediator subunits have been identified in S. cerevisiae Nine Mediator subunits (Srb2, Srb4, Srb5, Srb6, Srb7, Srb8, Srb9, Srb10 a nd Srb11) were identified based on the

PAGE 17

17 suppression of S. cerevisiae RNA pol II CTD truncation mutations. All of these subunits were shown to be present in the holoenzyme (Thomp son et al., 1993; Kim et al., 1994; Koleske and Young, 1994; Liao et al., 1995; Hengartner et al ., 1995). Fifteen Mediator subunits (Med1, Med2, Pgd1 (Hrs1), Med4, Med7, Med8, Med11, Gal11, Si n4 Rgr1, Mtr32, Rox3, Nut1, Nut2, and Cse2) were detected in the R NA pol II holoenzyme and identified by peptide sequencing (Kim et al., 1994; Gustafsson et al., 1997; Gustafsson et al., 1998; Li et al., 1995; Myers et al., 1998). The mutant yeast strains for Gal11, Sin4 and Rg r1 showed similar mutant phenotypes, which suggested they may function in the same path way (Fassler et al., 1991, Jiang and Stillman, 1995, Suzuki et al., 1988; Chen et al., 1993, Sakai et al., 1990). More recently, Med31 was found to be a Mediator subunit in S. cerevisiae and S. pombe based on co-purifica tion with previously characterized Mediator subunits (Linder and Gustafsson, 2004). Identification of the Mediat or Complex in Human Cells Two methods were used to identify the Mediat or subunits in human cells: 1) Isolate the nuclear extract fraction that can stimulate the activator-dependent tran scription. Separate the proteins on the gel, and then identify the prot eins by peptide sequencing; and 2) Identify the proteins that co-immunoprecipita te with the transactivators (or their activation domains) or known Mediator subunits. Most id entified subunits are orthologs of the yeast Mediator subunits. However, various Mediator complexes with di fferent subunit compositions were isolated in different labs (Sato et al., 2004). A brief description of human Mediator types follows. TRAP Complex A thyroid hormone receptor-associated protei n (TRAP) complex was isolated based on co-precipitation with FLAG epitope-tagged hTRal phal (human thyroid hormone receptor alpha1) (Fondell et al., 1996).

PAGE 18

18 SMCC Complex The human SRB/MED Cofactor Comple x (SMCC) was purified by affinity chromatography of FLAG epitope-tagged hum an SRB proteins (Gu et al., 1999). DRIP Complex The DRIP (vitamin D receptor interacting prot ein) complex was isolated from the nuclear extract of human Namalwa B cells based on it s interaction with the VDR LBD (vitamin D3 receptor ligand-binding domain) in the presence of hormone. This complex contains 10 proteins and it can stimulate transcription by VDRRXR. It was shown that at least one of its subunits has histone acetyltransferase ac tivity (Rachez et al., 1998). ARC Complex The ARC (activator-recruited cofactor) comp lex was isolated by its affinity for the activation domains of SREBP-1a, VP16 and the p65 subunit of NF-kB, respectively, from HeLa cell nuclear extract (Naar et al., 1999). It can not only stim ulate transcription by activators such as SREBP-1a/Sp1, NF-kB/Sp1, Gal4-VP16/Sp1, but it also enhances basal transcription in vitro CRSP Complex The CRSP (cofactor required for Sp1 activa tion) complex was isolated from HeLa cell nuclear extract and shown to be required for Sp1dependent transcriptional activation (Ryu et al., 1999). This complex consists of 9 subunits and has a mass of approximately 0.7 MDa. PC2 Complex The PC2 (positive cofactor 2) complex was isolated from HeLa cell nuclear extracts based on its ability to stimulate HNF4 (hepatocyt e nuclear factor 4) and GAL4-AH dependent transcription (Malik et al., 2000). This complex consis ts of at least 15 subun its and is larger than 0.5MDa. The presence of these subunits w ithin the complex was confirmed by the

PAGE 19

19 co-immunoprecipitation of epit ope (FLAG and HA)-tagged MED 10. Both PC2 and CRSP were found to be subcompexes of ARC, DRIP, or TRAP/SMCC (Malik and Roeder, 2000). Despite being originally isolated by different approaches, some complexes found in human cells (ARC, DRIP, and TRAP/SMCC) were show n to be very similar in subunit composition (Naar et al., 1999; Mali k and Roeder, 2000). The finding that various closely related Mediator complexes have slightly different subunit composition raised the question of whether some of the proteins identified are true subunits, or just contaminants associated with a particular isolation strategy. Sato and colleagues (Sato et al., 2004) addressed this question by co-immunopreci pitation of human Mediator using six FLAG-tagged subunits to individually purify co mplexes for analysis of subunit composition by MudPIT (multidimensional protei n identification technology). Pr oteins present in all six independent Mediator preparations were consider ed to be true Mediator subunits. Their results support the conclusion that all prot eins identified previously are bona fide Mediator subunits. In addition, they identified the MED13L and the CDK8-like cyclin-dependent kinase CDK11 as putative Mediator-associated proteins. The inconsistency in Mediator subunit compos ition was thought to be due in part to the dissociation of Mediator subunits during chromatographic purificat ion and to insensitive protein detection methods. Another possibilit y is that the distinct Mediator types from different labs may have various functions, and theref ore, slightly different compos ition. For example, two distinct Mediator complexes were isolated using VP 16 and SREBP-1 (sterolresponsive enhancer binding protein) affinity resins, respectively (Taatjes et al., 2002) The larger one was named as ARC-L, which is almost identical to the TRAP/DRIP/ARC/SMCC complexes. The smaller complex was the CRSP complex. ARC-L and CRSP have many subunits in common, except that

PAGE 20

20 CRSP has a CRSP70 subunit not present in A RC-L and does not have the following four subunits present in the ARC-L: ARC240 /TRAP230/MED12, ARC250/ TRAP240/MED13, cdk8, and Cyclin C. In yeast, homologs (Srb8, -9, 10 and -11) of these four proteins comprise a distinct complex (Borggrefe et al., 2002), desi gnated as the CDK8 module. The ARC-L complex is transcriptionally inactive, whereas the CRSP complex is highly active in a reconstituted Sp1/SREBP-dependent transcripti on system (Ryu et al., 1999). Mediator Interacts with Transactivators Many Mediator subunits, such as Med1, Med12, Med14, Med15, Med16, Med17, Med23, Med25, Med29, Cdk8 were found to interact with transactivators in human, yeast, or Drosophila cells (Table 1-1). Some transactivators, such as the glucocorticoid receptor (Hittelman et al., 1999) and differentiation-inducing factor (Kim et al., 2004), can interact with multiple Mediator subunits suggesting a mechanism for more efficiently recruiting the Mediator. The interaction between transactivators and Mediator subunits is important in transcriptional regulation Conditions that result in reduced levels of particular subunits may have a negative influence on transcription. For example, Med1 (TRAP220) was shown to interact with PPAR which is a nuclear receptor essential for adipogenesis (Zhu et al., 1997). In TRAP220 null mouse embryos, the adipogenesis markers and PPAR 2 target genes were not expressed in the embryonic fibroblasts (MEFs), and the MEFs failed to differentiate into adipocytes via the PPAR pathway (Ge et al., 2002). The aut hors also showed that activated transcription by PPAR can be greatly increased by the TRAP complex in a reconstituted transcription system. In addition, RXR another Med1 interacting pa rtner (Zhu et al., 1997), was shown to be able to enhance the effects of PPAR

PAGE 21

21 Mediator Interacts with RNA pol II Many lines of evidence indicate that Mediator interacts directly with the CTD of RNA pol II. Yeast Mediator, without the CDK8 module, and the human CRSP co mplex were isolated through CTD-affinity chromatography (Myers et al., 1998; Naar et al., 2002). RNA pol II lacking a CTD (Pol II CTD) functions just as well as WT enzyme in basal transcription in vitro when Mediator is absent. But contrary to th e WT polymerase, this mutant RNA pol II cannot respond to Mediator in basal tr anscription and in Gal4VP16 or GCN4 activated transcription (Myers et al., 1998). Precise structural information has revealed that the three module s of Mediator (head, middle and tail) wrap around the RNA pol II in the holoenzyme. RNA pol II makes multiple contacts with the head and middle modules and on e with the tail. These in teractions are centered on the RNA pol II Rpb3/Rpb11 heterodimer, but also involve Rpb1, Rpb2, Rpb6 and Rpb12 subunits. These contacts between Mediator a nd RNA pol II only account for 35% of the RNA pol II surface; however, th e remaining part is available for interaction with other PIC factors (Davis et al., 2002, Chad ick and Asturias, 2005). Phosphorylation of RNA Pol II CTD The cycle of phosphorylation and dephosphorylati on of RNA pol II CTD is significant for gene transcription. During transcri ption initiation, the recruitment of RNA pol II requires that the CTD be hypophosphorylated. The Medi ators isolated from Fleischmann's yeast (Kim et al., 1994), S. pombe (Spahr et al., 2000), S. cerevisiae (Myers et al., 1998) and mouse (Jiang et al., 1998) all stimulate the phosphorylation of the CTD by the TFIIH after PIC formation (Hengartner et al., 1998). This phosphorylation of the CTD happens during the transition from the transcriptional initiation to elongation a nd is thought to trigge r promoter clearance (Hengartner et al., 1998; Oelgeschlager, 2002). An additional role of the hyperphosphorylated

PAGE 22

22 CTD is to promote interaction of the mRNA capping enzyme with the nascent transcript (Cho et al., 1997). The Kin28 protein is a subunit of TFIIH in S. cerevisiae and is the primary kinase involved in the phosphorylation of RNA pol II CTD. Its ki nase activity can be st imulated by Mediator in vitro (Guidi et al., 2004). It was speculated that the Gal11 subun it of Mediator may regulate the phosphorylation activity of Kin28 du e to the interaction of Gal 11 with TFIIH (Sakurai and Fukasawa, 2000). The CDK8 module of Mediator in yeast contains Srb8, Srb9, Srb10, and Srb11 subunits and seems to exert a negative effect on transc ription (Song et al., 1996; Samuelsen et al., 2003). A plausible mechanism is provided by the ac tion of Srb10, which was shown to phosphorylate the CTD prior to PIC formation and, thus, preven t the entry of RNA pol II (Hengartner et al., 1998). Mediator Interacts with Coactivators Mediator has been shown to interact with other coactivators such as mammalian p300 and TFIID (Black et al., 2006; Koleske et al., 1992; Thompson et al., 1993; Johnson et al., 2002; Johnson and Carey, 2003). p300 is a coactivator that contains HAT activity, and in addition to histones, it can acetylate transc ription factors, as well as itself (Roth et al., 2001). The consequence of its interaction with Mediator is an elev ation in histone acetyl ation (Black et al., 2006), which makes chromatin more accessibl e to other factors (Roth et al., 2001). Autophosphorylation of p300 reduces its associati on with Mediator. The association of TFIID with Mediator competes with p300 binding and results in a displacem ent of p300 from the promoter. The joining of Mediator with TFIID contributes to the assembly of the PIC and activating of the promoter (Black et al., 2006).

PAGE 23

23 Mediator Promotes the Form ation of a Stable PIC In vitro and genetic evidence suggest that Mediator contributes to the formation of a stable PIC. It has been shown by a template commitment assay that Srb2 (Med20) is essential for the formation of the PIC (Koleske et al., 1992). In addition, mutations in Srb2 (Med20), Srb4 (Med17), or Srb5 (Med18) prevent the formation of the PIC (Ranish et al., 1999), and mutations in Sin4 (Med16) and Pgd1 (Med3) decrease both th e rate and amount of PIC formation in yeast (Reeves and Hahn, 2003). Mediator is Required in the Reinitiation Scaffold The association of Mediator with RNA pol II CTD, Gal11 with TFIIH (Sakurai and Fukasawa, 2000), and Srb2 with TFIID (Koleske et al., 1992) facilitate the formation of a stable PIC and maintain the reinitiati on scaffold (Nair et al., 2005). Reinitiation and then multiple rounds of transcription occur afte r RNA pol II, TFIIB, and TFIIF join the scaffold to re-form the PIC (Nair et al., 2005). Mutation of Pgd1 results in dissociation of Mediator from the scaffold after initiation and, thus, impairs reinitiation in ye ast (Reeves and Hahn, 2003). Mediator Stimulates both Basal and Activated Transcription The Mediator fraction from yeast has been shown to stimulate GAL4-VP16 or GCN4-dependent transcription in a reconstitute d system, and has also been shown to increase basal transcription by 8-fold (Kim et al., 1994). The ARC (activator-recruited cofactor) complex not only stimulates transcription by activat ors such as SREBP-1a/Sp1, NF-kB/Sp1 and Gal4-VP16/Sp1, but also enha nces basal transcription in vitro (Naar et al., 1999). Genome-wide expression analysis showed that only 7% of genes were expressed in the Med17 mutant of S. cerevisiae (Holstege et al., 1998). Dimi nished Mediator leads to the reduction of basal and activator-dependent transcription in yeast and HeLa cells, which can be restored by addition of purified Mediator complex in vitro (Baek et al., 2002, Nair et al., 2005).

PAGE 24

24 Model for Mediator Function in Activated Transcription Formation of the PIC starts with the bindi ng of transactivators to the DNA, which is followed by recruitment of TFIID, TFIIA and TFIIB to the promoter (Ranish et al., 1999, Reeves and Hahn, 2003; Woychik et al., 2002). The Mediator is recruited by trans activators and possibly by coactivators, such as p300 and TFIID (Koleske et al., 1992; Thompson et al., 1993; Johnson et al., 2002; Johnson and Carey, 2003; Black et al., 2006). Mediator and TFIID form a platform for the entry of the following factors. Mediator recruits the RNA pol II through interaction with the CTD. TFIIF may be enlisted together with RNA pol II. Then TFIIE and TFIIH enter the preinitiation complex (Thomas and Chiang, 2006) Next, the DNA helicase activity of TFIIH causes promoter melting (Jiang et al., 1996; Kim et al., 2000), an essential step before the synthesis of RNA can begin. Mediator greatly en hances the kinase activity of Kin28 of TFIIH, which hyperphosphorylates the RNAP II CTD (Gui di et al., 2004). Afte r CTD phosphorylation, RNA pol II leaves the promoter w ith TFIIF to start transcriptio nal elongation (Yan et al., 1999; Shilatifard et al., 2003). Mediator, TFIIA, TFIID, TFIIH and TFIIE stay on the promoter forming a platform that supports reinitiati on. This scaffold structure, in turn, recruits new TFIIB, TFIIF and RNA pol II repeatedly to support multiple ro unds of transcription (Yudkovsky et al., 2000). Hypothesis for a Mediator Complex in Arabidopsis Many of the basic mechanisms of transcrip tion are conserved in plants, metazoans and yeast (Reviewed in Gurley et al., 2006). The structures of many promoters in these three kingdoms contain a TATA box, CAAT box, transcription start site and cis -elements for the binding of general transcription factors and transactivators. In addition, RNA pol II and many general transcription factors ar e conserved between plants, f ungi and metazoans (Coulson and Ouzounis, 2003). Arabidopsis also has homologs of the subunits of some coactivators such as SAGA and other HAT containing complexes (Hsi eh and Fischer, 2005). This wide array of

PAGE 25

25 evidence for a high degree of conservation in the ba sic mechanisms of tran scription suggests that plants may also contain the Mediator coactivat or. This view is strongly reinforced by the presence of many putative Mediator subunits in Arabidopsis based on DNA sequence similarity (Gurley et al., 2006; Boube et al., 2002). A comp ilation of Mediator subunits from yeast, Drosophila and humans is presented in Table 12, along with putative subunits from Arabidopsis This provides the best estimate for Mediator s ubunit composition in plan ts and indicates that plants may have at least 20 Mediator subunits present in other eukaryotes. Despite evidence suggesting such a vital role for Mediator in gene expression, the precise subunit composition and function of Mediator has not been determined in plants. Up to now, two putative Mediator subunits in Arabidopsis thaliana have been studied. SWP (Struwwelpter) is the orthologue of Med14 and is involved in patt ern formation at the shoot apical meristem, as well as defining the duration of cell prolifera tion (Autran et al., 2002) PFT1 (phytochrome and flowering time 1) is the orthol ogue of Med25. It acts downstream of phyB to regulate the gene expression and induce flowering under low-li ght conditions (Cerda n and Chory, 2003). The important functions of these two putative Mediat or subunits hint at th e significance of the Mediator in plants. To unravel the mechanism of gene transcription in plants, it is important to identify the Mediator complex and characterize its function.

PAGE 26

26 Table 1-1. Interaction of the transactivators with the Mediator subunits in different organisms Transactivator Homo sapiens Saccharomyces cerevisiae Drosophila melanogaster ER and ER estrogen receptor (ER) Zhu et al., 1999; Burakov et al., 2000; Warnmark et al., 2001 GATA family of transcription factors Crawford et al., 2002 Breast cancer susceptibility gene 1 (BRCA1) Wada et al., 2004 Thyroid hormone receptor (TR TR 1) Yuan et al., 1998, Zhu et al., 1997 Androgen receptor Wang et al., 2002 Glucocorticoid receptor (GR) Hittelman et al., 1999 Peroxisome proliferator-activated receptors (PPAR and PPAR ) Zhu et al., 1997 Retinoic acid receptor (RAR ) Zhu et al., 1997 Retinoid-X-receptor for 9cis -retinoic acid (RXR ) Zhu et al., 1997 Vitamin D receptor (VDR) Rachez et al., 1999 Hepatocyte nuclear factor 4 (HNF-4) Malik et al., 2002 Farnesoid X receptor (FXR) Pineda et al., 2004 Retinoid-related orphan receptor (ROR ) Atkins et al., 1999 p53 Drane et al., 1997 Med1 Aryl hydrocarbon receptor (AHR) Wang et al., 2004 Med3 General control nondepressible factor 4 (GCN4) Park et al., 2000 SRY-box containing gene 9 (Sox9) Zhou et al., 2002 Med12 Replication and transcription activator (RTA) Gwack et al., 2003 Glucocorticoid receptor (GR) Hittelman et al., 1999 Hepatocyte nuclear factor 4 (HNF-4) Malik et al., 2002 Signal transducer and activator of transcription (STAT2) Lau et al., 2003 Med14 Sterol regulatory element-binding protein-1a (SREBP-1a) Toth et al., 2004

PAGE 27

27 Table 1-1. Continued. Transactivator Homo sapiens Saccharomyces cerevisiae Drosophila melanogaster Small mothers against decapentaplegic 2/3/4 (SMAD2, SMAD3, SMAD4) Kato et al., 2002 VP16 Lee et al., 1999; Park et al., 2000 General control nondepressible factor 4 (GCN4) Lee et al., 1999; Park et al., 2000 Med15 Gal4 Park et al., 2000 Med16 Differentiation-inducing factor (DIF) Kim et al., 2004 p53 Ito et al., 1999 VP16 Ito et al., 1999 Signal transducer and activator of transcription (STAT2) Lau et al., 2003 Differentiation-inducing factor (DIF) Kim et al., 2004 Med17 (Srb4) Heat-shock factor (HSF) Kim et al., 2004 Early region 1A (E1A) Boyer et al., 1999; Wang and Berk, 2002 ETS-like kinase protein-1 (Elk-1) Stevens et al., 2002 Epithelial-restricted with serine box (ESX) Asada et al., 2002 CCAAT/enhancer binding protein (C/EBP) Mo et al., 2004 Differentiation-inducing factor (DIF) Kim et al., 2004 Med23 HSF (heat-shock factor) Kim et al., 2004 Differentiation-inducing factor (DIF) Kim et al., 2004 Heat-shock factor (HSF) Kim et al., 2004 Med25 VP16 Mittler et al., 2003 Med29 Doublesex (dsxF) Garrett-Engele et al., 2002 Cdk8 Myc Eberhardy and Farnham, 2002

PAGE 28

28 Table 1-2. Mediator subunits in yeast, Arabidopsis Drosophila and humans (Gurley et al., 2006; Boube et al., 2002) Unified nomenclature (Bourbon et al., 2004) Saccharomyces cerevisiae Arabidopsis thaliana Drosophila melanogaster Homo sapiens MED1 Med1 Trap220 TRAP220-ARC/DRIP205 MED2 Med2 MED3 Med3 MED4 Med4 At5g02850 Trap36 TRAP36-ARC/DRIP36 MED5 Nut1 MED6 Med6 At3g21350 Med6 hMed6-ARC/DRIP33 MED7 Med7 At5g03220 Med7 ARC/DRIP34-CRSP33 MED8 Med8 Arc32 ARC32 MED9 Cse2/Med9 MED10 Nut2/Med10 At5g41910/ At1g26665 Nut2 hNut2-hMed10 MED11 Med11 Med21 HSPC296 MED12 Srb8 At4g00450 Kto TRAP230 ARC/DRIP240 MED13 Srb9 At1g55325 Skd/Pap/Bli TRAP240 ARC/DRIP250 MED14 Rgr1 At3g04740 (SWP1) Trap170 TRAP170-DRIP/CRSP150 MED15 Gal11 At1g15780 Arc105 ARC105 MED16 Sin4 Trap95 TRAP95-DRIP92 MED17 Srb4 At5g20170 Trap80 TRAP80-ARC/DRIP77 MED18 Srb5 At2g22370 P28/CG14802 p28b MED19 Rox3 CG5546 LCMR1 MED20 Srb2 At4g09070/ At2g28230 Trfp hTrfp MED21 Srb7 At4g04780 Trap19 hSrb7 MED22 Srb6 At1g07950/ At1g16430 Med24 Surf5 MED23 At1g23230 Trap150 hSur2/CRSP130 MED24 Trap100 TRAP/CRSP/DRIP100 MED25 At1g25540 (PFT1) Arc92 ARC92 MED26 At3g48060/ At3g48050 Arc70 CRSP70-ARC70 MED27 At3g09180 Trap37 TRAP37-CRSP34 MED28 Med23 Fksg20 MED29 Intersex Hintersex MED30 Trap25 TRAP25 MED31 Soh1 At5g19910 Trap18 hSoh1 CDK8 Srb10 At5g63610 CDK8 CDK8 CycC Srb11 At5g48640 CycC CycC

PAGE 29

29 CHAPTER 2 MATERIALS AND METHODS Plant Growth Conditions The ecotype of Arabidopsis thaliana used in this study was Co lumbia-0. The plants were grown in soil with continuous light from 40 W fluorescent bulbs at 27C. To examine the germination and root length, the seeds were gr own on vertical agar plates. The seeds were surface sterilized with 70% ethanol for 3-5 min, and then with 10% bleach for 15-20 min. After rinsing with sterile water (3 X 5 min), the seeds were plated in petri di shes containing 1/2 MS (Murashige & Skoog) medium supplemented w ith 1% sucrose, 0.5g/L MES (2-(N-morpholino) ethanesulfonic acid) and 0.8% agar. The plates were sealed with parafilm a nd placed vertically in a growth chamber with a 16h light / 8h dark cycl e provided by 40 W fluore scent bulbs at 22C. For dark treatment, the plates were wrapped in aluminum foil and placed ve rtically in a growth chamber at 22C. Genotyping of the T-DNA Insertion Lines The med31 T-DNA insertion mutants ( med31-1 and med31-2 ) were obtained from Arabidopsis Biological Resource Center (ABRC). For the genotyping of med31-1 Med31 -specific primer 5TGGATGTAAGTAGGATTGGCG -3 was paired with the T-DNA-specific primer LBb1 5-GCGTGGACCG CTTGCTGCAACT-3 to produce a 628 base pair (bp) fragment by polymerase ch ain reaction (PCR), or with another Med31 -specific primer 5GAACTTGTCTTGGCAAGTTGG -3 to produce a 975 bp fragment. For the genotyping of med31-2 Med31 -specific primer 5TGATGTACTCTGGT CGCTGC -3 was paired with the T-DNA-specific primer LBb1 5-GCGTGGA CCGCTTGCTGCAACT-3 to produce a 714 bp fragment, or with another Med31 specific primer 5-TTGCGGGGATTACAACATTAC-3 to

PAGE 30

30 produce a 1008 bp fragment. The T-DNA insertion sites were determined by sequencing the PCR products. RNA Analysis The leaves of 40-day-old plants grown on soil were collected and R NA was isolated with the Concert Plant RNA Reagent (Invitrogen). RNA bl ots were prepared as described by Cao et al. (1994) and probed with full-length Med31 cDNA. Microscopy A Zeiss Axiocam HRm camera was used to examine the subcellular localization of Med31-GFP fusion proteins in the root tip. GFP fl uorescence was monitored with the Zeiss filter set 10 (excitation, 450 to 490; dichro ic, 510 LP; emission, 515 to 565). DAPI (4',6-diamidino-2-phenylindole) fluorescence was monitored with Zeiss filter set 02 (excitation, 365; dichroic, 395 LP; emission, 420 LP). A Zei ss LSM 5 Pascal confocal laser scanning microscope was used to localize the Med31::GFP fusion proteins in the plant tissues with an Argon 488 nm laser and a Band Pass 505-530 filter. A Helium Neon 543 nm laser with a 560 nm filter was used to record chlorophyll autofluorescence. Plasmid Construction The pBI101sGFP(S65T) vector wa s provided by Dr. Robert Ferl (Manak et al., 2002). This vector was constructed by removing the GUS ( -glucuronidase) gene by digestion with restriction endonucleases XbaI a nd SacI, and then inserting th e sGFP(S65T) gene between the two restriction sites (Manak et al., 2002). The Med31 gene, including the 1.2 kb upstream sequence and the entire exon and intron region without the stop codon, was amplified from the genomic DNA by PCR using the primers 5 -tatTGTCGACTCTAATTA ATCAGTCTTGGTC-3 and 5agaTCTAGATATACCCTTCCTGACATTATATG ACT -3. The fragment was inserted in-frame to the 5 end of the sGFP(S65T) gene in the pBI101sGFP(S65T) vector using the SalI

PAGE 31

31 and XbaI sites. The Med31 1.2 kb upstream sequence was generated from the genomic DNA by PCR using the pimers 5-tatTGTC GACTCTAATTAATCAGTCTTGGTC-3 and 5-ttataTCTAGAGAACGAACG GAACCTGAAGC-3. This fragment was inserted in-frame to the 5 end of the GUS gene in the pBI101 vector using the SalI and XbaI sites. All of the PCR amplified fragments were confirmed by DNA sequencing. GUS Staining The tissues were immersed in GUS Staining Solution (1 M sodium phosphate (pH 7.0), 0.5 M EDTA (ethylenediaminetetraacetic acid), 50 mM K+ ferricyanide, 50 mM K+ ferrocyanide, 10% Triton X-100 and 2 mM X-gluc) and vacuum infiltrated for 20 min. The samples were incubated at 37 C until blue color appeared. As a final step, 70% ethanol was used to clear the tissue. Agrobacterium Transformation Technique The binary vector was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation and the T-DNA transferred to Arabidopsis plants via the standard floral dip protocol (Clough and Bent, 1998). Agrobacterium starter cultures were grown in 30 ml LB (Loria broth) liquid culture medium with 25 g/ml gentamicin, 50 g/ml rifampicin and 50 g/ml kanamycin with shaking (250rpm) at 28 C overnight. A 15 ml aliquot of the starter culture was added to 150 ml of LB liquid medium containing 25 g/ml gentamicin, 50 g/ml rifampicin and 50 g/ml kanamycin, and the culture was in cubated with shaking (250 rpm) at 28 C until an OD600 of 0.8 was reached. The cells were collected by centrifugation (5000 g, 30 min) and resuspended in 150 ml of 5% sucrose. After addition of 30 l of Silwet L-77 detergent, the 3-week-old Arabidopsis plants were dipped in the Agrobacterium solution for several sec, with gentle agitation. The plants were covered overnight to keep high humidity. Transformants

PAGE 32

32 were selected by germinating the seeds on plat es containing 1/2 MS medium with 50 mg/L kanamycin. Chromatin Immunoprecipitation The putative Mediator subunits were ma pped to promoter DNA using chromatin immunoprecipitation (ChIP) according to Ge ndrel and colleagues (2005), with minor modifications. The aerial parts of Arabidopsis plants were harvested (1.5-2.0 g) and rinsed with water. The sample was then placed in 37 ml of 1% formaldehyde for cross-linking and vacuum infiltrated for 15 min at room temperature. Th e reaction was quenched by the addition of 2.5 ml of 2 M glycine, and the sample was placed under vacuum for an additional 5 min. The tissue was rinsed thoroughly, frozen in liquid nitrogen a nd stored at -80 C until further treatment. Chromatin was extracted by grinding the fro zen samples in 30 ml of Extraction Buffer 1 (0.4 M sucrose, 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM -mercaptoethanol, 0.1 mM PMSF (phenylmethylsulphonyl fluoride), 1 X pr otease inhibitor). (To make 200 X Protease Inhibitor, dissolve 0.16 g TPCK (tosyl phenyl alanyl chloromethyl ketone) and 0.16 g TLCK (tosyl-L-lysine chloromethyl ketone) in 5 ml of DMSO (dimethyl sulfoxide), then dissolve in 10 ml of 0.2 M PMSF in isopropanol.) Next, the sample solution was f iltered with Miracloth (CalBiochem) and then centrifuged at 3000 X g at 4 C for 20 min. The pellet was dissolved with 1 ml of Extraction Buffer 2 (0.25 M sucros e, 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1% Triton X-100, 5 mM -mercaptoethanol, 0.1 mM PMSF, 1 X protease inhibito r) and centrifuged at 12,000 X g at 4 C for 10 min. After that, the pellet was resuspended with 300 l of Extraction Buffer 3 (1.7 M sucrose, 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 0.15% Triton X-100, 5 mM -mercaptoethanol, 0.1 mM PMSF, 1 X prot ease inhibitor), placed on another 300 l of extraction buffer 3, and centrifuged at 14,000 X g at 4 C for 1 hr. The pellet was resuspended with 300 l of Nuclei Lysis Buffer (50 mM Tris-H Cl (pH8.0), 10 mM EDTA, 1% SDS (sodium

PAGE 33

33 dodecyl sulfate), 1 X protease inhibitor), and the chromatin was sheared to a size of 150 bp to 750 bp by sonication (10 times for 15 sec each at an amplitude setting of 20 using a Tekmar Sonicator). The sample was centrifuged at 12,00 0 X g for 10 min and ChIP Dilution Buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris -HCl (pH 8.0), 167 mM NaCl) was added to the supernatant to make a final volume of 3 ml. The solution was divided into three tubes and 40 l of protein A-agarose (Santa Cr uz) was added to each tube of sa mple for pre-clearing at 4 C for 1 hr with gentle agitati on. The protein A-agarose was re moved by centrifugation (12,000 X g at 4 C for 30 sec), and the supernatan t was transferred to fresh tubes. A 60 l aliquot was saved at -20 C as the Input DNA control. The immunoprecipitation was set up as follows: 10 l of IgG (Immunoglobulin G) Sepharose (Amersham Biosciences) and 10 l of c-Myc (cellular myelocytomatosis oncogene) antibody (Santa Cruz) were added, respectively, to two tubes to precipitate the TAP-tagged Mediator subunits. No antibody was added to the third sample, which served as the no antibody control. The tubes were incubated at 4 C overn ight with gentle agitati on. In order to purify Mediator-bound complexes, 50 l of protein A-agarose beads we re added to the tubes with c-Myc antibody and no antibody cont rol, respectively, and the thr ee tubes were incubated at 4 C for 1 hr with gentle agita tion. The agarose beads were pell eted by centrifugation at 3800 X g at 4 C for 30 sec and washed sequentially with Low Salt Wash Buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris -HCl (pH 8.0)), High Salt Wash Buffer (500 mM NaCl, 0.1% SDS, 1% Trit on X-100, 2 mM EDTA, 20 mM Tris -HCl (pH 8.0)), LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% DOC ( 21-hydroxyprogesterone), 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)) and TE buffer. In order to ex tract the immune complex from the beads, 250 l of elution buffer (1% SDS, 8.4 mg/ml NaHCO3) was added to each sample, followed by

PAGE 34

34 incubation at 65 C for 15 min with gentle agita tion. The elution step was repeated once to reach a final volume of 500 l. Elution buffer (440 l) was also added to the 60 l of the Input DNA control. After adding 20 l of 5 M NaCl to each sample, the cross-linking was reversed by incubation at 65C overnight. The pr oteins in the sample were di gested by incubation with 10 l of 0.5 M EDTA, 20 l of 1 M Tris-HCl (pH6.5), and 2 l of 10 mg/ml proteinase K at 45C for 1 hr. Then the proteins were removed from the DNA by phenol/chloroform extraction, and the DNA was precipitated with the a ddition of ethanol (2.5 volume), sodium acetate (1/10 volume, pH5.2) and 20 g of glycogen. The DNA was resuspended with 50 l of 10 mM Tris-HCl (pH7.5). PCR Analysis of Chromatin Immunoprecipitation The immunoprecipitated fraction was analyzed by PCR amplification to determine if the DNA fragments from various prom oters were present. The 25 l of PCR reaction system contained 12.5 pmol of each primer, 5 nmol of dNTP, 3 l of DNA sample, 2.5 l of 10 X PCR Buffer I and 1 unit of AmpliTaq Gold (Applied Biosystems, Foster Cit y, CA, USA). The cycling conditions were 8 min of thermal activation at 95 C, followed by 50 cycles of 94 C (30 sec), 55 C (30 sec), and 72 C (3 min). The prim ers used for PCR were as follows: 5cgtggcctagaatacaaagaag -3 and 5tcaaacaataagaaagaccatga ca -3 were used for the amplification of the CCA1 promoter; 5-agattgttgacattctcggaaatttagtgccaactgt-3 and 5-aaatgctcctttttctaaaacc ttcgcttggagtct-3 for the amplification of Hsp18.2 promoter; 5-acaccacggcgtgaccat-3 and 5-attatccagtcga catctgta-3 for the amplification of Adh1 promoter; and 5TGTTTCTTCCCTTTAAGCAACC-3 and 5AACATTTCTTTAGAACATTGAC TTGG-3 were used to amp lify the intergenic region between At2g32950 and AT2G32960

PAGE 35

35 Bioinformatics Multiple protein sequence alignments were pe rformed with AlignX, which is a component of Vector NTI Advance 10.3.0 from Invitrogen. The accession numbers at the NCBI (National Center for Biotechnology Information) for the Med31 homologs are XP_307924 ( Anopheles gambiae ), NP_197491 ( Arabidopsis thaliana ), BQ583133 ( Beta vulgaris ), CD834180 ( Brassica napus ), NP_492413 (Caenorhabditis elegans), EAK92332 ( Candida albicans ), DY287612 ( Citrus clementina ), CX051496 ( Citrus sinensis ), EAU91557 ( Coprinopsis cinerea ), XP_626881 ( Cryptosporidium parvum ), DR063080 ( Cycas rumphii ), XP_638330 ( Dictyostelium discoideum ), NP_649483 ( Drosophila melanogaster ), CAD25946 ( Encephalitozoon cuniculi ), DV154959 ( Euphorbia esula ), BM892402 ( Glycine max ), DT547393 ( Gossypium hirsutum ), CO126156 ( Gossypium raimondii ), NP_057144 ( Homo sapiens ), DW049205 ( Lactuca saligna ), DW126099 ( Lactuca sativa ), CF393635 ( Loblolly pine ), BQ147110 ( Medicago truncatula ), NP_080344 ( Mus musculus ), DY336178 ( Ocimum basilicum ), CA902198 ( Phaseolus coccineus ), CF808645 ( Phytophthora sojae ), DR501487 ( Picea sitchensis ), CV015282 ( Rhododendron catawbiense ), NP_011388 ( Saccharomyces cerevisiae ), NP_587859 ( Schizosaccharomyces pombe ) and CAD21541 ( Taenia solium ). The accession numbers for the Med6 homologs are XP_319180 ( Anopheles gambiae ), NP_188772 ( Arabidopsis thaliana ), NP_504791 ( Caenorhabditis elegans ), EAK97077 ( Candida albicans ), XP_638621 ( Dictyostelium discoideum ), NP_731403 ( Drosophila melanogaster ), XP_965884 ( Encephalitozoon cuniculi ), NP_005457 ( Homo sapiens), NP_081489 ( Mus musculus ), NP_001057150 ( Oryza sativa ), NP_011925 ( Saccharomyces cerevisiae ) and Q9US45 ( Schizosaccharomyces pombe ).

PAGE 36

36 CHAPTER 3 RESULTS Med31/Soh1 is a mediator subunit that has be en identified in humans (Gu et al., 1999), Drosophila (Park et al., 2001), S. pombe and S. cerevisiae (Linder and Gustafsson, 2004). Soh1 (suppressor of hpr1) was first id entified as a suppressor of the S. cerevisiae hpr1 mutant which is temperature-sensitive for growth and can reduce the hyperrecombination phenotype (Fan and Klein, 1994). Yeast two hybrid analysis showed Soh1 interacts with the Rad5p protein, and a Soh1 mutation exacerbated the DNA repair defect of a rad5-535 mutant (Fan et al., 1996). The Soh1 orthologue Sep10 in S. pombe was identified in screening mu tants for both sterility and for defects in cell separation. Sep10 mutants are temperature-sensitive. At a non-permissive temperature (36 C), the mutants formed multip le, ill-organized septa (Grallert et al., 1999). Med31/Soh1 was identified in the Mediator complex in humans (Gu et al., 1999) and Drosophila (Park et al., 2001), but its functi on has not been determined, and the exact relationship between mutations in Med31 and phenotype is still not clear. Analysis of Arabidopsis Med31 Gene by Multiple Sequence Alignments The Med31 homologs are pres ent in the protists ( Cryptosporidium parvum Dictyostelium discoideum Encephalitozoon cuniculi ), fungi ( Candida albicans Coprinopsis cinerea Saccharomyces cerevisiae Schizosaccharomyces pomb ), metazoans ( Anopheles gambiae Caenorhabditis elegans Drosophila melanogaster Homo sapiens Mus musculus Taenia. solium ) and plants ( Arabidopsis thaliana Oryza sativa ) (Figure 3-1). Based on sequence homology, we identified AT5G19910 in Arabidopsis as the putative Med31 gene (AtMed31), which contains 6 exons and en codes a protein of 196 amino acids (aa), with a calculated molecular mass of 22.8 kDa. There is a conserved block of 70 aa (aa R30 to R99 of AtMed31) (Figure 3-1) that shows high simila rity to Med31 in other species ( D. melanogaster 58.3%

PAGE 37

37 identity and 76.4% similarity; H. sapiens 61.1% identity and 72.2% similarity; S. cerevisiae 45.2% identity and 56.2% similarity; S. pombe 59.7% identity and 77.8% similarity), where its functional identity has been demonstrated. Linder and Gustaf sson (2004) showed that this region is required for its assembly within the Mediator complex in S. cerevisiae which suggests this conserved domain is important for inte raction with other Mediator subunits. A search of the NCBI EST database using TBLASTN (Altschul et al., 1997) for AtMed31 identified the Med31 homologs in many other plan t species (Figure 3-2). There is a conserved block of 139 aa (aa M1 to V139 of AT5G19910) between these plant Med31 homologs, which includes the 70 aa domain conserved between differe nt species (Figure 3-1). The C-termini of the Med31 homologs are less conserved compared with their N-termini (Figure 3-1 and Figure 3-2), and often contain regions that resemble transcriptional activation domains which have glutamine-rich or serine/proline-rich blocks. Since these domains function in transactivator proteins to make contact with ta rget transcription factors, it se ems reasonable to assume that the C-terminal region of Med31 containing these activa tor domain-like blocks may be at the outside of the complex and provides surface for interaction with transactivators or other transcriptional machinery. Phenotype Characterization of med31 Mutants The Mediator plays a vital role for RNA pol II-m ediated transcription; therefore, disruption of the highly conserved Med31 subunit is predicte d to have a disruptive effect on the expression of a large number of genes, some leading to abnormal phenotypes. To test this hypothesis and study the function of this putative subunit, we searched the SIGnAL (signal.salk.edu) T-DNA insertion collection for mutants (Alonso et al., 2003). Four T-DNA insertion lines were identified and ordered from the Arabidopsis Biological Resource Cent er (ABRC). The T-DNAs of Salk035522 and Salk051025 lines insert into the promoter and 3 UTR (untranslated region) of

PAGE 38

38 Med31 respectively, but we did not observe any mutant phenotype for these two lines. The med31-1 (Salk145479) mutant line has the T-DNA inse rtion in the promoter region, and the T-DNA of med31-2 (Salk 143815) mutant line is located wi thin the 5 UTR (Figure 3-3). The insertion sites for all Salk lines were confirmed by DNA sequencing. In contrast to the previo us two mutant lines, both med31-1 and med31-2 plants showed abnormalities in growth. Under our experiment al conditions, the germination rate of med31-1 seeds was 17% compared with 100% for the wild type (WT). In addition, their root length was 41.6% of the WT root length (Figure 3-4). The seeds of med31-2 plants germinated as well as the WT seeds; however, their root length was 47.7% of the WT root length (Figure 3-5). These differences in growth were not present under dark conditions, where the med31-2 seedlings grew as well as the WT seedlings (F igure 3-6). This result suggests that the function of Med31 during seed initial development may be dependent on light. Some of the med31-2 plants had aberrant patterns of co tyledon development, such as three cotyledons and three first true leaves, a single cotyledon, or fork ed cotyledons (Figure 3-7). However, these mutant phenotypes were only inherited by some of their progeny, and the med31-2 mutants with normal cotyledons also pr oduced progeny with abnormal cotyledons. The seedlings with abnormal cotyledons segregated 26% (5/19) progeny with abnormal cotyledons; whereas the seedlings with normal cotyledons se gregated 30% (5/20) progeny with abnormal cotyledons. In addition, the overall sizes of the med31-2 mutants were reduced, their leaves were smaller, and they had fewer rosette leaves compared with WT plants (Figure 3-8). Med31 Expression in the med31-2 Plants In med31-2 plants, the T-DNA inserts into the 5 UTR of the gene and may, therefore, influence the expression of Med31 gene at either the transcrip tional or translational level. Northern blotting was used to examine the expression of Med31 in the WT and med31-2 plants.

PAGE 39

39 In med31-2 mutant plants, the corresponding mRNA was mo re abundant than that in WT plants (Figure 3-9). The mutant phenotype of med31-2 plants may be caused by the overexpression of Med31 protein, which possibly sequesters the adj acent Mediator subunits or other components of the transcriptional apparatus. Alternatively, Me d31 translation may be inhibited due to the missing, or changed nucleotides at the 5 end of the transcript. Subcellular Localization and Tissue Expression Pattern of Med31::GFP Fusion Proteins To investigate the subcellu lar localization of Med31, the 1.2 kb upstream sequence and the entire exon and intron region of Med31 (without the stop codon) was amplified from the genomic DNA by PCR. The resulting DNA was ligated in-frame to the 5 end of the sGFP(S65T) gene in the pBI101 sGFP(S65T) vector. We obser ved that the Med31::GFP fusion proteins were expressed in the tip of primary roots and that the signal was c onfined to the nucleus (Figure 3-10). In addition to being expre ssed in the lateral root tips, pr imordia (Figure 3-11) and root hairs (Figure 3-12), the Med31::GFP signal was also found to be pres ent in the aerial portions of the plants including leaves (Fi gure 3-13), trichomes (Figure 314) and petioles (Figure 3-15). Free GFP has been shown to be present in both th e nucleus and cytosol (Li et al., 2001; Ye et al., 2002; Zhong et al., 2005). To further complicate anal ysis, GFP has been shown to move to other cells and tissues via the plasmodesmata (Crawfor d and Zambryski, 2001). However, in each type of tissue we observed, signal from the Med31::G FP fusion protein was almost exclusively found in the nucleus. The nuclear local ization exhibited by Med31::GFP is consistent with its presence in the nucleus being a property conferred by the Med31 portion of th e protein, as contrasted with the more general subcellular localiza tion previously shown for GFP alone. Tissue Expression Pattern of Med31 Promoter::GUS Fusions To investigate the tissue expres sion pattern of Med31 protein, the Med31 promoter was fused to the 5 end of the -glucuronidase (GUS) gene and transferred to Arabidopsis The GUS

PAGE 40

40 signal was detected in the shoot apexes (Figure 3-16A) and lateral root primordia (Figure 3-16B) of young seedlings (16 days old) of transformed plants. It was also de tected in the whole young inflorescences (Figure 3-17A), anthers (Figure 317B) and stigmas (Figure 3-17C) of adult plants (46 days old) and in developing seeds (Figur e 3-17D). This pattern differs from where Med31::GFP signal was detected in that no GUS si gnal was detected in the primary root tips, leaves and petioles. Two possibl e explanations for this appare nt inconsistency are that GUS staining sensitivity may be less than that of GFP. Alternatively, the promoter DNA alone as present in the Med31::GUS cons truct (without the e xons, introns and untranslated regions) is insufficient to fully reproduce the expression pattern of the endogenous Med31 gene. Co-immunoprecipitation Maps Med6 and Med31 to Promoter DNA Chromatin immunoprecipitation (ChIP) is a powerful tool to explore in vivo protein-DNA interactions. The ChIP assays conducted here involves the cross-linking of proteins and DNA in chromatin, followed by co-immunoprecipitation of DNA fragments associated with the epitope-tagged Mediator subunits. After the pr oteins have been removed, the pool of DNA fragments can be queried by PCR amplification for the presence of specific promoter regions. Mediator associates with pr omoter DNA indirectly by binding with transactivators and RNA pol II. Previous studies in yeast using th e ChIP technique (Andrau et al., 2006, Zhu et al., 2006) showed that Mediator could not only bind to the core promoters and upstream activating sequences, but also to the coding regions of ma ny genes, as well. It can associate with the promoters of both active and some inactive genes, but genes with higher transcriptional activity usually have higher promoter occupancy by Mediator It is thought that th e presence of Mediator at inactive promoters may be required for quick response to environmental changes. If Med31 is a genuine mediator subunit, it should be found associated with promoter DNA. We used the ChIP assay to test this hypot hesis. In addition, we checked if another Arabidopsis

PAGE 41

41 putative Mediator subunit, Med6 (AT3G21350), was also localized to promoter DNA. As with Med31, the assignment of Arabidopsis Med6 as a putative subunit of Mediator was based strictly on protein sequence homology (Figure 3-18). Ther e is a conserved block of 129 aa (aa M33 to S161 of AtMed6) that shows similar ity to Med6 in other species ( D. melanogaster 36.4% identity and 51.2% similarity; H. sapiens 42.6% identity and 55.0% similarity; S. pombe 31.1% identity and 48.1% similarity) where its functi onal identity has been demonstrated. It was predicted to be localized in the nucleus by two web tools (Hua and Sun, 2001; Nair and Rost, 2002). Our prediction is that both proteins we have tentativel y identified as AtMed31 and 6, respectively, should be associated with the pr omoter regions of a wide array of genes. The Med6 and Med31 cDNAs were introduced in to the pC-TAPa vectors (Rubio et al., 2005) and individually transformed into Arabidopsis by Dr. Kevin OGra dy (Gurley laboratory, University of Florida). Their C-termini were fused with nine repeats of the myc epitope, followed by six histidine residues, the 3C protease cleavage site and two copies of the protein A IgG binding domain. The fusions of Med6 or Me d31 with the epitope tags were confirmed by Western blots. Immunoglobulin G Sepharo se and c-Myc antibody were used to immunoprecipitate the tagged Med6 or Med31 proteins, respectivel y, in the ChIP experiment. ChIP Analysis for Med31 Immunoglobulin G Sepharose was used to immunoprecipitate Med31-DNA complexes from the T1 generation of Med31-pC-TAPa transgenic Arabidopsis plants. Primer pairs for the promoters of CCA1 (AT2g46830), Hsp18.2 (AT5g59720), Adh1 (AT1g77120) and a fragment in the intergenetic region (betw een AT2g32950 and AT2g32960) were used to test if Med31 binds to these sequences. The CCA1 ( circadian clock associated 1 ) gene encodes a MYB-related transcription factor and its expression oscillat es with a circadian rhythm (Wang and Tobin, 1998). Hsp18.2 ( heat shock protein 18.2 ) is a heat inducible gene. Adh ( alcohol dehydrogenase ) is also

PAGE 42

42 an inducible gene regulated by environmental stresses, such as low oxygen, dehydration, and low temperature (Dolferus et al., 1994). Both Hsp18.2 and Adh genes are expressed at low levels under normal conditions (Volkov et al., 2003; Dolferus et al., 1994). Mining of Arabidopsis EST database failed to find the transcripts of th e intergenetic region (>4kb) between At2g32950 and AT2G32960, suggesting this region is not transcribed. Therefore, we used this region as a negative control which Mediator may not bind. The promoters of CCA1 Hsp18.2 and Adh1 were all co-immunoprecipitated with the epitope tagged Med31 prot ein by IgG Sepharose (Figure 3-19), demonstrating the localization of Med31 to th ese promoters. As pred icted, the intergenetic region was not co-immunoprecipitated with the tagged Med31 by IgG Sepharose. ChIP Analysis for Med6 Immunoglobulin G Sepharose and c-Myc an tibody were used individually to immunoprecipitate Med6DNA complexe s from the T2 generation of Med6-pC-TAPa transgenic plants. The same set of primer pairs were used for the amplification from the DNA pool derived from co-immunoprecipitation with epit ope tagged Med6. The promoters of CCA1 Hsp18.2 and Adh1 were all co-immunoprecipita ted with Med6 by both IgG Sepharose and c-Myc antibody (Figure 3-20), demonstrating the localization of Med6 with these promoters. Again, as predicted, the intergenetic region was not co-immunoprecipitated with Med6. It should be noted that both Med6 and Med31 were independently found to be localized to the promoters of three unrelated genes, CCA1 Hsp18.2 and Adh1, a finding consistent with both proteins belonging to a Mediator complex. Wild type plants were also included in ChIP experiment as a negativ e control to check if IgG Sepharose and c-Myc anti body can immunopr ecipitate the CCA1 promoter in the absence of epitope tagged Mediator subunits. No PCR product of this promot er was amplified (Figure 3-21),

PAGE 43

43 validating the conclusion that ou r ChIP protocol serves as a re liable indicator that Med6 and Med31 can specifically immunopr ecipitate promoter DNA. Conclusion Two T-DNA insertion lines in either the Med31 promoter or 5 unt ranslated region were identified. The germination rate of med31-1 plants was lower, and th eir root length was much shorter than that of WT pl ants. The root length of med31-2 plants was also shorter than that of WT plants. The med31-2 mutants exhibited a dwarfed phenotype with fewer rosettes leaves than the WT plants, and some of them had aberrant pa tterns of cotyledon development, such as three cotyledons and three first true leaves, a single cotyledon, or fork ed cotyledons. These mutant phenotypes imply that Med31 plays an important role in many aspects of plant development, such as germination, root elongation and cotyledon development. Using Med31::GFP constructs, we found that the Med31 protein was localized in the nucleus. The Med31::GFP signal was detected in all the tissues that were examined, including roots, root hairs, leaves trichomes and petioles. In another experiment, the Med31 promoter was fused to GUS gene to study its tissue expression pattern. The Med31 promoter::GUS reporter was detected in the shoot apexes and lateral r oots of young seedlings (16 days old) and in the young inflorescences, anthers, stigma s of the adult plants (46 days old) and in developing seeds. The promoters of three unrelated genes ( CCA1 Hsp18.2 and Adh1 ) were all co-immunoprecipitated with Med31 by IgG Sepha rose and with Med6 by both IgG Sepharose and c-Myc antibody. These results demonstrate the localizatio n of Med6 and Med31 to these promoters, which is consistent with the function of Mediator. Taken together, this study provides eviden ce that Med6 and Med31 are both Mediator subunits because 1) Med31 was localized in the nucleus; 2) Med31 was e xpressed in every type of tissue that were examined; 3) Disruption of Med31 resulted in abnormal plant growth; and 4)

PAGE 44

44 Both Med6 and Med31 proteins were localized to promoters. Th ese data strongly support our hypothesis that the Mediator complex found in fungi and metazoans is also present in plants.

PAGE 45

45 1 120 10 20 30 40 50 60 70 80 90 100 110 (1) M A S P E E M G D D A S E I P S P P K N T Y K D P D G G R Q R F L L E L E F I Q C L A N P T Y I H Y L A Q N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q NArabidopsis_thaliana(1) M E E A E A R P A P P D P N D A R Q R F L L E L E F I Q C L A N P T Y I H Y L A Q N R Y F E D E A F I G Y L K Y L K Y W Q R P E Y I K Y I M Y P H C L F F L E L L Q NOryza_sativa(1) M S S S S P I N E N D N G N I E N N N E T N I T E N G D N G E S I D K K D D N I V L P Y E N D E E E A N Y L R F I M E L E F I Q C L S N P R Y L N Y L A Q N R Y F Q D K A F V N Y L V Y L Q Y W K K P E Y A K F I V Y P Q S L Y F L D L L Q EDictyostelium_discoideum(1) M L S E G E S E L L I E D E N P I A R F S L E L E F V Q C L S N P D Y L Q W L S K E G Y F E D E S F V N Y L K Y L L Y W C E F P Y V K Y I S Y P H C I K M L R L L Q ICryptosporidium_parvum(1) L R F Q V E L E F V Q C L A N P N Y L H F L A Q R G Y F K D A A F V N Y L K Y L L Y W K E P E Y A K Y L K F P M C L Y F L D L L Q YAnopheles_gambiae(1) M A K M Y G K G K T A I E S E E L Q K R R W Q I E L E F V Q C L S N P N Y L N F L A Q R G F F K D Q S F I N Y L K Y L Q Y W K E P D Y A K Y L M Y P M C L Y F L D L L Q YDrosophila_melanogaster(1) M A A A V A M E T D D A G N R L R F Q L E L E F V Q C L A N P N Y L N F L A Q R G Y F K D K A F V N Y L K Y L L Y W K D P E Y A K Y L K Y P Q C L H M L E L L Q YHomo_sapiens(1) M A A A V A M E T D D A G N R L R F Q L E L E F V Q C L A N P N Y L N F L A Q R G Y F K D K A F V N Y L K Y L L Y W K E P E Y A K Y L K Y P Q C L H M L E L L Q YMus_musculus(1) M E S V E S E K T R F E V E C E F V Q A L A N P N Y L N F L A Q R G Y F K E E Y F V N Y L K Y L L Y W K D P Q Y A R C L K F P Q C L H M L E A L Q SCaenorhabditis_elegans(1) M Q N R P K S V L T P A R L G T S G V V R N T L E D P W V R F Q I E L E F V Q S L G N P D Y L T F L A Q Q G C F D K P E F I N Y L S Y L Q Y W K S P S Y S R F I T Y P F C L H M L D L L Q STaenia_solium(1) M S A Q T D Q P I T E Q Q K K E Q E Q Y T N L I N S L P T R W E I E L E F V Q S L S N I P Y V N Y L A Q N N Y F N D E N F I N Y L N Y L Q Y W T Q P E Y S K F L V Y P N C L H I L K L L Q DCandida_albicans(1) M S S T N G N A P A T P S S D Q N P L P T R F E V E L E F I Q S L A N I Q Y V T Y L L T Q Q Q I W K S P N F K N Y L K Y L E Y W C N P P Y S Q C I V Y P N C L F I L K L L N GSaccharomyces_cerevisiae(1) M S G S R F E R E L E F V Q L L C N P D Y L R W L T R E G H F E S E E F R S Y L R Y L E Y W R S P E Y S R F L T Y P Q C L A V L E H L N SEncephalitozoon_cuniculi(1) M E T K W L L S K V P D D K S R F E I E L E F V Q M L S N P W Y L N F L A Q H K Y F E D E A F L Q Y L E Y M E Y W R E P E Y V K F I I Y P T C L H M L T L L K NSchizosaccharomyces_pombe(1) M S A H P G Q T P G V S A P T D P K S A N R A R F E L E L E F V Q A L A N P Y Y L H S L A Q Q N I L E K P A F V N Y L K Y L L Y W K D K D Y A R F I H Y P H A L H H L E L L Q NCoprinopsis_cinerea(1) R R F L E L E F V Q C L A N P Y L N F L A Q G Y F D A F V N Y L K Y L Y W K E P E Y A K F I Y P C L H M L E L L Q Consensus(1) 121 240 130 140 150 160 170 180 190 200 210 220 230 (121) P N F R T A M A H P A N K E L A H R Q Q F Y Y W K N Y R N N R L K H I L P R P L P E P V P P Q P P V A P S T S L P P A P S A T A A L S P A L S P M Q Y N N M L S K N D T R N M G A T G I D R R K R K K G I -Arabidopsis_thaliana(96) A N F R N A M A H P A S K E V A H R Q Q Y F F W K N Y R N N R L K H I L P R P P P E P T P T P A P A P A A V P P S A S V P S T V V P P V A A P P S A L L P M S A A G A S A M S P M Q F A G T P G T N I P K N D M R N V M G G Q G G R K R K I GOryza_sativa(84) E R F R Q E L N H S Q S T D F I H E Q Q F Y H W Q Y Y R N N R M S I K E Q E L Q Q Q Q Q Q Q Q Q Q Q V Q P P T T V -Dictyostelium_discoideum(120) E D F R K N L S K E E V I Q I I R E Q Q T Y Q W I Y S D I K K E H L K L -Cryptosporidium_parvum(84) E H F R R E I V S A Q C C K F I D D Q A I L L W Q H Y T R R R T R L T A L G T T S L T G L A V G G Q P V G -Anopheles_gambiae(67) E H F R R E I V N S Q C C K F I D D Q A I L Q W Q H Y T R K R I K L I E N V T A A Q Q Q Q Q Q L Q Q Q Q Q Q A N G M E A A T G G E S A A P T P N V N G S A S T A D S Q Q T S S A L Q P V Q A Q P G N P Q Q Q Q Q I N G V A S G A N I K L E L NDrosophila_melanogaster(86) E H F R K E L V N A Q C A K F I D E Q Q I L H W Q H Y S R K R M R L Q Q A L A E Q Q Q Q N N T S G K -Homo_sapiens(82) E H F R K E L V N A Q C A K F I D E Q Q I L H W Q H Y S R K R V R L Q Q A L A E Q Q Q Q N N T A G K -Mus_musculus(82) Q Q F R D S M A Y G P S A K F V E D Q V V L Q W Q F Y L R K R H R L C M M P D E G Q E L E E S E D E A D I R Q K D T E D E D D E E T M K K P D A D T A E K N S T T S T V S K K E K -Caenorhabditis_elegans(75) P D F R R E V A H E S V T R F I D D Q M L L H W K N Y L R K R A E M V N K H V Q S L D A M A T P G P G P S S -Taenia_solium(95) E N F R K N I I N Q D F M N S L M N D M V K R W Q S N A N D Q D E N K E K E E N K E V P E V R I N G T N -Candida_albicans(95) F M E S A I V N E D G L L E G L D E L P K I I Q L Q G P Q W M N E M V E R W A N -Saccharomyces_cerevisiae(88) E N I N D M L S D E N F F L A L G E Q Q Y F I W L N K H K E G W N K -Encephalitozoon_cuniculi(70) P Q F R N D I S R A D L S K Q V N D E I Y Y E W L G K G L Q Q Y G S A D D A T L S Q P Q Q E E D E K K V D V K K E N E -Schizosaccharomyces_pombe(81) A Q F R A A L K K D E F L R D Y L Q Q K Q F D H W R T W R D P K H L N P S T S N T S N E A P A D E T A A D K Q Q Q A I -Coprinopsis_cinerea(89) E F R L K I E Q Q W Y R L Consensus(121) Figure 3-1. Multiple sequence alignments of Med31 homologs in different species. Identical amino acids are indicated in yellow, cons ervative amino acids in light blue and similar amino acids in green.

PAGE 46

46 1 120 10 20 30 40 50 60 70 80 90 100 110 (1) M A S P E E M G D D A S E I P S P P K N T Y K D P D G G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QArabidopsis_thalina(1) M I F R L R L F S P G D Y S Q P S P W A C L P S S V S V S S M A S P E E M V D A S E T P S T P K S T Y K D P D V G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QBrassica_napus(1) N S R V C R F C S C S E S T N S M A S K I E S E N S T D T S P S S P K N I Y K D P D D G Q Q R F L L E L E F V Q C L A N P T Y I H Y L A QGlycine_max(1) V P S S N P A N Y I S L N R C V F V L V V E D S V S M A S K T E S G S P R D T P P S P P K S I Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QMedicago_truncatula(1) M A S K I E S E N S T D T S P S S P K N I Y K D P D D G Q Q R F L L E L E F V Q C L A N P T Y I H Y L A QPhytophthora_sojae(1) I D L G L I F I Q R R E Q G R I A V L T P P D F V F H T L C W F C S E S T D S M A S K N E S D N S T D T S P S S P K N I Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QPhaseolus_coccineus(1) Q T A I S V N L P I D I I F N F S I C K K N K M A A S K D N E E A S D A P S S P K K V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QCitrus_clementina(1) F S I C K K N K M A A A K D G E E A S D A P S S P K K V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QCitrus_sinensis(1) I R H E G F F P V L G F I T S M A S N Q E T D A S A N T P S S P K N V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QRhododendron_catawbiense(1) R X X X N A R A G F H H P X X S S K E S D S A P D T P S S P K S L Y K D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QEuphorbia_esula(1) R I S F K P F C T V T I E F L R F N C F V I S R V G V L K S L R D S L N F T V W H S T D Q P L K L N H R L F S V F H I V K V V V S M A S T K E S D N A S D T P S S P K N V Y K D P D D G R Q R F L L E L E F L Q C L A N P T Y I H Y L A QGossypium_raimondii(1) V D F L S I L S N F L G L N F M I S T V N R F Q E L C K F A A R Q V K V D S S S E F I C L M A S S N D A D D T S N S P S L T Q N V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QBeta_vulgaris(1) G G A D R W W I L R Q R L L P R T R R E S N L P S E I C N S M A S S H E D D D S S N T H S S P K K V Y Q D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QLactuca_saligna(1) G N N D A D R W I L R R L L P R T V T G V Y S L L K Q R R E S N L P S E V A N S M A S S H E D D D S S N T H S S P K K V Y Q D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QLactuca_sativa(1) H P F R C R I G N E D S M E I P S P P P S P P K T V Y K D P D D G R Q R F F L E L E F V Q C L A K P T Y I H Y L A QOcimum_basilicum(1) G T R K G G M D L L L A P S I P K E P Y N D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QCycas_rumphii(1) G K G S S D S S P L P S I P K E P Y K D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QPicea_sitchensis(1) C L S R T F N Q I R R R D L D L V E D D W C N K S G R R T V Q S R C I G A R N R P E Y F V R P K D I E V T R H F Q A K N M E P G K G N S D S S P H P S I P K E P Y K D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QPinus_taeda(1) M A S E D D A S T P S S P K V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QConsensus(1) 121 240 130 140 150 160 170 180 190 200 210 220 230 (121) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N P N F R T A M A H P A N K E L A H R Q Q F Y Y W K N Y R N N R L K H I L P R P L P E P V P P Q P P V A P S T S L P P A P S A T A A L S PArabidopsis_thalina(54) N R Y F E D E A F I E Y L K Y L Q Y G Q R P E Y I K F I M Y P H C L Y F L E L L Q N P N F R S A M A H P A N K E L A H R Q Q F Y Y W K N Y R N N R L K H I L P R P L P E P V A P Q P P P V P S S S L P P A P P A T A A PBrassica_napus(83) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P T N K E L A H R Q Q F Y F W K N Y R N N R L K H I L Q R -Glycine_max(70) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P T N K E L T H R Q Q F Y F W K N Y R N N R L K H I L P R S L A E P S A A L P A P A S T Q P Q P P V P A L P P V P A T S V A V T T S S S QMedicago_truncatula(80) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P T N K E L A H R Q Q F Y F W K N Y R N N R L K H I L P R S L P E L S A T P A A P A S T S S Q A P V S A L P P V P A T S V A V T A T P S QPhytophthora_sojae(54) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P T N K E L A H R Q Q F Y F W K N Y R N N R L K H I X P R S L P E P S A T S A V P A P V S T T -Phaseolus_coccineus(93) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E P S E A P P P A A A P P L P P A P P V L T P V T A A P G PCitrus_clementina(76) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E P A E A P P P A A A P P L P P A P P V P T P V T A A S G PCitrus_sinensis(61) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N S N F R N A M A H P G N K E L A H R Q Q F Y Y W K N Y R N N R M K H I L P K P P P E P V A A P P A S V P P P P P I P P S T I P V S A V P P P Q P A P S PRhododendron_catawbiense(68) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E P A P A A P V S A P P P P V Q P M P P V P P T T I G G P A G S A SEuphorbia_esula(66) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E V A H R Q Q F F F W K N Y R N N R L K F I L P K P P P E E V P T P A P L P P A S A P P Q Q S L P A S N I A M T T A P P A P A SGossypium_raimondii(118) N R Y F D D E A F I G Y L K Y L Q Y W Q Q P E Y I K F I M Y P H C L F F L E L L Q N A N F R N A M A H P G S K E L A H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E P D P -Beta_vulgaris(98) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K Y I M Y P H C L Y F L E L L Q N A S F R N A M A H P A N K E L T H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E T T A P P P S N A V P P P P T T T I A A A S S G G P V A V P PLactuca_saligna(83) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K Y I M Y P H C L Y F L E L L Q N A S F R N A M A H P A N K E L T H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E T T A P P P S N A V P P P P T T T L L L L L L V V L W L C R Q Y S R L CLactuca_sativa(93) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y L K F I M Y P H C L F F L E L L Q N P N F R N A M A H P A N K E L A H R Q Q F Y F W K N Y R N N R L K H I L P K P L P E S S T T A T S A S V A P L A L P P T T V P A A V S N I P P A P P P QOcimum_basilicum(59) N R Y F D D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L F F L E L L Q N A N F R T A M A H P A N K E L A H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E A A P P P P L L A -Cycas_rumphii(52) N R Y F D D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L F F L E L L Q N A N F R S A M A H P T N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E A A P A P P P A G A A T A P A P A A A A L P V P P T A V A V S S S Q K TPicea_sitchensis(50) N R Y F D D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L F F L E L L Q N A N F R S A M A H P A N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E A A P A P S P A V A A T A P A P A A A A L P A P Q T A V A V S S A Q K TPinus_taeda(113) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E L A H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E P A P P A P P A Consensus(121) 241 360 250 260 270 280 290 300 310 320 330 340 350 (241) A L S P M Q Y N N M L S K N D T R N M G A T G I D R R K R K K G I -Arabidopsis_thalina(164) S P S P M Q Y N N M L A K N E T R N M V S A G I D R R K R K K G P A Y L A L K Q T P W D L A Y A S C V -Brassica_napus(191) -Glycine_max(150) A P S P M P Y G I P P G S G I A K N D M X N T S A D -Medicago_truncatula(200) A P S P M P Y G M P P G S G L A K N D M R N P T V D N R R K R K L Y N T T C K L I I K A M Q W W L D Q A I S L F F L -Phytophthora_sojae(174) -Phaseolus_coccineus(191) A L S P M Q Y G I P P G S A L M K N D M R S S S I D R R K R K K D G I G I T F L C V K L M R N A Y Y K D Y K G Y I R A Y E W R S F F R P I I Y L S R G F H F G R W E A R N H H I L T P P N S Y F S Q G Q R I V N F W S A L P C W Citrus_clementina(189) A L S P M Q Y G I P P G S A L M K N D M R S S S I D R R K R K K D G I G I T F L C L K L M R S F L F Q S V Y V I R G N S I V I Y S I C I D F E I V L A A S R S N M S F L F G Y F V K L I SCitrus_sinensis(174) A L S P M Q Y A I P H G S A L P K N D P R T S G G D R -Rhododendron_catawbiense(186) A L S P M P Y G M P A G S T L A K N D M R N T G M D R R K R K K E G P N I R S S E S N W P N I S -Euphorbia_esula(183) T H S P M P Y G L P S G S A L A K N D M R N S G I D R R K R K H E R S L N P T I Y -Gossypium_raimondii(237) -Beta_vulgaris(185) V L S P M Q Y G V P S G P P L K S D P R S G I D R R K R K K D G F D L R Y S C N T S W L X E H K T R G L V F P F V V L S K F Y I L L L A M R A -Lactuca_saligna(198) S M V Y L L V H L K V T L G V G L I E E R E S K I F L S E R W I V F E V F V Q Y F M V E R T N S R F S V S F C S V V V L Y F T I G N E S L I F C L C V F F Y I K Y T F V F V Y I T L K S W I Y R E I M Y P L M M -Lactuca_sativa(213) V P S P M Q Y G I G S G S T F V K N D P R N S G V E K R K R K S L L T S A V N F D I L K F K F L V N F Y N -Ocimum_basilicum( 177) -Cycas_rumphii(145) E N T R G S T G E R R K R K Y N N L L K P Y F L D V V I L F I V S E N Y L C F R F C L L S S G S G V F A T E I T Q I C S V P L S G G G -Picea_sitchensis(170) E N T R G S A V E R R K R K Y I I D Y R R T F -Pinus_taeda(233) A S P M Y G I G L K N D R I D R R K R K Consensus(241) 361 4 62 370 380 390 400 410 420 430 440 450 (361) -Arabidopsis_thalina(197) -Brassica_napus(242) -Glycine_max(150) -Medicago_truncatula(226) -Phytophthora_sojae(232) -Phaseolus_coccineus(191) A S V V F I F V C L S R S T Y I G M C A L S P S C I D L L K I L F S N L S P T G Q I Q N R N D L N Q S N R G S F G G K R Y P G G K R E R F E F Q I L A L N S L A F S N L G K P P L F S F P P C P K I K L K Citrus_clementina(301) L F N L A L F F L K K K K K -Citrus_sinensis(267) -Rhododendron_catawbiense(213) -Euphorbia_esula(231) -Gossypium_raimondii(278) -Beta_vulgaris(185) -Lactuca_saligna(269) -Lactuca_sativa(317) -Ocimum_basilicum(230) -Cycas_rumphii(145) -Picea_sitchensis(237) -Pinus_taeda(256) Consensus(361) Figure 3-2. Multiple alignments of AtMed31 with the deduced amino acid sequences of its homologs in other plant species. Identical amino acids are indicated in yellow, conservative amino acids in light blue and similar amino acids in green.

PAGE 47

47 Figure 3-3. Diagrammatic representation of the insertions of the T-DNA in med31-1 and med31-2 The Med31 gene contains six exons, whic h are represented by red boxes. UTR regions are indicated by blue boxes. The position of triangle represents the T-DNA insertion site. Figure 3-4. Germination rate a nd root length of WT and med31-1 seedlings (9-day-old). A) WT. B) med31-1 The size bars represent 0.5 cm. Figure 3-5. Nine-day-old WT and med31-2 seedlings grown under cont inuous light. A) WT. B) med31-2 The size bars represent 0.5 cm.

PAGE 48

48 Figure 3-6. Nine-day-old WT and med31-2 seedlings grown under dark. A) WT. B) med31-2 The size bars represent 0.5 cm. Figure 3-7. Ten-da y-old WT and med31-2 seedlings. A) WT. B) med31-2 with three cotyledons and three first true leaves. C) med31-2 with forked cotyledon. D) med31-2 with a single cotyledon. Figure 3-8. Comparison of adult WT plants and med31-2 plants. A) 30-day-old WT and med31-2 plants. B) 55-day-old WT and med31-2 plants. In both panels, the left plant is med31-2 and the right plant is WT.

PAGE 49

49 Figure 3-9. Northern blot analysis of Med31 expression in WT and med31-2 plants. Figure 3-10. Subcellula r localization of Med31::GFP fusion pr oteins in the root tip of a 35-day-old plant. A) Image of G FP. B) Image of DAPI staining.

PAGE 50

50 Figure 3-11. Expression of Med31::G FP fusion proteins in lateral ro ots. A) A late ral root. B) A lateral root primordium. Figure 3-12. Expression of Med31::G FP fusion proteins in a root ha ir. A) Image of GFP signal. B) DIC image. C) Overlay of the DIC and GFP images.

PAGE 51

51 Figure 3-13. Expression of Med31::G FP fusion proteins in a leaf. A) Image of GFP signal. B) DIC image. C) Image of autofluorescence. The chloroplasts are red because of autofluorescence of chlorophyll. D) Overla y of the GFP and autofluorescence images.

PAGE 52

52 Figure 3-14. Expression of Med31::G FP fusion proteins in a trichom e. A) Image of GFP signal. B) DIC image. C) Image of autofluorescen ce. The chloroplasts are red because of autofluorescence of chlorophyll. D) Overla y of the GFP and autofluorescence images.

PAGE 53

53 Figure 3-15. Expression of Med31::G FP fusion proteins in a petiole. A) Image of GFP signal. B) DIC image. C) Image of autofluorescence. The chloroplasts are red because of autofluorescence of chlorophyll. D) Overla y of the GFP and autofluorescence images.

PAGE 54

54 Figure 3-16. Med31 promoter directed GUS tissue e xpression pattern in young plants (16-day-old). GUS signal was detected in A) A shoot apex. B) Lateral root primordia and tips. Figure 3-17. Med31 promoter directed GUS tissue expression pattern in adult pl ants (46-day-old). GUS signal was detected in A) A young inflor escence. B) Anthers. C) A stigma. D) Developing seeds.

PAGE 55

55 1 100 10 20 30 40 50 60 70 80 90 (1) M D S S L L S A A T A D T F N G N A A D Q I P P P L Q P P G T D M T G I S F R D Q L W I N S Y P L D R N Y I F D Y F A L S P F Y D TArabidopsis_thalina(1) M S G T P L P P P A L P P P P G T D M T G I C F R D Q L W L N T Y P L D R N L V F D Y F A L S P F Y D LOryza_sativa(1) M E D F N N D D P M N F D K D D M I N N N N D N N N N N N D N D N N N E N N E D S N N N S N L E N M I S D T S T P R I L E E E P D L T Q V M W R D P L W L Q M Y P L N P Q T I L Q Y F S Y S Q F Y D K Dictyostelium_discoideum(1) L A P L L Q E N P L W I S W H D S N W I P V L N P G N V M D Y F S E K S N P F Y D R Anopheles_gambiae(1) M A S R Q M T N D H L R L S W H D T Q M M A T L S P Q T V M D Y F C R K S N P F Y D HDrosophila_melanogaster(1) M A A V D I R D N L L G I S W V D S S W I P I L N S G S V L D Y F S E R S N P F Y D R Homo_sapiens(1) -Mus_musculus(1) M M P R M G P P A A A R Q D N P L H V S F R N P Q W P P N F I N K D N V L D Y F C N Q A N A F Y E M Caenorhabditis_elegans(1) M E S L D E I Q W K S P E F I Q E R G L N T N N V L E Y F S L S P F Y D R Candida_albicans(1) M N V T P L D E L Q W K S P E W I Q V F G L R T E N V L D Y F A E S P F F D K Saccharomyces_cerevisiae(1) M A S G A P P S V D L T S I Q W R M P E W V Q S M G G L R T E N V L E Y F S Q S P F Y S HSchizosaccharomyces_pombe(1) M E E R E E S I S F V D Q R F L G S K P L D D T N V L E Y F S G S P F Y D K Encephalitozoon_cuniculi(1) L I S W R D W I Q L V Y D Y F S S P F Y D K Consensus(1) 101 200 110 120 130 140 150 160 170 180 190 (101) T C N N E I L R R R S I H P L D L S H L S K M T G L E Y M L T D A T E P N L F V F R K Q K R D G P E -Arabidopsis_thalina(67) T C N N E S L R S R Q I H P L D M S H L T K M T G M E Y V L S D V M E P H L F V I R K Q R R E S P E -Oryza_sativa(53) N C N N E Q L K M Q R L D L S A L K N M D G L E Y E L I K F V E P S F F L I A K Q T R I S P T -Dictyostelium_discoideum(100) T C N N E I V R M Q R Q S L E L L N N M T G V E Y I P L H V Q D P I L Y V I R K Q H R H S P T -Anopheles_gambiae(43) M C N N E T V R M Q R L G P E H L H N M I G L E Y I L L H V A E P I L Y V I R K Q H R H N P S -Drosophila_melanogaster(44) T C N N E V V K M Q R L T L E H L N Q M V G I E Y I L L H A Q E P I L F I I R K Q Q R Q S P A -Homo_sapiens(44) M Q R L T L E H L N Q M V G I E Y I L L H A Q E P I L F I I R K Q Q R Q S P A -Mus_musculus(1) N S C N Q Q I R M Q N I V N R T V E E C L R T M P G I Q Y V L W Y S Q P P L F I I C K Q R R N N V T -Caenorhabditis_elegans(51) T S N N Q V L M M Q F Q Y Q Q I Q I P P G V S F H Q Y F Q S R L S E M T G I E F V I A Y T K E P D F W I I R K Q K R Q D P Q N Candida_albicans(38) T S N N Q V I K M Q R Q F S Q L N D P N A A V N M T Q N I M T L P D G K N G N L E E E F A Y V D P A R R Q I L F K Y P M Y M Q L E E E L M K L D G T E Y V L S S V R E P D F W V I R K Q R R T N N S G Saccharomyces_cerevisiae(40) K S N N E M L K M Q S Q F N A L D L G D L N S Q L K R L T G I Q F V I I H E R P P F L W V I Q K Q N R L N E N -Schizosaccharomyces_pombe(46) S C N N E I L K M Q T Q F R G L D Q K S K L F S M V G I F Y E V E S S N H E K T L F V I R K A Y N H G D T -Encephalitozoon_cuniculi(39) T C N N E I L K M Q R L L S L M G I E Y V L L H E P L F V I R K Q R P T Consensus(101) 201 300 210 220 230 240 250 260 270 280 290 (201) K V T P M L T Y Y I L D G S I Y Q A P Q L C S V F A A R V S R T I Y N I S K A F T D A A S K L E T I R Q V D T E N Q N E P A E S K P A S E T V D L K E M K R V Arabidopsis_thalina(117) K S N A M L A Y Y I L D G S I Y Q A P Q L C S V F A S R I S R A M H H I S K A F T T A C S K L E K I G H V E T E P D T A A S E S K T Q K E A I D L K E L K R V Oryza_sativa(103) D V L I N T L Y Y V I N G N I Y Q A P E L H V V F K S R V S Q S I S H L S E A F N S I S S I V N W D I V N G Y S L N L D P S N Q Q E K S K L A A Y S R K Q I E D T K R LDictyostelium_discoideum(147) E A T P M A D Y Y I I A G T V Y Q A P D L A S V F N S R I L S T V H H L Q T A F D E A S S Y S R Y H P S K G Y S W D F S S N K A I A E K T K T Q T K K E A P V K E E P S S I F Q R Q R V Anopheles_gambiae(90) E A T P I A D Y Y I I G G T V Y K A P D L A N V I N S R I L N T V V N L Q S A F E E A S S Y A R Y H P N K G Y T W D F S S N K V F S D R S K S D K K D A N S A K D E N S G T L F Q K Q R V Drosophila_melanogaster(91) Q V I P L A D Y Y I I A G V I Y Q A P D L G S V I N S R V L T A V H G I Q S A F D E A M S Y C R Y H P S K G Y W W H F K D H E E Q D K V R P K A K R K E E P S S I F Q R Q R V Homo_sapiens(91) Q V I P L A D Y Y I I A G V I Y Q A P D L G S V I N S R V L T A V H G I Q S A F D E A M S Y C R Y H P S K G Y W W H F K D H E E Q E K V K P K A K R K E E P S S I F Q R Q R V Mus_musculus(40) N V S P I A Y Y Y V I N G S V H Q A P D M Y S L V Q S R L L G A L E P L R N A F G E V T N Y S R Y N T A K G Y Y W E F K N K P N V K K R E E E K K E D E E E K L E D R S T N F Q K T R TCaenorhabditis_elegans(101) T V T L Q D Y Y I I G A N V Y Q A P R I Y D V L S S R L L A S V L S I K N S T D L L N D M T S Y H I S D G G H S Y I N S I H G S S S K P S Q S S A V S K P S S T N T G T N A T T T PCandida_albicans(101) V G S A K G P E I I P L Q D Y Y I I G A N I Y Q S P T I F K I V Q S R L M S T S Y H L N S T L E S L Y D L I E F Q P S Q G V H Y K V P T D T S T T A T A A T N G N N A G G G S N K S S V R P T G G A Saccharomyces_cerevisiae(139) E V K P L T V Y F V C N E N I Y M A P N A Y T L L A T R M L N A T Y C F Q K A L T K I E K F P Q Y N P Q E G Y T Y P K L S N D N L E V D H S N T N E P A D E N K Schizosaccharomyces_pombe(101) A E T L G M Y Y I I H G H V Y A A P T N Y S I Y R C R M G D S M W Q L N S F I D R M M E K R R F N P F S P P K G R R L A K S L E D S K DEncephalitozoon_cuniculi(92) V P L A D Y Y I I A G I Y Q A P D L S V I N S R V L A V H L Q S A F D E A S Y R Y P S G Y W K S K S I R V Consensus(201) 301 3 77 310 320 330 340 350 360 (301) D V I L T S L Y R K L A P P P P P P P F P E G Y V S Q E A L G E K E E L G T Q G G E S Q P P Q V D P I I D Q G P A K R M K F Arabidopsis_thalina(196) D H I L M S L Q R K L Q P A P P P P P F P E G Y V P S E Q E K A S D D L L A S E A L P P Q V D P I I D Q G P A K R P R F Q Oryza_sativa(182) D Q L I N S L F I K F P A I N R P V E N P Q Q M G G G I T P Q Q Q P S Q P Dictyostelium_discoideum(231) D M L L G D L L R K F P L P L P Q M T N N P T G G N P S D T A N A S N N H G G A A G D S D H V G A D A T L I K Q E P T E G G V Anopheles_gambiae(182) D M L L A E L L R K F P P P I P P M L Q N L Q Q P P P A G D D L N T A R N A S E M N N A T G P L D I K T E G V D M K P P P E K K S K Drosophila_melanogaster(184) D A L L L D L R Q K F P P K F V Q L K P G E K P V P V D Q T K K E A E P I P E T V K P E E K E T T K N V Q Q T V S A K G P P E K R M R L Q Homo_sapiens(178) D A L L I D L R Q K F P P R F V Q Q K S G E K P V P V D Q A K K E A E P L P E T V K S E E K E S T K N I Q Q T V S T K G P P E K R M R L Q Mus_musculus(127) M M L L N Q L F S E M P A E D A L E R E E K E E V E E E E E E T L K T E E P T T S T D E P K F A E P T A R T T S K Q Caenorhabditis_elegans(193) I T L T T P S G A T V P S T V S N G I S T S T E I A S G V F D T L L N D V V M N D D H L Y I D E I P L Y G E G S T L E R L G L K G N K D A G L S L Candida_albicans(191) N M A T V P S T T N V N M T V N T M G T G G Q T I D N G T G R T G N G N M G I T T E M L D K L M V T S I R S T P N Y I Saccharomyces_cerevisiae(237) N Q S I E N A D Y S F S P E D F S V V R A F M Q S L H S S K E A P D V K Schizosaccharomyces_pombe(181) L D F M M E I F N D F K K E Q A E S Encephalitozoon_cuniculi(160) D L L L K F P P M V K P K R Consensus(301) Figure 3-18. Multiple sequence alignments of Me d6 homologs in different species. Identical amino acids are indicated in yellow, conser vative amino acids are indicated in light blue, and similar amino acids are indicated in green.

PAGE 56

56 Figure 3-19. Med31 associates with the promoters of CCA1 Hsp18.2 and Adh1 but not with the intergenetic region. The promoters used ar e indicated above the gels. Lane M was loaded with 100 bp DNA Ladder from New En gland Biolabs. The templates for each PCR are as follows: Lane 1: Genomic DNA from wild-type plants; Lane 2: Input DNA control (sonicated genomic DNA from Med31-pC-TAPa transgenic plants); Lane 3: Negative control (chromatin ex tract without antibody immunoprecipitation from Med31-pC-TAPa transgenic plants); Lane 4: Chromatin immunoprecipitated with IgG Sepharose from Med31-pC-TAPa transgenic plants. Figure 3-20. Med6 associates with the promoters of CCA1 Hsp18.2 and Adh1 but not with the intergenetic region. The promoters used ar e indicated above the gels. Lane M was loaded with 100 bp DNA Ladder from New En gland Biolabs. The templates for each PCR are as follows: Lane 1: genomic DNA from wild-type plants; Lane 2: Input DNA control (sonicated genomic DNA from Med6-pC-TAPa transgenic plants); Lane 3: Negative control (chromatin extrac t without antibody immunoprecipitation from Med6-pC-TAPa transgenic plants); Lane 4: Ch romatin immunoprecipitated with IgG Sepharose from Med6-pC-TAPa transgenic plants. Lane 5: Chromatin immunoprecipitated with c-Myc antibody from Med6-pC-TAPa transgenic plants.

PAGE 57

57 Figure 3-21. Immunoglobulin G Sepharose and c-Myc antibody cannot immunoprecipitate the CCA1 promoter from WT Arabidopsis The templates for each PCR are as follows. Lane M was loaded with 100 bp DNA Ladder from New England Biolabs. Lane 1: genomic DNA from wild-type plants; Lane 2: Input DNA control (sonicated genomic DNA from wild-type plants); Lane 3: Negative control (c hromatin extract without antibody immunoprecipitation from wild-t ype plants); Lane 4: Chromatin immunoprecipitated with IgG Sepharose from wild-type plants. Lane 5: Chromatin immunoprecipitated with c-Myc an tibody from wild-type plants.

PAGE 58

58 CHAPTER 4 DISCUSSION Phenotype Characterization of med31 Mutants The Med31 promoter or 5 UTR were disrupted by T-DNA insertion in med31-1 and med31-2 lines. Both mutant lines had shorter root s than WT plants under our experimental conditions. In addition, seeds were examined in pr eliminary studies (data not included) for their responses to a variety of hormones. The med31-2 seedlings were insensitive to ABA, kinetin, and 2, 4-D, compared with WT seedlings. A pos sible cause for the mutant phenotypes of these two insertion lines is due to the disruption of eith er transcriptional or tr anslational expression of Med31 The T-DNA in med31-1 breaks a GT-1 cis -element (identified by AthaMap web tools; www.athamap.de), which has been shown in other prom oters to play a role in the gene regulation by light, pathogens and salt (Vil lain et al., 1994; Park et al ., 2004). Likewise, the T-DNA in med31-2 breaks the CCAAT BOX1 (identified in the PLACE database; www.dna.affrc.go.jp/PLACE), which has been repor ted to be involved in transcriptional expression by heat stress (Rieping and Scho ffl, 1992; Haralampidis et al., 2002). T-DNA insertions not only disrupt the inserted cis -elements, but also impede the function of the cis -elements upstream of the insertion sites. Th e location of the two T-DNA insertions found in med311 and -2 are predicted to strongly interfere with th e regulation of Med31 gene expression. Med31 is a subunit of Mediator complex, which is important in gene transcription mediated by RNA pol II. Defective Med31 expression has the potential to influence the binding of the transactivators to Med31 subunit, alter the structure of the medi ator, hinder the entry of other subunits into the Mediator, or the entry of general transc ription factor or RNA pol II into the PIC, and thus, cause pleiotropic effects by impeding RNA pol II-dependent transcription. Consistent

PAGE 59

59 with this hypothesis, our preliminary data show ed multiple aspects of plant development were influenced for the med31-2 mutant. Evidence for a Mediator Complex in Arabidopsis The transcription apparatus of plants, metazoans and yeast are conserved (Gurley et al., 2006). Many of the promoters in these three ki ngdoms contain the TATA motif and CAAT box for the binding of RNA pol II and general transcription factors. Transactivators generally bind the upstream cis -elements to regulate gene expression. The RNA pol II in a ll the three kingdoms contains 12 conserved subunits. In addition, plants possess the genes coding for all the general transcription factors (TFIIA, B, D, E, F, and H) that are presen t in metazoans and fungi (Coulson and Ouzounis, 2003). Arabidopsis also has the homologs of th e subunits of some coactivators (Hsieh and Fischer, 2005), such as the SAGA (S pt-Ada-Gcn5-acetyltransfe rase) (Stockinger et al., 2001) and SWI/SNF complexes (Brzeski et al ., 1999; Eshed et al., 19 99; Ogas et al., 1999). This high degree in conservation of the transcription machinery suggests that the plants may also have the Mediator coactivator which has been shown to play an essential role in RNA pol II-mediated transcription in other eukaryotes. Iden tification of the homologs of most of the yeast and metazoan Mediator subunits in Arabidopsis strongly supports this hypothesis (Gurley et al., 2006; Boube et al., 2002). The experiments described here explore va rious aspects of gene expression for two putative Mediator subunits from Arabidopsis Med31 and Med6. By all measures tested, these two proteins behaved as expected for bona fide members of plant Mediator. AtMed31 was localized in the nucleus, and was widely e xpressed throughout the plants. Both AtMed6 and AtMed31 were localized to the prom oters of three unrelated genes: CCA1 Hsp18.2 and Adh1 Together with the sequence homology between Arabidopsis proteins and known Mediator subunits from other eukaryotes, these data str ongly support the presence of a Mediator complex

PAGE 60

60 in Arabidopsis, and higher plants in gene ral, that shows strong conservation in both form and function with analogous complexes in fungi and metazoans.

PAGE 61

61 LIST OF REFERENCES Alonso J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., D een, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Cr osby, W.L., Berry, C.C. and Ecker, J.R. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science 301 653-657. Altschul, S.F., Madden, T.L., Schaffer, A.A ., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25 3389-3402. Andrau, J.C., van de Pasch, L., Lijnzaad, P., Bijma, T., Koerkamp, M.G ., van de Peppel, J., Werner, M. and Holstege F.C. (2006) Genome-wide location of the coactivator mediator: Binding without activation and tr ansient Cdk8 interaction on DNA. Mol Cell 22 179-192. Asada, S., Choi, Y., Yamada, M., Wang, S.C., Hung, M.C., Qin, J. and Uesugi, M. (2002) External control of Her2 expression and can cer cell growth by ta rgeting a Ras-linked coactivator. Proc. Natl. Acad. Sci. USA 99 12747-12752. Atkins, G.B., Hu, X., Guenther, M.G., Rach ez, C., Freedman, L.P. and Lazar, M.A. (1999) Coactivators for the orphan nuclear receptor RORalpha. Mol. Endocrinol. 13 1550. Autran, D., Jonak, C., Belcram, K., Beemster, G.T., Kronenberger, J., Grandjean, O., Inze, D. and Traas, J. (2002) Cell numbers and leaf development in Arabidopsis : a functional analysis of the STRUWWELPETER gene. EMBO J. 21 6036-6049. Baek, H.J., Malik, S., Qin, J. and Roeder, R.G. (2002) Requirement of TRAP/mediator for both activator-independent and activator-depen dent transcription in conjunction with TFIID-associated TAF(II)s. Mol. Cell Biol. 22 2842-2852. Black, J.C., Choi, J.E,. Lo mbardo, S.R. and Carey, M. (2006) A mechanism for coordinating chromatin modification and prei nitiation complex assembly. Mol. Cell, 23 809-818. Borggrefe, T., Davis, R., Erdjument-Bro mage, H., Tempst, P. and Kornberg, R.D. (2002) A complex of the Srb8, -9, -10, and -11 transcri ptional regulatory pr oteins from yeast. J. Biol. Chem. 277 44202-44207. Boube, M., Joulia, L., Cribbs, D.L. and Bourbon, H.M. (2002) Evidence for a mediator of RNA polymerase II transcri ptional regulation conser ved from yeast to man. Cell 110 143.

PAGE 62

62 Bourbon, H.M., Aguilera, A., Ansari, A.Z ., Asturias, F.J., Berk, A.J., Bjorklund, S., Blackwell, T.K., Borggrefe, T., Carey, M., Carlson, M., Conaway, J.W., Conaway, R.C., Emmons, S.W., Fondell, J.D., Freedman, L.P., Fukasawa, T., Gustafsson, C.M., Han, M., He, X., Herman, P.K., Hinnebusch, A.G., Holmberg, S., Holstege, F.C., Jaehning, J.A., Kim, Y.J., Kuras, L., Leutz, A., Lis, J.T., Meisterernest, M., Naar, A.M., Nasmyth, K., Parvin, J.D., Ptashne, M ., Reinberg, D., Ronne, H., Sadowski, I., Sakurai, H., Sipiczki, M., Sternberg, P .W., Stillman, D.J., Strich, R., Struhl, K., Svejstrup, J.Q., Tuck, S., Winston, F., Roeder, R.G. and Kornberg, R.D. (2004) A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II. Mol. Cell 14 553-557. Boyer, T.G., Martin, M.E., Lees, E., Ricciardi, R.P. and Berk, A.J. (1999) Mammalian Srb/Mediator complex is target ed by adenovirus E1A protein. Nature 399 276-279. Brzeski, J., Podstolski, W., Olcza k, K. and Jerzmanowski, A. (1999) Identification and analysis of the Arabidopsis thaliana BSH gene, a member of the SNF5 gene family. Nucleic Acids Res. 27 2393. Burakov, D., Wong, C.W., Rachez, C., Cheskis, B.J. and Freedman, L.P. (2000) Functional interactions between the estrogen receptor and DRIP205, a subunit of the heteromeric DRIP coactivator complex. J. Biol. Chem. 275 20928. Cao, H., Bowling, S.A., Gordon, A.S. and Dong, X. (1994) Characterization of an Arabidopsis Mutant That Is Nonresponsive to In ducers of Systemic Acquired Resistance. Plant Cell 6 1583-1592. Cerdan, P.D. and Chory, J. (2003). Regulation of flowering time by light quality. Nature 423 881-885. Chadick, J.Z. and Asturias, F.J. (2005) Structure of eukary otic Mediator complexes. Trends Biochem. Sci. 30 264-271. Chen, S., West, R.W.J., Johnston, S.L., Gans, H. and Ma, J. (1993) TSF3, a global regulatory protein that silences transc ription of yeast GAL genes, also mediates repression by 2 repressor and is id entical to SIN4. Mol. Cell. Biol. 13 831-840. Cho, E.J., Takagi, T., Moore, C.R. and Buratowski, S. (1997) mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev. 11 3319. Clayton, A.L., Hazzalin, C.A. and Mahadevan, L.C. (2006) Enhanced histone acetylation and transcription: a dynamic perspective. Mol. Cell, 23 289-296. Conaway, R.C., Sato, S., Tomomori-S ato, C., Yao, T. and Conaway, J.W. (2005) The mammalian Mediator complex and its ro le in transcrip tional regulation. Trends Biochem. Sci. 30 250-255.

PAGE 63

63 Coulson, R.M. and Ouzounis, C.A. (2003) The phylogenetic diversity of eukaryotic transcription. Nucleic Acids Res. 31 653-660. Crawford, K.M. and Zambryski, P.C. (2001) Non-targeted and targeted protein movement through plasmodesmata in leaves in differe nt developmental and physiological states. Plant Physiol. 125 1802-1812. Crawford, S.E., Qi, C., Misra, P., Stellmach, V., Rao, M.S., Engel, J.D., Zhu, Y. and Reddy J.K. (2002) Defects of the heart, eye, and me gakaryocytes in peroxisome proliferator activator receptor-binding protein (PBP) null embryos implicate GATA family of transcription factors. J. Biol. Chem. 277 3585-3592. Davis, J.A., Takagi, Y., Kornberg, R.D. and Asturias, F.A. (2002) Structure of the yeast RNA polymerase II holoenzyme: Mediator c onformation and polymerase interaction. Mol. Cell, 10 409-415. Dolferus, R., Jacobs, M., Peacock, W.J. and Dennis, E.S. (1994) Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene. Plant Physiol. 105 1075-1087. Drane, P., Barel, M., Balbo, M. and Frade, R. (1997) Identification of RB18A, a 205 kDa new p53 regulatory protein which shares antig enic and functional properties with p53. Oncogene 15 3013. Dvir, A., Conaway, R.C. and Conaway, J.W. (1997) A role for TFIIH in controlling the activity of early RNA polymerase II elongation complexes. Proc. Natl. Acad. Sci. USA 94 9006. Eberhardy, S.R. and Farnham, P.J. (2002) Myc recruits P-TEFb to mediate the final step in the transcriptional activation of the cad promoter. J Biol Chem 277 40156-40162. Eshed, Y., Baum, S.F. and Bowman, J.L. (1999) Distinct mechanisms promote polarity establishment in carpels of Arabidopsis Cell 99 : 199. Fan, H.Y., Cheng, K.K. and Klein, H.L. (1996) Mutations in the RNA polymerase II transcription machinery suppre ss the hyperrecombination mutant hpr1 delta of Saccharomyces cerevisiae Genetics 142 749-759. Fan, H.Y. and Klein, H.L. (1994) Characterization of mutations that suppress the temperature-sensitive growth of the hpr1 delta mutant of Saccharomyces cerevisiae Genetics 137 945-956. Fassler, J.S., Gray, W., Lee, J.P ., Yu, G.Y. and Gingerich, G. (1991) The Saccharomyces cerevisiae SPT14 gene is essential for normal e xpression of the yeast transposon, Ty, as well as for expression of the HIS4 gene and several genes in the mating pathway. Mol. Gen. Genet. 230 310.

PAGE 64

64 Featherstone, M. (2002) Coactivators in transcription in itiation: here are your orders. Curr. Opin. Genet. Dev. 12 149-155. Flanagan, P.M., Kelleher, R.J. 3rd, Sayre, M.H., Tschochner, H. and Kornberg, R.D. (1991). A mediator required for activa tion of RNA polymerase II transcription in vitro Nature 350 436. Fondell, J.D., Ge, H. and Roeder, R.G. (1996) Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc. Natl. Acad. Sci. USA 93 8329-8233. Friedl, E.M., Lane, W.S., Erdjument-Broma ge, H., Tempst, P. and Reinberg, D. (2003) The C-terminal domain phosphatase and transc ription elongation activ ities of FCP1 are regulated by phosphorylation. Proc. Natl. Acad. Sci. USA 100 2328-2333. Garrett-Engele CM, Siegal ML, Manoli DS, Williams BC, Li H, Baker BS. (2002) intersex a gene required for female sexual development in Drosophila is expressed in both sexes and functions together with doublesex to regulate terminal differentiation. Development 129 4661-4675. Gavin, I., Horn, P.J. and Peterson, C.L. (2001) SWI/SNF chromatin remodeling requires changes in DNA topology. Mol. Cell, 7 97-104. Ge, K., Guermah, M., Yuan, C. X., Ito, M., Wallberg, A.E., Spiegelman, B.M. and Roeder, R.G. (2002) Transcription coactivator TR AP220 is required for PPAR gamma 2-stimulated adipogenesis. Nature 417 563-567. Gendrel, A.V., Lippman, Z., Martienssen, R. and Colot, V. (2005) Profiling histone modification patterns in plants using genomic tiling microarrays. Nat Methods 2 213-218. Grallert, A., Grallert, B., Zilahi E., Szilagyi, Z. and Sipiczki, M. (1999) Eleven novel sep genes of Schizosaccharomyces pombe required for efficient cell separation and sexual differentiation. Yeast 15 669-686. Gu, W., Malik, S., Ito, M., Yuan, C.X.,, Fond ell J.D., Zhang, X., Martinez, E., Qin, J. and Roeder, R.G. (1999) A novel human SRB/MED-cont aining cofactor complex, SMCC, involved in transcription regulation. Mol. Cell, 3 97-108. Guidi, B.W., Bjornsdottir, G., Hopkins, D. C., Lacomis, L., Erdjument-Bromage, H., Tempst, P. and Myers, L.C. (2004) Mutual targeting of me diator and the TFIIH kinase Kin28. J. Biol. Chem. 279 29114-29120. Gurley, W.B., O'Grady, K., Czarnecka-Verner, E. and Lawit, S.J. (2007) General transcription factors and the core promoter: ancient roots. In Regulation and Transcription in Plants (Grasser, K.D., ed). Annual Plant Reviews vol. 29, Blackwell Publishing: Oxford, UK, pp.368.

PAGE 65

65 Gustafsson, C.M., Myers, L.C., Beve, J., Spa hr, H., Lui, M., Erdjument-Bromage, H., Tempst, P. and Kornberg, R.D. (1998) Identification of new mediator subunits in the RNA polymerase II holoenzyme from Saccharomyces cerevisiae J Biol Chem 273 30851-30854. Gustafsson, C.M., Myers, L.C., Li, Y., Re dd, M.J., Lui, M., Erdjument-Bromage, H., Tempst, P. and Kornberg, R.D. (1997) Identification of Rox3 as a component of mediator and RNA polymerase II holoenzyme. J Biol Chem 272 48-50. Gwack, Y., Baek, H.J., Nakamura, H., Lee, S. H., Meisterernst, M., Roeder, R.G. and Jung, J.U. (2003) Principal role of TRAP/media tor and SWI/SNF complexes in Kaposis sarcoma-associated herpesvirus RTA-mediated lytic reactivation. Mol. Cell Biol. 23 2055. Haralampidis, K., Milioni, D., Rigas, S. and Hatzopoulos, P. (2002) Combinatorial interaction of cis elements specifie s the expression of the Arabidopsis AtHsp90-1 gene. Plant Physiol 129 1138-1149. Havas, K., Flaus, A., Phelan, M., Kingston, R ., Wade, P.A., Lilley, D.M. and Owen-Hughes, T. (2000) Generation of superhelical tors ion by ATP-dependent chromatin remodeling activities. Cell 103 1133-1142. Hengartner, C.J., Thompson, C.M., Zhang, J., Chao, D.M., Liao, S.M., Koleske, A.J., Okamura, S. and Young, R.A. (1995) Association of an activator with an RNA polymerase II holoenzyme. Genes Dev. 9 897-910. Hengartner, C.J., Myer, V.E., Liao, S.M., Wilson, C.J., Koh, S.S. and Young, R.A. (1998) Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases. Mol. Cell, 2 43-53. Hittelman, A.B., Burakov, D., Iniguez-Lluhi, J.A., Freedman, L.P. and Garabedian, M.J. (1999) Differential regulation of glucocorticoid receptor tr anscriptional activation via AF-1-associated proteins. EMBO J. 18 5380-5388. Holstege, F.C., Jennings, E.G., Wyrick, J.J., Lee, T.I., Hengartner, C.J., Green, M.R., Golub, T.R., Lander, E.S. and Young, R.A. (1998). Dissecting the re gulatory circuitry of a eukaryotic genome. Cell 95 717-728. Hsieh, T.F. and Fischer, R.L. (2005) Biology of chromatin dynamics. Annu. Rev. Plant Biol. 56 327-351. Hua, S. and Sun, Z. (2001) Support vector machine a pproach for protein subcellular localization prediction. Bioinformatics 17 721-728. Ito, M., Yuan, C.X., Malik, S., Gu, W., Fonde ll, J.D., Yamamura, S., Fu, Z.Y., Zhang, X., Qin, J. and Roeder, R.G. (1999) Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear re ceptors and diverse mammalian activators. Mol. Cell, 3 361-370.

PAGE 66

66 Jeoung, J.M., Krishnaveni, S., Muthukrish nan, S., Trick, H.N. and Liang, G.H. (2002) Optimization of sorghum transformation para meters using genes for green fluorescent protein and beta-glucuronida se as visual markers. Hereditas 137 20-28. Jiang, Y., Yan, M. and Gralla, J.D. (1996) A three-step pathway of transcription initiation leading to promoter clearance at an activation RNA polymerase II promoter. Mol. Cell Biol. 16 1614-1621. Jiang, Y.W. and Stillman, D.J. (1995) Regulation of HIS4 Expression by the Saccharomyces cerevisiae SIN4 Transcriptional Regulator. Genetics 140 103-114. Jiang, Y.W., Veschambre, P., Erdjument-B romage, H., Tempst, P., Conaway, J.W., Conaway, R.C. and Kornberg, R.D. (1998) Mammalian mediator of transcriptional regulation and its possible role as an e nd-point of signal tr ansduction pathways. Proc. Natl. Acad. Sci. USA 95 8538-8543. Johnson, K.M. and Carey, M. (2003) Assembly of a mediat or/TFIID/TFIIA complex bypasses the need for an activator. Curr. Biol. 13 772-777. Johnson, K.M., Wang, J., Smallwood, A., Arayata, C. and Carey, M. (2002) TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA. Genes Dev. 16 1852-1863. Kato, Y., Habas, R., Katsuyama, Y., Naar, A.M. and He, X. (2002) A component of the ARC/Mediator complex required for TGF beta/Nodal signaling. Nature 418 641. Kelleher, R.J., Flanagan, P.M. and Kornberg, R.D. (1990) A novel mediator between activator proteins and the RNA polym erase II transcription apparatus. Cell 61 1209-1215. Kim, T.K., Ebright, R.H. and Reinberg, D. (2000) Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science 288 1418-1422. Kim, T.W., Kwon, Y.J., Kim, J.M., So ng, Y.H., Kim, S.N. and Kim, Y.J. (2004) MED16 and MED23 of Mediator are coactivators of lipopolysaccharideand heat-shock-induced transcriptional activators. Proc. Natl. Acad. Sci. USA 101 ,12153. Kim, Y.J., Bjorklund, S., Li, Y., S ayre, M.H. and Kornberg, R.D. (1994) A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77 599-608. Koleske, A.J., Buratowski, S., Nonet, M. and Young, R.A. (1992) A novel transcription factor reveals a functional link between th e RNA Polymerase II CTD and TFIID. Cell 69 883. Koleske, A.J., Buratowski, S., Nonet, M. and Young, R.A. (1992) A novel transcription factor reveals a functional link between th e RNA polymerase II CTD and TFIID. Cell 69 883-894.

PAGE 67

67 Koleske, A.J. and Young, R.A. (1994) An RNA polymerase II holoenzyme responsive to activators. Nature 368 466-469. Kornberg, RD. (2005) Mediator and the mechanis m of transcrip tional activation. Trends Biochem. Sci. 30 235-239. Krogan, N.J., Kim, M., Tong, A., Golshani, A., Cagney, G., Canadien, V., Richards, D.P., Beattie, B.K., Emili, A., Boone, C., Shilati fard, A., Buratowski, S. and Greenblatt, J. (2003) Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol. Cell Biol. 23 4207-4218. Kugel, J.F. and Goodrich, J.A. (1998) Promoter escape limits the rate of RNA polymerase II transcription and is enhanced by TFIIE, TF IIH, and ATP on negatively supercoiled DNA. Proc. Natl. Acad. Sci. USA 95 9232. Kumar, K.P., Akoulitchev, S. and Reinberg, D. (1998) Promoter-proximal stalling results from the inability to recruit transcription fact or IIH to the transcri ption complex and is a regulated event. Proc. Natl. Acad. Sci. USA 95 9767. Lau, J.F., Nusinzon, I., Burakov, D., Freedman, L.P. and Horvath, C.M. (2003) Role of metazoan mediator proteins in interferon-responsive transcription. Mol. Cell Biol 23 620-628. Lee, Y.C., Park, J.M., Min, S., Han, S.J. and Kim, Y.J. (1999) An activator binding module of yeast RNA polymerase II holoenzyme. Mol. Cell Biol. 19 2967-2976. Li, L., Tutone, A.F., Drummond, R.S., Gardner, R.C. and Luan, S. (2001) A novel family of magnesium transport genes in Arabidopsis Plant Cell 13 2761-2775. Li, Y., Bjorklund, S., Jiang, Y.W., Kim, Y.J., La ne, W.S., Stillman, D.J. and Kornberg, R.D. (1995) Yeast global tran scriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme. Proc. Natl. Acad. Sci. USA 92 10864-10868. Liao, S.M., Zhang, J., Jeffery, D.A., Koleske, A.J., Thompson, C.M., Chao, D.M., Viljoen, M., van Vuuren, H.J. and Young, R.A. (1995) A kinase-cyclin pair in the RNA polymerase II holoenzyme. Nature 374 193-196. Linder, T. and Gustafsson, C.M. (2004) The Soh1/MED31 protein is an ancient component of Schizosaccharomyces pombe and Saccharomyces cerevisiae Mediator. J. Biol. Chem. 279 49455-49459. Malik, S., Gu, W., Wu, W., Qin, J. and Roeder, R.G. (2000) The USA-deri ved transcriptional coactivator PC2 is a submodule of TRAP/SMCC and acts synerg istically with other PCs. Mol. Cell, 5 753-760. Malik, S. and Roeder, R.G. (2000) Transcriptional regu lation through Mediator-like coactivators in yeast and metazoan cells. Trends Biochem. Sci. 25 277.

PAGE 68

68 Malik, S., Wallberg, A.E., Ka ng, Y.K. and Roeder, R.G. (2002) TRAP/SMCC/mediator-dependent transcrip tional activation from DNA and chromatin templates by orphan nuclear recepto r hepatocyte nuclear factor 4. Mol. Cell Biol. 22 5626. Manak, M.S., Paul, A.L., Sehnke, P.C. and Ferl, R.J. (2002) Remote sensing of gene expression in Planta: transgenic plants as monitors of exogenous stress perception in extraterrestrial environments. Life Support Biosph. Sci. 8 83-91. Mittler, G., Stuhler, T., Santo lin, L., Uhlmann, T., Kremmer, E., Lottspeich, F., Berti, L. and Meisterernst, M. (2003) A novel docking site on Mediat or is critical for activation by VP16 in mammalian cells. EMBO J 22 6494-6504. Mo, X., Kowenz-Leutz, E., Xu, H. and Leutz, A. (2004) Ras induces mediator complex exchange on C/EBP beta. Mol. Cell 13 241. Myers, L.C., Gustafsson, C.M., Bushnell, D.A ., Lui, M., Erdjument-Bromage, H., Tempst, P. and Kornberg, R.D. (1998) The Med proteins of y east and their function through the RNA polymerase II carboxy-terminal domain. Genes Dev. 12 45-54. Naar, A.M., Beaurang, P.A., Zhou, S., Ab raham, S., Solomon, W. and Tjian, R. (1999) Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398 828-832. Naar, A.M., Lemon, B.D. and Tjian, R. (2001) Transcriptional coactivator complexes. Annu. Rev. Biochem. 70 475-501. Naar, A.M., Taatjes, D.J., Zhai, W., Nogales, E. and Tjian, R. (2002) Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation. Genes Dev. 16 1339-1344. Nair, D., Kim, Y. and Myers, L.C. (2005) Mediator and TFIIH govern carboxyl-terminal domain-dependent transcri ption in yeast extracts. J. Biol Chem. 280 33739-33748. Nair, R. and Rost, B. (2002) Inferring sub-cellular local ization through automated lexical analysis. Bioinformatics 18 S78S86 Navarro, A., Rondon, G., Aguilera, A ., Struhl, K., Reed, R. and Hurt, E. (2002) TREX is a conserved complex coupling transcri ption with messenger RNA export. Nature 417 304. Nevado, J., Tenbaum, S.P. and Aranda, A. (2004) hSrb7, an esse ntial human Mediator component, acts as a coactivator for the thyroid hormone receptor. Mol. Cell Endocrinol. 222 41. Oelgeschlager, T. (2002) Regulation of RNA polymeras e II activity by CTD phosphorylation and cell cycle control. J. Cell Physiol. 190 160-169.

PAGE 69

69 Ogas, J., Kaufmann, S., Henderson, J. and Somerville, C. (1999) PICKLE is a CHD3 chromatin-remodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis Proc. Natl. Acad. Sci. USA 96 13839. Onodera, Y., Haag, J.R., Ream, T., Nunes, P.C., Pontes, O. and Pikaard, C.S. (2005) Plant nuclear RNA polymerase IV mediates siRNA and DNA methyl ation-dependent heterochromatin formation. Cell 120 613-622. Park, H.C., Kim, M.L., Kang, Y.H., Jeon, J.M., Yoo, J.H., Kim, M.C., Park, C.Y., Jeong, J.C., Moon, B.C., Lee, J.H., Yoon, H.W., Lee, S.H, Chung, W.S., Lim, C.O., Lee, S.Y., Hong, J.C. and Cho, M.J. (2004) Pathogenand NaCl-indu ced expression of the SCaM-4 promoter is mediated in part by a GT-1 box th at interacts with a GT-1-like transcription factor. Plant Physiol. 135 2150-2161. Park, J.M., Gim, B.S., Kim, J.M., Yoon, J.H., Kim, H.S., Kang, J.G. and Kim, Y.J. (2001) Drosophila Mediator complex is broadly utilized by diverse gene-specific transcription factors at different type s of core promoters. Mol. Cell. Biol. 21 2312-2323. Park, J.M., Kim, H.S., Han, S. J., Hwan g, M.S., Lee, Y. C. and Kim, Y.J. (2000) In vivo requirement of activator-specifi c binding targets of Mediator. Mol. Cell. Biol. 20 8709. Pineda Torra, I., Freedman, L.P. and Garabedian, M.J. (2004) Identification of DRIP205 as a coactivator for the Farnesoid X receptor. J. Biol. Chem. 279 36184. Pugh, B.F. (2000) Control of gene expression thr ough regulation of the TATA-binding protein. Gene 255 1-14. Rachez, C., Lemon, B.D., Suldan, Z., Br omleigh, V., Gamble, M., Naar, A.M., Erdjument-Bromage, H., Tempst, P. and Freedman, L.P. (1999) Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature, 398 824. Rachez, C., Suldan, Z., Ward, J., Chang, C. P., Burakov, D., Erdjument-Bromage, H., Tempst, P. and Freedman, L.P. (1998) A novel protein comple x that interacts with the vitamin D3 receptor in a ligand-dependent mann er and enhances VDR transactivation in a cell-free system. Genes Dev. 12 1787-800. Ranish, J.A., Yudkovsky, N. and Hahn, S. (1999) Intermediates in formation and activity of the RNA polymerase II preinitiation co mplex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. Genes Dev. 13 49-63. Reeves, W.M. and Hahn, S. (2003) Activator-independent functi ons of the yeast mediator sin4 complex in preinitiation complex form ation and transcription reinitiation. Mol. Cell Biol. 23 349-358.

PAGE 70

70 Rieping, M. and Schoffl, F. (1992) Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced e xpression of chimaeric heat shock genes in transgenic tobacco. Mol. Gen. Genet. 231 226-232. Roth, S.Y., Denu, J.M. and Allis CD. (2001) Histone acetyltransferases. Annu. Rev. Biochem. 70 81. Rubio, V., Shen, Y., Saijo, Y., Liu, Y., Gusmar oli, G., Dinesh-Kumar, S.P. and Deng, X.W. (2005) An alternative tandem affin ity purification st rategy applied to Arabidopsis protein complex isolation. Plant J. 41 767-778. Ryu, S., Zhou, S., Ladurner, A.G. and Tjian, R. (1999) The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1. Nature 397 446-450. Sakai, A., Shimizu, Y., Kondou, S ., Chibazakura, T. and Hishinuma, F. (1990) Structure and molecular analysis of RGR1 a gene required for glucose repression of Saccharomyces cerevisiae Mol. Cell. Biol. 10 4130-4138. Sakurai, H. and Fukasawa, T. (2000) Functional connections between mediator components and general transcri ption factors of Saccharomyces cerevisiae J. Biol. Chem. 275 37251-37256. Samuelsen, C.O., Baraznenok, V., Khorosjutina, O., Sphr, H ., Kieselbach, T., Holmberg, S. and Gustafsson, C.M. (2003) TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits are functionally c onserved through evolution. Proc. Natl. Acad. Sci. USA 100 6422. Sato, S., Tomomori-Sato, C., Parmely, T.J., Floren s, L., Zybailov, B., Swanson, S.K., Banks, C.A., Jin, J., Cai, Y., Washburn, M.P., Conaway, J.W. and Conaway, R.C. (2004) A set of consensus mammalian mediator s ubunits identified by multidimensional protein identification technology. Mol. Cell 14 685-691. Shilatifard, A., Conaway, R.C. and Conaway, J.W. (2003) The RNA polymerase II elongation complex. Annu. Rev. Biochem. 72 693. Song, W., Treich, I., Qian, N., Kuchin, S. and Carlson, M. (1996) SSN genes that affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SRB proteins associated with RNA polymerase II. Mol. Cell. Biol. 16 115. Spahr, H., Beve, J., Larsson, T., Bergstrom, J., Karlsson, K.A. and Gustafsson, C.M. (2000) Purification and characterization of RNA polymerase II holoenzyme from Schizosaccharomyces pombe J. Biol. Chem. 275 1351-1356. Stevens, J.L., Cantin, G.T., Wang, G., Shevchenko, A. and Berk, A.J. (2002) Transcription control by E1A and MAP kinase path way via Sur2 mediator subunit. Science 296 755.

PAGE 71

71 Stockinger, E.J., Mao, Y., Regier, M.K., Triezenberg, S.J. and Thomashow, M.F. (2001) Transcriptional adaptor and histon e acetyltransferase proteins in Arabidopsis and their interactions with CBF1, a transcriptional ac tivator involved in cold-regulated gene expression. Nucleic Acids Res. 29 1524-1533. Suzuki, Y., Nogi, Y., Abe, A. and Fukasawa, T. (1988) GAL11 protein, an auxiliary transcription activator for genes enco ding galactose-metabolizing enzymes in Saccharomyces cerevisiae Mol. Cell Biol. 8 4991-4999. Taatjes, D.J., Naa,r A.M., Andel, F. 3rd, Nogales, E. and Tjian, R. (2002) Structure, function, and activator-induced conformations of the CRSP coactivator. Science 295 1058-1062. Thomas, M.C. and Chiang, C.M. (2006) The general transcri ption machinery and general cofactors. Crit. Rev. Biochem. Mol. Biol. 41 105-178. Thompson, C.M., Koleske, A .J., Chao, D.M. and Young, R.A. (1993) A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast. Cell 73 1361-1375. Toth, J.I., Datta, S., Athanikar, J.N., Freedman, L.P. and Osborne, T.F. (2004) Selective coactivator interactions in gene activation by SREBP-1a and -1c. Mol. Cell Biol. 24 8288. Villain, P., Clabault, G., Mache, R. and Zhou, D.X. (1994) S1F binding site is related to but different from the light-responsive GT-1 bi nding site and differentially represses the spinach rps1 promoter in transgenic tobacco. J. Biol. Chem. 269 16626-16630. Volkov, R.A., Panchuk, I.I. and Schoffl, F. (2003) Heat-stress-depend ency and developmental modulation of gene expression: the potential of hous e-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR. J. Exp. Bot. 54 2343-2349. Wada, O., Oishi, H., Takada, I., Yan agisawa, J., Yano, T. and Kato, S. (2004) BRCA1 function mediates a TRAP/DRIP complex th rough direct interaction with TRAP220. Oncogene 23 6000-6005. Wang, G. and Berk, A.J. (2002) In vivo association of adenovirus large E1A protein with the human mediator complex in adenoviru s-infected and -transformed cells. J Virol 76 9186-9193. Wang, Q., Sharma, D., Ren, Y. and Fondell, J.D. (2002) A coregulatory role for the TRAP-mediator complex in androgen receptor-mediated gene expression. J. Biol. Chem. 277 42852. Wang, S., Ge, K., Roeder, R.G. and Hankinson, O. (2004) Role of mediat or in transcriptional activation by the aryl hydrocarbon receptor. J. Biol. Chem. 279 13593.

PAGE 72

72 Wang, Z.Y. and Tobin, E.M. (1998) Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 ( CCA1 ) gene disrupts circadian r hythms and suppresses its own expression. Cell 93 1207-1217. Warnmark, A., Almlof, T., Leers, J., Gustafsson, J.A. and Treuter, E. (2001) Differential recruitment of the mammalian mediator s ubunit TRAP220 by estroge n receptors ERalpha and ERbeta. J. Biol. Chem. 276 23397. Woychik, N.A. and Hampsey, M. (2002) The RNA polymerase II machinery: structure illuminates function. Cell 108 453-463. Yan, Q., Moreland, R.J., Co naway, J.W. and Conaway, R.C. (1999) Dual roles for transcription factor IIF in promoter escape by RNA polymerase II. J. Biol. Chem. 274 35668. Ye, Z.H., Freshour, G., Hahn, M.G., Burk, D.H. and Zhong, R. (2002) Vascular development in Arabidopsis Int. Rev. Cytol. 220 225-256. Yuan, C.X., Ito, M., Fondell, J.D., Fu, Z.Y. and Roeder, R.G. (1998) The TRAP220 component of a thyroid hormone receptorasso ciated protein (TRAP) coactivator complex interacts directly with nuclear rece ptors in a ligand-dependent fashion. Proc. Natl. Acad. Sci USA 95 7939-7944. Yudkovsky, N., Ranish, J.A. and Hahn, S. (2000) A transcription rein itiation intermediate that is stabilized by activator. Nature 408 225-229. Zhong, R., Burk, D.H., Nairn, C.J., Wood-Jon es, A., Morrison, W.H. 3rd and Ye, Z.H. (2005) Mutation of SAC1 an Arabidopsis SAC domain phosphoinositide phosphatase, causes alterations in cell mo rphogenesis, cell wall synthesis, and actin organization. Plant Cell 17 1449-1466. Zhou, R., Bonneaud, N., Yuan, C.X., de Sa nta Barbara, P., Boizet, B., Schomber, T., Schere,r G., Roeder, R.G., Poulat ,F., Berta, P. and Tibor, S. (2002) SOX9 interacts with a component of the human thyroid hor mone receptor-associated protein complex. Nucleic Acids Res. 30 3245. Zhu, X., Wiren, M., Sinha, I., Rasmussen, N.N., Linder, T., Holmberg, S., Ekwall, K. and Gustafsson, C.M. (2006) Genome-wide occupancy prof ile of mediator and the Srb8-11 module reveals interacti ons with coding regions. Mol Cell 22 169-178. Zhu, Y., Qi, C., Jain, S., Le Beau, M.M., Es pinosa, R. 3rd, Atkins, G.B., Lazar, M.A., Yeldandi, A.V., Rao, M.S. and Reddy, J.K. (1999) Amplification and overexpression of peroxisome proliferator-activated receptor binding protein (PBP/PPARBP) gene in breast cancer. Proc. Natl. Acad. Sci. USA 96 10848. Zhu, Y., Qi, C., Jain, S., Rao, M.S. and Reddy, J.K. (1997) Isolation and characterization of PBP, a protein that interacts with per oxisome proliferator-activated receptor. J. Biol. Chem. 272 25500.

PAGE 73

73 BIOGRAPHICAL SKETCH Wei Pan received his Bachelor of Scien ce degree in biology from Northeast Normal University in Changchun, China, and then a Mast er of Science degree in biophysics from the Chinese Academy of Agricultural Sciences in Be ijing, China. His current research interests are centered on genetics, devel opment and molecular biology.


Permanent Link: http://ufdc.ufl.edu/UFE0018461/00001

Material Information

Title: Molecular Analysis of Two Putative Mediator Subunits in Arabidopsis thaliana
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: UFE0018461:00001

Permanent Link: http://ufdc.ufl.edu/UFE0018461/00001

Material Information

Title: Molecular Analysis of Two Putative Mediator Subunits in Arabidopsis thaliana
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: UFE0018461:00001


This item has the following downloads:


Full Text












MOLECULAR ANALYSIS OF TWO PUTATIVE MEDIATOR SUBUNITS INTArabidopsis
thaliana











By

WEl PAN
















A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE


UNIVERSITY OF FLORIDA

2007





























Copyright 2007

by

Wei Pan



























To my family









ACKNOWLEDGMENTS

I thank Dr. Bill Gurley for his kindness, commitment and great mentoring. He is an

excellent advisor because he always encouraged me to develop independent thinking and gave

me the opportunity to pursue my research interests. His careful reading and editing greatly

improved this thesis. I thank Dr. Robert Ferl for his suggestion on my work and giving me the

pBIl01sGFP vector. I thank Dr. Kevin O'Grady for his continuous help with my experiments

and teaching me many molecular biology techniques. I extend thanks to Dr. Eva

Czarnecka-Verner for her suggestions on my research.

I thank Dr. Zhonglin Mou and Ms. Xudong Zhang for their valuable assistance with some

techniques such as Northern blotting and GUS staining, and generously letting me share some of

their facilities. I thank Ms. Donna Williams for her assistance with the con-focal observation.

I thank Dr. Masaharu Suzuki for helping me get started in research in the Plant Molecular

and Cellular Biology Program (PMCB); Dr. Alice Harmon for the invaluable training I gained in

her lab; and Dr. David Clark for his kindness and support for my study in PMCB.

I also wish to express my gratitude to the entire PMCB faculty who taught me in classes

and journal clubs. I benefited a lot from the wonderful courses.

I am extremely grateful for all my family and friends for their understanding and support

over the years.











TABLE OF CONTENTS


page

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


LI ST OF T ABLE S .........__.. ..... .__. ...............7....


LI ST OF FIGURE S .............. ...............8.....


LI ST OF AB BREVIAT IONS ........._.___..... ..._. ............... 10...


AB S TRAC T ............._. .......... ..............._ 12...


CHAPTER


1 INTRODUCTION ................. ...............14.......... ......


Assembly of the Preinitiation Complex ................. ...............14........... ...
Identification of the Mediator Complex in Yeast ................. ...............16........... ..
Identification of the Mediator Complex in Human Cells ................ ......... ................17
TRAP Complex .............. ...............17....
SM CC Complex .............. ...............18....
DRIP Complex .............. ...............18....
ARC Complex .............. ...............18....
CRSP Complex............... ...............18
PC2 Complex............... ... .................1
Mediator Interacts with Transactivators .............. ...............20....
Mediator Interacts with RNA pol II............... ...............21...
Phosphorylation of RNA Pol II CTD ..........._... ...__.....__....._ ..............21
Mediator Interacts with Coactivators ................... .......... ...............22......
Mediator Promotes the Formation of a Stable PIC ................. ...............23..............
Mediator is Required in the Reinitiation Scaffold ....__ ......_____ ...... ....__........2
Mediator Stimulates both Basal and Activated Transcription ....._____ ..... .. ...__...........23
Model for Mediator Function in Activated Transcription ................ .......... ...............24
Hypothesis for a Mediator Complex in Arabidopsis ................ .............. ......... .....24

2 MATERIALS AND METHODS .............. ...............29....


Plant Growth Conditions .............. .. ...............29...
Genotyping of the T-DNA Insertion Lines ................. ...............29...............
RN A Analy si s............... ...............3
M icroscopy .............. ...............30....
Plasmid Construction................ .............3
GU S Staining ................. .... ... ...... .......... ............3
Agrobacterium Transformation Technique .............. ...............3 1....
Chromatin Immunoprecipitation .............. ...... ...............32.
PCR Analysis of Chromatin Immunoprecipitation............... ..........3












Bioinf orm atics .............. ...............3 5....


3 RE SULT S .............. ...............36....


Analysis of Arabidopsis 2ed31 Gene by Multiple Sequence Alignments ............._..__.........36
Phenotype Characterization of med31 Mutants ................. ...............37........... ...
M~ed31 Expression in the med31-2 Plants .................. ........... ........ .. .............. ......3
Subcellular Localization and Tissue Expression Pattern of Med3 1::GFP Fusion Proteins....3 9
Tissue Expression Pattern of2~ed31 Promoter::GUS Fusions ........_............. ...... .........39
Co-immunoprecipitation Maps Med6 and Med31 to Promoter DNA..........._._... ...............40
ChlP Analysis for Med31 .............. ...............41....
ChlP Analysis for Med6 ........._._ ...... .... ...............42...
Conclusion ........._.___..... .__ ...............43....


4 DI SCUS SSION ............ ..... ._ ............... 8....


Phenotype Characterization of med31 Mutants ................. ...............58........... ...
Evidence for a Mediator Complex in Arabidopsis .............. ...............59....

LIST OF REFERENCES ................. ...............61................


BIOGRAPHICAL SKETCH .............. ...............73....










LIST OF TABLES


Table page

1-1 Interaction of the transactivators with the Mediator subunits in different organisms ..........26

1-2 Mediator subunits in yeast, Arabidopsis, Drosophila and humans ............ ...................28










LIST OF FIGURES


Figure page

3-1 Multiple sequence alignments of Med3 1 homologs in different species. ................... .45

3-2 Multiple alignments of AtMed3 1 with the deduced amino acid sequences of its
homologs in other plant species .............. ...............46....

3-3 Diagrammatic representation of the insertions of the T-DNA in med31-1 and
med31-2............... ...............47

3-4 Germination rate and root length of WT and med31-1 seedlings (9-day-old). ............47

3-5 Nine-day-old WT and med31-2 seedlings grown under continuous light ................... .47

3-6 Nine-day-old WT and med31-2 seedlings grown under dark ................. ................ .48

3-7 Ten-day-old WT and med31-2 seedlings............... ...............4

3-8 Comparison of adult WT plants and med31-2 plants .............. .....................4

3-9 Northern blot analysis of Med31 expression in WT and med31-2 plants ....................49

3-10 Subcellular localization of Med3 1::GFP fusion proteins in the root tip of a
35-day-old plant .............. ...............49....

3-11 Expression of Med3 1::GFP fusion proteins in lateral roots ................ ................ ...50

3-12 Expression of Med3 1::GFP fusion proteins in a root hair ................. ............... .....50

3-13 Expression of Med3 1::GFP fusion proteins in a leaf ................. ................. ......5 1

3-14 Expression of Med3 1::GFP fusion proteins in a trichome. ........._._... ........_._........52

3-15 Expression of Med3 1::GFP fusion proteins in a petiole. .........._.... ......_._...........53

3-16 M~ed31 promoter directed GUS tissue expression pattern in young plants
(16-day-old) ................. ...............54........... ....

3-17 M~ed31 promoter directed GUS tissue expression pattern in adult plants
(46-day-old)................ .............5

3-18 Multiple sequence alignments of Med6 homologs in different species .....................55

3-19 Med3 1 associates with the promoters of CCA1~, Hspl8. 2 and Adhl, but not with
the inter genetic region ................. ...............56........... ....










3-20 Med6 associates with the promoters of CCA1~, Hspl8. 2 and Adhl, but not with
the inter genetic region ................. ...............56........... ....

3-21 Immunoglobulin G Sepharose and c-Myc antibody cannot immunoprecipitate the
CCA 1 promoter from WT Arabidopsis ..........._..__......__ ....._._ ...........5









LIST OF ABBREVIATIONS

Adh alcohol dehydrogenase

ARC activator-recruited cofactor

CCAl circadian clock associated 1

ChlP chromatin immunoprecipitation

c-Myc cellular myelocytomatosis oncogene

CRSP cofactor required for Spl activation

CTD carboxy-terminal domain

DRIP vitamin D receptor interacting protein

EST expressed sequence tag

GFP green fluorescent protein

GUS P-glucuronidase

HAT histone acetyltransferase

Hsp l 8.2 heat shock protein 18.2

IgG immunoglobulin G

PC2 positive cofactor 2

PIC preinitiation complex

RNA pol RNA polymerase

Sepl0 Separationl0

SMCC SRB/MED Cofactor Complex

Sohl suppressor of hprl1

SWI/SNF switching/sucrose non-fermenting

TAP tandem affinity purification

TFIIA Transcription Factor II A

TFIIB Transcription Factor II B









TFIID Transcription Factor II D

TFIIE Transcription Factor II E

TFIIF Transcription Factor II F

TFIIH Transcription Factor II H

TRAP thyroid hormone receptor-associated protein

UTR untranslated region

VPl16 herpes simplex virus protein 16

WT wild type









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

MOLECULAR ANALYSIS OF TWO PUTATIVE MEDIATOR SUBUNITS INTArabidopsis
thaliana


By

Wei Pan

May 2007

Chair: William B. Gurley
Maj or Department: Plant Molecular and Cellular Biology

Mediator is a conserved coactivator complex that has been identified in yeast, Drosophila

and humans. It plays a critical role in gene transcription mediated by RNA polymerase II (RNA

pol II) by serving as a bridge between activators bound to the promoter and other transcription

machineries, including RNA pol II. Despite evidence suggesting such a vital role of Mediator in

gene expression, the subunit composition and function of Mediator has not been determined in

plants. Based on the conserved transcriptional machineries (RNA pol II, general transcription

factors and some coactivators) in plants, metazoans and yeast, we hypothesized the plant also has

the Mediator coactivator. Identification of the homologs of most of the yeast and metazoan

Mediator subunits in Arabidopsis supported this hypothesis.

This study characterized the function of two putative Mediator subunits, Med6 and Med3 1.

Two T-DNA insertion lines in the M~ed31 promoter or 5' untranslated region were identified. The

med31-1 mutant line had shorter root length and a reduced germination rate. The med31-2 plants

had shorter root length, aberrant patterns of cotyledon development, and smaller size compared

with wild type plants. We found the Med31::GFP (green fluorescent protein) fusion proteins

were localized to the nucleus. The Med31::GFP signal was detected in the roots, leaves,









trichomes and petioles. In addition, we found the M~ed31 promoter::GUS fusions were expressed

in the shoot apexes and lateral roots of the young seedlings (16 days old), and in the young

inflorescences, anthers, stigmas of adult plants (46 days old) and in developing seeds. Both

Med6 and Med3 1 proteins were localized to the promoters of three unrelated genes (CCA1,

Hspl8.2 and Adhl). These results strongly support the conclusion that Med6 and Med31 are

members of the Mediator complex in Arabidopsis.









CHAPTER 1

INTRODUCTION

Assembly of the Preinitiation Complex

Transcription is one of the most significant steps that occur during gene expression. It is

carried out by RNA polymerases and additional factors. There are four kinds of RNA

polymerases in plants, RNA polymerase (RNA pol) I, II, III and IV. RNA pol I is located in the

nucleolus, and it transcribes rRNA genes, except 5S rRNA. RNA pol II is located in the

nucleoplasm and transcribes hnRNA, the precursor of mRNA. RNA pol III is also located in the

nucleoplasm and is responsible for the synthesis of tRNA, 5 S rRNA and other small RNAs

(Thomas and Chiang, 2006). And last, an RNA polymerase unique to plants, RNA polymerase

IV, is involved in the siRNA silencing pathway, RNA-dependent DNA methylation and the

formation of heterochromatin (Onodera et al., 2005).

Transcription by RNA pol II can be broadly categorized as basal transcription

(activator-independent) and activated transcription (activator-dependent) A simplified sequence

of activated transcription initiation for RNA pol II has been postulated as follows. Activators

(transactivators or transcription factors) bind the regulatory motifs of DNA and then recruit a

variety of additional factors that prepare the promoter for the arrival of RNA pol II and the

formation of the preinitiation complex (PIC) (Thomas and Chiang, 2006). One of the first

components to arrive is a kinase which phosphorylates histone H3 (Featherstone, 2002). Then

coactivators that can modify chromatin structures are recruited. For example, HAT (histone

acetyltransferase) arrives at the promoter early in the activation process and its role is to

acetylate specific lysines in histone amino-termini and other transcription factors (Roth et al.,

2001; Naar et al., 2001; Clayton et al., 2006). Another complex that is recruited early in the

process of gene activation is SWI/SNF (switching/sucrose non-fermenting), which remodels the









chromatin structure and facilitates the accessibility of other members of the transcriptional

apparatus to the DNA (Gavin et al., 2001, Havas et al., 2000). After the promoter is made

accessible, the TFIID complex is recruited to the TATA box in the promoter (Pugh, 2000).

Activators also recruit Mediator complex which facilitates the formation of pol II PIC, which

consists of RNA pol II and general transcription factors (TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH)

(Conaway et al., 2005).

Next, TFIIH facilitates promoter melting and phosphorylates the CTD (carboxy-terminal

domain) of the largest subunit of RNAP II (Jiang et al., 1996; Kim et al., 1994). This

phosphorylation event is thought to be required for promoter clearance and the start of

transcriptional elongation (Dvir et al., 1997; Kugel and Goodrich, 1998; Kumar et al., 1998).

After the synthesis of the initial transcript, most members of the PIC (with the exception of

TFIIB and TFIIF) remain at the promoter and form a structure know as the "scaffold" that

facilitates the reentry of RNA pol II, TFIIB and TFIIF for subsequent rounds of synthesis

(Yudkovsky et al., 2000). In the process of reinitiation, the CTD of RNA pol II is

dephosphorylated by a CTD phosphatase that is stimulated by TFIIF (Friedl et al., 2003). This

cycle of phosphorylation and dephosphorylation of the CTD is essential to the entry of RNA pol

II to the PIC (with a hypophosphorylated CTD) and subsequent promoter clearance

(hyperphosphorylated CTD) (Oelgeschlager, 2002).

One of the key regulatory complexes involved in the process of promoter activation is the

Mediator. This large assemblage of proteins (~2MDa) is conserved from yeast to humans and is

composed of 25-29 subunits (Boube et al., 2002). It is becoming increasingly clear that Mediator

plays a critical role in both activated and basal transcription mediated by RNA pol II in yeast and

metazoans (Back et al., 2002, Nair et al., 2005), because it serves as a bridge between activators









bound to the promoter and other general transcription factors, as well as RNA pol II (Kornberg,

2005).

Identification of the Mediator Complex in Yeast

The presence of Mediator was proposed because of the discovery of activator inhibition

in yeast. The activator GAL4-VPl16 was found to repress the activation effect of another

activator (a factor binding to a thymidine-rich DNA element) both in vivo and in vitro. This

phenomenon led to the hypothesis that the two activators competed for a common intermediate

factor. Activator interference was relieved in vitro with the addition of the fraction containing

this intermediate factor, which was named Mediator (Kelleher et al., 1990).

The Mediator fraction from column chromatography was shown to be required for

GAL4-VPl16 and GCN4-dependent gene transcription in an in vitro transcription system

(Flanagan et al., 1991). This was the initial direct evidence that Mediator was involved in gene

transcription. The In vitro transcription system was reconstituted with purified RNA pol II and

general transcription factors from yeast and was widely used for checking the presence of

Mediator, thereafter.

Three experimental approaches were originally used to identify proteins as Mediator

subunits: 1) Identify the suppressors of RNA pol II CTD truncation mutations; 2) Isolate the

fraction (RNA pol II holoenzyme) that can stimulate the activator-dependent transcription.

Separate the proteins by electrophoresis in a gel, and then identify the proteins by peptide

sequencing; and 3) Identify the proteins that co-immunoprecipitate with known Mediator

subunits. The presence in the RNA pol II holoenzyme and support of activator-dependent

transcription were two criteria that were used to confirm the identities of Mediator subunits.

In total, 25 Mediator subunits have been identified in S. cerevisiae. Nine Mediator subunits

(Srb2, Srb4, Srb5, Srb6, Srb7, Srb8, Srb9, Srbl0 and SrbI1) were identified based on the










suppression of S. cerevisiae RNA pol II CTD truncation mutations. All of these subunits were

shown to be present in the holoenzyme (Thompson et al., 1993; Kim et al., 1994; Koleske and

Young, 1994; Liao et al., 1995; Hengartner et al., 1995). Fifteen Mediator subunits (Medl, Med2,

Pgdl (Hrsl), Med4, Med7, Med8, Med11, Gallli, Sin4 Rgrl, Mtr32, Rox3, Nutl, Nut2, and

Cse2) were detected in the RNA pol II holoenzyme and identified by peptide sequencing (Kim et

al., 1994; Gustafsson et al., 1997; Gustafsson et al., 1998; Li et al., 1995; Myers et al., 1998).

The mutant yeast strains for Galll, Sin4 and Rgrl showed similar mutant phenotypes, which

suggested they may function in the same pathway (Fassler et al., 1991, Jiang and Stillman, 1995,

Suzuki et al., 1988; Chen et al., 1993, Sakai et al., 1990). More recently, Med31 was found to be

a Mediator subunit in S. cerevisiae and S. pombe based on co-purification with previously

characterized Mediator subunits (Linder and Gustafsson, 2004).

Identification of the Mediator Complex in Human Cells

Two methods were used to identify the Mediator subunits in human cells: 1) Isolate the

nuclear extract fraction that can stimulate the activator-dependent transcription. Separate the

proteins on the gel, and then identify the proteins by peptide sequencing; and 2) Identify the

proteins that co-immunoprecipitate with the transactivators (or their activation domains) or

known Mediator subunits. Most identified subunits are orthologs of the yeast Mediator subunits.

However, various Mediator complexes with different subunit compositions were isolated in

different labs (Sato et al., 2004). A brief description of human Mediator types follows.

TRAP Complex

A thyroid hormone receptor-associated protein (TRAP) complex was isolated based on

co-precipitation with FLAG epitope-tagged hTRalphal (human thyroid hormone receptor alphal)

(Fondell et al., 1996).









SMCC Complex

The human SRB/MED Cofactor Complex (SMCC) was purified by affinity

chromatography of FLAG epitope-tagged human SRB proteins (Gu et al., 1999).

DRIP Complex

The DRIP (vitamin D receptor interacting protein) complex was isolated from the nuclear

extract of human Namalwa B cells based on its interaction with the VDR LBD (vitamin D3

receptor ligand-binding domain) in the presence of hormone. This complex contains 10 proteins

and it can stimulate transcription by VDR-RXR. It was shown that at least one of its subunits has

histone acetyltransferase activity (Rachez et al., 1998).

ARC Complex

The ARC (activator-recruited cofactor) complex was isolated by its affinity for the

activation domains of SREBP-la, VPl16 and the p65 subunit of NF-kB, respectively, from HeLa

cell nuclear extract (Naar et al., 1999). It can not only stimulate transcription by activators such

as SREBP-la/Spl, NF-k
CRSP Complex

The CRSP (cofactor required for Spl activation) complex was isolated from HeLa cell

nuclear extract and shown to be required for Spl-dependent transcriptional activation (Ryu et al.,

1999). This complex consists of 9 subunits and has a mass of approximately 0.7 MDa.

PC2 Complex

The PC2 (positive cofactor 2) complex was isolated from HeLa cell nuclear extracts based

on its ability to stimulate HNF4 (hepatocyte nuclear factor 4) and GAL4-AH dependent

transcription (Malik et al., 2000). This complex consists of at least 15 subunits and is larger than

0.5MDa. The presence of these subunits within the complex was confirmed by the









co-immunoprecipitation of epitope (FLAG and HA)-tagged MED 10. Both PC2 and CRSP were

found to be subcompexes of ARC, DRIP, or TRAP/SMCC (Malik and Roeder, 2000).

Despite being originally isolated by different approaches, some complexes found in human

cells (ARC, DRIP, and TRAP/SMCC) were shown to be very similar in subunit composition

(Naar et al., 1999; Malik and Roeder, 2000).

The finding that various closely related Mediator complexes have slightly different subunit

composition raised the question of whether some of the proteins identified are true subunits, or

just contaminants associated with a particular isolation strategy. Sato and colleagues (Sato et al.,

2004) addressed this question by co-immunoprecipitation of human Mediator using six

FLAG-tagged subunits to individually purify complexes for analysis of subunit composition by

MudPIT (multidimensional protein identification technology). Proteins present in all six

independent Mediator preparations were considered to be true Mediator subunits. Their results

support the conclusion that all proteins identified previously are bona fide Mediator subunits. In

addition, they identified the MED13L and the CDK8-like cyclin-dependent kinase CDKll as

putative Mediator-associated proteins.

The inconsistency in Mediator subunit composition was thought to be due in part to the

dissociation of Mediator subunits during chromatographic purification and to insensitive protein

detection methods. Another possibility is that the distinct Mediator types from different labs may

have various functions, and therefore, slightly different composition. For example, two distinct

Mediator complexes were isolated using VPl6 and SREBP-1 (sterol-responsive enhancer

binding protein) affinity resins, respectively (Taatj es et al., 2002). The larger one was named as

ARC-L, which is almost identical to the TRAP/DRIP/ARC/SMCC complexes. The smaller

complex was the CRSP complex. ARC-L and CRSP have many subunits in common, except that









CRSP has a CRSP70 subunit not present in ARC-L and does not have the following four

subunits present in the ARC-L: ARC240 /TRAP230O/MED 12, ARC250/ TRAP240/MED 13, cdk8,

and Cyclin C. In yeast, homologs (Srb8, -9, -10 and -1 1) of these four proteins comprise a

distinct complex (Borggrefe et al., 2002), designated as the CDK8 module. The ARC-L complex

is transcriptionally inactive, whereas the CRSP complex is highly active in a reconstituted

Spl/SREBP-dependent transcription system (Ryu et al., 1999).

Mediator Interacts with Transactivators

Many Mediator subunits, such as Medl, Medl2, Medl4, Medl5, Medl6, Medl7, Med23,

Med25, Med29, Cdk8 were found to interact with transactivators in human, yeast, or Drosophila

cells (Table 1-1). Some transactivators, such as the glucocorticoid receptor (Hittelman et al.,

1999) and differentiation-inducing factor (Kim et al., 2004), can interact with multiple Mediator

subunits suggesting a mechanism for more efficiently recruiting the Mediator.

The interaction between transactivators and Mediator subunits is important in

transcriptional regulation. Conditions that result in reduced levels of particular subunits may

have a negative influence on transcription. For example, Medl (TRAP220) was shown to

interact with PPARy, which is a nuclear receptor essential for adipogenesis (Zhu et al., 1997). In

7RAP220 null mouse embryos, the adipogenesis markers and PPARy2 target genes were not

expressed in the embryonic fibroblasts (MEFs), and the MEFs failed to differentiate into

adipocytes via the PPARy pathway (Ge et al., 2002). The authors also showed that activated

transcription by PPARy can be greatly increased by the TRAP complex in a reconstituted

transcription system. In addition, RXRa, another Med1 interacting partner (Zhu et al., 1997), was

shown to be able to enhance the effects of PPARy.









Mediator Interacts with RNA pol II

Many lines of evidence indicate that Mediator interacts directly with the CTD of RNA pol

II. Yeast Mediator, without the CDK8 module, and the human CRSP complex were isolated

through CTD-affinity chromatography (Myers et al., 1998; Naar et al., 2002). RNA pol II

lacking a CTD (Pol II ACTD) functions just as well as WT enzyme in basal transcription in vitro

when Mediator is absent. But contrary to the WT polymerase, this mutant RNA pol II cannot

respond to Mediator in basal transcription and in Gal4-VPl6 or GCN4 activated transcription

(Myers et al., 1998).

Precise structural information has revealed that the three modules of Mediator (head,

middle and tail) wrap around the RNA pol II in the holoenzyme. RNA pol II makes multiple

contacts with the head and middle modules and one with the tail. These interactions are centered

on the RNA pol II Rpb3/Rpb 11 heterodimer, but also involve Rpbl1, Rpb2, Rpb6 and Rpbl12

subunits. These contacts between Mediator and RNA pol II only account for 3 5% of the RNA

pol II surface; however, the remaining part is available for interaction with other PIC factors

(Davis et al., 2002, Chadick and Asturias, 2005).

Phosphorylation of RNA Pol II CTD

The cycle of phosphorylation and dephosphorylation of RNA pol II CTD is significant for

gene transcription. During transcription initiation, the recruitment of RNA pol II requires that the

CTD be hypophosphorylated. The Mediators isolated from Fleischmann's yeast (Kim et al.,

1994), S. pombe (Spahr et al., 2000), S. cerevisiae (Myers et al., 1998) and mouse (Jiang et al.,

1998) all stimulate the phosphorylation of the CTD by the TFIIH after PIC formation

(Hengartner et al., 1998). This phosphorylation of the CTD happens during the transition from

the transcriptional initiation to elongation and is thought to trigger promoter clearance

(Hengartner et al., 1998; Oelgeschlager, 2002). An additional role of the hyperphosphorylated









CTD is to promote interaction of the mRNA capping enzyme with the nascent transcript (Cho et

al., 1997).

The Kin28 protein is a subunit of TFIIH in S. cerevisiae and is the primary kinase involved

in the phosphorylation of RNA pol II CTD. Its kinase activity can be stimulated by Mediator in

vitro (Guidi et al., 2004). It was speculated that the Gall11 subunit of Mediator may regulate the

phosphorylation activity of Kin28 due to the interaction of Gall11 with TFIIH (Sakurai and

Fukasawa, 2000).

The CDK8 module of Mediator in yeast contains Srb8, Srb9, Srbl0, and Srbl1 subunits

and seems to exert a negative effect on transcription (Song et al., 1996; Samuelsen et al., 2003).

A plausible mechanism is provided by the action of Srbl10, which was shown to phosphorylate

the CTD prior to PIC formation and, thus, prevent the entry of RNA pol II (Hengartner et al.,

1998).

Mediator Interacts with Coactivators

Mediator has been shown to interact with other coactivators such as mammalian p300 and

TFIID (Black et al., 2006; Koleske et al., 1992; Thompson et al., 1993; Johnson et al., 2002;

Johnson and Carey, 2003). p300 is a coactivator that contains HAT activity, and in addition to

histones, it can acetylate transcription factors, as well as itself (Roth et al., 2001). The

consequence of its interaction with Mediator is an elevation in histone acetylation (Black et al.,

2006), which makes chromatin more accessible to other factors (Roth et al., 2001).

Autophosphorylation of p300 reduces its association with Mediator. The association of TFIID

with Mediator competes with p300 binding and results in a displacement of p300 from the

promoter. The j oining of Mediator with TFIID contributes to the assembly of the PIC and

activating of the promoter (Black et al., 2006).









Mediator Promotes the Formation of a Stable PIC

In vitro and genetic evidence suggest that Mediator contributes to the formation of a stable

PIC. It has been shown by a template commitment assay that Srb2 (Med20) is essential for the

formation of the PIC (Koleske et al., 1992). In addition, mutations in Srb2 (Med20), Srb4

(Medl7), or Srb5 (Medl8) prevent the formation of the PIC (Ranish et al., 1999), and mutations

in Sin4 (Medl6) and Pgdl (Med3) decrease both the rate and amount of PIC formation in yeast

(Reeves and Hahn, 2003).

Mediator is Required in the Reinitiation Scaffold

The association of Mediator with RNA pol II CTD, Gall11 with TFIIH (Sakurai and

Fukasawa, 2000), and Srb2 with TFIID (Koleske et al., 1992) facilitate the formation of a stable

PIC and maintain the reinitiation scaffold (Nair et al., 2005). Reinitiation and then multiple

rounds of transcription occur after RNA pol II, TFIIB, and TFIIF j oin the scaffold to re-form the

PIC (Nair et al., 2005). Mutation of Pgd1 results in dissociation of Mediator from the scaffold

after initiation and, thus, impairs reinitiation in yeast (Reeves and Hahn, 2003).

Mediator Stimulates both Basal and Activated Transcription

The Mediator fraction from yeast has been shown to stimulate GAL4-VPl6 or

GCN4-dependent transcription in a reconstituted system, and has also been shown to increase

basal transcription by 8-fold (Kim et al., 1994). The ARC (activator-recruited cofactor) complex

not only stimulates transcription by activators such as SREBP-la/Spl, NF-k
Gal4-VPl16/Spl, but also enhances basal transcription in vitro (Naar et al., 1999). Genome-wide

expression analysis showed that only 7% of genes were expressed in the Medl7 mutant of S.

cerevisiae (Holstege et al., 1998). Diminished Mediator leads to the reduction of basal and

activator-dependent transcription in yeast and HeLa cells, which can be restored by addition of

purified Mediator complex in vitro (Back et al., 2002, Nair et al., 2005).









Model for Mediator Function in Activated Transcription

Formation of the PIC starts with the binding of transactivators to the DNA, which is

followed by recruitment of TFIID, TFIIA and TFIIB to the promoter (Ranish et al., 1999, Reeves

and Hahn, 2003; Woychik et al., 2002). The Mediator is recruited by transactivators and possibly

by coactivators, such as p300 and TFIID (Koleske et al., 1992; Thompson et al., 1993; Johnson

et al., 2002; Johnson and Carey, 2003; Black et al., 2006). Mediator and TFIID form a platform

for the entry of the following factors. Mediator recruits the RNA pol II through interaction with

the CTD. TFIIF may be enlisted together with RNA pol II. Then TFIIE and TFIIH enter the

preinitiation complex (Thomas and Chiang, 2006). Next, the DNA helicase activity of TFIIH

causes promoter melting (Jiang et al., 1996; Kim et al., 2000), an essential step before the

synthesis of RNA can begin. Mediator greatly enhances the kinase activity of Kin28 of TFIIH,

which hyperphosphorylates the RNAP II CTD (Guidi et al., 2004). After CTD phosphorylation,

RNA pol II leaves the promoter with TFIIF to start transcriptional elongation (Yan et al., 1999;

Shilatifard et al., 2003). Mediator, TFIIA, TFIID, TFIIH and TFIIE stay on the promoter forming

a platform that supports reinitiation. This scaffold structure, in turn, recruits new TFIIB, TFIIF

and RNA pol II repeatedly to support multiple rounds of transcription (Yudkovsky et al., 2000).

Hypothesis for a Mediator Complex in Arabidopsis

Many of the basic mechanisms of transcription are conserved in plants, metazoans and

yeast (Reviewed in Gurley et al., 2006). The structures of many promoters in these three

kingdoms contain a TATA box, CAAT box, transcription start site and cis-elements for the

binding of general transcription factors and transactivators. In addition, RNA pol II and many

general transcription factors are conserved between plants, fungi and metazoans (Coulson and

Ouzounis, 2003). Arabidopsis also has homologs of the subunits of some coactivators such as

SAGA and other HAT containing complexes (Hsieh and Fischer, 2005). This wide array of









evidence for a high degree of conservation in the basic mechanisms of transcription suggests that

plants may also contain the Mediator coactivator. This view is strongly reinforced by the

presence of many putative Mediator subunits in Arabidopsis based on DNA sequence similarity

(Gurley et al., 2006; Boube et al., 2002). A compilation of Mediator subunits from yeast,

Drosophila and humans is presented in Table 1-2, along with putative subunits from Arabidopsis.

This provides the best estimate for Mediator subunit composition in plants and indicates that

plants may have at least 20 Mediator subunits present in other eukaryotes.

Despite evidence suggesting such a vital role for Mediator in gene expression, the precise

subunit composition and function of Mediator has not been determined in plants. Up to now, two

putative Mediator subunits in Arabidopsis thaliana have been studied. SWP (Strawwelpter) is

the orthologue of Medl4 and is involved in pattern formation at the shoot apical meristem, as

well as defining the duration of cell proliferation (Autran et al., 2002). PFT 1 (phytochrome and

flowering time 1) is the orthologue of Med25. It acts downstream of phyB to regulate the gene

expression and induce flowering under low-light conditions (Cerdan and Chory, 2003). The

important functions of these two putative Mediator subunits hint at the significance of the

Mediator in plants. To unravel the mechanism of gene transcription in plants, it is important to

identify the Mediator complex and characterize its function.










Table 1-1. Interaction of the transactivators with the Mediator subunits in different organisms


Saccharomyces
cerevisiae


Drosophila
melanogaster


Transactivator


ERa and ER 13 estrogen receptor (ER)


GATA family of transcription factors
Breast cancer susceptibility gene 1
(BRCA1i)
Thyroid hormone receptor (TRu, TRBl)

Androgen receptor

Glucocorticoid receptor (GR)
Peroxisome proliferator-activated
Med1
receptors (PPAR aand PPARy)
Retinoic acid receptor (RARu)
Retinoid-X-receptor for 9-cis-retinoic
acid (RXRu)
Vitamin D receptor (VDR)

Hepatocyte nuclear factor 4 (HNF-4)

Farnesoid X receptor (FXR)
Retinoid-related orphan receptor
(RORu)
p53

Aryl hydrocarbon receptor (AHR)


Me3 General control nondepressible factor 4
(GCN4)


Homo sapiens
Zhu et al., 1999;
Burakov et al.
2000: Warnmark
et al., 2001
Crawford et al.
2002

Wada et al., 2004
Yuan et al., 1998,
Zhu et al., 1997
Wang et al., 2002
Hittelman et al.,
1999

Zhu et al., 1997

Zhu et al., 1997

Zhu et al., 1997
Rachez et al.,
1999
Malik et al., 2002

Pineda et al., 2004

Atkmns et al., 1999

Drane et al., 1997
Wang et al., 2004


Park et al., 2000


SRY-box containing gene 9 (Sox9)

Replication and transcription activator
(RTA)


Zhou et al., 2002

Gwack et al., 2003


Hittelman et al.,
1999
Malik et al., 2002

Lau et al., 2003

Toth et al., 2004


Medl2


Glucocorticoid receptor (GR)

Hepatocyte nuclear factor 4 (HNF-4)
Medl4 Signal transducer and activator of
transcription (STAT2)
Sterol regulatory element-binding
protein-l a (SREBP-la)










Table 1-1. Continued.


Saccharomyces
cerevisiae


Drosophila
melanogaster


Transactivator


Homo sapiens


Kato et al., 2002


Small mothers against
decapentaplegic 2/3/4 (SMAD2,
SMAD3, SMAD4)
Medl5 VPl6
General control nondepressible factor
4 (GCN4)
Gal4


Medl6 Differentiation-inducing factor (DIF)

p53
VPl6
Medl7 Signal transducer and activator of
(Srb4) transcription (STAT2)
Differentiation-inducing factor (DIF)
Heat-shock factor (HSF)



Early region 1A (E1A)

ETS-like kinase protein-1 (Elk-1)

Med23 Epithelial-restricted with serine box
(ESX)
CCAAT/enhancer binding protein
(C/EBP)
Differentiation-inducing factor (DIF)
HSF (heat-shock factor)

Differentiation-inducing factor (DIF)
Med25 Heat-shock factor (HSF)
VPl6


Lee et al., 1999-'
Park et al., 2000
Lee et al., 1999;
Park et al., 2000
Park et al., 2000


Kim et al., 2004


Ito et al., 1999
Ito et al., 1999
Lau et al., 2003


Kim et al., 2004
Kim et al., 2004


Boyer et al.,
1999; Wang and
Berk, 2002
Stevens et al.
2002

Asada et al., 2002

Mo et al., 2004


Kim et al., 2004
Kim et al., 2004


Kim et al., 2004
Kim et al., 2004



Garrett-Engele et
al., 2002


Mittler et al., 2003


Med29 Doublesex (dsx )


Eberhardy and
Farnham, 2002


Cdk8


Myc










Table 1-2. Mediator subunits in yeast, Arabidopsis, Drosophila and humans (Gurley et al., 2006;
Boube et al., 2002)
Unified


nomenclature
(Bourbon et al.,
2004)
MED1
MED2
MED3
MED4
MED5
MED6
MED7
MED8
MED9


Arabidopsis
thaliana


Drosophila
melanogaster


Homo sapiens


Saccharomyces
cerevisiae

Med1
Med2
Med3
Med4
Nut1
Med6
Med7
Med8
Cse2/Med9

Nut2/Med 10


Trap220


Trap36

Med6
Med7
Arc32


TRAP220-ARC/DRIP205


TRAP36-ARC/DRIP36

hMed6-ARC/DRIP33
ARC/DRIP34-CRSP33
ARC32


At5g02850

At3g21350
At5g03220


At5g41910/
Atlg26665

At4g00450
Atlg55325
At3g04740
(SWP1)
Atl1g5780

At5g20170
At2g22370

At4g09070/
At2g28230
At4g04780
Atlg07950/
Atl1g6430
Atlg23230

Atlg25540
(PFT1)
At3g48060/
At3g48050
At3g09180


MED10

MED11
MED12
MED13

MED14

MED15
MED16
MED17
MED18
MED19

MED20

MED21

MED22

MED23
MED24

MED25


MED26

MED27
MED28
MED29
MED30
MED31
CDK8
CycC


Nut2


hNut2-hMed 10


Med11
Srb8
Srb9

Rgrl

Gall1
Sin4
Srb4
Srb5
Rox3


Med21
Kto
Skd/Pap/Bli

Trapl170

Arcl05
Trap95
Trap80
P28/CG14802
CG5546


HSPC296
TRAP230 ARC/DRIP240
TRAP240 ARC/DRIP250

TRAPl70-DRIP/CRSPl50

ARC105
TRAP95-DRIP92
TRAP80-ARC/DRIP77
p28b
LCMR1


Srb2

Srb7

Srb6


Trfp


hTrfp


Trap19

Med24

Trapl150/7
Trapl100

Arc92


Arc70

Trap37
Med23
Intersex
Trap25
Trap18
CDK8
CycC


hSrb7

SurfS

hSur2/CRSPl30
TRAP/CRSP/DRIP 100

ARC92


CRSP70-ARC70


TRAP3 7-CRSP3 4
Fksg20
Hintersex
TRAP25
hSoh1
CDK8
CycC


Soh1
Srbl0
Srbl1


At5gl9910
At5g63610
At5g48640









CHAPTER 2

MATERIALS AND METHODS

Plant Growth Conditions

The ecotype of Arabidopsis thalianaiiii~~~~~~iiiiii used in this study was Columbia-0. The plants were

grown in soil with continuous light from 40 W fluorescent bulbs at 27+10C. To examine the

germination and root length, the seeds were grown on vertical agar plates. The seeds were

surface sterilized with 70% ethanol for 3-5 min, and then with 10% bleach for 15-20 min. After

rinsing with sterile water (3 X 5 min), the seeds were plated in petri dishes containing 1/2 MS

(Murashige & Skoog) medium supplemented with 1% sucrose, 0.5g/L MES (2-(N-morpholino)

ethanesulfonic acid) and 0.8% agar. The plates were sealed with parafilm and placed vertically in

a growth chamber with a 16h light / 8h dark cycle provided by 40 W fluorescent bulbs at 220C.

For dark treatment, the plates were wrapped in aluminum foil and placed vertically in a growth

chamber at 220C.

Genotyping of the T-DNA Insertion Lines

The nzed31 T-DNA insertion mutants (nzed31-1 and nzed31-2) were obtained from

Arabidopsis Biological Resource Center (ABRC). For the genotyping of nzed31-1,

M~ed31-specific primer 5'- TGGATGTAAGTAGGATTGGCG -3' was paired with the

T-DNA-specific primer LBb 1 5' -GCGTGGACCGCTTGCTGCAACT-3 to produce a 628 base

pair (bp) fragment by polymerase chain reaction (PCR), or with another M~ed31-specific primer

5'- GAACTTGTCTTGGCAAGTTGG -3' to produce a 975 bp fragment. For the genotyping of

nzed31-2, M~ed31-specific primer 5'- TGATGTACTCTGGTCGCTGC -3' was paired with the

T-DNA-specific primer LBb 1 5' -GCGTGGACCGCTTGCTGCAACT-3 to produce a 714 bp

fragment, or with another Med31 specific primer 5' -TTGCGGGGATTACAACATTAC-3 to









produce a 1008 bp fragment. The T-DNA insertion sites were determined by sequencing the

PCR products.

RNA Analysis

The leaves of 40-day-old plants grown on soil were collected and RNA was isolated with

the Concert Plant RNA Reagent (Invitrogen). RNA blots were prepared as described by Cao et al.

(1994) and probed with full-length M~ed31 cDNA.

Microscopy

A Zeiss Axiocam HRm camera was used to examine the subcellular localization of

Med31-GFP fusion proteins in the root tip. GFP fluorescence was monitored with the Zeiss filter

set 10 (excitation, 450 to 490; dichroic, 510 LP; emission, 515 to 565). DAPI

(4',6-diamidino-2-phenylindole) fluorescence was monitored with Zeiss filter set 02 (excitation,

365; dichroic, 395 LP; emission, 420 LP). A Zeiss LSM 5 Pascal confocal laser scanning

microscope was used to localize the Med31::GFP fusion proteins in the plant tissues with an

Argon 488 nm laser and a Band Pass 505-530 fi1ter. A Helium Neon 543 nm laser with a 560 nm

fi1ter was used to record chlorophyll autofluorescence.

Plasmid Construction

The pBIl01sGFP(S65T) vector was provided by Dr. Robert Ferl (Manak et al., 2002). This

vector was constructed by removing the GUS (P-glucuronidase) gene by digestion with

restriction endonucleases Xbal and SacI, and then inserting the sGFP(S65T) gene between the

two restriction sites (Manak et al., 2002). The M~ed31 gene, including the 1.2 kb upstream

sequence and the entire exon and intron region without the stop codon, was amplified from the

genomic DNA by PCR using the primers 5' -tatTGTCGACTCTAATTAATCAGTCTTGGTC-3 '

and 5'- agaTCTAGATATACCCTTCCTGACATTATATGACT -3'. The fragment was inserted

in-frame to the 5' end of the sGFP(S65T) gene in the pBIl01sGFP(S65T) vector using the Sall









and Xbal sites. The M~ed31 1.2 kb upstream sequence was generated from the genomic DNA by

PCR using the pimers 5' -tatTGTCGACTCTAATTAATCAGTCTTGGTC-3 and

5'-ttataT CTAGAGAAC GAAC GGAAC CTGAAGC -3'. Thi s fragm ent was i n serte d i n-frame to

the 5' end of the GUS gene in the pBI101 vector using the Sall and Xbal sites. All of the PCR

amplified fragments were confirmed by DNA sequencing.

GUS Staining

The tissues were immersed in GUS Staining Solution (1 M sodium phosphate (pH 7.0), 0.5

M EDTA (ethylenediaminetetraacetic acid), 50 mM K' ferricyanide, 50 mM K' ferrocyanide,

10% Triton X-100 and 2 mM X-gluc) and vacuum infiltrated for 20 min. The samples were

incubated at 37 OC until blue color appeared. As a final step, 70% ethanol was used to clear the

tissue.

Agrobacterium Transformation Technique

The binary vector was transformed into Agrobacteriunt tunrefaciens strain GV3101 by

electroporation and the T-DNA transferred to Arabidopsis plants via the standard floral dip

protocol (Clough and Bent, 1998). Agrobacterium starter cultures were grown in 30 ml LB

(Loria broth) liquid culture medium with 25 Cpg/ml gentamicin, 50 Cpg/ml rifampicin and 50

Cpg/ml kanamycin with shaking (250rpm) at 28 OC overnight. A 15 ml aliquot of the starter

culture was added to 150 ml of LB liquid medium containing 25 Cpg/ml gentamicin, 50 Cpg/ml

rifampicin and 50 Cpg/ml kanamycin, and the culture was incubated with shaking (250 rpm) at 28

OC until an OD600 of 0.8 was reached. The cells were collected by centrifugation (5000 g, 30

min) and resuspended in 150 ml of 5% sucrose. After addition of 30 Cl~ of Silwet L-77 detergent,

the 3-week-old Arabidopsis plants were dipped in the Agrobacterium solution for several sec,

with gentle agitation. The plants were covered overnight to keep high humidity. Transformants









were selected by germinating the seeds on plates containing 1/2 MS medium with 50 mg/L

kanamycin.

Chromatin Immunoprecipitation

The putative Mediator subunits were mapped to promoter DNA using chromatin

immunoprecipitation (ChIP) according to Gendrel and colleagues (2005), with minor

modifications. The aerial parts ofArabidopsis plants were harvested (1.5-2.0 g) and rinsed with

water. The sample was then placed in 37 ml of 1% formaldehyde for cross-linking and vacuum

infiltrated for 15 min at room temperature. The reaction was quenched by the addition of 2.5 ml

of 2 M glycine, and the sample was placed under vacuum for an additional 5 min. The tissue was

rinsed thoroughly, frozen in liquid nitrogen and stored at -80 OC until further treatment.

Chromatin was extracted by grinding the frozen samples in 30 ml of Extraction Buffer 1

(0.4 M sucrose, 10 mM Tris-HCI (pH 8.0), 10 mM MgCl2, 5 mM P-mercaptoethanol, 0.1 mM

PMSF (phenylmethylsulphonyl fluoride), 1 X protease inhibitor). (To make 200 X Protease

Inhibitor, dissolve 0.16 g TPCK (tosyl phenylalanyl chloromethyl ketone) and 0.16 g TLCK

(tosyl-L-lysine chloromethyl ketone) in 5 ml of DMSO (dimethyl sulfoxide), then dissolve in 10

ml of 0.2 M PMSF in isopropanol.) Next, the sample solution was filtered with Miracloth

(CalBiochem) and then centrifuged at 3000 X g at 4 OC for 20 min. The pellet was dissolved with

1 ml of Extraction Buffer 2 (0.25 M sucrose, 10 mM Tris-HCI (pH 8.0), 10 mM MgCl2, 1%

Triton X-100, 5 mM P-mercaptoethanol, 0.1 mM PMSF, 1 X protease inhibitor) and centrifuged

at 12,000 X g at 4 OC for 10 min. After that, the pellet was resuspended with 300 Cl1 of Extraction

Buffer 3 (1.7 M sucrose, 10 mM Tris-HCI (pH 8.0), 2 mM MgCl2, 0.15% Triton X-100, 5 mM

P-mercaptoethanol, 0.1 mM PMSF, 1 X protease inhibitor), placed on another 300 Cl1 of

extraction buffer 3, and centrifuged at 14,000 X g at 4 OC for 1 hr. The pellet was resuspended

with 300 Cl1 of Nuclei Lysis Buffer (50 mM Tris-HCI (pH8.0), 10 mM EDTA, 1% SDS (sodium









dodecyl sulfate), 1 X protease inhibitor), and the chromatin was sheared to a size of 150 bp to

750 bp by sonication (10 times for 15 sec each at an amplitude setting of 20 using a Tekmar

Sonicator). The sample was centrifuged at 12,000 X g for 10 min and ChlP Dilution Buffer

(1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCI (pH 8.0), 167 mM NaC1) was added to

the supernatant to make a final volume of 3 ml. The solution was divided into three tubes and 40

Cl1 of protein A-agarose (Santa Cruz) was added to each tube of sample for pre-clearing at 4 OC

for 1 hr with gentle agitation. The protein A-agarose was removed by centrifugation (12,000 X g

at 4 OC for 30 sec), and the supernatant was transferred to fresh tubes. A 60 Cl1 aliquot was saved

at -20 oC as the "Input DNA control."

The immunoprecipitation was set up as follows: 10 Cl1 of IgG (Immunoglobulin G)

Sepharose (Amersham Biosciences) and 10 Cl1 of c-Myc (cellular myelocytomatosis oncogene)

antibody (Santa Cruz) were added, respectively, to two tubes to precipitate the TAP-tagged

Mediator subunits. No antibody was added to the third sample, which served as the "no antibody

control." The tubes were incubated at 4 OC overnight with gentle agitation. In order to purify

Mediator-bound complexes, 50 Cl1 of protein A-agarose beads were added to the tubes with

c-Myc antibody and "no antibody control," respectively, and the three tubes were incubated at 4

OC for 1 hr with gentle agitation. The agarose beads were pelleted by centrifugation at 3800 X g

at 4 OC for 30 sec and washed sequentially with Low Salt Wash Buffer (150 mM NaC1, 0.1%

SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCI (pH 8.0)), High Salt Wash Buffer (500

mM NaC1, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCI (pH 8.0)), LiCl wash

buffer (0.25 M LiC1, 1% NP-40, 1% DOC (21-hydroxyprogesterone), 1 mM EDTA, 10 mM

Tris-HCI (pH 8.0)) and TE buffer. In order to extract the immune complex from the beads, 250

Cl1 of elution buffer (1% SDS, 8.4 mg/ml NaHCO3) WAS added to each sample, followed by









incubation at 65 OC for 15 min with gentle agitation. The elution step was repeated once to reach

a final volume of 500 Cl1. Elution buffer (440 Cl) was also added to the 60 Cl1 of the "Input DNA

control." After adding 20 Cl1 of 5 M NaCl to each sample, the cross-linking was reversed by

incubation at 650C overnight. The proteins in the sample were digested by incubation with 10 Cl1

of 0.5 M EDTA, 20 Cl1 of 1 M Tris-HCI (pH6.5), and 2 Cl1 of 10 mg/ml proteinase K at 450C for 1

hr. Then the proteins were removed from the DNA by phenol/chloroform extraction, and the

DNA was precipitated with the addition of ethanol (2.5 volume), sodium acetate (1/10 volume,

pH5.2) and 20 Cpg of glycogen. The DNA was resuspended with 50 Cl~ of 10 mM Tris-HCI

(pH7.5).

PCR Analysis of Chromatin Immunoprecipitation

The immunoprecipitated fraction was analyzed by PCR amplification to determine if the

DNA fragments from various promoters were present. The 25 Cl1 of PCR reaction system

contained 12.5 pmol of each primer, 5 nmol of dNTP, 3 Cl1 of DNA sample, 2.5 Cl1 of 10 X PCR

Buffer I and 1 unit of AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA). The cycling

conditions were 8 min of thermal activation at 95 OC, followed by 50 cycles of 94 OC (30 sec), 55

OC (30 sec), and 72 OC (3 min). The primers used for PCR were as follows: 5'-

cgtggcctagaatacaaagaag -3' and 5'- tcaaacaataagaaagaccatgaca -3' were used for the

amplification of the CCA1 promoter; 5'-agattgttgacattctcggaaatttagtgccaactgt-3' and

5' -aaatgctcctttttctaaaaccttcgcttggagtct- 3' for the amplifi cati on of Hspl~8. 2 promoter;

5' -acaccacggcgtgaccat-3 and 5' -attatccagtcgacatctgta-3 for the amplification ofAdh1 promoter;

and 5'- TGTTTCTTCCCTTTAAGCAACC-3' and 5'-

AACATTTCTTTAGAACATTGACTTGG-3 were used to amplify the intergenic region

between At2g32950 and AT2G32960.









Bioinformatics

Multiple protein sequence alignments were performed with AlignX, which is a component

of Vector NTI Advance 10.3.0 from Invitrogen. The accession numbers at the NCBI (National

Center for Biotechnology Information) for the Med31 homologs are XP_307924 (Anopheles

ga~nbiae), NP_197491 (Arabidopsis thaliana), BQ583133 (Beta vulgaris), CD834180 (Bra~ssica

napus), NP_492413 (Caenorhabditis elegans), EAK92332 (Candida albicans), DY287612

(Citrus clententina), C X05 1496 (Citrus sinensis), E AU9 1557 (Coprinopsis cinerea), X P_62688 1

(Cryptosporidium parvum), DRO63 080 (Cyca~s runsphii), XP_63 83 30 (Dictyosteliunt

discoideunt), NP_649483 (Drosophilanzelan2oga~ster), CAD25946 (Encephalitozoon cuniculi),

DV154959 (Euphorbia esula), BM892402 (Glycine nzax), DT547393 (Gossypium hirsutunt),

CO126156 (Gossypium raintondii), NP_057144 (Homo sapiens), DWO49205 (Lactuca saligna),

DW126099 (Lactuca sativa), CF39363 5 (Loblolly pine), BQ1471 10 (M~edicago truncatula),

NP_080344 (M~us nauscuhts), DY336178 (Ocinsun ba~silicunz), CA902198 (Pha~seobts coccineus),

CF808645 (Phytophthora sojae), DR501487 (Picea sitchensis), CV015282 (Rhododendron

cataw/biense), NP_011388 (Saccharonzyces cerevisiae), NP_587859 (Schizosaccharonzyces

ponabe) and CAD21541 (Taenia solium). The accession numbers for the Med6 homologs are

XP_319180 (Anopheles ga~nbiae), NP_188772 (Arabidopsis thaliana), NP_504791

(Caenorhabditis elegan2s), EAK97077 (Candida albicans), XP_638621 (Dictyosteliunt

discoideunt), NP_731403 (Drosophilanzelan2oga~ster), XP_965884 (Encephalitozoon cuniculi),

NP_005457 (Homo sapiens), NP_081489 (M~us nauscuhts), NP_001057150 (Oryza sativa),

NP_ 1 1925 (Saccharonzyces cerevisiae) and Q9US45 (Schizosaccharonyces ponabe).









CHAPTER 3

RESULTS

Med31/Sohl is a mediator subunit that has been identified in humans (Gu et al., 1999),

Drosophila (Park et al., 2001), S. pombe and S. cerevisiae (Linder and Gustafsson, 2004). Soh1

(suppressor of hprl) was first identified as a suppressor of the S. cerevisiae hprl A mutant which

is temperature-sensitive for growth and can reduce the hyperrecombination phenotype (Fan and

Klein, 1994). Yeast two hybrid analysis showed Sohl interacts with the Rad~p protein, and a

Soh1 mutation exacerbated the DNA repair defect of a rad5-535 mutant (Fan et al., 1996). The

Soh1 orthologue Sepl0 in S. pombe was identified in screening mutants for both sterility and for

defects in cell separation. Sepl0 mutants are temperature-sensitive At a non-permissive

temperature (36 OC), the mutants formed multiple, ill-organized septa (Grallert et al., 1999).

Med31/Sohl was identified in the Mediator complex in humans (Gu et al., 1999) and Drosophila

(Park et al., 2001), but its function has not been determined, and the exact relationship between

mutations in M~ed31 and phenotype is still not clear.

Analysis of Arabidopsis Med31 Gene by Multiple Sequence Alignments

The Med3 1 homologs are present in the protests (Cryptosporidium parvum, Dictyostelium

discoideum, Encephalitozoon cuniculi), fungi (Candida albicans, Coprinopsis cinerea,

Saccharomyces cerevisiae, Schizosaccharomyces pomb), metazoans (Anopheles gamnbiae,

Caenorhabditis elegan2s, Drosophila melan2oga~ster, Homo sapiens, M~us musculus, Taenia.

solium) and plants (Arabidopsis thaliana, Oryza sativa) (Figure 3-1). Based on sequence

homology, we identified AT5Gl9910 in Arabidopsis as the putative M~ed31 gene (AtMed31),

which contains 6 exons and encodes a protein of 196 amino acids (aa), with a calculated

molecular mass of 22.8 kDa. There is a conserved block of 70 aa (aa R30 to R99 of AtMed3 1)

(Figure 3-1) that shows high similarity to Med31 in other species (D. melan2oga~ster, 58.3%









identity and 76.4% similarity; H. sa-piens, 61.1% identity and 72.2% similarity; S. cerevisiae,

45.2% identity and 56.2% similarity; S. pombe, 59.7% identity and 77.8% similarity), where its

functional identity has been demonstrated. Linder and Gustafsson (2004) showed that this region

is required for its assembly within the Mediator complex in S. cerevisiae, which suggests this

conserved domain is important for interaction with other Mediator subunits.

A search of the NCBI EST database using TBLASTN (Altschul et al., 1997) for AtMed3 1

identified the Med31 homologs in many other plant species (Figure 3-2). There is a conserved

block of 139 aa (aa Ml to V139 of AT5Gl9910) between these plant Med3 1 homologs, which

includes the 70 aa domain conserved between different species (Figure 3-1). The C-termini of

the Med31 homologs are less conserved compared with their N-termini (Figure 3-1 and Figure

3-2), and often contain regions that resemble transcriptional activation domains which have

glutamine-rich or serine/proline-rich blocks. Since these domains function in transactivator

proteins to make contact with target transcription factors, it seems reasonable to assume that the

C-terminal region of Med3 1 containing these activator domain-like blocks may be at the outside

of the complex and provides surface for interaction with transactivators or other transcriptional

machinery.

Phenotype Characterization of med31 Mutants

The Mediator plays a vital role for RNA pol II-mediated transcription; therefore, disruption

of the highly conserved Med3 1 subunit is predicted to have a disruptive effect on the expression

of a large number of genes, some leading to abnormal phenotypes. To test this hypothesis and

study the function of this putative subunit, we searched the SIGnAL (signal.salk.edu) T-DNA

insertion collection for mutants (Alonso et al., 2003). Four T-DNA insertion lines were identified

and ordered from the Arabidopsis Biological Resource Center (ABRC). The T-DNAs of

SalkO3 5522 and Salk05 1025 lines insert into the promoter and 3' UTR (untranslated region) of









M~ed31, respectively, but we did not observe any mutant phenotype for these two lines. The

med31-1 (Salkl45479) mutant line has the T-DNA insertion in the promoter region, and the

T-DNA of med31-2 (Salk 143 815) mutant line is located within the 5' UTR (Figure 3-3). The

insertion sites for all Salk lines were confirmed by DNA sequencing.

In contrast to the previous two mutant lines, both med31-1 and med31-2 plants showed

abnormalities in growth. Under our experimental conditions, the germination rate of med31-1

seeds was 17% compared with 100% for the wild type (WT). In addition, their root length was

41.6% of the WT root length (Figure 3-4). The seeds of med31-2 plants germinated as well as the

WT seeds; however, their root length was 47.7% of the WT root length (Figure 3-5). These

differences in growth were not present under dark conditions, where the med31-2 seedlings grew

as well as the WT seedlings (Figure 3-6). This result suggests that the function of Med3 1 during

seed initial development may be dependent on light.

Some of the med31-2 plants had aberrant patterns of cotyledon development, such as three

cotyledons and three first true leaves, a single cotyledon, or forked cotyledons (Figure 3-7).

However, these mutant phenotypes were only inherited by some of their progeny, and the

med31-2 mutants with normal cotyledons also produced progeny with abnormal cotyledons. The

seedlings with abnormal cotyledons segregated 26% (5/19) progeny with abnormal cotyledons;

whereas the seedlings with normal cotyledons segregated 30% (5/20) progeny with abnormal

cotyledons. In addition, the overall sizes of the med31-2 mutants were reduced, their leaves were

smaller, and they had fewer rosette leaves compared with WT plants (Figure 3-8).

Med31 Expression in the med31-2 Plants

In med31-2 plants, the T-DNA inserts into the 5' UTR of the gene and may, therefore,

influence the expression of2~ed31 gene at either the transcriptional or translational level.

Northern blotting was used to examine the expression of2~ed31 in the WT and med31-2 plants.









In med31-2 mutant plants, the corresponding mRNA was more abundant than that in WT plants

(Figure 3-9). The mutant phenotype of med31-2 plants may be caused by the overexpression of

Med3 1 protein, which possibly sequesters the adj acent Mediator subunits or other components of

the transcriptional apparatus. Alternatively, Med31 translation may be inhibited due to the

missing, or changed nucleotides at the 5' end of the transcript.

Subcellular Localization and Tissue Expression Pattern of Med31::GFP Fusion Proteins

To investigate the subcellular localization of Med3 1, the 1.2 kb upstream sequence and the

entire exon and intron region of2~ed31 (without the stop codon) was amplified from the

genomic DNA by PCR. The resulting DNA was ligated in-frame to the 5' end of the sGFP(S65T)

gene in the pBI101 sGFP(S65T) vector. We observed that the Med31::GFP fusion proteins were

expressed in the tip of primary roots and that the signal was confined to the nucleus (Figure

3-10). In addition to being expressed in the lateral root tips, primordia (Figure 3-11) and root

hairs (Figure 3-12), the Med31::GFP signal was also found to be present in the aerial portions of

the plants including leaves (Figure 3-13), trichomes (Figure 3-14) and petioles (Figure 3-15).

Free GFP has been shown to be present in both the nucleus and cytosol (Li et al., 2001; Ye et al.,

2002; Zhong et al., 2005). To further complicate analysis, GFP has been shown to move to other

cells and tissues via the plasmodesmata (Crawford and Zambryski, 2001). However, in each type

of tissue we observed, signal from the Med3 1::GFP fusion protein was almost exclusively found

in the nucleus. The nuclear localization exhibited by Med31::GFP is consistent with its presence

in the nucleus being a property conferred by the Med3 1 portion of the protein, as contrasted with

the more general subcellular localization previously shown for GFP alone.

Tissue Expression Pattern of Med31 Promoter::GUS Fusions

To investigate the tissue expression pattern of Med3 1 protein, the M~ed31 promoter was

fused to the 5' end of the P-glucuronidase (GUS) gene and transferred to Arabidopsis. The GUS









signal was detected in the shoot apexes (Figure 3-16A) and lateral root primordia (Figure 3-16B)

of young seedlings (16 days old) of transformed plants. It was also detected in the whole young

inflorescences (Figure 3-17A), anthers (Figure 3-17B) and stigmas (Figure 3-17C) of adult plants

(46 days old) and in developing seeds (Figure 3-17D). This pattern differs from where

Med31::GFP signal was detected in that no GUS signal was detected in the primary root tips,

leaves and petioles. Two possible explanations for this apparent inconsistency are that GUS

staining sensitivity may be less than that of GFP. Alternatively, the promoter DNA alone as

present in the Med31::GUS construct (without the exons, introns and untranslated regions) is

insufficient to fully reproduce the expression pattern of the endogenous M~ed31 gene.

Co-immunoprecipitation Maps Med6 and Med31 to Promoter DNA

Chromatin immunoprecipitation (ChIP) is a powerful tool to explore in vivo protein-DNA

interactions. The ChlP assays conducted here involves the cross-linking of proteins and DNA in

chromatin, followed by co-immunoprecipitation of DNA fragments associated with the

epitope-tagged Mediator subunits. After the proteins have been removed, the pool of DNA

fragments can be queried by PCR amplification for the presence of specific promoter regions.

Mediator associates with promoter DNA indirectly by binding with transactivators and

RNA pol II. Previous studies in yeast using the ChlP technique (Andrau et al., 2006, Zhu et al.,

2006) showed that Mediator could not only bind to the core promoters and upstream activating

sequences, but also to the coding regions of many genes, as well. It can associate with the

promoters of both active and some inactive genes, but genes with higher transcriptional activity

usually have higher promoter occupancy by Mediator. It is thought that the presence of Mediator

at inactive promoters may be required for quick response to environmental changes.

If Med3 1 is a genuine mediator subunit, it should be found associated with promoter DNA.

We used the ChlP assay to test this hypothesis. In addition, we checked if another Arabidopsis









putative Mediator subunit, Med6 (AT3G21350), was also localized to promoter DNA. As with

Med3 1, the assignment of Arabidopsis Med6 as a putative subunit of Mediator was based strictly

on protein sequence homology (Figure 3-18). There is a conserved block of 129 aa (aa M33 to

S161 of AtMed6) that shows similarity to Med6 in other species (D. melan2oga~ster, 36.4%

identity and 51.2% similarity; H. sa-piens, 42.6% identity and 55.0% similarity; S. pombe, 31.1%

identity and 48. 1% similarity) where its functional identity has been demonstrated. It was

predicted to be localized in the nucleus by two web tools (Hua and Sun, 2001; Nair and Rost,

2002). Our prediction is that both proteins we have tentatively identified as AtMed31 and 6,

respectively, should be associated with the promoter regions of a wide array of genes.

The Med6 and Med31 cDNAs were introduced into the pC-TAPa vectors (Rubio et al.,

2005) and individually transformed into Arabidopsis by Dr. Kevin O'Grady (Gurley laboratory,

University of Florida). Their C-termini were fused with nine repeats of the myc epitope,

followed by six histidine residues, the 3C protease cleavage site and two copies of the protein A

IgG binding domain. The fusions of Med6 or Med3 1 with the epitope tags were confirmed by

Western blots. Immunoglobulin G Sepharose and c-Myc antibody were used to

immunoprecipitate the tagged Med6 or Med31 proteins, respectively, in the ChlP experiment.

ChlP Analysis for Med31

Immunoglobulin G Sepharose was used to immunoprecipitate Med31-DNA complexes

from the T1 generation of2~ed31-pC-TAPa transgenic Arabidopsis plants. Primer pairs for the

promoters of CCA1 (AT2g46830), Hspl8.2 (AT5g59720), Adh1 (ATlg77120) and a fragment in

the intergenetic region (between AT2g32950 and AT2g32960) were used to test if Med3 1 binds

to these sequences. The CCA1 circadiann clock associated 1) gene encodes a MYB-related

transcription factor and its expression oscillates with a circadian rhythm (Wang and Tobin, 1998).

Hspl8.2 (heat shock protein 18.2) is a heat inducible gene. Adh (alcohol dehydrogenase) is also









an inducible gene regulated by environmental stresses, such as low oxygen, dehydration, and low

temperature (Dolferus et al., 1994). Both Hspl8.2 and Adh genes are expressed at low levels

under normal conditions (Volkov et al., 2003; Dolferus et al., 1994). Mining of Arabidopsis EST

database failed to find the transcripts of the intergenetic region (>4kb) between At2g32950 and

AT2G32960, suggesting this region is not transcribed. Therefore, we used this region as a

negative control which Mediator may not bind. The promoters of CCA1, Hspl8.2 and Adh1 were

all co-immunoprecipitated with the epitope tagged Med31 protein by IgG Sepharose (Figure

3 -19), demonstrating the localization of Med3 1 to these promoters. As predicted, the intergenetic

region was not co-immunoprecipitated with the tagged Med31 by IgG Sepharose.

ChlP Analysis for Med6

Immunoglobulin G Sepharose and c-Myc antibody were used individually to

immunoprecipitate Med6- DNA complexes from the T2 generation of2~ed6-pC-TAPa transgenic

plants. The same set of primer pairs were used for the amplification from the DNA pool derived

from co-immunoprecipitation with epitope tagged Med6. The promoters of CCA1, Hspl8. 2, and

Adh1 were all co-immunoprecipitated with Med6 by both IgG Sepharose and c-Myc antibody

(Figure 3-20), demonstrating the localization of Med6 with these promoters. Again, as predicted,

the intergenetic region was not co-immunoprecipitated with Med6. It should be noted that both

Med6 and Med3 1 were independently found to be localized to the promoters of three unrelated

genes, CCA1, Hspl8.2 and Adhl, a finding consistent with both proteins belonging to a Mediator

complex.

Wild type plants were also included in ChlP experiment as a negative control to check if

IgG Sepharose and c-Myc antibody can immunoprecipitate the CCA1 promoter in the absence of

epitope tagged Mediator subunits. No PCR product of this promoter was amplified (Figure 3-21),









validating the conclusion that our ChlP protocol serves as a reliable indicator that Med6 and

Med31 can specifically immunoprecipitate promoter DNA.

Conclusion

Two T-DNA insertion lines in either the M~ed31 promoter or 5' untranslated region were

identified. The germination rate of med31-1 plants was lower, and their root length was much

shorter than that of WT plants. The root length of med31-2 plants was also shorter than that of

WT plants. The med31-2 mutants exhibited a dwarfed phenotype with fewer rosettes leaves than

the WT plants, and some of them had aberrant patterns of cotyledon development, such as three

cotyledons and three first true leaves, a single cotyledon, or forked cotyledons. These mutant

phenotypes imply that M~ed31 plays an important role in many aspects of plant development,

such as germination, root elongation and cotyledon development.

Using Med31::GFP constructs, we found that the Med31 protein was localized in the

nucleus. The Med31::GFP signal was detected in all the tissues that were examined, including

roots, root hairs, leaves, trichomes and petioles. In another experiment, the M~ed31 promoter was

fused to GUS gene to study its tissue expression pattern. The M~ed31 promoter::GUS reporter

was detected in the shoot apexes and lateral roots of young seedlings (16 days old) and in the

young inflorescences, anthers, stigmas of the adult plants (46 days old) and in developing seeds.

The promoters of three unrelated genes (CCA1~, Hspl8. 2 and Adhl) were all

co-immunoprecipitated with Med31 by IgG Sepharose and with Med6 by both IgG Sepharose

and c-Myc antibody. These results demonstrate the localization of Med6 and Med3 1 to these

promoters, which is consistent with the function of Mediator.

Taken together, this study provides evidence that Med6 and Med31 are both Mediator

subunits because 1) Med31 was localized in the nucleus; 2) Med31 was expressed in every type

of tissue that were examined; 3) Disruption of Med3 1 resulted in abnormal plant growth; and 4)









Both Med6 and Med31 proteins were localized to promoters. These data strongly support our

hypothesis that the Mediator complex found in fungi and metazoans is also present in plants.


















(1) 1 3 5 7 9 0 1 2
Arabd~opss~thalpna (1) M S E M D A EPPIT K EG ~RLEEl
Orymautwa (1) MEEnAP ELnn LEElCP E YHL
Drtyostelum~dscod~eum (1) M S S IELG I N N T I E G N E I K D N
Cryptorpor~um~parvum (1) MS G SLIL N I RSEE~CS EYaL
Anophezs~gambpe (1) L~V LFaLNN LF~RYKP F~L
aorophibamelnogaster (1) Mi~cGTIs ~an ~ aEE~CS E Y
Homo~saprzns (1) MEVMTL GRRaEE~CPEYNLa
Mus~murcubs (1) MEVMTL GRRaEE~CPEYNLa
Caenorhabdas~elgans (1) MSEETF VCF(LN NLF ~ R YKE
Taenapsolum (1) M~Ps LPnGsW NLLk RaEEVS
Cand~a~abrans (1) M~ Ta I~a (~~Y NI SE RoILF
Saccharomyces cerevena e (1) MsNNPTs oNr TFV LF(LN ( T
Encephaltozooncunrul (1) MGREEEVL CEYRL RGF SERY
Schaosaccharomyces pombe (1) M T WL K EDSFILF(LN kLF ~ H
Coprnopsscnerea (1) M P Pa P V A T P S NAFLLF(LN Y
Consensus (1) nn EEVCEE YNL Y FIYK LYKEYKIY LME~
(121) 2 3 4 5 6 7 8 9 0 1 2 3 0
Arabd~opss~thalpna (96) PF T M H EKLH (Fro NI~LIIP
Orymautwa (84) PF NMHEKVH(Y~KY~RIHLR
Drtyostelum~dscodeum(120)RRENH(TFH((YFoYI~R SKa
Cryptorpor~um~parvum (84) DRNSEV(I(T aolDKEH L
Anophezs~gambpe (67) EFRIS(CFD(ILoHTRTL AG
aorophibamelnogaster (86) EF R I N (CFD (Iao H T KILE V
Homo~saprzns (82) EFKLN(AFD(II~(YRRRaA
Mus~murcubs (82) EFKLN(AFD(II~(YRRRaA
Caenorhabdas~elgans (75) arLM Y E AF E (VaoFLK HLM P
Taenapsolum (95) onvHsnro(rIuKYR AE\T
Cand~a~abrans (95) EFKINLMSM LVRoSP~DNEE
Saccharomyces cere vena e (88) MSIN LLED LKI(G aNMVRP
Encephaltozooncunrull (70) NNMSE FLG(YlkIHKGK
Schaosaccharomyces pombe (81) PrNIR LSaNEY ~ LK ~ aGADT
Coprnopsscnerea (89) ArELKELL~(( L~o~~D iHN
Conrenur(121) F I (




Figure 3-1. Multiple sequence alignments of Med3 1 homologs in different species. Identical

amino acids are indicated in yellow, conservative amino acids in light blue and


similar amino acids in green.



















(1)1 1 2 3 4 5 6 7 8 9 10 ~ 1 2
ARabd~opss thalna (1) MSEM EPPKTKPG~RLEE CPPYH LC
Brassra~napus (1) lRRFED aPPA LSVVSAPE LE KTKPV aRLEE CNTYHA,
G ocne~max (1) svnsssTssKEESL SP YDDaaFLLFaPNYI La
Medrago~truncatuba (1) ESENILRVVWDVMSTSSRT YDDGaFEEFCPPYH La
Phytophthora~solae (1) AKEESL SP YDDaaFEE FCPNTI La
Phaseobs coceneus (1) IL~r(R(RE LELVH LWCETS SPNYDDGaFLLFaLNTIY~
Cirus clmentna (1) (~s~ ~or~sCKIM KN SASPKYDDGaFLLFaLNTIY~
Cirus~snenss (1) SCKIM K SASP YDDGaFLLFCPNTH La
Rhododendron~catawbrz nse (1) REFP LFTMS aTE SP YDDGRLEE FCPNYI La
Euphorbapesuba (1) RXNRGHPXSE DPSK KPDRRLEE CPPYH LC
Gossvpum~ralmondll (1) IFPCVI FRN F SVVKLLLFkHT( LI ILSFIK PSK KPDRRLE F LNTYYA,
Betauubars (1) VFSLNLLFITNFECFE aKDS S STNYDDGaFLLFaLNTIY~
Lactuca~ulgna (1) GLklRR LRRENPECSASE HSK OPDRRLEE LNTYYA,
Lactuca~satwa (1) G~A~lRLETTVS LaRSLSVN NHSK OPDRRLEE CPPYHLC
Ocimum~basilrum (1) HFCINDM S P S P TYDDGaFLLFaLKTIY~
Cycas rumphil (1) GRGMLLPIKPNPD~RLEE~CPPYH
P rea~srchenss (1) GG EPIKPKPD~RLEE CPPYH LC
Pnus taeda (1) CST~lR DDVL kNSRT(RIEN EPIKPKPD~RLEE CPPYHLC
Consensus (1) A L TSE YLLG( FLLFaPN YILa
(121) 11 ~ 3 10 ~ 5 10 ~ 7 10 ~ 9 20 ~ 1 20 ~ 3 4
Arabd~opss thalna (54) RFDAIYKaaREIF MPCYLL WWIRLHLRLEV aEESTPEST P
Brassra~napus (83) RFDAIYKaYa PYKIYHLF E WWIRLHLRLEV aEVSSPEPT
G ocne~max (70) N YEEFGLYaa P YKI YHLFE~
Medrago~truncatuba (80) RF D AI YKaa REIFM P CYLL ELEETEPV LPPTSVT a
Phytophthora~solae (54) RF D AI YKaa R EIFM P CYLL EEEETSAV LPPTVVAPa
Phaseobs coccneus (93) RF D AIYKara P YKI YHLLE TSVEST
Cirus clmentna (76) NYEEFGL YaaPYKIYHLF E ~ WWI~LHLRLE AEPEEEEPLPT G
Cirus~snenss (61) NYEEFGL YaaPYKIYHLF E ~ WWI~LHLRLE AEPEEEEPPPT G
Rhododendron~catawbrz nse (68) RFDAIYK aaREI FMPCY LL NRAA NEA aa WWI KI EPEEPEV EEESTVAPPP P
Euphorbapesuba (66) NYEEF GL YaaPYKIYHLF E ~ WWI~LHLRLE EESPP~PPVPTGPGA
Gossvpum~ralmondl(118) RFDAIYK~~REIFMPCYLL a A~a WIY~LFL PEVTELESE~SPSIMTP A
Betauubars (98) NY EFG LY aaPYKIYH LFE~NNRAAP SEP~aYWW I~LHLRLEL
Lactuca~ulgna (83) NYEEFG LY aaPYK I Y HLF E~N SR P P P N ET~aYW P
Lactuca~satwa (93) NYEEFG LY aaPYK I Y HLF E~N SR P P P N ET~aYW L
Ocimum~basilrum (59) RFDAIYK aaRE FMPC LLoPFNMHPK LH arFIY~R PP TES ELLEVPVNPAP a
Cycas rumphil (52) Nu Er cLuaaPu~lYHLFE~NNRPPPNEP ~aYWWI~L PEL
Prea~sTchenss (50) R DAIYK aaREI FMPC LLoPFSMHTK LH a FIY~RKIPPP EPE EAAEEPVTVVS T
Pnus taeda(113) NY EFG LY aaPYKIYH LFE~NNRPPPNEP ~ a WWI~LHLRLE ASEETEEPAP~TVVS a(
Consenus(121) NuEEr cLuaaPY~rYHrr~r EE E
(241) 4 5 6 7 8 9 0 1 2 3 4 5 0
Arabdopss thalna( 164) LP OTM SWTNGT RKKG
Brassra~napur(191) SMTL W IVE RKKGEA LaPDYSC

MedK asornaua Zo SMY PS IDXTE
Phytophtoaoau llSPPGPGGAWMNE NRR LNTKIIP oU LoI LF
Phass~Locns ..en .9101


Rhododendronctwrne16 LSMO PHS IIPTG L

Gosq rpeuania~ P LSMY :S IDRT R KKEPISElnPI











edr ago mrunatu( 226) -


Ctmas snens(267O) LFRGTG RR RK NNLFFLIIIFISE YL FR
Rhddnr nucatawense(213) --R S VE R R YI LR T
Euporbaesub(231)A -- G WDR I RR

AlBeostauar(2185) -
Latuass alna (269) --

Ldastruncatutwa(37) -
vOcimumobasilaeu(230) -

Cyru cas rumnzphl5) --~ r CS S Y GCLS SILKLSLE~
Prearsncenss(237) --LLFLKK

Cuponbenag36Z1)



Figure 3-.Mutpl linensofAme3 wt tedeuedain ci eqece f t

homlog inohe latsece. dniclamn aisar ndctd nyelw

cosevtie mnoacd i lgt lu ndsiiaramn aid n ren












I I I I


med31-2


100 bp


Figure 3-3. Diagrammatic representation of the insertions of the T-DNA in nzed31-1 and
nzed31-2. The M~ed31 gene contains six exons, which are represented by red boxes.
UTR regions are indicated by blue boxes. The position of triangle represents the
T-DNA insertion site.


Figure 3-4. Germination rate and root length of WT and nzed31-1 seedlings (9-day-old). A) WT.
B) nzed31-1. The size bars represent 0.5 cm.


Figure 3-5. Nine-day-old WT and nzed31-2 seedlings grown under continuous light. A) WT. B)
nzed31-2. The size bars represent 0.5 cm.

























Figure 3-6. Nine-day-old WT and med31-2 seedlings grown under dark. A) WT. B) med31-2.
The size bars represent 0.5 cm.


U


U


Figure 3-7. Ten-day-old WT and med31-2 seedlings. A) WT. B) med31-2 with three cotyledons
and three first true leaves. C) med31-2 with forked cotyledon. D) med31-2 with a
single cotyledon.


Figure 3-8. Comparison of adult WT plants and med31-2 plants. A) 30-day-old WT and med31-2
plants. B) 55-day-old WT and med31-2 plants. In both panels, the left plant is
med31-2, and the right plant is WT.










WT mdd31-2


Figure 3-9. Northern blot analysis of2~ed31 expression in WT and med31-2 plants.




















Figure 3-10. Subcellular localization of Med3 1::GFP fusion proteins in the root tip of a
35-day-old plant. A) Image of GFP. B) Image of DAPI staining.





























Figure 3-11i. Expression of Med3 1::GFP fusion proteins in lateral roots. A) A lateral root. B) A
lateral root primordium.


:S*;;


V.~~';' ;d
ii r ,~flr*~",~'!
..~..
d"i..* *e ..":~?*Y~1
I~
,r b=

-;~* -.


Figure 3-12. Expression of Med3 1::GFP fusion proteins in a root hair. A) Image of GFP signal.
B) DIC image. C) Overlay of the DIC and GFP images.



















A 11 IIB













Figure 3-13. Expression of Med3 1::GFP fusion proteins in a leaf. A) Image of GFP signal. B)
DIC image. C) Image of autofluorescence. The chloroplasts are red because of
autofluorescence of chlorophyll. D) Overlay of the GFP and autofluorescence images.













~g~,
-
T,


Figure 3-14. Expression of Med3 1::GFP fusion proteins in a trichome. A) Image of GFP signal.
B) DIC image. C) Image of autofluorescence. The chloroplasts are red because of
autofluorescence of chlorophyll. D) Overlay of the GFP and autofluorescence images.





































Figure 3-15. Expression of Med3 1::GFP fusion proteins in a petiole. A) Image of GFP signal. B)
DIC image. C) Image of autofluorescence. The chloroplasts are red because of
autofluorescence of chlorophyll. D) Overlay of the GFP and autofluorescence images.




















A L~


Figure 3-16. M~ed31 promoter directed GUS tissue expression pattern in young plants
(16-day-old). GUS signal was detected in A) A shoot apex. B) Lateral root primordia
and tips.















A B C D

Figure 3-17. M~ed31 promoter directed GUS tissue expression pattern in adult plants (46-day-old).
GUS signal was detected in A) A young inflorescence. B) Anthers. C) A stigma. D)
Developing seeds.
















(1) 1 ,10 ,20 ,30 ,40 5 O 7 0 9 0
Arabidopss~thalina (1) -----------------MDS S LSAATADTFGEU PPCPTM RULIS LDN YAS YT
Oryza~sativa (1) --TLPAPPGDTGICRaWNY LRNTF~F L F~
Dictyostellu m~dsco de um (1) MEDFNNDDPMNFDKDDMINNNN~NDNNNN~NNDNDNNNENNEDSNNNSNEEDTC~W~PWCMPN~T ILCFYSF~
Anopheles_gambae (1) --LCNLISH~~~ TLPNV~F KNF~
Drosophila~mehnogaster (1) --RMNHRSHTIMEL~T~~FRS
Homo~saplens (1) --T~IRNL ST~ WPILSGV~F RNF~
Mus~musculus (1) -
Caenorhabdits~elgans (1) --RGPERDPH~SRPPNIKNI
Candda~albrans (1) --LECKPFCR LNNT LYSSF~
Saccharomyces_cerevsne (1) --~PD LWSEIT~GRET LYASF
Schmosaccharomyces~pombe (1) --GPSDTIWMEWCMGRET LY~
Encephalitozoon~cun cull (1) --RE SF~~R LSPDTT~EFG F~
Consensus (1) LIW~WC LTYYSSF~


(101) 13 ,140 150
Arabidopss~thalina (67) TN EL RSHLLHS~TLYLDTPL
Oryza~sativa (53) TN ELSaHLMHT~T M YLD~~P
Dictyostellum~dscoldeum(100) N N~L ~ RDSL~GE ELKVPF IK~R P
Anopheles_gambae (43) T N EV~~SEL~GEIL~DIYI~
Drosophib~melanogaster (44) C N TRaLPHHILY HVE YIKCRNS
Homo~saplens (44) C N V KC R LHNMrGE LHCE F IaaSP
Mus_musculus (1) CR LHNMrGE LHCE F IaaSP
Caenorhabditis_elegans (51) N Ca I~NV R V ELTP~YLYaP F
Candida~albicans (38) T NaL~r~aa P G S~YaRS M GE
Saccharomyces~cerevislae (40) TS~VKaa~LD NA~aIMTLPDGKNGNLEEEAVPRQL
Sch mosaccharomyces~pombe (46) KN EL~~rADGL~LRT~rIH R
Encephalitozoon~cunicul (39) S N EL~~rG~ISLS~GFEES H
Consensus(101) CNIK R GEVL FIK P


(201) 201 ,210 ,220 ,230 ,240 ,250 ,260 ,270 ,280 ,290 300
Arabidopss~thalina(117) ----KVTPMLTYYI LDGSIYQAPQLCSVFAARVSRTI YNI SKAFTDAASKLETIRQVDTENQNEPAE----SKPASET----VD LKEM KR
Oryza~sativa(103) ----KSNAMLAYYI LDGSIYQAPQLCSVFASRI SRAMHHI SKAFTTACSKLEKIGHVETEPDTAASE----SKTQK Dictyostellum~dscoldeum(147) ----DVLINT LYYVINGNIYQ~APE LHVVFKSRVSQ~SISHLSEAFNSI SSIVNWUDIVNGYS LNLDPSN---QEKSKLAAYS--RK--IEDTKRL
Anopheles_gambae (90) ----EATPMADYYIIAGTVYQAPDLASVFNSRI LSTVHHLQTAFDEASSYSRYHPSKGYSWDFSSNKAIAEKTKQKEPEPSIFRV
Drosophib~melanogaster (91) ----EATPIADYYI IGGTVYKAPDLANVrINSRI LNTVVNILQSAFEEASSYARYHPNKGYTWD FSSNK VFSRSKDKDNSKENGLFQ~KQRV
Homo~saplens (91) -----VIPLADYYIIAGVIYQAPDLGSVINSRVLTAVHGIQSAFDEMYRHSGWHKH--QKRKK-KESSFRR
Mus_musculus (40) -----VIPLADYYIIAGVIYQAPDLGSVINSRVLTAVHGIQSAFDEMYRHSGWHKH--QKKKK-KESSFRR
Caenorhabditis_elegans(101) ----NVSPIAYYYVINGSVHQAPDMYSLVQSRLLGALEPLRNAFETYRNAGWFKPVKEEKDE-LDSNQTT
Candida~albicans(101) ----TVTLQDYYIIGANVYQAPRIYDVLSSRLL ASVLSIKNSTDLNMSHDGSYSHSSKQSAKP-TTTATP
Saccharomyces~cerevislae(139) VGSAKGPEIIPLQ DYYIIGANIYQSPTIFKIVQSRLMSTSYLSELDIFQQGHKPTTAATNNG-GNSSRGA
Schmosaccharomyces_pombe(101) ----EVKPLTVYFVCNENIYMAPNAYTLL ATRMLNATYCFQALTIKPYPEGTPLNNEVHNNPD-------N
Encephalitozoon~cunicul (92) ---AETLGMYYIIHGHVYAAPTNYSIYRCRMGDSMWQLNS FID RMK FN S- --P---GRL S------ED D
Consensus(201) V PLADYYIIAG IYQ)APDL SVINSRVL AVH LQ)SAFDEA SY RY PS GY W KSK SI RV


(0)301 ,310 ,320 ,330 ,340 ,350 ,360
Arab dopss5thalina(19 6) -----------QVDPP GY~CELGKEL~aG S PP V P I IDQGPAKRMKF-
O ryzasativa(18 2) -----------QVDPP GT~S CEASD LA EL P~ P I IDQGPAKRPRFQ-
Drtyostellum~dscod eu m(231) -----------Q------QQ~GGGI TU C PSQP--
Anophees~gamblae(182) -------GDSDHVGADATLIKQEPTEGGVASNNHG
Drosoph la~melanogaster(184) -----P-PI P~N LCPEG~NARA MNEG LDIKTEGVDMKPPPEKKSK--
Homoaplens(178) -----ETTKEVQQTVSAKGPPEKRNRLQ-KEAEPI
Mus_musculus(127) -----ESTKNIQQTVSEPT~T~CEKEAPLTKGPPEKRNRLQ-
Caenorhabdits~elegans(193) --------TEAEREKEVEEE TSTDEPEPTTRTSQ
Candida~albicans(191) ITIPLYG--EGSTLERLGLKGNKDAGLSL--NDVV
Saccharomyces~cerev slae(237) -----------LMVTSIRSTPNYI--RTG GNMG
Sch izosaccharomyces~po mbe(181) ---------------SW AF US HSS-KEAPD~K --
Encephalitozoon~cunicul(160) LFMINFK~
Consensus(301) KP K P KR




Figure 3-18. Multiple sequence alignments of Med6 homologs in different species. Identical

amino acids are indicated in yellow, conservative amino acids are indicated in light

blue, and similar amino acids are indicated in green.










CCAl Equl.2 Adlh Inerge esreg
ML2 4M234M123 Y1Z44M~I 2 34








Figure 3 -19. Med3 1 associates with the promoters of CCA1~, Hspl8. 2 and Adhl, but not with the
intergenetic region. The promoters used are indicated above the gels. Lane M was
loaded with 100 bp DNA Ladder from New England Biolabs. The templates for each
PCR are as follows: Lane 1: Genomic DNA from wild-type plants; Lane 2: Input
DNA control (sonicated genomic DNA from M~ed31-pC-TAPa transgenic plants);
Lane 3: Negative control chromatinn extract without antibody immunoprecipitation
from M~ed31-pC-TAPa transgenic plants); Lane 4: Chromatin immunoprecipitated
with IgG Sepharose from M~ed31-pC-TAPa transgenic plants.




(TAl I~slB. Adh In~eimpEh ~eH@D
M 12394 5 1M l 2 3 4 5 Ml 2 34 5 ~M I2 34









Figure 3-20. Med6 associates with the promoters of CCA1, Hspl8.2 and Adhl, but not with the
intergenetic region. The promoters used are indicated above the gels. Lane M was
loaded with 100 bp DNA Ladder from New England Biolabs. The templates for each
PCR are as follows: Lane 1: genomic DNA from wild-type plants; Lane 2: Input
DNA control (sonicated genomic DNA from M~ed6-pC-TAPa transgenic plants); Lane
3: Negative control chromatinn extract without antibody immunoprecipitation from
M~ed6-pC-TAPa transgenic plants); Lane 4: Chromatin immunoprecipitated with IgG
Sepharose from M~ed6-pC-TAPa transgenic plants. Lane 5: Chromatin
immunoprecipitated with c-Myc antibody from M~ed6-pC-TAPa transgenic plants.










O4C~I
Mu 1 2 4 5









Figure 3-21. Immunoglobulin G Sepharose and c-Myc antibody cannot immunoprecipitate the
CCA1 promoter from WT Arabidopsis. The templates for each PCR are as follows.
Lane M was loaded with 100 bp DNA Ladder from New England Biolabs. Lane 1:
genomic DNA from wild-type plants; Lane 2: Input DNA control (sonicated genomic
DNA from wild-type plants); Lane 3: Negative control chromatinn extract without
antibody immunoprecipitation from wild-type plants); Lane 4: Chromatin
immunoprecipitated with IgG Sepharose from wild-type plants. Lane 5: Chromatin
immunoprecipitated with c-Myc antibody from wild-type plants.









CHAPTER 4

DISCUSSION

Phenotype Characterization of med31 Mutants

The M~ed31 promoter or 5' UTR were disrupted by T-DNA insertion in med31-1 and

med31-2 lines. Both mutant lines had shorter roots than WT plants under our experimental

conditions. In addition, seeds were examined in preliminary studies (data not included) for their

responses to a variety of hormones. The med31-2 seedlings were insensitive to ABA, kinetin,

and 2, 4-D, compared with WT seedlings. A possible cause for the mutant phenotypes of these

two insertion lines is due to the disruption of either transcriptional or translational expression of

M~ed31. The T-DNA in med31-1 breaks a GT-1 cis-element (identified by AthaMap web tools;

www.athamap.de), which has been shown in other promoters to play a role in the gene regulation

by light, pathogens and salt (Villain et al., 1994; Park et al., 2004). Likewise, the T-DNA in

med31-2 breaks the CCAAT BOX1 (identified in the PLACE database;

www. dna. affrc.go.j p/PLACE), which has been reported to be involved in transcriptional

expression by heat stress (Rieping and Schoffl, 1992; Haralampidis et al., 2002). T-DNA

insertions not only disrupt the inserted cis-elements, but also impede the function of the

cis-elements upstream of the insertion sites. The location of the two T-DNA insertions found in

med31-1 and -2 are predicted to strongly interfere with the regulation of2~ed31 gene expression.

Med3 1 is a subunit of Mediator complex, which is important in gene transcription mediated by

RNA pol II. Defective M~ed31 expression has the potential to influence the binding of the

transactivators to Med3 1 subunit, alter the structure of the mediator, hinder the entry of other

subunits into the Mediator, or the entry of general transcription factor or RNA pol II into the PIC,

and thus, cause pleiotropic effects by impeding RNA pol II-dependent transcription. Consistent









with this hypothesis, our preliminary data showed multiple aspects of plant development were

influenced for the med31-2 mutant.

Evidence for a Mediator Complex in Arabidopsis

The transcription apparatus of plants, metazoans and yeast are conserved (Gurley et al.,

2006). Many of the promoters in these three kingdoms contain the TATA motif and CAAT box

for the binding of RNA pol II and general transcription factors. Transactivators generally bind

the upstream cis-elements to regulate gene expression. The RNA pol II in all the three kingdoms

contains 12 conserved subunits. In addition, plants possess the genes coding for all the general

transcription factors (TFIIA, B, D, E, F, and H) that are present in metazoans and fungi (Coulson

and Ouzounis, 2003). Arabidopsis also has the homologs of the subunits of some coactivators

(Hsieh and Fischer, 2005), such as the SAGA (Spt-Ada-Gen5-acetyltransferase) (Stockinger et

al., 2001) and SWI/SNF complexes (Brzeski et al., 1999; Eshed et al., 1999; Ogas et al., 1999).

This high degree in conservation of the transcription machinery suggests that the plants may also

have the Mediator coactivator which has been shown to play an essential role in RNA pol

II-mediated transcription in other eukaryotes. Identification of the homologs of most of the yeast

and metazoan Mediator subunits in Arabidopsis strongly supports this hypothesis (Gurley et al.,

2006; Boube et al., 2002).

The experiments described here explore various aspects of gene expression for two

putative Mediator subunits from Arabidopsis, Med31 and Med6. By all measures tested, these

two proteins behaved as expected for bona fide members of plant Mediator. AtMed3 1 was

localized in the nucleus, and was widely expressed throughout the plants. Both AtMed6 and

AtMed3 1 were localized to the promoters of three unrelated genes: CCA1, Hspl8.2 and Adhl.

Together with the sequence homology between Arabidopsis proteins and known Mediator

subunits from other eukaryotes, these data strongly support the presence of a Mediator complex









in Arabidopsis, and higher plants in general, that shows strong conservation in both form and

function with analogous complexes in fungi and metazoans.










LIST OF REFERENCES


Alonso J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K.,
Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema,
E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M.,
Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman,
P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T.,
Rissecuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, C.C. and Ecker, J.R. (2003)
Genome-wide insertional mutagenesis ofArabidopsis thaliana. Science, 301, 653-657.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman,
D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res. 25, 3389-3402.

Andrau, J.C., van de Pasch, L., Lijnzaad, P., Bijma, T., Koerkamp, M.G., van de Peppel, J.,
Werner, M. and Holstege F.C. (2006) Genome-wide location of the coactivator mediator:
Binding without activation and transient Cdk8 interaction on DNA. M~olCell, 22, 179-192.

Asada, S., Choi, Y., Yamada, M., Wang, S.C., Hung, M.C., Qin, J. and Uesugi, M. (2002)
External control of Her2 expression and cancer cell growth by targeting a Ras-linked
coactivator. Proc. Natl. Acad. Sci. USA, 99, 12747-12752.

Atkins, G.B., Hu, X., Guenther, M.G., Rachez, C., Freedman, L.P. and Lazar, M.A. (1999)
Coactivators for the orphan nuclear receptor RORalpha. Mol1. Endocrinol. 13, 1550-1557.

Autran, D., Jonak, C., Belcram, K., Beemster, G.T., Kronenberger, J., Grandjean, O., Inze,
D. and Traas, J. (2002) Cell numbers and leaf development in Arabidopsis: a functional
analysis of the STRUWWELPETER gene. EM~BO J. 21, 6036-6049.

Baek, H.J., Malik, S., Qin, J. and Roeder, R.G. (2002) Requirement of TRAP/mediator for
both activator-independent and activator-dependent transcription in conjunction with
TFIID-associated TAF(II)s. Mol1. CellBiol. 22, 2842-2852.

Black, J.C., Choi, J.E,. Lombardo, S.R. and Carey, M. (2006) A mechanism for coordinating
chromatin modification and preinitiation complex assembly. Mol1. Cell, 23, 809-818.

Borggrefe, T., Davis, R., Erdjument-Bromage, H., Tempst, P. and Kornberg, R.D. (2002) A
complex of the Srb8, -9, -10, and -1 1 transcriptional regulatory proteins from yeast. J. Biol.
Chem. 277, 44202-44207.

Boube, M., Joulia, L., Cribbs, D.L. and Bourbon, H.M. (2002) Evidence for a mediator of
RNA polymerase II transcriptional regulation conserved from yeast to man. Cell, 110,
143-151.










Bourbon, H.M., Aguilera, A., Ansari, A.Z., Asturias, F.J., Berk, A.J., Bjorklund, S.,
Blackwell, T.K., Borggrefe, T., Carey, M., Carlson, M., Conaway, J.W., Conaway,
R.C., Emmons, S.W., Fondell, J.D., Freedman, L.P., Fukasawa, T., Gustafsson, C.M.,
Han, M., He, X., Herman, P.K., Hinnebusch, A.G., Holmberg, S., Holstege, F.C.,
Jaehning, J.A., Kim, Y.J., Kuras, L., Leutz, A., Lis, J.T., Meisterernest, M., Naar,
A.M., Nasmyth, K., Parvin, J.D., Ptashne, M., Reinberg, D., Ronne, H., Sadowski, I.,
Sakurai, H., Sipiczki, M., Sternberg, P.W., Stillman, D.J., Strich, R., Struhl, K.,
Svejstrup, J.Q., Tuck, S., Winston, F., Roeder, R.G. and Kornberg, R.D. (2004) A
unified nomenclature for protein subunits of mediator complexes linking transcriptional
regulators to RNA polymerase II. Mol1. Cell, 14, 553-557.

Boyer, T.G., Martin, M.E., Lees, E., Ricciardi, R.P. and Berk, A.J. (1999) Mammalian
Srb/Mediator complex is targeted by adenovirus E1A protein. Nature, 399, 276-279.

Brzeski, J., Podstolski, W., Okztak, K. and Jerzmanowski, A. (1999) Identification and
analysis of the Arabidopsis thaliana BSH gene, a member of the SNF5 gene family.
Nucleic Acids Res. 27, 2393-2399.

Burakov, D., Wong, C.W., Rachez, C., Cheskis, B.J. and Freedman, L.P. (2000) Functional
interactions between the estrogen receptor and DRIP205, a subunit of the heteromeric
DRIP coactivator complex. J. Biol. Chem. 275, 20928-20934.

Cao, H., Bowling, S.A., Gordon, A.S. and Dong, X. (1994) Characterization of an
Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance.
Plant Cell, 6, 1583-1592.

Cerdan, P.D. and Chory, J. (2003). Regulation of flowering time by light quality. Nature, 423,
881-885.

Chadick, J.Z. and Asturias, F.J. (2005) Structure of eukaryotic Mediator complexes. Trends
Biochem. Sci. 30, 264-271.

Chen, S., West, R.W.J., Johnston, S.L., Gans, H. and Ma, J. (1993) TSF3, a global regulatory
protein that silences transcription of yeast GAL genes, also mediates repression by 2
repressor and is identical to SINT4. Mol1. Cell. Biol. 13, 831-840.

Cho, E.J., Takagi, T., Moore, C.R. and Buratowski, S. (1997) mRNA capping enzyme is
recruited to the transcription complex by phosphorylation of the RNA polymerase II
carboxy-terminal domain. Genes Dev. 11, 3319-3326.

Clayton, A.L., Hazzalin, C.A. and Mahadevan, L.C. (2006) Enhanced histone acetylation and
transcription: a dynamic perspective. Mol1. Cell, 23, 289-296.

Conaway, R.C., Sato, S., Tomomori-Sato, C., Yao, T. and Conaway, J.W. (2005) The
mammalian Mediator complex and its role in transcriptional regulation. Trends Biochem.
Sci. 30, 250-255.










Coulson, R.M. and Ouzounis, C.A. (2003) The phylogenetic diversity of eukaryotic
transcription. Nucleic Acids Res. 31, 653-660.

Crawford, K.M. and Zambryski, P.C. (2001) Non-targeted and targeted protein movement
through plasmodesmata in leaves in different developmental and physiological states.
Plan2tPhysiol. 125, 1802-1812.

Crawford, S.E., Qi, C., Misra, P., Stellmach, V., Rao, M.S., Engel, J.D., Zhu, Y. and Reddy
J.K. (2002) Defects of the heart, eye, and megakaryocytes in peroxisome proliferator
activator receptor-binding protein (PBP) null embryos implicate GATA family of
transcription factors. J. Biol. Chem. 277, 3585-3592.

Davis, J.A., Takagi, Y., Kornberg, R.D. and Asturias, F.A. (2002) Structure of the yeast RNA
polymerase II holoenzyme: Mediator conformation and polymerase interaction. Mol1. Cell,
10, 409-415.

Dolferus, R., Jacobs, M., Peacock, W.J. and Dennis, E.S. (1994) Differential interactions of
promoter elements in stress responses of the Arabidopsis Adh gene. Plant Physiol. 105,
1075-1087.

Drane, P., Barel, M., Balbo, M. and Frade, R. (1997) Identification of RE 8A, a 205 kDa new
p53 regulatory protein which shares antigenic and functional properties with p53.
Oncogene, 15, 3013-3024.

Dvir, A., Conaway, R.C. and Conaway, J.W. (1997) A role for TFIIH in controlling the
activity of early RNA polymerase II elongation complexes. Proc. Natl. Acad. Sci. USA, 94,
9006-9010.

Eberhardy, S.R. and Farnham, P.J. (2002) Myc recruits P-TEFb to mediate the final step in
the transcriptional activation of the cad promoter. JBiol Chem. 277, 40156-40162.

Eshed, Y., Baum, S.F. and Bowman, J.L. (1999) Distinct mechanisms promote polarity
establishment in carpels of Arabidopsis. Cell, 99: 199-209.

Fan, H.Y., Cheng, K.K. and Klein, H.L. (1996) Mutations in the RNA polymerase II
transcription machinery suppress the hyperrecombination mutant hprl delta of
Saccharomyces cerevisiae. Genetics, 142, 749-759.

Fan, H.Y. and Klein, H.L. (1994) Characterization of mutations that suppress the
temperature-sensitive growth of the hprl delta mutant of Saccharomyces cerevisiae.
Genetics, 137, 945-956.

Fassler, J.S., Gray, W., Lee, J.P., Yu, G.Y. and Gingerich, G. (1991) The Saccharomyces
cerevisiae SPT14 gene is essential for normal expression of the yeast transposon, Ty, as
well as for expression of the HIS4 gene and several genes in the mating pathway. Mol1. Gen.
Genet. 230, 310-320.










Featherstone, M. (2002) Coactivators in transcription initiation: here are your orders. Curr.
Opin. Genet. Dev. 12, 149-155.

Flanagan, P.M., Kelleher, R.J. 3rd, Sayre, M.H., Tschochner, H. and Kornberg, R.D.
(1991). A mediator required for activation of RNA polymerase II transcription in vitro.
Nature, 350, 436-438.

Fondell, J.D., Ge, H. and Roeder, R.G. (1996) Ligand induction of a transcriptionally active
thyroid hormone receptor coactivator complex. Proc. Natl. Acad'. Sci. USA, 93, 8329-8233.

Friedl, E.M., Lane, W.S., Erdjument-Bromage, H., Tempst, P. and Reinberg, D. (2003) The
C-terminal domain phosphatase and transcription elongation activities of FCP1 are
regulated by phosphorylation. Proc. Natl. Acad'. Sci. USA, 100, 2328-2333.

Garrett-Engele CM, Siegal ML, Manoli DS, Williams BC, Li H, Baker BS. (2002) intersex, a
gene required for female sexual development in Drosophila, is expressed in both sexes and
functions together with doublesex to regulate terminal differentiation. Development, 129,
4661-4675.

Gavin, I., Horn, P.J. and Peterson, C.L. (2001) SWI/SNF chromatin remodeling requires
changes in DNA topology. Mol1. Cell, 7, 97-104.

Ge, K., Guermah, M., Yuan, C.X., Ito, M., Wallberg, A.E., Spiegelman, B.M. and Roeder,
R.G. (2002) Transcription coactivator TRAP220 is required for PPAR gamma
2-stimulated adipogenesis. Nature, 417, 563-567.

Gendrel, A.V., Lippman, Z., Martienssen, R. and Colot, V. (2005) Profiling histone
modification patterns in plants using genomic tiling microarrays. Nat2~ethods, 2, 213-218.

Grallert, A., Grallert, B., Zilahi, E., Szilagyi, Z. and Sipiczki, M. (1999) Eleven novel sep
genes of Schizosaccharonzyces ponabe required for efficient cell separation and sexual
differentiation. Yeast, 15, 669-686.

Gu, W., Malik, S., Ito, M., Yuan, C.X.,, Fondell J.D., Zhang, X., Martinez, E., Qin, J. and
Roeder, R.G. (1999) A novel human SRB/MED-containing cofactor complex, SMCC,
involved in transcription regulation. Mol1. Cell, 3, 97-108.

Guidi, B.W., Bjornsdottir, G., Hopkins, D.C., Lacomis, L., Erdjument-Bromage, H.,
Tempst, P. and Myers, L.C. (2004) Mutual targeting of mediator and the TFIIH kinase
Kin28. J. Biol. Chent. 279, 29114-29120.

Gurley, W.B., O'Grady, K., Czarnecka-Verner, E. and Lawit, S.J. (2007) General
transcription factors and the core promoter: ancient roots. In Regulation and' Transcription
in Plants (Grasser, K.D., ed). Annual Plant Reviews, vol. 29, Blackwell Publishing: Oxford,
UK, pp.368.










Gustafsson, C.M., Myers, L.C., Beve, J., Spahr, H., Lui, M., Erdjument-Bromage, H.,
Tempst, P. and Kornberg, R.D. (1998) Identification of new mediator subunits in the
RNA polymerase II holoenzyme from Saccharonzyces cerevisiae. JBiol Chent. 273,
30851-30854.

Gustafsson, C.M., Myers, L.C., Li, Y., Redd, M.J., Lui, M., Erdjument-Bromage, H.,
Tempst, P. and Kornberg, R.D. (1997) Identification of Rox3 as a component of
mediator and RNA polymerase II holoenzyme. JBiol Chent. 272, 48-50.

Gwack, Y., Baek, H.J., Nakamura, H., Lee, S.H., Meisterernst, M., Roeder, R.G. and Jung,
J.U. (2003) Principal role of TRAP/mediator and SWI/SNF complexes in Kaposis
sarcoma-associated herpesvirus RTA-mediated lytic reactivation. Mol1. Cell Biol. 23,
2055-2067.

Haralampidis, K., Milioni, D., Rigas, S. and Hatzopoulos, P. (2002) Combinatorial interaction
of cis elements specifies the expression of the Arabidopsis AtHsp90-1 gene. Plant Physiol.
129, 1138-1149.

Havas, K., Flaus, A., Phelan, M., Kingston, R., Wade, P.A., Lilley, D.M. and Owen-Hughes,
T. (2000) Generation of superhelical torsion by ATP-dependent chromatin remodeling
activities. Cell, 103, 1133-1142.

Hengartner, C.J., Thompson, C.M., Zhang, J., Chao, D.M., Liao, S.M., Koleske, A.J.,
Okamura, S. and Young, R.A. (1995) Association of an activator with an RNA
polymerase II holoenzyme. Genes Dev. 9, 897-910.

Hengartner, C.J., Myer, V.E., Liao, S.M., Wilson, C.J., Koh, S.S. and Young, R.A. (1998)
Temporal regulation of RNA polymerase II by Srbl10 and Kin28 cyclin-dependent kinases.
Mol1. Cell, 2, 43-53.

Hittelman, A.B., Burakov, D., Iniguez-Lluhi, J.A., Freedman, L.P. and Garabedian, M.J.
(1999) Differential regulation of glucocorticoid receptor transcriptional activation via
AF-1-associated proteins. E IBO J. 18, 5380-5388.

Holstege, F.C., Jennings, E.G., Wyrick, J.J., Lee, T.I., Hengartner, C.J., Green, M.R.,
Golub, T.R., Lander, E.S. and Young, R.A. (1998). Dissecting the regulatory circuitry of
a eukaryotic genome. Cell, 95, 717-728.

Hsieh, T.F. and Fischer, R.L. (2005) Biology of chromatin dynamics. Annu. Rev. Plant Biol. 56,
327-351.

Hua, S. and Sun, Z. (2001) Support vector machine approach for protein subcellular
localization prediction. Bioinfornzatics, 17, 721-728.

Ito, M., Yuan, C.X., Malik, S., Gu, W., Fondell, J.D., Yamamura, S., Fu, Z.Y., Zhang, X.,
Qin, J. and Roeder, R.G. (1999) Identity between TRAP and SMCC complexes indicates
novel pathways for the function of nuclear receptors and diverse mammalian activators.
M~ol. Cell, 3, 361-370.










Jeoung, J.M., Krishnaveni, S., Muthukrishnan, S., Trick, H.N. and Liang, G.H. (2002)
Optimization of sorghum transformation parameters using genes for green fluorescent
protein and beta-glucuronidase as visual markers. Heredita~s, 137, 20-28.

Jiang, Y., Yan, M. and Gralla, J.D. (1996) A three-step pathway of transcription initiation
leading to promoter clearance at an activation RNA polymerase II promoter. Mol1. Cell Biol.
16, 1614-1621.

Jiang, Y.W. and Stillman, D.J. (1995) Regulation of HIS4 Expression by the Saccharomyces
cerevisiae SINT4 Transcriptional Regulator. Genetics, 140, 103-114.

Jiang, Y.W., Veschambre, P., Erdjument-Bromage, H., Tempst, P., Conaway, J.W.,
Conaway, R.C. and Kornberg, R.D. (1998) Mammalian mediator of transcriptional
regulation and its possible role as an end-point of signal transduction pathways. Proc. Natl.
Acad. Sci. USA, 95, 8538-8543.

Johnson, K.M. and Carey, M. (2003) Assembly of a mediator/TFIID/TFIIA complex bypasses
the need for an activator. Curr. Biol. 13, 772-777.

Johnson, K.M., Wang, J., Smallwood, A., Arayata, C. and Carey, M. (2002) TFIID and
human mediator coactivator complexes assemble cooperatively on promoter DNA. Genes
Dev. 16, 1852-1863.

Kato, Y., Habas, R., Katsuyama, Y., Naar, A.M. and He, X. (2002) A component of the
ARC/Mediator complex required for TGF beta/Nodal signaling. Nature, 418, 641-646.

Kelleher, R.J., Flanagan, P.M. and Kornberg, R.D. (1990) A novel mediator between
activator proteins and the RNA polymerase II transcription apparatus. Cell, 61, 1209-1215.

Kim, T.K., Ebright, R.H. and Reinberg, D. (2000) Mechanism of ATP-dependent promoter
melting by transcription factor IIH. Science, 288, 1418-1422.

Kim, T.W., Kwon, Y.J., Kim, J.M., Song, Y.H., Kim, S.N. and Kim, Y.J. (2004) MED16 and
MED23 of Mediator are coactivators of lipopolysaccharide- and heat-shock-induced
transcriptional activators. Proc. Natl. Acad. Sci. USA, 101,12153-12158.

Kim, Y.J., Bjorklund, S., Li, Y., Sayre, M.H. and Kornberg, R.D. (1994) A multiprotein
mediator of transcriptional activation and its interaction with the C-terminal repeat domain
of RNA polymerase II. Cell, 77, 599-608.

Koleske, A.J., Buratowski, S., Nonet, M. and Young, R.A. (1992) A novel transcription factor
reveals a functional link between the RNA Polymerase II CTD and TFIID. Cell, 69,
883-894.

Koleske, A.J., Buratowski, S., Nonet, M. and Young, R.A. (1992) A novel transcription factor
reveals a functional link between the RNA polymerase II CTD and TFIID. Cell, 69,
883-894.










Koleske, A.J. and Young, R.A. (1994) An RNA polymerase II holoenzyme responsive to
activators. Nature, 368, 466-469.

Kornberg, RD. (2005) Mediator and the mechanism of transcriptional activation. Trends
Biochem. Sci. 30, 235-239.

Krogan, N.J., Kim, M., Tong, A., Golshani, A., Cagney, G., Canadien, V., Richards, D.P.,
Beattie, B.K., Emili, A., Boone, C., Shilatifard, A., Buratowski, S. and Greenblatt, J.
(2003) Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to
transcriptional elongation by RNA polymerase II. Mol1. Cell Biol. 23, 4207-4218.

Kugel, J.F. and Goodrich, J.A. (1998) Promoter escape limits the rate of RNA polymerase II
transcription and is enhanced by TFIIE, TFIIH, and ATP on negatively supercoiled DNA.
Proc. Natl. Acad. Sci. USA, 95, 9232-9237.

Kumar, K~P., Akoulitchev, S. and Reinberg, D. (1998) Promoter-proximal stalling results
from the inability to recruit transcription factor IIH to the transcription complex and is a
regulated event. Proc. Natl. Acad. Sci. USA, 95, 9767-9772.

Lau, J.F., Nusinzon, I., Burakov, D., Freedman, L.P. and Horvath, C.M. (2003) Role of
metazoan mediator proteins in interferon-responsive transcription. Mol1. Cell Biol. 23,
620-628.

Lee, Y.C., Park, J.M., Min, S., Han, S.J. and Kim, Y.J. (1999) An activator binding module
of yeast RNA polymerase II holoenzyme. Mol1. CellBiol. 19, 2967-2976.

Li, L., Tutone, A.F., Drummond, R.S., Gardner, R.C. and Luan, S. (2001) A novel family of
magnesium transport genes in Arabidopsis. Plant Cell, 13, 2761-2775.

Li, Y., Bjorklund, S., Jiang, Y.W., Kim, Y.J., Lane, W.S., Stillman, D.J. and Kornberg, R.D.
(1995) Yeast global transcriptional regulators Sin4 and Rgrl are components of mediator
complex/RNA polymerase II holoenzyme. Proc. Natl. Acad. Sci. USA, 92, 10864-10868.

Liao, S.M., Zhang, J., Jeffery, D.A., Koleske, A.J., Thompson, C.M., Chao, D.M., Viljoen,
M., van Vuuren, H.J. and Young, R.A. (1995) A kinase-cyclin pair in the RNA
polymerase II holoenzyme. Nature, 374, 193-196.

Linder, T. and Gustafsson, C.M. (2004) The Sohl/MED31 protein is an ancient component of
Schizosacch aromyces~~hh~~hh~~ pombe and Saccharomyces cerevisiae Mediator. J. Biol. Chem. 279,
49455-49459.

Malik, S., Gu, W., Wu, W., Qin, J. and Roeder, R.G. (2000) The USA-derived transcriptional
coactivator PC2 is a submodule of TRAP/SMCC and acts synergistically with other PCs.
M~ol. Cell, 5, 753-760.

Malik, S. and Roeder, R.G. (2000) Transcriptional regulation through Mediator-like
coactivators in yeast and metazoan cells. Trends Biochem. Sci. 25, 277-283.










Malik, S., Wallberg, A.E., Kang, Y.K. and Roeder, R.G. (2002)
TRAP/SMCC/mediator-dependent transcriptional activation from DNA and chromatin
templates by orphan nuclear receptor hepatocyte nuclear factor 4. Mol1. Cell Biol. 22,
5626-5637.

Manak, M.S., Paul, A.L., Sehnke, P.C. and Ferl, R.J. (2002) Remote sensing of gene
expression in Planta: transgenic plants as monitors of exogenous stress perception in
extraterrestrial environments. Life Support Biosph. Sci. 8, 83-91.

Mittler, G., Stuhler, T., Santolin, L., Uhlmann, T., Kremmer, E., Lottspeich, F., Berti, L.
and Meisterernst, M. (2003) A novel docking site on Mediator is critical for activation by
VPl16 in mammalian cells. EM~BO J. 22, 6494-6504.

Mo, X., Kowenz-Leutz, E., Xu, H. and Leutz, A. (2004) Ras induces mediator complex
exchange on C/EBP beta. Mol1. Cell, 13, 241-250.

Myers, L.C., Gustafsson, C.M., Bushnell, D.A., Lui, M., Erdjument-Bromage, H., Tempst,
P. and Kornberg, R.D. (1998) The Med proteins of yeast and their function through the
RNA polymerase II carboxy-terminal domain. Genes Dev. 12, 45-54.

Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W. and Tjian, R. (1999)
Composite co-activator ARC mediates chromatin-directed transcriptional activation.
Nature, 398, 828-832.

Naar, A.M., Lemon, B.D. and Tjian, R. (2001) Transcriptional coactivator complexes. Annu.
Rev. Biochem. 70, 475-501.

Naar, A.M., Taatjes, D.J., Zhai, W., Nogales, E. and Tjian, R. (2002) Human CRSP interacts
with RNA polymerase II CTD and adopts a specific CTD-bound conformation. Genes Dev.
16, 1339-1344.

Nair, D., Kim, Y. and Myers, L.C. (2005) Mediator and TFIIH govern carboxyl-terminal
domain-dependent transcription in yeast extracts. J. Biol Chem. 280, 33739-33748.

Nair, R. and Rost, B. (2002) Inferring sub-cellular localization through automated lexical
analysis. Bioinformatics, 18, S78-S86

Navarro, A., Rondon, G., Aguilera, A., Struhl, K., Reed, R. and Hurt, E. (2002) TREX is a
conserved complex coupling transcription with messenger RNA export. Nature, 417,
304-308.

Nevado, J., Tenbaum, S.P. and Aranda, A. (2004) hcrb7, an essential human Mediator
component, acts as a coactivator for the thyroid hormone receptor. Mol1. Cell Endocrinol.
222, 41-51.

Oelgeschlager, T. (2002) Regulation of RNA polymerase II activity by CTD phosphorylation
and cell cycle control. J. CellPhysiol. 190, 160-169.










Ogas, J., Kaufmann, S., Henderson, J. and Somerville, C. (1999) PICKLE is a CHD3
chromatin-remodeling factor that regulates the transition from embryonic to vegetative
development in Arabidopsis. Proc. Natl. Acad. Sci. USA, 96, 13839-13844.

Onodera, Y., Hang, J.R., Ream, T., Nunes, P.C., Pontes, O. and Pikaard, C.S. (2005) Plant
nuclear RNA p olym erase IV m edi ate s siRNA and DNA m ethyl ati on- dep endent
heterochromatin formation. Cell, 120, 613-622.

Park, H.C., Kim, M.L., Kang, Y.H., Jeon, J.M., Yoo, J.H., Kim, M.C., Park, C.Y., Jeong,
J.C., Moon, B.C., Lee, J.H., Yoon, H.W., Lee, S.H, Chung, W.S., Lim, C.O., Lee, S.Y.,
Hong, J.C. and Cho, M.J. (2004) Pathogen- and NaCl-induced expression of the SCaM-4
promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription
factor. Plant Physiol. 135, 2150-2161.

Park, J.M., Gim, B.S., Kim, J.M., Yoon, J.H., Kim, H.S., Kang, J.G. and Kim, Y.J. (2001)
Drosophila Mediator complex is broadly utilized by diverse gene-specific transcription
factors at different types of core promoters. Mol1. Cell. Biol. 21, 2312-2323.

Park, J.M., Kim, H.S., Han, S. J., Hwang, M.S., Lee, Y. C. and Kim, Y.J. (2000) In vivo
requirement of activator-specific binding targets of Mediator. Mol1. Cell. Biol. 20,
8709-8719.

Pineda Torra, I., Freedman, L.P. and Garabedian, M.J. (2004) Identification of DRIP205 as
a coactivator for the Farnesoid X receptor. J. Biol. Chem. 279, 3 61 84-3 6191.

Pugh, B.F. (2000) Control of gene expression through regulation of the TATA-binding protein.
Gene, 255, 1-14.

Rachez, C., Lemon, B.D., Suldan, Z., Bromleigh, V., Gamble, M., Naar, A.M.,
Erdjument-Bromage, H., Tempst, P. and Freedman, L.P. (1999) Ligand-dependent
transcription activation by nuclear receptors requires the DRIP complex. Nature, 398,
824-828.

Rachez, C., Suldan, Z., Ward, J., Chang, C.P., Burakov, D., Erdjument-Bromage, H.,
Tempst, P. and Freedman, L.P. (1998) A novel protein complex that interacts with the
vitamin D3 receptor in a ligand-dependent manner and enhances VDR transactivation in a
cell-free system. Genes Dev. 12, 1787-800.

Ranish, J.A., Yudkovsky, N. and Hahn, S. (1999) Intermediates in formation and activity of
the RNA polymerase II preinitiation complex: holoenzyme recruitment and a
postrecruitment role for the TATA box and TFIIB. Genes Dev. 13, 49-63.

Reeves, W.M. and Hahn, S. (2003) Activator-independent functions of the yeast mediator sin4
complex in preinitiation complex formation and transcription reinitiation. Mol1. CellBiol.
23, 349-358.










Rieping, M. and SchoM, F. (1992) Synergistic effect of upstream sequences, CCAAT box
elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in
transgenic tobacco. Mol1. Gen. Genet. 231, 226-232.

Roth, S.Y., Denu, J.M. and Allis CD. (2001) Histone acetyltransferases. Annu. Rev. Biochem.
70, 81-120.

Rubio, V., Shen, Y., Saijo, Y., Liu, Y., Gusmaroli, G., Dinesh-Kumar, S.P. and Deng, X.W.
(2005) An alternative tandem affinity purification strategy applied to Arabidopsis protein
complex isolation. Plant J. 41, 767-778.

Ryu, S., Zhou, S., Ladurner, A.G. and Tjian, R. (1999) The transcriptional cofactor complex
CRSP is required for activity of the enhancer-binding protein Spl. Nature, 397, 446-450.

Sakai, A., Shimizu, Y., Kondou, S., Chibazakura, T. and Hishinuma, F. (1990) Structure and
molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces
cerevisiae. Mol1. Cell. Biol. 10, 4130-4138.

Sakurai, H. and Fukasawa, T. (2000) Functional connections between mediator components
and general transcription factors of Saccharomyces cerevisiae. J. Biol. Chem. 275,
37251-37256.

Samuelsen, C.O., Baraznenok, V., Khorosjutina, O., Sp~hr, H., Kieselbach, T., Holmberg,
S. and Gustafsson, C.M. (2003) TRAP230/ARC240 and TRAP240/ARC250 Mediator
subunits are functionally conserved through evolution. Proc. Natl. Acad. Sci. USA, 100,
6422-6427.

Sato, S., Tomomori-Sato, C., Parmely, T.J., Florens, L., Zybailov, B., Swanson, S.K., Banks,
C.A., Jin, J., Cai, Y., Washburn, M.P., Conaway, J.W. and Conaway, R.C. (2004) A
set of consensus mammalian mediator subunits identified by multidimensional protein
identification technology. Mol1. Cell, 14, 685-691.

Shilatifard, A., Conaway, R.C. and Conaway, J.W. (2003) The RNA polymerase II elongation
complex. Annu. Rev. Biochem. 72, 693-715.

Song, W., Treich, I., Qian, N., Kuchin, S. and Carlson, M. (1996) SSN genes that affect
transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SRB
proteins associated with RNA polymerase II. Mol1. Cell. Biol. 16, 115-120.

Spahr, H., Beve, J., Larsson, T., Bergstrom, J., Karlsson, K.A. and Gustafsson, C.M. (2000)
Purification and characterization of RNA polymerase II holoenzyme from
Schizosacch aromyces~~hh~~hh~~ pombe. J. Biol. Chem. 275, 1351-13 56.

Stevens, J.L., Cantin, G.T., Wang, G., Shevehenko, A. and Berk, A.J. (2002) Transcription
control by E1A and MAP kinase pathway via Sur2 mediator subunit. Science, 296,
755-758.










Stockinger, E.J., Mao, Y., Regier, M.K., Triezenberg, S.J. and Thomashow, M.F. (2001)
Transcriptional adaptor and histone acetyltransferase proteins in Arabidopsis and their
interactions with CBF l, a transcriptional activator involved in cold-regulated gene
expression. Nucleic Acids Res. 29, 1524-1533.

Suzuki, Y., Nogi, Y., Abe, A. and Fukasawa, T. (1988) GAL11 protein, an auxiliary
transcription activator for genes encoding galactose-metabolizing enzymes in
Saccharomyces cerevisiae. Mol1. Cell Biol. 8, 4991-4999.

Taatjes, D.J., Naa,r A.M., Andel, F. 3rd, Nogales, E. and Tjian, R. (2002) Structure, function,
and activator-induced conformations of the CRSP coactivator. Science, 295, 1058-1062.

Thomas, M.C. and Chiang, C.M. (2006) The general transcription machinery and general
cofactors. Crit. Rev. Biochem. Mol1. Biol. 41, 105-178.

Thompson, C.M., Koleske, A.J., Chao, D.M. and Young, R.A. (1993) A multisubunit
complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast.
Cell, 73, 1361-1375.

Toth, J.I., Datta, S., Athanikar, J.N., Freedman, L.P. and Osborne, T.F. (2004) Selective
coactivator interactions in gene activation by SREBP-la and -lc. Mol1. Cell Biol. 24,
8288-8300.

Villain, P., Clabault, G., Mache, R. and Zhou, D.X. (1994) S1F binding site is related to but
different from the light-responsive GT-1 binding site and differentially represses the
spinach rps1 promoter in transgenic tobacco. J. Biol. Chem. 269, 16626-16630.

Volkov, R.A., Panchuk, I.I. and Schoffl, F. (2003) Heat-stress-dependency and developmental
modulation of gene expression: the potential of house-keeping genes as internal standards
in mRNA expression profiling using real-time RT-PCR. J. Exp. Bot. 54, 2343-2349.

Wada, O., Oishi, H., Takada, I., Yanagisawa, J., Yano, T. and Kato, S. (2004) BRCAl
function mediates a TRAP/DRIP complex through direct interaction with TRAP220.
Oncogene, 23, 6000-6005.

Wang, G. and Berk, A.J. (2002) In vivo association of adenovirus large E1A protein with the
human mediator complex in adenovirus-infected and -transformed cells. J Virol. 76,
9186-9193.

Wang, Q., Sharma, D., Ren, Y. and Fondell, J.D. (2002) A coregulatory role for the
TRAP-mediator complex in androgen receptor-mediated gene expression. J. Biol. Chem.
277, 42852-42858.

Wang, S., Ge, K., Roeder, R.G. and Hankinson, O. (2004) Role of mediator in transcriptional
activation by the aryl hydrocarbon receptor. J. Biol. Chem. 279, 13593-13600.










Wang, Z.Y. and Tobin, E.M. (1998) Constitutive expression of the CIRCADIAN CLOCK
ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own
expression. Cell, 93, 1207-1217.

Warnmark, A., Almlof, T., Leers, J., Gustafsson, J.A. and Treuter, E. (2001) Differential
recruitment of the mammalian mediator subunit TRAP220 by estrogen receptors ERalpha
and ERbeta. J. Biol. Chem. 276, 23397-23404.

Woychik, N.A. and Hampsey, M. (2002) The RNA polymerase II machinery: structure
illuminates function. Cell, 108, 453-463.

Yan, Q., Moreland, R.J., Conaway, J.W. and Conaway, R.C. (1999) Dual roles for
transcription factor IIF in promoter escape by RNA polymerase II. J. Biol. Chem. 274,
35668-35675.

Ye, Z.H., Freshour, G., Hahn, M.G., Burk, D.H. and Zhong, R. (2002) Vascular development
in Arabidopsis. Int. Rev. Cytol. 220, 225-256.

Yuan, C.X., Ito, M., Fondell, J.D., Fu, Z.Y. and Roeder, R.G. (1998) The TRAP220
component of a thyroid hormone receptor- associated protein (TRAP) coactivator complex
interacts directly with nuclear receptors in a ligand-dependent fashion. Proc. Natl. Acad.
Sci. USA, 95, 7939-7944.

Yudkovsky, N., Ranish, J.A. and Hahn, S. (2000) A transcription reinitiation intermediate that
is stabilized by activator. Nature, 408, 225-229.

Zhong, R., Burk, D.H., Nairn, C.J., Wood-Jones, A., Morrison, W.H. 3rd and Ye, Z.H.
(2005) Mutation of SAC1, an Arabidopsis SAC domain phosphoinositide phosphatase,
causes alterations in cell morphogenesis, cell wall synthesis, and actin organization. Plant
Cell, 17, 1449-1466.

Zhou, R., Bonneaud, N., Yuan, C.X., de Santa Barbara, P., Boizet, B., Schomber, T.,
Schere,r G., Roeder, R.G., Poulat ,F., Berta, P. and Tibor, S. (2002) SOX9 interacts
with a component of the human thyroid hormone receptor-associated protein complex.
Nucleic Acids Res. 30, 3245-3252.

Zhu, X., Wiren, M., Sinha, I., Rasmussen, N.N., Linder, T., Holmberg, S., Ekwall, K. and
Gustafsson, C.M. (2006) Genome-wide occupancy profile of mediator and the Srb8- 11
module reveals interactions with coding regions. Mol1 Cell, 22, 169-178.

Zhu, Y., Qi, C., Jain, S., Le Beau, M.M., Espinosa, R. 3rd, Atkins, G.B., Lazar, M.A.,
Yeldandi, A.V., Rao, M.S. and Reddy, J.K. (1999) Amplification and overexpression of
peroxisome proliferator-activated receptor binding protein (PBP/PPARBP) gene in breast
cancer. Proc. Natl. Acad. Sci. USA, 96, 10848-10853.

Zhu, Y., Qi, C., Jain, S., Rao, M.S. and Reddy, J.K. (1997) Isolation and characterization of
PBP, a protein that interacts with peroxisome proliferator-activated receptor. J. Biol. Chem.
272, 25500-25506.









BIOGRAPHICAL SKETCH

Wei Pan received his Bachelor of Science degree in biology from Northeast Normal

University in Changchun, China, and then a Master of Science degree in biophysics from the

Chinese Academy of Agricultural Sciences in Beijing, China. His current research interests are

centered on genetics, development and molecular biology.