Evolution and divergence of the mammalian SAMD9/SAMD9L gene family

MISSING IMAGE

Material Information

Title:
Evolution and divergence of the mammalian SAMD9/SAMD9L gene family
Physical Description:
Mixed Material
Language:
English
Creator:
Matos, Ana Lemos de
McFadden, Grant
Liu, Jia
Esteves, Pedro J.
Publisher:
BioMed Central (BMC Evolutionary Biology)

Notes

Abstract:
Background: The physiological functions of the human Sterile Alpha Motif Domain-containing 9 (SAMD9) gene and its chromosomally adjacent paralogue, SAMD9-like (SAMD9L), currently remain unknown. However, the direct links between the deleterious mutations or deletions in these two genes and several human disorders, such as inherited inflammatory calcified tumors and acute myeloid leukemia, suggest their biological importance. SAMD9 and SAMD9L have also recently been shown to play key roles in the innate immune responses to stimuli such as viral infection. We were particularly interested in understanding the mammalian evolutionary history of these two genes. The phylogeny of SAMD9 and SAMD9L genes was reconstructed using the Maximum Likelihood method. Furthermore, six different methods were applied to detect SAMD9 and SAMD9L codons under selective pressure: the site-specific model M8 implemented in the codeml program in PAML software and five methods available on the Datamonkey web server, including the Single Likelihood Ancestor Counting method, the Fixed Effect Likelihood method, the Random Effect Likelihood method, the Mixed Effects Model of Evolution method and the Fast Unbiased Bayesian AppRoximation method. Additionally, the house mouse (Mus musculus) genome has lost the SAMD9 gene, while keeping SAMD9L intact, prompting us to investigate whether this loss is a unique event during evolution. Results: Our evolutionary analyses suggest that SAMD9 and SAMD9L arose through an ancestral gene duplication event after the divergence of Marsupialia from Placentalia. Additionally, selection analyses demonstrated that both genes have been subjected to positive evolutionary selection. The absence of either SAMD9 or SAMD9L genes from some mammalian species supports a partial functional redundancy between the two genes. Conclusions: To the best of our knowledge, this work is the first study on the evolutionary history of mammalian SAMD9 and SAMD9L genes. We conclude that evolutionary selective pressure has acted on both of these two genes since their divergence, suggesting their importance in multiple cellular processes, such as the immune responses to viral pathogens. Keywords: SAMD9, SAMD9-like, Mammals, Evolutionary history, Positive selection
General Note:
Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/UFir) at the time of funding. The institutional Repository at the University of Florida community, with research, news, outreach, and educational materials.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved by the source institution.
System ID:
AA00016100:00001

Full Text


SAMD9L_Orcu
100 | SAMD9L_Caja
-- SAMD9L_Mamu
10 SA
1 SAMD9L_Poab
100 52 SAMD9L_Nole
100 SAMD9L_Patr
100 D SAMD9L Hosa
69 SAMD9L-Gogo
100 100 SAMD9L_Rano
SAMD9L_Mumu
-8 fI SAMD9LCrgr
M SAMD9LCapo
1001 SAMD9L_Eqca
100 SAMD9LCalu
100 )"T I-SAMD9L_Aime
100.-- 1 S100MD SAMD9LaSoar

9SAMD9LEreu
54 SAMD9_Mylu
68 SAMD9_Soar
SASAMD9_Eqca
94 9 |SAMD9_Susc A
100 SAMD9._Bota
SASAMD9_Orcu
100 SAMD9_Capo
100 SAMD9_Crgr
AM o SAMD9_Rano
969i SAMD9_Mamu
SAMD9_Nole
74 SAMD9_Poab
10o SAMD9_Gogo
96 SAMD9_Patr
SAMD9_Hosa
SAMD9LModo

0.08








(.urilla
Orangulan
- Macaque


Pika


chnlincating bal


Shrew


Tearc


- Armadillo
Sluolh


Platypus


Lao ,'p" I.w I


Eutheria




SAMD9_Nole


SAMD9_Mamu


- SAMD9_Poab


I- SAMD9_Gogo


SAMD9_Mylu


SAMD9_Soar


)_Eqca


SAMD9.Bota


- SAMD9..Crgr


SAMD9_Rano


SAMD9_Capo


0.03




SAMD9LCaja


- SAMD9LNole

SAMD9LPoab


SAMD9L_Hosa

- SAMD9LGogo


SAMD9L_Orcu

- SAMD9LLoaf


SAMD9L.Soar


SAMD9LCalu


SAMD9LCrgr

SAMD9L_Mumu

- SAMD9LRano


0.03




Full Text

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Table S4 : SAMD9 and SAMD9L amino acid properties under positive selection determined in TreeSAAP a Between 6 and 8 (the most radical values denoting positive destabilizing selection). b z Score > 3.09, level of significance p<0.001 (***). Protein Property Category a z S core b SAMD9 Isoelectric point 8 8.218*** Equilibrium constant (ionization of COOH) 8 5.001*** Power to be at the C terminal 7 4.032*** Alpha helical tendencies 6 5.581*** SAMD9L Isoelectric point 8 6.922*** Equilibrium constant (ionization of COOH) 8 4.797*** Solvent accessible reduction ratio 7 4.366*** Power to be at the middle of alpha helix 7 3.554*** Power to be at the C terminal 6 10.442*** Alpha helical tendencies 6 5.228*** Surrounding hydrophobicity 6 3.921***



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Table S1: Detection of recombination breakpoints from SAMD9 and SAMD9 L genes alignment using GARD analysis a LHS: segment to the left of the breakpoint b RHS: segment to the right of the breakpoint SAMD9 + SAMD9L alignment Breakpoint LHS a Raw p LHS a adjusted p RHS b Raw p RHS b adjusted p 1179 0.94650 1.00000 0.00010 0.00060 2404 0.00010 0.00060 0.40590 1.00000 4755 0.00010 0.00060 0.00010 0.00060


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10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa MSKQVSLPEMIKDWTKE HVKKWVNEDLKINEQYGQILLSEEVTGLVLQELTEKDLVEMGLPWGPALLIKRSYNKLNSKSPESDNHDPGQLDNSKPSKTEH SAMD9L_Patr .......................T...............G...............I............................................ SAMD9L_Gogo .............................S.......F..... ............I............................................ SAMD9L_Poab ...........E...........T...............................I.....R............FY........................ SAMD9L_Nole .......................T...............................I.....R....... ...............R............... SAMD9L_Caja .....T.................T.....D.K......N................I.....R..............NT.A..........SH..S..... SAMD9L_Mamu .........I.............TD..............................I.....R........A....................H... ..R.. SAMD9L_Loaf ..E..T....TQ...........T.....D.K......N................TD....R........G.....NS....H.Q.FE...HT.T..K.Q SAMD9L_Eqca .NE.AN....T.........Q..TK....G.K......N................I.....R...I....A..R..NS....N.Q.S....HT.S..E.. SAMD9L_Calu .NEE.N...VVD........Q..TKH.NVD.K......R................R.......S......K.....NS.S..N.Q.S....HT....K.. SAMD9L_Aime ..E..N...I.N........Q..TK....D.K......T.............N..R.....R........A..R..NS.S..N.Q.S..V.HT....K.. SAMD9L_Ereu .DE..N....TQ........Q..TN. .Q.D.K......C.....R...V......I.....R........KFKS..NL.SKNS.QNS....HAE...K.. SAMD9L_Orcu .NE..T...LV............T....VD.K......N........K....D..K.....R........AC...LNS......Q.S.K...I....KKQ SAMD9L_Mumu ..G..TQ.KL.........R...T...N.V.K.A...FK.....M....... E..R.....R........M....I. ....H.Q.SRE.NDK.L.TK.Q SAMD9L_Crgr .NE..TA.KLV......Q....IT...N.D.K.AE..FN.....M..........R.....R........A....SN AT....Q.SK..H.K.L.IK.. SAMD9L_Rano .DRH.TQ.KL.........R..IT.....D.K.A..VFE.....M..........R.....R........M....I. ...GH.Q.SR..N.KTL.IN.Q SAMD9L_Capo .NE.GT..........D.....IT.E...D.K.....FN................K.....R........M.....TSF.....PNSR.VHDT.S..K.. SAMD9L_Soar ..EHTN....T.A....D..Q..T.V.Q.D.E......N.K.S..A...I..E..R.....R........T.....NI....NSPNSRP..STNV..KKS 110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa QKNPKHTKKEEENSMSSNIDYDPREIRDIKQEESILMKENVLDEVANAKHKKKGKLKPEQLTCMPYPFDQFHDSHRYIEHYTLQ PETGALNLIDPIHEF SAMD9L_Patr .................................................D.................................. ............... SAMD9L_Gogo ..............T........... .......................D.................................. ............... SAMD9L_Poab .....Q........................EQ.................D.................................. ............... SAMD9L_Nole .....Q........K.............V..Q....... ..........D.................................. ............... SAMD9L_Caja H.K..Q...K.K.....S.......V....EQK...........AE...D...D.....R.............GQ......... ....S.......... SAMD9L_Mamu ..D..Q........T..........V....ER..........E......D....E.......... ................... ............... SAMD9L_Loaf .KA. ----K.K.I..S..H.LT..G...ER........ATN...AT.D...N.V.T................Q..T...I.. ....P.......... SAMD9L_Eqca KEK.QQ....K.K.T.....HNL..T..T.EQ........A.N..V T.D.Q.N..QA................Q......I.. ....P. ......... SAMD9L_Calu P.K.QQM.....K.VL....H.L..A..T.EQ.......DA.N.G.T.EDQNED..GIK.............N..C...NSV.. ....P.......... SAMD9L_Aime P.K.QKM.....K.VL..N.H.L..M..T.EQ....V..DA.N.EVTTEDQNEDRTET..............N.Q.....SI.. ....P.......... SAMD9L_Ereu .R K -. --KT..T L..N.H.V...QNA.AQ.LAATGK.AQ...GITEE. .K.R.IV.S...........A..H.T...I.. ....P.......... SAMD9L_Orcu ..K..QKS...GT...L...H.L..TTE.EVQ...PL..KA...TV..AD. ENAIQT.R..............Q......I.. ....P.......... SAMD9L_Mumu .TK --..N.....V...S.HGL..TGQ NEEQ.PS.T...M.GD.V T.DMEDN.P....MS.T.....S.C.VKQ....SI.RVA...P.......... SAMD9L_Crgr P.K --.NN...KLI...SGH.L..MGLNTEQ.PS.L..KA.SD.L T.DMEGNTA....MS.......S...DR....R.I..VA...P......V... SAMD9L_Rano P.K --SNS.....I...S.FGL..TGQNEEQ.PSI..V.T.GD.E T.DM.DN MP.S..MS...H..NFA..AK.....SI.RVA...P......V... SAMD9L_Capo ..K. ---NL.KEL.P.S..Q.LS.S.N..DQD..P.E..AAN..S.TID...N...T.N...P.....N...GQ......I.. ....P.......... SAMD9L_Soar K..SQ -NTK..KLV...S.H.LK.SVNT.EQ..VPIEKDS..YQGIPEDQ ..... K..S..V..........R..V..QI.. ....PR.........

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210 220 230 240 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa KALTNTETATEVDIKMKFSNEVFRFASACMNSRTNGTIHFGVKDKPHGEIVGVKITSKAAFIDHFNVMIKKYFEESEINEAKKCIREPRFVEVLLQNNTP SAMD9L_Patr .............. ...................................................................................... SAMD9L_Gogo .................................................................................................... SAMD9L_Poab ...........A..N......................... ................N..S.........R.............................. SAMD9L_Nole ...........A................................N....................................................... SAMD9L_Caja .......R...E..........L..............................N....DV...... L.........D....................... SAMD9L_Mamu ...........A..............................................D.......L............Q..................M. SAMD9L_Loaf ......D....K..H.......................................VA..DD..N...L...Q...D.D.K..E....Q..... ..Q..... SAMD9L_Eqca ...........K.........A.....................N..........V...DS..........Q.......KV.................... SAMD9L_Calu .......A...K...............................NR...Q.....VAN.D.......I..RQ.........V..................L SAMD9L_Aime ... ....A...K...............................N....Q.....V...D..........RQ...GN........................ SAMD9L_Ereu ...........K..............................L...........VR..D.......L...Q.......K..................Y.. SAMD9L_Orcu ......AA...E...........................................S..DVL.N...........D.D....................SM. SAMD9L_Mumu ..F...KK...E.........T....A..........................QV...DI.VN...T..T....D...S..RA................Q SAMD9L_Crgr ...........E.........I.... ......H......................P..DV.V....K.......D.D.K..RE................. SAMD9L_Rano ..F.......QEQM.......T....A....T.....................QV...DI..N......T....D.D.H..RA............L...Q SAMD9L_Capo .......N...E.L.......T.............................. ..V...D.......L...........S..Q....Q...ID.......L SAMD9L_Soar .......SVE.K..............................L...Q.......V.R.DS......K...H...D...K..R.................S 310 320 330 340 350 360 370 380 390 400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa SDRFVIEVDTIPKHSICNDKYFYIQMQICKDKIWKQNQNLSLFVREGASSRDILANSKQRDVDFKAFLQNLKSLVASRKEAEEEYGMKA MKKESEGLKLV SAMD9L_Patr .................................................................................................... SAMD9L_Gogo ...........................................I........................................................ SAMD9L_Poab .........I.......K..............T........................................................I.......... SAMD9L_Nole .........IN......K.....................T............................................................ SAMD9L_Caja .......I.I.....V.K.M..C.R. .SYT..K....K.S...............DPNK............E.............R...K.......... SAMD9L_Mamu .........I...Y...K..........F..........S................................................T........... SAMD9L_Loaf ..K......V..R..V.K......N......GS.R.DKDY.....Y....K. ....VR...A.....TL.FE.V............V..N.........A SAMD9L_Eqca .........V.......KE.....K..N..NEK....EDH.....D....K.....G....T.....T.......T.........EV..N......Q... SAMD9L_Calu .........V.....V.EK...L.R..N..SET..P..D......D.P..K.....V...ELNL.......T.V.... .......E...DR.....Q... SAMD9L_Aime .........V...Y.V.GE...L.R..S..NET..P..D......D.P..K.....V....IAY.E.S..F..VAT...V.....EV..N......Q... SAMD9L_Ereu .........V.......KE....TN..SY.NDT.....ES.....D....V.....G....KN..T........AED..D..K.HE...N...C..Q..I SAMD9L_Orcu .........V.....V.KE.....K..S...NV.....EY...I............A....P.....T..................R..T.......... SAMD9L_Mumu .N.......V..R....QE.....M..SSTG.T...SKDT..........KN..G.PN...RE..K..ED..MWT....A....LR. VT.........S SAMD9L_Crgr .........V.......KE.....I..SYT..T...SKENC......T..KN....A....RE.RK..E...AWT........QQR. VT.........A SAMD9L_Rano .N.......V.......QE....VML.T.TGTT...SKDT..............G.P....RE..K..E...MSI....A. ..CMV VS..D......S SAMD9L_Capo .........I...Y.V.KE.....KL .SLT..T............D....K.....V.R.EIE....YE....CA.A..D....HQ..TT.R........ SAMD9L_Soar .........V.......QE....TK..EL..G....TE.T.........TK.....ERT..AG......D....ITM......KDEV.VN.Q....Q..I

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410 420 430 440 450 460 470 480 490 500 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa KLLIGNRDSLDNSYYDWYILVTNKCHPNQIKHLDFLKEIKWFAVLEFDPESMINGVVKAY KESRVANLHFPNQYEDKTTNMWEKISTLNLYQQPSWIFCN SAMD9L_Patr ......................................M..........................................R.................. SAMD9L_Gogo .................................................................................R.... .............. SAMD9L_Poab .................................................................................R.................. SAMD9L_Nole ..................V..........................................Q...................R.................. SAMD9L_Caja ...T.................I.............................QS.......R.................M..R....A............. SAMD9L_Mamu ...................................................V.............................R....A............. SAMD9L_Loaf .....S.....D............S....T.....................VSK.................D...K.AATIR.M.F.....E........ SAMD9L_Eqca ...............N.....I.......T.N...................QSK..A..F.K.........S...E.S.P.R....S............. SAMD9L_Calu ...............N.......... ...T.N......M.L..........VSK.......K..I........F.E..N.IR....S......T...... SAMD9L_Aime ...............S.............T.N......M............VSK.......K...........F.E.SN.IR....S....E........ SAMD9L_Ereu ......Q........N.........N.S.R -...................E SK..A.T.R........ ....AE...T.R....SXS.W --------SAMD9L_Orcu ...............S.............T.....................G...................S......SSLR..............V... SAMD9L_Mumu ...TRHQG...E...........T.A.T.LE..E.I..M.L....D...Y.H.K......R...I....L.SH..E. .TIA......K..E........ SAMD9L_Crgr ...TRHQG...............N.A....E.........L....D...Y.Q.K......R...I....L.S...E. STIE......K.FE........ SAMD9L_Rano ...TRHQG...K...........S.V.T.ME....I....L....D...Y.H.K......R........L.SH..E. .TIE......K..E........ SA MD9L_Capo ...............NS............MQ....................R....A...QK.....M.......E.K.T.RQ...S....E.T...... SAMD9L_Soar ...........S...N.............T.Y.......S.....D.....VSK....VFQ..........S..VEGKSTQN.TVCS......T...... 510 520 530 540 550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa GRSDL KSETYKPLEPHLWQRERASEVRKLILFLTDENIMTRGKFLVVFLLLSSVESPGDPLIETFWAFYQALKGMENMLCISVNSHIYQRWKDLLQTRMK SAMD9L_Patr .................................................................................................... SAMD9L_Gogo ..................H............ ..................................................................... SAMD9L_Poab .........H.......................................................................................... SAMD9L_Nole ...............Y......................................... ........................................... SAMD9L_Caja ..............A..........................R.........FT....V.........................S...............N SAMD9L_Mamu ..................................................... ............................................... SAMD9L_Loaf .....NN......D..........................K.........F..............C...........I...C.K.Q.........R..LT SAMD9L_Eqca ..T...D.RH...............I....Q......V....R.........A..........S.C............. ..C...Q............LT SAMD9L_Calu ......N.S.........H.................................P............C...........I...C................LT SAMD9L_Aime ......N.S.........H..............................................C...........I...C...Q............LT SAMD9L_Ereu ------------..RQ..E....GRA..NY.....................P..........P.CT.FKV.N..Q.IM..C.TPQ............FV SAMD9L_Orcu .....T......F.................F........A.........................C...............C...Q.........K..LT SAMD9L_Mumu ..V.. --SCQ.. .......D...G..R..S.......IVK..V........PI.NQK.......C....VFN..D.....C...A...Q.S....V.LE SAMD9L_Crgr ..L...NDSCQ.........D...G..R..S........V...V........P.NNQK.......C....VFN..D.....C..P......S....V.LE SAMD9L_Rano ..V.. --SCQ.........D...G.....S....G..I A...V........P..NQK...L...C....VFN..D.....C..PS...Q.S....V.LE SAMD9L_Capo .....QN.......A.....D..C......S......L.AK...............L...FT...C......R........C...K............LT SAMD9L_Soar ......GAV.........NKN.....T.........S..A............P............ CV..K..N....I...C..LD..KQ.....KA.LI

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610 620 630 640 650 660 670 680 690 700 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|.. ..|....|....|....|....|....| SAMD9L_Hosa MEDELTNHSISTLNIELVNSTILKLKSVTRSSRRFLPARGSSSVILEKKKEDVLTALEILCENECTETDIEKDKSKFLEFKKSKEEHFYRGGKVSWWNFY SAMD9L_Patr .................................................................................................... SAMD9L_Gogo .................................................................................................... SAMD9L_Poab .......................... ...Q........H..........................KD......E....................C..... SAMD9L_Nole .............................Q.P......H..........................RD......E.......................... SAMD9L_Caja I...................S........Q........H.......K.....A............RD......E......R................... SAMD9L_Mamu .............................Q...................................RD......E.......................... SAMD9L_Loaf IA............L.QI........ ..IQ.......S...........E..LF...........KD.............R...........R....... SAMD9L_Eqca VA...A........L..I........P..E.......SS.L........E..I............RD......EF..Q.......KY............. SAMD9L_Calu IA............L..I........P..Q.L.....SH.F..I.....E.. I............KD..........Q....L................. SAMD9L_Aime AA............L..I........P..Q.......SH.FA.......E..T............KD......E...Q...T.................. SAMD9L_Ereu .S...........SL..L...........Q..K....S...........E..F............GD......EC..E ..R................... SAMD9L_Orcu VAH..S........L......................S..............T............RD......E.......................... SAMD9L_Mumu IK.D.AK............N........IQ.......SC.........MD..IMS..........KD......E.Q.......R........R....... SA MD9L_Crgr IKG..AE........Q...N........IQ.......SC.........IE..T............RD.....NE.E..K...LR........RA...... SAMD9L_Rano IK.D.AK.....................IQ.......SC.........MD..IMS..........RD......E.Q......LR........R....... SAMD9L_Capo IG...SK..V..... ..I.........L.Q...K...SY..........E..L....Q..S....K..............R..R................ SAMD9L_Soar E.S..ED.T.....L.FL..............K....SK.....T....E..TF.T.........KD.....NA...Q..I..N..Q....S........ 710 720 730 740 750 760 770 780 790 800 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa FSSENYSSDFVKRDSYEKLKDLIHCWAESPKPIFAKIINLYHHPGCGGTTLAMHVLWDLKKNFRCAVLKNKTTDFAEIAEQVINLVTYRAKSHQDYIPVL SAMD9L_Patr .................................................................................................... SAMD9L_Gogo ......................V... .......................................................................... SAMD9L_Poab .......................R............................................................................ SAMD9L_Nole ....................................... .................................................K........... SAMD9L_Caja ......P................Q.L..................................EK.............V..G...TS....K........... SAMD9L_Mamu .......................Q......................................... ........G....G.........K........... SAMD9L_Loaf ........A........H.....Q........V........................N.............A...T..GD..T..I..K.T...E..... SAMD9L_Eqca ........A..............L.R.Q....P....V...............N.................A......G...TS.I..K.T ......... SAMD9L_Calu ........A...........V..K........E....................N.....................E..V...TK.I..K.T..E..F... SAMD9L_Aime ........A..............Q...D....V....................N.......S.............G..V...TK.I..K.T.PE..F... SAMD9L_Ereu .. ...... .I...R..E.RS..ED.......V....................N....FR.K.........A......G...T..IA.K.S.Y....... SAMD9L_Orcu ........A.......KE..N..Q......R.V......................................A......G...T..I..K.TC........ SAMD9L_Mumu ........A......F.E.TT..QQC.D ....V.V.V.......................QK.........A...V..G...SK.MS.K.T..E.F.... SAMD9L_Crgr ........A.....G..E.TT..QQC.D..E.V...V........................K.........A...L..G...SK.IS.K.T......... SAMD9L_Rano ........A.....NF.E.TT..QQC.D....V...V................. ......QK.........A...I..G...SK.IS.K.S......... SAMD9L_Capo ........A.....M.KN.....ERC..C...E.....................I.....QKY........AI..E.VG..I.H....K.T......... SAMD9L_Soar .....HT.A.....R..N..K..ED.......L....................N...E.R.K........I.A.....G. ..TK.I..K.S.........

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810 820 830 840 850 860 870 880 890 900 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa LLVDDFEEQENVYFLQNAIHSVLAEKDLRYEKTLVIILNCMRSRNPDESAKLADSIALNYQLSSKEQRAFGAKLKEIEKQHKNCENFYSFMIMKSNFDET SAMD9L_Patr .................................................................................................... SAMD9L_Gogo .................................................................................................... SAMD9L_Poab .....................L.... ...........................................................K.IP........... SAMD9L_Nole ....................T............................................................................... SAMD9L_Caja .............I...V.............N...........Q.......S ................................................ SAMD9L_Mamu .................................................................................................... SAMD9L_Loaf ........L..ICI..D..N.S..K.G................Q...R...S...V..K....P......E...E... ....................DM SAMD9L_Eqca ........P...CV.....QTIF....................Q.......S.Y....MHR..P.......D...........................I SAMD9L_Calu .......D....CV.....D.I.G..G................Q....T......V..T...........E...E......................NKM SA MD9L_Aime .......D.....V.....D.I....G................Q....T......V..T....P......E...E.................L....N.I SAMD9L_Ereu ............FV.......I.....................Q........D...V.K....P..K...ST...........F...............M SAMD9L_Orcu .............I. ....N.I.....................Q........I.....K....P..........E.......D.........L.R..... SAMD9L_Mumu ...........A.I.....NAFI...G................Q.........N..S.K....P......E...Q....E............L.G...T. SAMD9L_Crgr ...........T.I...T.N.FI...G.............. ..Q...A........S.K....P......E...Q....E...........ML.GS.ET. SAMD9L_Rano .........Q.T.I.....N.FI...GV...............Q............S.K....P..K...E...Q....EY.D.........L.D...T. SAMD9L_Capo ...........I.I..........KR..Q......V.......Q...Q........S.K.A..A... ...E...E...................G....K SAMD9L_Soar ........L....N..D....S.....V...............Q..E...........TN...L..R............E...F........V.....D. 910 920 930 940 950 960 970 980 990 1000 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa YIENVVRNILKGQD VDSKEAQLISFLALLSSYVTDSTISVSQCEIFLGIIYTSTPWEPESLEDKMGTYSTLLIKTEVAEYGRYTGVRIIHPLIALYCLKE SAMD9L_Patr .................................................................................................... SAMD9L_Gogo ........................................ ............................................................ SAMD9L_Poab ................R............N................................................................V..... SAMD9L_Nole .............................N.............................N...... .................................. SAMD9L_Caja ...................V.........N...............................................S................V..... SAMD9L_Mamu .............................N.........................................................V.... ........ SAMD9L_Loaf .............N...............N...IN.........F....TSAR......T..................................IA.... SAMD9L_Eqca ....L.........A....T.........N...I...............MC..I..V.....N...A......N...T..R.............IS.... SAMD9L_Calu ... K.........N.....G.........N....E..................M............A......N...T................IS.... SAMD9L_Aime .............N.....G.........N...............................D....A......N.....F..............IF.... SAMD9L_Ereu ......G.......G....T......... N..........P........T.IK...KQ...............NADI.... FP..........IF.... SAMD9L_Orcu ..K............Y.............NT.........................K................N...S................IH.... SAMD9L_Mumu ..K...K.T..DL.AK.RR.....Y....N...................T..KKY GK..TV.KN.........R...SD......I........TH.... SAMD9L_Crgr ......K.T..DL.IH.RR.....Y....N.........L............NKHGK...V..N.......V.R...LD......I........IH.... SAMD9L_Rano ..K...K.T..DL.AH.RK..F..Y....NT..I...............T..IKRGK..TV..N.........R...SD.. ..A.I........IH.... SAMD9L_Capo ...........E.NIN.............N.........L.............V..K......R....Y....S....................IH.... SAMD9L_Soar .........F...NA....T....Y....N..........P........RH.PS..V...I..........I.....K... .K......S....C....

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1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa LERSYHLDKCQIALNILEENLFYDSGIGRDKFQHDVQTLLLTRQRKVYGDETDTLFSPLMEALQNKD IEKVLSAGSRRFPQNAFICQALARHFYIKEKD SAMD9L_Patr ..............................................E.................... ................................ SAMD9L_Gogo .......................... ....................E.................... ................................ SAMD9L_Poab ..............................................E.................... ................................ SAMD9L_Nole ..............................................E..... ............... ................................ SAMD9L_Caja ..............K.........................V....RE.................... ..M............................. SAMD9L_Mamu .................D............................E.................... .......... ...................... SAMD9L_Loaf ..K......S.....L.R.K...V.....E..L............RE............I...P.SK ..N..T...IQ.............Y....... SAMD9L_Eqca ..K..D....K...K..K...............Q............EH...........I.....EE .....VE..I.....................K SAMD9L_Calu ..E..D.N......K..N.....V.....E...............RE.........A..I.....E. .....I..AT..............Y......N SAMD9L_Aime ..N..N.N......K..K.D...V.....E................E.........A..I...E.EE .....M..TL..............Y......N SAMD9L_Ereu .KK......SR.. .KL.S.....N....K.................G..G... M....ID ..XXX XXXXXXXX.N.............. L..H.RN SAMD9L_Orcu ..EKH..N.HH...KL.N......F..................R..E............I.D....E ..E..V..CS.....................N SAMD9L_Mumu ..M..RM........M....VL....L.....KY..... .......EH.A.........I.E...EE T....I...D.....................N SAMD9L_Crgr ..M..GMG....T......KV...........K.............EH.A....R....I.E.K.EE T..I.TV..D.....................N SAMD9L_Rano ..M.HGM........M....I...........K.............EH.A.I......FI.E... EQ T....T...D.................L...N SAMD9L_Capo ......M..........N.TV.........................E..A.........I.T.K.EE VK...TR..S...E.................N SAMD9L_Soar ..K..Q..MSR....M.R.K........K..............H..EH...........I.D.K.E.IV.N..T...N...K......... ......K.N 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa FNTALDWARQAKMKAPKNSYISDTLGQVYKSEIKWWLDGNKNCRSITVNDLTHLLEAAEKASRAFKESQRQTDSKNYET -ENWSPQKSQRRYDMYNTAC SAMD9L_Patr ............................................................................... -................... SAMD9L_Gogo ............................................................................... -................... SAMD9L_Poab ............................................................................... -.......Y........... SAMD9L_Nole ........H................. ..................................................... -................... SAMD9L_Caja ........C.............................E.....N...SQ..........................C.. -.T..........GT..... SAMD9L_Mamu .................................................... ........................... -.E................. SAMD9L_Loaf .....H..N...KI..........V......K......E..T..EV..K....F..T.....G........N...D..P -.V.PL..Y..K.......G SAMD9L_Eqca ..I..E..E..QKR..............H..K.....GE....KD........F............K..E...R.... -.A.L......K.V.....G SAMD9L_Calu .G...E..N...K..................Q......E...STD......IC......N..K...K..E..ER.G...WADT.AK.TL..K..T....G SAMD9L_Aime .S......NE..K..................R...R..EM..AKD......IC......N..K...K..E.AER.AC.. -.P.G...L..K..T....G SA MD9L_Ereu ........Y...R.................N.V.....E.RS.KE..ID..M.C.D..........T..E...K.DS.. -.A..T...LK..E.....G SAMD9L_Orcu .E...E..NL..TR.................K......E...SKV...K.............G......Q......... -.A..A........T....G SAMD9L_Mumu .S...V..NL..R.. .................L.Q..GK..T.GN.S.D..AYF..V.....K......N.S...D.G. -.A....N......TF...G SAMD9L_Crgr .S...V..NL..R.....................Q..GK.E...L.S.D.......I....TK......E...N.D... -.A...R.......T....G SAMD9L_Rano .S...V..N...R....................RQ.. ----------------KV.....K......E.....D..I -.V...........T....G SAMD9L_Capo ..D..T..NL..ER....................C. ------------------.T.G..A...R...Q.....D... -.A.....P.....T....G SAMD9L_Soar .D...F..T...T...N....................EK....KI.SAS...YF. .......K...M..E...K.DC.R -GV.LQ.RFR.K.......G

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1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 ....|....|....|....|....|....|. ...|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa FLGEIEVGLYTIQILQLTPFFHKENELSKKHMVQFLSGKWTIPPDPRNECYLALSKFTSHLKNLQSDLKRCFDFFIDYMVLLKMRYTQKEIAEIMLSKKV SAMD9L_Patr .......................................................... .......................................... SAMD9L_Gogo .................................................................................................... SAMD9L_Poab .................................................................................... ..........T.G... SAMD9L_Nole ...........................................................................................T........ SAMD9L_Caja .....................Q......ENP...............K..Y............H.......................A....V.V...... SAMD9L_M amu ............................I.T..................Y............T............................V..T..... SAMD9L_Loaf ..........S.E......L.......AQ.PL......IGD.LA..E..YH.T.KN...Y.Q..RL...K.....V.......T.N.H...V.SI..... SAMD9L_Eqca ..........A........C. .........S..E.....GN.LTNSKS.Y..V.R....Y.Q..........E..A......E.KN....T..LS.N..I SAMD9L_Calu .F........A........C.L.Q......A..E.....GM..TN.KC.Y....N....Y.E.............D..L.....KN....TG..S....I SAMD9L_Aime .V........A........C.L....F...A.AD.....GMT.TN.K C.Y.........Y.E.............N..I...Q..N....TV..S.N..I SAMD9L_Ereu ..........A........C.....K..QR.T.E.....GN..EHSKGKY...I.......R.............A....F..T.N....T...I..... SAMD9L_Orcu .F.....................D.VSFL.P........GD..F.A...Y.........L.Q....N...... ............N....M...L..... SAMD9L_Mumu .F.......D...L.....L......I..ES.AE.....G..LS..KG.YCVV......L.Q..H...E...H..G...GF..P.N.P..LT.LL..... SAMD9L_Crgr .F.......D.........P....S.IAIEA........G..S ..KD.YGAV....A...Q....E........L..IG...P.I.P..TT.LS.I... SAMD9L_Rano .F.......D...L.....L......M..ES.A......G......KG.Y.VV.N..K.F.Q.............L...G...P.N.P..LT.LS..... SAMD9L_Capo .......S................D.S.LES........EN.S...K..Y........A..QS.....IK..H..S.......T.NF...MT.LK..... SAMD9L_Soar .........FA......I.C.....D..E.R..K..L..VNV.T..K.DY....RQ...Y.Q...P.........A....F..T.TVH...T..S.R..I 1310 1 320 1330 1340 1350 1360 1370 1380 1390 1400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9L_Hosa SRCFRKYTELFCHLDPCLLQSKESQLLQEENCRKKLE ALRADRFAGLLEYLNPNYKDA TTMESIVNEYAFLLQQNSKKP ---MTNEKQNSILANIILS SAMD9L_Patr ........................H................................. ..................... ---................ SAMD9L_Gogo .......................................................... .... ................. ---................ SAMD9L_Poab .......................................................... A.........T.........H ---................ SAMD9L_Nole ..............E........................................... A...................R ---..... ........... SAMD9L_Caja ...............SDQ..N.G..............................K...SA.I............H.....C ---................ SAMD9L_Mamu ...................R......F...........................F.N.A....I..D......K...N.R ---...............N SAMD9L_Loaf ..YY.....I....GLDP..... -......Y..S.........S.........H...AN.V.R...Q..L.F...LN.K ---LIK....F........ SAMD9L_Eqca .......M.......LGV...R.....K......A.........S.......S.H.EVA....NV..K.T......PN.Q ---L.R....F.......N SAMD9L_Calu T...K..V.......SGP.HR .... ........A.........S.........HRE.A.N..N...K.N......P..Q ---..K....F.......N SAMD9L_Aime T......G....C..LG........FF...SF..G.........S.........HRE.A.N..N...K.S...R..PN.Q ---L.K..L.F.......N SAMD9L_Ereu ..Y..Q.I.....S.VGI..N.G.......YY..S...K.....S........SHQETGNI..N..E.........PN.R ---.IK....F........ SAMD9L_Orcu ...YK..RD...I...SP.H.......L..........W.....S......SL.H.E.TS...N..D......K..PN.R ---L.K....F........ SAMD9L_Mumu ....K..V.......TN.V.G..DL...K.....RIQ.W...T.S.........H.E. NNI.N..GN.T....DILN.QLSKVL.KDI..F........ SAMD9L_Crgr A.Y.K..AG...RMNTN...G..NV.........RIV.W...T.S.........H.EV DN.....KD.T....HSLS.RVTKGL.K.T..F........ SAMD9L_Rano ....K..AD...Q E --..G..DL. ........RIK.W...T.S.........H.E. NN..N..EH.T....HTLN.QLSKALIKDT..F........ SAMD9L_Capo IF.YKT..K......STP..G.............R...W.....S.......S.H...A.I..N............PSIR ---SIK....F........ SAMD9L_Soar GH..MS.ISI.N..ELGTT.AQ.NL.........R..KC.....SL...... ......A..I....KD.T......LQN. ---.RK....F........

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1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 ....|....|....|....|....|....|....|....|....|....|....|.... |....|....|....|....|....|....|....|....| SAMD9L_Hosa CLKPNSKLIQPLTTLKKQLREVLQFVGLSHQYPGPYFLACLLFWPENQELDQDSKLIEKYVSSLNRSFRGQYKRMCRSKQASTLFYLGKRKGLNSIVHKA SAMD9L_Patr ...................................................................................... .............. SAMD9L_Gogo .................................................................................................... SAMD9L_Poab .......F...................P........................................................................ SAMD9L_Nol e ....K...................C........................................................................... SAMD9L_Caja .......S....N....................N.......................................H...........F.............. SAMD9L_Mamu ....S.RF.....M...................D........................................................R......... SAMD9L_Loaf ....S..Y.R.FNI.........EL.EPT....D......................M...........GR..RH...............S...H...... SAMD9L_Eqca ....T..S....SK.....Q...HI. .PR....D..........L......E..Q.M...........KR..SN....R....V.....K...H.L.... SAMD9L_Calu ....H..F....PI.........LSI.P.....N...........Q.....E...FM.........N.KR..RS...............Q...H.L.C.. SAMD9L_Aime ....H..S....SI...L...A.LSI.P.Y...D.................E....M.........T.KR..RS...............K...H.L.C.. SAMD9L_Ereu .......F....D...........L.EINR...D...........D..........M.......KK..NRH.RL..............N......L.... SAMD9L_Orcu ....T..S....NM..........L. ..H...SD......................M.........T................F........F.GL.L.. SAMD9L_Mumu ....S..Y.L.FS....K......I...T.S..D.............K...E..T..............R...H....R.P.......QK.....L.... SAMD9L_Crgr ....S..Y.L.FN...TK.....EL...G.P..D.............K.... ..T..............R...H.....KP.......QK...H.L.... SAMD9L_Rano ....S..H.L.FS....K......I...T.S..D.............K......S..............R...H....R.P.......QK.....L.... SAMD9L_Capo .V.....F....SI..Q.......L...NY...D..............K.......M....V................ .....F....TK.....L.... SAMD9L_Soar ..N...VF.E..D...EL......LAK.D.S..D.F...........EV.....Q.L.......KK..NR..R...............NG...KGL...T 1510 1520 1530 1540 1550 1560 1570 1580 1590 ....|....|....|....|....|....|....|....|....|....|....|....|.. ..|....|....|....|....|....|... SAMD9L_Hosa KIEQYFDKAQNTNSLWHSGDVWKKNEVKDLLRRLTGQAEGKLISVEYGTEEKIKIPVISVYSGPLRSGRNIERVSFYLGFSIEGPLAYDIEVI SAMD9L_Patr E........................ ...................I................................................ SAMD9L_Gogo ............................................I................................................ SAMD9L_Poab ........................................ ....I................................................ SAMD9L_Nole E...........................................I............T................................K.. SAMD9L_Caja E.....G.VH......Q.................I.........I....K.............................. ..........Q.. SAMD9L_Mamu E.......V...............H...H..C..I.........I................................................ SAMD9L_Loaf E.....S.E.......Q.....................V.....I........R..............G........................ SAMD9L_Eqca ......G.V... ....QN....E.K......C.........R..I....K...............Q...............M........... SAMD9L_Calu E.....N.....S..SQ.A..C.. K.....C............M.....K.V.....P.........G............M..LQ..E..I. SAMD9L_Aime E.....N....V...SP.......K......C............M.....K.......P.........G.........A......Q.....I. SAMD9L_Ereu E....IS.VK....F.Q..H....E...G.....N.R......LM..........................Q.........M........... SAMD9L_Orcu E.....S....I....Q.......K......H..V..... ....I............T..............K...................V SAMD9L_Mumu E..R..SEV.DS..F....V..E.R.......L.D.........L.....A......T....A.........................G.K.. SAMD9L_Crgr E..R.VSEV..S..F.Q..V..E.R.......L.D...............A......T................V..... ........G.K.. SAMD9L_Rano E..R..SEV.DS..F.Q..V..E.G....I..L.D.........L.....A......T..............................G.K.. SAMD9L_Capo EL.KHIS.TK....F.Q...I..T ...H..H............I...IN...T..ITPAFF.Q.....S..K.........G.......QIL SAMD9L_Soar E.....S.VP.. SI..QN......K...S..C............IQ.......N...............H.QS........M.........L.



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Table S2 : SAMD9 and SAMD9L likelihood ratio test (LRT) for PARRIS analysis from HyPhy software *, significant; n.s., not significant Hypothesis LRT Null Hypothesis Alternative Hypothesis df p Value PARRIS SAMD9 M1: no selection M2: selection 0.2 2 n.s. SAMD9L M1: no selection M2: selection 8.4 2 < 0.05*



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RESEARCHARTICLEOpenAccessEvolutionanddivergenceofthemammalian SAMD9 / SAMD9L genefamilyAnaLemosdeMatos1,2,3,JiaLiu3,GrantMcFadden3andPedroJEsteves1,4*AbstractBackground: ThephysiologicalfunctionsofthehumanSterileAlphaMotifDomain-containing9( SAMD9 )geneand itschromosomallyadjacentparalogue, SAMD9 -like( SAMD9L ),currentlyremainunknown.However,thedirectlinks betweenthedeleteriousmutationsordeletionsinthesetwogenesandseveralhumandisorders,suchasinherited inflammatorycalcifiedtumorsandacutemyeloidleukemia,suggesttheirbiologicalimportance.SAMD9and SAMD9Lhavealsorecentlybeenshowntoplaykeyrolesintheinnateimmuneresponsestostimulisuchasviral infection.Wewereparticularlyinterestedinunderstandingthemammalianevolutionaryhistoryofthesetwogenes. Thephylogenyof SAMD9 and SAMD9L geneswasreconstructedusingtheMaximumLikelihoodmethod. Furthermore,sixdifferentmethodswereappliedtodetectSAMD9andSAMD9Lcodonsunderselectivepressure:the site-specificmodelM8implementedinthecodemlprograminPAMLsoftwareandfivemethodsavailableonthe Datamonkeywebserver,includingtheSingleLikelihoodAncestorCountingmethod,theFixedEffectLikelihood method,theRandomEffectLikelihoodmethod,theMixedEffectsModelofEvolutionmethodandtheFastUnbiased BayesianAppRoximationmethod.Additionally,thehousemouse( Musmusculus )genomehaslostthe SAMD9 gene whilekeeping SAMD9L intact,promptingustoinvestigatewhetherthislossisauniqueeventduringevolution. Results: Ourevolutionaryanalysessuggestthat SAMD9 and SAMD9L arosethroughanancestralgeneduplication eventafterthedivergenceofMarsupialiafromPlacentalia.Additionally,selectionanalysesdemonstratedthatboth geneshavebeensubjectedtopositiveevolutionaryselection.Theabsenceofeither SAMD9 or SAMD9L genesfrom somemammalianspeciessupportsapartialfunctionalredundancybetweenthetwogenes. Conclusions: Tothebestofourknowledge,thisworkisthefirststudyontheevolutionaryhistoryofmammalian SAMD9 and SAMD9L genes.Weconcludethatevolutionaryselectivepressurehasactedonbothofthesetwogenes sincetheirdivergence,suggestingtheirimportanceinmultiplecellularprocesses,suchastheimmuneresponsesto viralpathogens. Keywords: SAMD9,SAMD9-like,Mammals,Evolutionaryhistory,PositiveselectionBackgroundTheSterileAlphaMotifDomain-containing9( SAMD9 ) geneislocatedinchromosome7q21.2ofthehumangenome,andisadjacenttoitscloseparalogue, SAMD9 -like ( SAMD9L ),inahead-to-tailposition[1,2]andseparated byapproximately12kb.Thephysiologicalfunctionsof both SAMD9 and SAMD9L currentlyremainpoorly understood,buttheimportanceofhuman SAMD9 has beenrecentlyemphasizedduringthediscoveryofthe geneticcauseofararelife-threateninghumandisease, normophosphatemicfamiliartumoralcalcinosis(NFTC) [3,4].PatientswithNFTCexhibitednormalcalciumand phosphatemetabolismwhiledevelopingcalcifiedtumorous nodulesattheirextremities,accompaniedbyseveregingivitis.Twoindependentfoundergeneticeventsleadingto thedeleteriousmutationsin SAMD9 areresponsiblefor theautosomalrecessivediseaseofNFTC[3,4].Interestingly,thesepatientsandtheirkindredarefromaculturally isolatedethicalgroup,namelyJewish-Yemenite,suggesting apotentialselectionpressureassociatedwiththispopulation[3,4].InadditiontoNFTC,misregulatedhuman *Correspondence: pjesteves@cibio.up.pt1CIBIO-CentrodeInvestigaoemBiodiversidadeeRecursosGenticos/ InBioLaboratrioAssociado,UniversidadedoPorto,4485-661Vairo, Portugal4CentrodeInvestigaoemTecnologiasdaSade,IPSN,CESPU,4585-116 Gandra,Portugal Fulllistofauthorinformationisavailableattheendofthearticle 2013LemosdeMatosetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121 http://www.biomedcentral.com/1471-2148/13/121

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SAMD9 expressionwasalsoshowntobeassociatedwith aggressivefibromatosis,breast,andcoloncancers[1]. Human SAMD9 expressioncanbeupregulatedbytumor necrosisfactor( TNF )[4]orbytypeI[5]andtypeIIinterferons( IFN s)[6],anditisclassifiedasaninterferonstimulatedgene(ISG).Recently,aninterferonregulatory factor( IRF-1 )bindingelementwasidentifiedinthepromoterregionofthe SAMD9 geneinhumans[6],and overexpressionof IRF-1 canleadtoelevated SAMD9 gene expression[7].Alltheseobservationssuggestakeyroleof SAMD9 asasignallinghubinresponsetoinnateimmune stimulations.Mostimportantly,human SAMD9 alsohas veryrecentlybeenshowntopossessanti-viralpropertiesin culturedcells[8,9]emphasizingitscrucialroleinhost defenceagainstviralpathogens. Ontheotherhand,thehuman SAMD9L genewas showntoexhibitlowerexpressionlevelsinbreast cancertissuethaninnormalbreasttissuefromthe samepatient[1].Itwasalso identifiedtobeaninduciblegenefortypeI IFN s( IFN and ),andinactivated humanTcellsthefunctionof SAMD9L iscorrelated withits IFN -inducedinhibitoryeffectsoncellmigration [10].Themurine SAMD9L geneexpressionwasalsofound tobeupregulatedbycalcitonin[11],suggestingapotential involvementincalciumhomeostasisaswell. Lastly,thehuman SAMD9 and SAMD9L geneswere bothclassifiedasmyeloidtumorsuppressors,asthey arelocalizedwithinamicrodeletionclusterassociated withmyeloiddisorders,suc hasjuvenilemyelomonocytic leukemia(JMML),acutemyeloidleukemia(AML),and myelodysplasticsyndrome(MDS)[2].Inanotherstudyinvestigatingalteredimmuneresponsesinpatientswith metastaticmelanoma,both SAMD9 and SAMD9L expressionwereshowntobesignificantlyreducedinTandBcell populationswhencomparedwiththosefromhealthycontrolindividuals[12].Ithasbeensuggestedthatsincethese twoproteinsexhibitconsiderablesequencesimilarity,they mayfunctionredundantlyorinrelatedpathways,butit shouldbenotedthatpatientswithNFTCpossessmutationsonlyin SAMD9 andthusitislikelythatthetwoproteinsperformnon-identicaltasksinhumans. Evolutionarily,theorthologousgenesforboth SAMD9 and SAMD9L arehighlyconservedinmanymammalian genomes,suchasrat,primatesandrabbit,butnotin chicken,frogandfishspecies,orinsects[1].Thissuggeststhattheoriginofthesetworelatedgenes,possibly fromanancestralduplicationevent,occurredatsome pointafterbranchingofthemammalianspecies.In addition,oneintriguingfactisthatthehousemouse genome( Musmusculus ,Mumu)haslostthe SAMD9 genewhilemaintaining SAMD9L, afteranevolutionary chromosomebreakageevent[1]. Theabsenceof SAMD9 fromthehousemouse(Mumu) genomeledustoquestionifitwasauniqueevent restrictedtothistaxona ndstimulatedthestudyofSAMD9 and SAMD9L evolutionanddivergenceindifferentmammaliangenomes.Wehaveexaminedtheevolutionaryhistoryandphylogenyof SAMD9 and SAMD9L, usingalltheavailableandc ompletemammaliangenomic sequencesofbothgenesinNCBIandEnsembldatabases, inordertoobtainabroaderunderstandingoftheoriginof thesetwogenes.Ourdeducedphylogenetictreesuggests that SAMD9 and SAMD9L indeedresultedfromanancestralgeneduplicationeventthatoccurredafterthedivergenceofMarsupialiafromPlacentalia.Atthesame time,weappliedsixdifferentMaximumLikelihood(ML) methodstotestforpotentialpositiveselectivepressures exertedatthegenelevel,andwealsolookedforevidence ofpositiveselectionatthededucedproteinlevel.The analysesrevealedthatSAMD9andSAMD9L,atboththe genomeanddeducedproteinsequencelevels,wereunder theeffectsofwhatappearstobesustainedpositiveselectivepressures.Ourresultssuggestthatthesetwoproteins havebeenselectedbylongtermenvironmentalpressures, suchasthoseexertedbypathogenresponsesthatare underthecontrolofinnateimmuneregulatorslikethe typeIinterferons.ResultsSAMD9 and SAMD9L genesprevalenceinmammalsAlltheavailableandcompletemammalian SAMD9 and SAMD9L genescodingsequencesintheNCBIand Ensembldatabaseswerecollected,resultinginatotal offifteen SAMD9 andnineteen SAMD9L genomicsequencesofdifferentspeciesindicatedinTable1.Thespeciescollectedfor SAMD9 genesfitintosevenEutheria orders,commonlydesignatedasplacentalmammals,while thetaxacollectedfor SAMD9L genesfitintoeightplacentalorders.Thegreyshort-tailedopossum,arepresentative oftheorderDidelphimorphiatraditionallyincludedin Marsupialia(pouchmammals),wastheonlymarsupial genometopossessacomplete SAMD9L sequence. Besidesthecomplete SAMD9 and SAMD9L coding sequences,severalothernon-complete SAMD9 and SAMD9L mammaliangenes,includingfulllengthmRNAderivedtranscriptswithmanystill-undeterminednucleotides(forexample,thelargeflyingfoxorthewest Europeanhedgehog SAMD9 codingsequences,orthe Americanpika SAMD9L sequence)orpartialgenesequences(forexample,theOrd ’ skangaroorat SAMD9L or theHoffmann ’ stwo-toedsloth SAMD9 genes),havebeen alreadyidentifiedandannotatedinEnsembldatabase. However,theseincompletesequenceswerenotusedin thephylogeneticandselectionanalysesperformedinthis study.Boththecompleteandthenon-complete SAMD9 and SAMD9L genesannotatedinEnsemblarerepresented inFigure1,allowingabroaderviewintothisgenefamily distributionwithinthemammaliancontext.LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page2of16 http://www.biomedcentral.com/1471-2148/13/121

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Specialreferencehastobemadetotwoparticular completesequencesthatwereincludedinourevolutionaryanalyses:thenorthernwhite-cheekedgibbon (Nole) SAMD9 andthedomesticdog(Calu) SAMD9L Thenorthernwhite-cheekedgibbonhasno SAMD9 genecurrentlyannotatedinEnsembl.However,bycomparing SAMD9 sequencesofotherprimatestothegibbon genomeinEnsemblusingBLASTanalysis,weobtaineda perfectmatchwithaneighboringdesignatedpseudogene of SAMD9L .Despitethisbiotypeclassification,wecould Table1Mammalian SAMD9 and SAMD9L genesaccessionnumbersfromspeciesusedinphylogeneticandselection analysesSAMD9 MammalianorderCommonnameSpeci esnameDatabaseIDAbbreviation ArtiodactylaCow Bostaurus Chromosome4:10,302,667-10,307,412aSAMD9_Bota Pig Susscrofa Chromosome9:79,679,836-79,684,587aSAMD9_Susc ChiropteraLittlebrownmyotis Myotislucifugus ScaffoldAAPE02063303:7,766-12,520aSAMD9_Mylu LagomorphaEuropeanrabbit Oryctolaguscuniculus Chromosome10:35,728,133-35,732,926aSAMD9_Orcu PerissodactylaHorse Equuscaballus Chromosome4:36,749,161-36,753,927aSAMD9_Eqca PrimatesCommonchimpanzee Pantroglodytes Chromosome7:92,731,148-92,735,917aSAMD9_Patr Human Homosapiens Chromosome7:92,728,829-92,747,336aSAMD9_Hosa Northernwhite-cheekedgibbon Nomascusleucogenys SuperContigGL397261.1:24,263,901-24,268,665aSAMD9_Nole Rhesusmonkey Macacamulatta Chromosome3:124,130,532-124,147,894aSAMD9_Mamu Sumatranorangutan Pongoabelii Chromosome7:83,034,053-83,038,819aSAMD9_Poab Westerngorilla Gorillagorilla Chromosome7:90,353,240-90,358,009aSAMD9_Gogo RodentiaBrownrat Rattusnorvegicus XM_575365.2bSAMD9_Rano Chinesehamster Cricetulusgriseus AFTD01024384.1bSAMD9_Crgr DomesticGuineapig Caviaporcellus AAKN02016823.1bSAMD9_Capo SoricomorphaCommonshrew Sorexaraneus Scaffold_257382:52,686-57,449aSAMD9_Soar SAMD9L MammalianorderCommonnameSpeci esnameDatabaseIDAbbreviation CarnivoraDomesticdog Canislupusfamiliaris XM_539422.3bSAMD9L_Calu Giantpanda Ailuropodamelanoleuca ScaffoldGL192585.1:1,477,672-1,482,429aSAMD9L_Aime Didelphimorphia (Marsupialia) Greyshort-tailedopossum Monodelphisdomestica XM_001378475.1bSAMD9L_Modo ErinaceomorphaWestEuropeanhedgehog Erinaceuseuropaeus GeneScaffold_8766:48,007-52,945aSAMD9L_Ereu LagomorphaEuropeanrabbit Oryctolaguscuniculus Chromosome10:35,699,236-35,703,990aSAMD9L_Orcu PerissodactylaHorse Equuscaballus Chromosome4:36,788,011-36,792,765aSAMD9L_Eqca PrimatesCommonchimpanzee Pantroglodytes Chromosome7:92,759,911-92,778,202aSAMD9L_Patr Commonmarmoset Callithrixjacchus Chromosome8:54,405,622-54,420,907aSAMD9L_Caja Human Homosapiens Chromosome7:92,759,368-92,777,682aSAMD9L_Hosa Northernwhite-cheekedgibbon Nomascusleucogenys SuperContigGL397261.1:24,263,209-24,320,238aSAMD9L_Nole Rhesusmonkey Macacamulatta Chromosome3:124,099,607-124,117,554aSAMD9L_Mamu Sumatranorangutan Pongoabelii Chromosome7:83,003,315-83,008,287aSAMD9L_Poab Westerngorilla Gorillagorilla Chromosome7:90,382,062-90,397,829aSAMD9L_Gogo ProboscideaAfric anbushelephant Loxodontaafricana XM_003407146.1bSAMD9L_Loaf RodentiaBrownrat Rattusnorvegicus Chromosome4:28,180,812-28,185,536aSAMD9L_Rano Chinesehamster Cricetulusgriseus XM_003496952.1bSAMD9L_Crgr DomesticGuineapig Caviaporcellus scaffold_11:24,689,192-24,742,963aSAMD9L_Capo Housemouse Musmusculus Chromosome6:3,322,257-3,349,571aSAMD9L_Mumu SoricomorphaCommonshrew Sorexaraneus scaffold194773:6,206-10,964aSAMD9L_SoarDatabaseID:aEnsembl;bNCBIGenBank.LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page3of16 http://www.biomedcentral.com/1471-2148/13/121

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notexcludethisSAMD9sequencefrombeingconsidered asa bonafide gibbon SAMD9 gene.Regardingthedomesticdog SAMD9L ,thisgeneispresentinNCBIandisannotatedinEnsembl,butinthislatterdatabasethesequence wasmissingseventy-fournucleotideswhencomparedto thesequenceinNCBI.Thus,forthesubsequentanalyses weusedonlythesequencefromNCBI.Itshouldalsobe notedthat,despitenotbeingannotatedinEnsembl,an incomplete SAMD9 sequenceforthedomesticdogis availableinNCBI.However,whentheNCBIsequence (XM_003639470.1)wasanalyzedbyBLAST,itpossessed 99to100%identitywithanon-annotatedregionof chromosome14.Sinceitisano n-completenucleotidesequence,itwasnotusedfurtherforthestudyreportedhere. When SAMD9 and SAMD9L weremappedinhuman chromosome7,orthologouscounterpartsofbothgenes wereidentifiedinthechimpanzee(Patr),dog(Calu)and rat(Rano),butinthehousemouse(Mumu)genomethere wasonlyasinglegeneticcorrespondencetothe SAMD9L openreadingframeinchromosome6[1].Fromwhatis currentlyavailableinEnsembldatabase,theabsenceof SAMD9 forthehousemouse(Mumu)isconfirmed.We checkedtheotheravailablerodentstoconfirmthepresenceorabsenceof SAMD9 inthisspecificlineage.In Ensemblthereisasingle SAMD9 annotationforthe thirteen-linedgroundsquirrel(Ictr).Inaddition,whatappeartobeintact SAMD9 geneshavebeendepositedin NCBIdatabaseforthebrownrat(Rano),theChinesehamster(Crgr)andthedomesticGuineapig(Capo).Onthe otherhand,likethehousemouse(Mumu),theOrd ’ s kangaroorat(Dior)doesnothave SAMD9 geneannotated inEnsembldatabase. ORDER SAMD9 SAMD9L Didelphimorphia Modo Chromosome8 < CDK6 < SAMD9L Afrosoricida Ecte GeneScaffold_7273 Artiodactyla Bota Chromosome4 < CDK6 < SAMD9 < HEPACAM2 CCDC132 > < CALCR Susc Chromosome 9 < CDK6 < SAMD9 < HEPACAM2 CCDC132 > < CALCR VipaGeneScaffold_1246 < CDK6 < SAMD9 < HEPACAM2 CCDC132 > < CALCR Carnivora Aime Scaffold GL192585.1 CALCR > < CCDC132 HEPACAM2 > SAMD9L > CDK6 > Calu Chromosome14 < CDK6 < SAMD9L < HEPACAM2 CCDC132 > < CALCR Feca GeneScaffold_4062 Cingulata Dano GeneScaffold_7428 Chiroptera Mylu Scaffold AAPE02063303Scaffold GL429782 < CDK6 < SAMD9L < HEPACAM2 CCDC132 > < CALCR PtvaGeneScaffold_3953GeneScaffold_3953 Erinaceomorpha EreuGeneScaffold_8766 GeneScaffold_8766 Hyracoidea Prca GeneScaffold_6298 Lagomorpha Ocpr GeneScaffold_4408 Orcu Chromosome10 Chromosome10 CALCR > < CCDC132 HEPACAM2 > SAMD9L > SAMD9 > CDK6 > Perissodactyla Eqca Chromosome4 Chromosome4 < CDK6 < SAMD9 < SAMD9L < HEPACAM2 CCDC132 > < CALCR Pilosa ChhoScaffold_109991Scaffold_65211 Primates Caja Chromosome8 CALCR > < CCDC132 HEPACAM2 > SAMD9L > CDK6 > Gogo Chromosome7 Chromosome7 < CDK6 < SAMD9 < SAMD9L < HEPACAM2 CCDC132 > < CALCR Hosa Chromosome7 Chromosome7 < CDK6 < SAMD9 < SAMD9L < HEPACAM2 CCDC132 > < CALCR Mamu Chromosome3 Chromosome3 CALCR > < CCDC132 HEPACAM2 > SAMD9L > SAMD9 > CDK6 > Nole SuperContig GL397261.1 SuperContig GL397261.1 < CDK6 < SAMD9 < SAMD9L < HEPACAM2 CCDC132 > < CALCR OtgaScaffold GL873524.1 < CDK6 < SAMD9 < HEPACAM2 CCDC132 > < CALCR Patr Chromosome7 Chromosome7 < CDK6 < SAMD9 < SAMD9L < HEPACAM2 CCDC132 > < CALCR Poab Chromosome7 Chromosome7 CALCR > < CCDC132 HEPACAM2 > SAMD9L > SAMD9 > CDK6 > TasyScaffold_62435 Proboscidea Loaf SuperContig scaffold_5 CALCR > < CCDC132 HEPACAM2 > SAMD9L > CDK6 > Rodentia Capo Scaffold_11 < CDK6 < SAMD9L < HEPACAM2 CCDC132 > < CALCR Dior GeneScaffold_5660 < SAMD9L < HEPACAM2 CCDC132 > IctrScaffold JH393613.1Scaffold JH393613.1 CALCR > < CCDC132 HEPACAM2 > SAMD9L > SAMD9 > CDK6 > Mumu Chromosome6 < SAMD9L < HEPACAM2 CCDC132 > < CALCR Rano Chromosome4 < CDK6 < SAMD9L < HEPACAM2 CCDC132 > < CALCR Soricomorpha Soar Scaffold_257382 Scaffold_194773 Forward strand Figure1 Ensemblannotationofmammalian SAMD9 and SAMD9L genes,andneighboringgenes. Ensemblannotationoftheavailable mammalian SAMD9 and SAMD9L genes,bothcompleteandincompletecodingregionsequences,arerepresented.Completesequencesforboth genesarehighlighted(darkyellowfor SAMD9 andlightyellowfor SAMD9L ).Other SAMD9 and SAMD9L genesarealreadyannotatedinEnsembl, butatthetimeoftheseanalyseswerestillincomplete,correspondingtothenon-highlightedlocationsinthefigureandwereexcludedfrom posterioranalyses.Ensemblspeciesbyorderofappearance:Modo-Opossum;Ecte-Lesserhedgehogtenrec;Bota-Cow;Susc-Pig;Vipa-Alpaca; Aime-Panda;Calu-Dog;Feca-Cat;Dano-Armadillo;Mylu-Microbat;Ptva-Megabat;Ereu-Hedgehog;Prca-Hyrax;Ocpr-Pika;Orcu-Rabbit; Eqca-Horse;Chho-Sloth;Caja-Marmoset;Gogo-Gorilla ; Hosa-Human;Mamu-Macaque;Nole-Gibbon;Otga-Bushbaby;Patr-Chimpanzee; Poab-Orangutan;Tasy-Tarsier;Loaf-Elephant;Capo-Guineapig;Dior-Kangaroorat;Ictr-Squirrel;Mumu-Mouse;Rano-Rat;Soar-Shrew.To accessthecompletespeciesname,thelistofabbreviationsshouldbeconsulted.Basedonhumanchromosome7mapping, SAMD9 and SAMD9L neighboringgeneswereidentifiedandrepresented( CDK6 HEPACAM2 CCDC132 and CALCR ).UsingEnsembldatabase,thesamesearchwas performedfortheremainingspeciesandtheidentifiedgenesarerepresentedundertheforwardstrainarrow.Therepresentedgenesare,inmost cases,theimmediateneighboringgenes,whileforareducednumberofspeciessomeothergenesarelocatedinthesameregion,butwere excludedforthispurpose. “ < ” symbol:genelocatedonthereversestrand; “ > ” symbol:genelocatedontheforwardstrand. LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page4of16 http://www.biomedcentral.com/1471-2148/13/121

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Completemammalian SAMD9 and SAMD9L genesequences: recombinationandphylogeneticanalysesThecompletenucleotidecodingsequencesfrom SAMD9 and SAMD9L werealignedtogether( SAMD9 + SAMD9L ) andtranslatedintodeducedproteinsequences(Additional file1:FigureS1).Beforefurtherphylogeneticanalyses,we usedthesoftwareGARD[13,14]tolookforanyevidence ofrecombinationinthealignment.Threebreakpointswere identified,butonlyonewasstronglysupportedbythe Kishino-Hasegawa(KH)test(Additionalfile2:TableS1), whichshouldresultintheestimationofaphylogenetic treeforeachsegment.However,sincethebreakpoint waslocatedonnucleotide4755,thegenomicsegment totherightofthebreakpointwasonlycomposedof 150nucleotides. AMaximumLikelihood(ML)treewasestimatedfor thesmallestgeneticsegment(notshown),butthenodes wereweaklysupportedbylowbootstrapvalues.Therefore,onlythelargesegmentwith4755nucleotideswas usedtoreconstructaMLphylogenetictreeunderthe GTR+I+Gnucleotidesubstitutionmodel.Theresulting treeisrepresentedinFigure2.AnotherMLphylogenetic treewasestimated,butwithouttestingrecombination, tocomparedifferencesinthetreetopologies.Themodel Figure2 Mammalian SAMD9 and SAMD9L genesestimatedMaximumLikelihoodtree. Forthemammalian SAMD9 and SAMD9L genes alignmentandafterGARDanalysis[13,14],asignificantrecombinationbreakpointwasdetected(nucleotideposition4755)definingaleftanda rightsegment.AphylogenetictreewasestimatedforeachsegmentusingtheMaximumLikelihood(ML)method.However,theresultingtree fromtherightsegmentpresentedweaklysupportednodesandwasdiscarded.Ontheotherhand,theleftsegmentwith4755nucleotideswas usedtoreconstructaMLphylogenetictreeundertheGTR+I+Gnucleotidesubstitutionmodel.Theanalyseswereperformedwith1,000,000 generationsand1,000bootstrapsearches.Thebootstrapvaluesareindicatedonthebranches.Theabbreviationscorrespondtothefollowing speciescommonnames:Aime-Giantpanda;Bota-Cow;Caja-Commonmarmoset;Calu-Domesticdog;Capo-DomesticGuineapig; Crgr-Chinesehamster;Eqca-Horse;Ereu-WestEuropeanhedgehog;Gogo-Westerngorilla;Hosa-Human;Loaf-Africanbushelephant; Mamu-Rhesusmonkey;Modo-Greyshor t-tailedopossum;Mumu-Housemouse;Mylu-Littlebro wnmyotis;Nole-Northernwhite-cheekedgibbon; Orcu-Europeanrabbit;Patr-Commo nchimpanzee;Poab-Sumatranorangutan;Rano-Brow nrat;Soar-Commonshrew;Susc-Pig.Toaccessthe speciesscientificnames,thelistof abbreviationsshou ldbeconsulted. LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page5of16 http://www.biomedcentral.com/1471-2148/13/121

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usedwasagaintheGTR+I+Gandresultedinatree (Additionalfile3:FigureS2)withasimilaroveralltopologytothegenesegmentcontaining4755nucleotides. IntheestimatedMLphylogenetictree(Figure2), SAMD9 and SAMD9L formedtwowelldefinedmonophyleticgroups,andwithineachcladeweobservedaconcordanttopologywiththeacceptedevolutionaryrelationships ofeutherianmammals[15](Additionalfile4:FigureS3). Interestingly,themarsupialgreyshort-tailedopossum (Modo) SAMD9L representedahighlydivergentoutgroup, evenfromtheremaining SAMD9L species.Ageneduplicationeventafterthesplitofmarsupialand placentalmammalsoriginated SAMD9 / SAMD9L genefamilyIthasbeenpreviouslysuggestedthat SAMD9 andits paralogous SAMD9L mayhaveoriginatedfromacommonancestorbyageneduplicationevent[1].Inour study,theMLtree(Figure2)topologysupportsthis view.However,theopossum(Modo)geneannotatedas SAMD9L inNCBIdatabase(XM_001378475.1)does notclusterintheplacentalmammal SAMD9L group. Infact,theopossumsequencecanberecognizedasbeinginabasalposition.Twohighlysupportedeutherian monophyleticcladesintheMLtree,onecorresponding toall SAMD9 genesandtheotheronetoall SAMD9L genes,wereobserved.Themostlikelyevolutionaryscenariocanbedescribedasfollowing:anancestralgeneis presentbeforetheseparationofmarsupialfromplacental mammalsinthecommonancestorthatoriginatedtheextant SAMD9L geneinthemarsupialopossum(Modo)and theancestralgeneofplacental SAMD9 / SAMD9L gene family.Later,inplacentalmammals,thisancestralgene sufferedaneventofgeneduplicationresultinginthecontemporary SAMD9 and SAMD9L genes. Theconservationofsimilararrangementofgenesin thesamerelativelocationsonthechromosomesofdifferentspecies,denominatedassharedsynteny,canindicatetheexistenceofacommonancestor.InEnsembl, amongthemammalianspecieswherethepresenceof SAMD9 and/or SAMD9L hasbeenannotated,shared syntenycanbereadilyobservedinchromosomesand ‘ gene-scaffolds ’ .Theconsistentpresenceofthesame commonflankinggenes( CALCR CCDC132 CDK6 and HEPACAM2 )indifferentspeciessupportstheideathat SAMD9 and SAMD9L arelocatedinhighlyconservedregionsthroughoutplacentalmammals ’ divergenceanddiversification(Figure1).Inferenceofpositiveselectionat SAMD9 and SAMD9L geneslevelPlacentalSAMD9andSAMD9Ldeducedproteinsequenceswerealignedindependently(Additionalfile5: FigureS4;Additionalfile6:FigureS5)andMLtrees wereestimatedforeachgene(Additionalfile7:FigureS6; Additionalfile8:FigureS7).Afterwards,wedetermined whetherthe SAMD9 and SAMD9L genesmighthavebeen subjecttopositiveselectionpressuresbycomparingPAML codon-basednestedmodelswithandwithoutpositiveselectionusinglikelihoodratiotests(LRTs)[16,17].Both comparisonsofM1(nearlyneutral)versusM2(positiveselection)andM7(beta)versusM8(betaand >1)resulted intherejectionofthenullhypothesis,stronglysupporting thefindingofpositiveselectionforboth SAMD9 and SAMD9L (<0.001;Table2).WealsousedthePARRIS[18] methodtodetectifaproportionofsitesineachgenealignmentevolvedunderpositiveselectionafteraccountingfor thepotentiallyconfoundingeffectsofrecombinationand synonymoussitevariation.Interestingly,only SAMD9L wasfoundtobeunderselectionwhenusingthismethod (<0.05;Additionalfile9:TableS2). Sixdifferentmethodswereusedtodetectsitesunder selectionfor SAMD9 and SAMD9L (Additionalfile10: TableS3).ForPAMLsoftware,weusedM8modelto detectsitesunderselectionfor SAMD9 and SAMD9L phylogenetictrees,andtheBEBapproachwasusedto identifycodonswithaposteriorprobability>90%.The otherfiveappliedmethodstodetectsitesunderpositive selectionareavailableintheDatamonkeywebserver. Inthisstudy,weonlyconsideredacodonwithevidenceof selectionwhenitwasidentifiedbyatleastthreeofthesix usedmethods[19,20](Additionalfile10:TableS3).SeventeensitesforSAMD9andnineteensitesforSAMD9Lwere identifiedascandidatesforsitesunderpositiveselection (Figure3and4;Additionalfile10:TableS3). Aminoacidsubstitutionscanbeeitherconservativeor radical,dependingonwhethertheyleadtoachangeina certainphysicochemicalproperty[21].Forthecodons identifiedasbeingunderselection,weinvestigatedthe alterationsofchargeandpolaritybetweenmammalian taxa.ForSAMD9allthedetectedcodons(Figure3) exhibitedatleastonephysicochemicalalterationacross speciesandamaximumoffivedifferentcombinationsof propertieswereidentifiedforcodon331.Primatespecies Table2 SAMD9 and SAMD9L likelihoodratiotest(LRT)for foursitemodelsfromPAMLsoftwareHypothesisLRT NullHypothesisAlternativeHypothesis 2 lnLdf pValue SiteModels SAMD9 M1:nearlyneutralM2:positiveselection25.552<0.001*** M7:betaM8:betaand >177.762<0.001*** SAMD9L M1:nearlyneutralM2:positiveselection51.102<0.001*** M7:betaM8:betaand >197.442<0.001******,highlysignificant.LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page6of16 http://www.biomedcentral.com/1471-2148/13/121

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Codons 4888279331352383491513731872993100611161258132013291398AnalysesPAML M8 SLAC FEL REL MEME FUBARSpeciesHosaWMLYKTPLAQNEGPVSSPatrWMLYKTPLAQNEGPVSSGogoWMLYKTPLAQNEGPVSSPoabWMLYKTPLAQNKDPVSSNoleWMLYKTSLAQNKGPISSMamuWMLYKTPSAQNKGQTSSBotaYSLVPR-LEQNIDAALISuscYRMQPK-LTNNTNPASSEqcaWKLDSTSIENKDSSVSPMyluFKLHKAPLRQKITPALQOrcuFQEHSTTSAHMS-SLILRanoWKLEPAVLKQSTELVLQCrgrWRLESELSKQSSKLVSQCapoWASHSSTSEKTINLISLSoarLKQHSTTLDQCLESGTT Figure3 Positively-selectedSAMD9codonsandrespectivephysicochemicalpropertiesforeachmammalianspecies. SAMD9sitesunder positiveselectionidentifiedbyatleastthreeofthesixusedMaximumLikelihoodmethods.CodonsarenumberedaccordingtotheSAMD9 deducedproteinsalignment(Additionalfile5:FigureS4).Theabbreviationscorrespondtothefollowingspeciescommonnames:Hosa-Human; Patr-Commonchimpanzee;Gogo-Westerngorilla;Poab-Sumatranorangutan;Nole-Northernwhite-cheekedgibbon;Mamu-Rhesus monkey;Bota-Cow;Susc-Pig;Eqca-Horse;Mylu-Littlebrownmyotis;Orcu-Europeanrabbit;Rano-Brownrat;Crgr-Chinesehamster; Capo-DomesticGuineapig;Soar-Commonshrew.Toaccessthespeciesscientificnames,thelistofabbreviationsshouldbeconsulted. Thebackgroundcolorsrepresentaminoacidproperties:polarpositive(yellow),polarnegative(orange),polarneutral(green),non-polar neutral(purple),non-polaraliphatic(blue)andnon-polararomatic(pink). Codons 39156260267340357362452586606653776978118612291276130814291474AnalysesPAML M8 SLAC FEL REL MEME FUBARSpeciesHosaSLAVLSVMHTVAAPKITSRPatrSLAVLSVMHTVAAPKITSRGogoSLAVLSVMHTVAAPKITSRPoabSLSVLSVMHTVAAPKITSRNoleSLAVTSVMHTVAAPKITSRCajaNLVLSPVQHTAVSPEITSHMamuSLALSSVVHTVAAPIITSRLoafNVDLYVAVQTLTALQVTTHEqcaNLSVHGTQQAIATPKAMRNCaluRLAILVLVHTIETKKDVSSAimeTTAVLVIVQTTGAPKNGSSEreuCRALSGKEQTFAATQAINLOrcuNIVVYAPGQSTASALIRHRMumuKPITTPRHAAIVSPKGVTHCrgrNAVKNARQHATLLPILAGHRanoEPIVTPRHSAIISPKLATHCapoNLALLVIRKSLEAPLSTNRSoarNLSKTEAVDETAKQEAIDR Figure4 Positively-selectedSAMD9Lcodonsandrespectivephysicochemicalpropertiesforeachmammalianspecies. SAMD9Lsites underpositiveselectionidentifiedbyatleas tthreeofthesixusedMaximumLikelihoodmethod s.CodonsarenumberedaccordingtotheSAMD9L deducedproteinsalignment(Additionalfile6:FigureS5).Theabbrev iationscorrespondtothefollowingspeciescommonnames:Hosa-Human; Patr-Commonchimpanzee;Gogo-Westerngorilla;Poab-Sumatra norangutan;Nole-Northernwhite-cheekedgibbon;Caja-Common marmoset;Mamu-Rhesusmonkey;Loaf-Africa nbushelephant;Eqca-Horse;Calu-Domesticdog;Aime-Giantpanda;Ereu-WestEuropean hedgehog;Orcu-Europeanrabbit;Mumu-Housemouse;Crgr-Chinese hamster;Rano-Brownrat;Capo-D omesticGuineapig;Soar-Common shrew.Toaccessthespeciesscientificnames,thelistofabbreviationss houldbeconsulted.Thebackgroundcol orsrepresentaminoacidproperties :polar positive(yellow),polarnegative(orange),pol arneutral(green),non-polarneutral(purple), non-polaraliphatic(blue)andnon-polararomati c(pink). LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page7of16 http://www.biomedcentral.com/1471-2148/13/121

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SAMD9aminoacidchangeswerequiteconservative, sinceelevencodonsexhibitedthesameaminoacid.DespitethelownumberofspeciesavailableforArtiodactyla andRodentia,weverifiedineachorderagreatnumberof aminoacidphysicochemicalalterations per codoninthe SAMD9genes.Inaddition,allSAMD9Lcodonsunder presumptiveselection(Figure4)exhibitedphysicochemical alterationsacrosstaxaandatleastthreepropertieswere representedineachcodon.Amaximumoffivedifferent physicochemicalproperties wereidentifiedforcodon position452.InPrimates,aminoacidsubstitutionsin SAMD9Lwereonceagainquiteconservative,giventhat thirteenpositionskeptthesamephysicochemicalpropertiesevenwhenaminoacidsubstitutionshappened.Onthe contrary,amongthefourRodentiaspecies,onlythreepositionsinSAMD9Lpresentedthesamephysicochemical properties,butjustonewasinfactthesameaminoacid. Todetectwhethersomesitesalongparticular SAMD9 and SAMD9L lineageswereunderpositiveselection,we employedbranch-siteModelA(Table3).Onthe SAMD9 phylogenetictreeweidentifiedsixbranches(foreground branches)with ratiogreaterthan1,butonlythecommonshrew(Soar)branchhadastatisticalsignificantLRT (<0.01). SAMD9L branch-siteanalysisrevealedatotal oftwelvebrancheswith ratiogreaterthan1,yetonly fourofthosebranchespresentedastatisticalsignificant LRT.BoththeSumatranorangutan(Poab)andthedomesticGuineapig(Capo)brancheshadstatisticalsignificance<0.05,whilethewestEuropeanhedgehog(Ereu) andthecommonshrew(Soar)brancheshadstatisticalsignificance<0.01.InferenceofpositiveselectionatSAMD9andSAMD9L deducedproteinslevelTheevaluationofdestabilizingradicalchangesthatmay occurinspecificregionsofproteinsshouldcomplement theinformationobtainedfrompositiveselectionanalysesatthegenelevel.UsingTreeSAAPsoftware,itis possibletoestimate,fromaphylogenetictree,theamino acidpropertiesunderselectionfromthethirty-oneavailableinthesoftware[22](seeMethodssectionforfull listofthethirty-oneproperties). Table3 SAMD9 and SAMD9L parameterestimatesandlikelihoodratiotest(LRT)forbranch-sitemodelA(PAML)Branch-siteModelALRT Foreground branchesaParameterestimates 2 lnLbdfcp -ValuePositivelyselected sitesdSAMD9 Gogo p0=0.693 p1=0.291 p2a=0.011 p2b=0.005 0=0.096 1=1.000 2= 6.192 0.201n.s.none Poab p0=0.701 p1=0.293 p2a=0.004 p2b=0.002 0=0.096 1=1.000 2= 21.339 3.651n.s.none Nole p0=0.692 p1=0.291 p2a=0.012 p2b=0.005 0=0.095 1=1.000 2= 2.555 0.121n.s.none Orcu p0=0.696 p1=0.290 p2a=0.010 p2b=0.004 0=0.094 1=1.000 2= 4.818 3.301n.s.none Capo p0=0.702 p1=0.292 p2a=0.005 p2b=0.002 0=0.096 1=1.000 2= 5.728 1.251n.s.none Soar p0=0.696 p1=0.286 p2a=0.013 p2b=0.005 0=0.094 1=1.000 2= 8.1659.56 1 <0.01 288,572 SAMD9L Poab p0=0.729 p1=0.270 p2a=0.001 p2b=0.000 0=0.139 1=1.000 2= 409.279 6.59 1 <0.05 888 Caja p0=0.714 p1=0.263 p2a=0.016 p2b=0.006 0=0.138 1=1.000 2= 3.169 1.121n.s.none Mamu p0=0.727 p1=0.268 p2a=0.004 p2b=0.001 0=0.140 1=1.000 2= 11.372 1.221n.s.none Loaf p0=0.717 p1=0.262 p2a=0.015 p2b=0.006 0=0.139 1=1.000 2= 2.244 0.801n.s.none Calu p0=0.730 p1=0.269 p2a=0.013 p2b=0.000 0=0.140 1=1.000 2= 20.273 0.521n.s.none Aime p0=0.728 p1=0.269 p2a=0.003 p2b=0.001 0=0.139 1=1.000 2= 16.318 2.511n.s.none Ereu p0=0.725 p1=0.266 p2a=0.006 p2b=0.002 0=0.139 1=1.000 2= 998.998 7.45 1 <0.01 none Mumu p0=0.716 p1=0.264 p2a=0.015 p2b=0.005 0=0.138 1=1.000 2= 3.755 0.341n.s.none Crgr p0=0.728 p1=0.268 p2a=0.003 p2b=0.001 0=0.139 1=1.000 2= 38.672 2.621n.s.none Rano p0=0.727 p1=0.268 p2a=0.004 p2b=0.001 0=0.139 1=1.000 2= 11.843 2.141n.s.none Capo p0=0.722 p1=0.261 p2a=0.012 p2b=0.004 0=0.139 1=1.000 2= 6.9846.36 1 <0.05 861 Soar p0=0.717 p1=0.264 p2a=0.014 p2b=0.005 0=0.137 1=1.000 2= 7.75910.49 1 <0.01 84,1338,1346aSpeciesnamesontheforegroundbranchesbyorderofappearance:Gogo-Westerngorilla;Poab-Sumatranorangutan;Nole-Northernwhite-cheekedgib bon; Orcu-Europeanrabbit;Capo-DomesticGuineapig;Soar-Commonshrew;Caja-Commonmarmoset;Mamu-Rhesusmonkey;Loaf-Africanbushelephant; Calu-Domesticdog;Aime-Giantpanda;Ereu-WestEuropeanhedgehog;Mumu-Housemouse;Crgr-Chinesehamster;Rano-Brownrat.b-2 lnL:likelihoodratiotest(LRT)todetectpositiveselection.cdf:degreesoffreedom.dPositivelyselectedsites:posteriorprobabilities>90%intheBEB(BayesEmpiricalBayes)analyses.LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page8of16 http://www.biomedcentral.com/1471-2148/13/121

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ForbothSAMD9andSAMD9Lphylogenetictrees, thetwoaminoacidpropertieswiththemostradical value(category8)denotingpositivedestabilizingselection weretheisoelectricpoint(pI )andtheequilibriumconstant (ionizationofCOOH)(Additionalfile11:TableS4).When comparingthepIvaluesamongspeciesforeachprotein, weobservedahighvariabilityacrossthem,especiallyfor SAMD9Ltaxa(Figure5).ForSAMD9proteins,boththe cow(Bota)andthedomesticGuineapig(Capo)exhibited thelowestpI(7.60),whileapIof8.11forthenorthern white-cheekedgibbonwasthehighestobservedinSAMD9 proteins.SAMD9Lproteinsfromplacentalmammals exhibitedalargerrangeforthepIvalueswiththegiant panda(Aime)presentingthelowestpI(6.85)andthehorse (Eqca)exhibitingthehighestpI(8.22).Interestingly,the marsupialgreyshort-tailedopossumSAMD9Ldeduced proteinpresentedthelowestpI(6.74)ofall.ThedifferencesinthepI,andespeciallyinSAMD9Lproteins,may causedramaticeffectsonproteinsfolding,sincethose changesarecausedbysignificantdifferencesinthepolarity oftheaminoacidsthatcomposetheproteins.Besidesthe pIandequilibriumconstant,SAMD9presentedtwoother propertiesunderstrongpositivedestabilizingselection, whilefivemorepropertieswereidentifiedasbeingunder positivedestabilizingselec tionfortheSAMD9Lalignment (Additionalfile11:TableS4). RegardingtheSAMD9slidingwindow,thefouramino acidpropertieswithsignificantz-Scorevalues(>3.09) wereevenlydistributedalongtheSAMD9proteinsalignment(Figure6).However,asuperiorconcentrationof higherz-Scorevalueswasobservedintheregionbetween aminoacid660and910,specificallyforthepI.The SAMD9Lslidingwindowshowedadensepatternfor thesevenaminoacidpropertiesunderdestabilizing selection(Figure7).Yet,tworegionsofSAMD9Lproteinsalignmentpresentedanevenlargerdensityof propertiesandthehighestz-Scorevaluesforsomeof thoseproperties:aminoacidrangeof208 – 431andthe rangeof863 – 1430.DiscussionFromapreviousstudy, SAMD9 anditsparalogue SAMD9L havebeenidentifiedinavarietyofspecies,namelyinhuman,chimpanzee,dogandrat.However,inthehouse mouse( Musmusculus ,Mumu)genome, SAMD9 was uniquelylost[1].Thesamestudyindicatedtheabsenceof bothgenesinchicken,frogandallcurrentlysequencedfish species,suggestingthatthe SAMD9 / SAMD9L genes 6,00 6,507,007,50 8,00 8,50 Figure5 MammalianSAMD9andSAMD9Ldeducedproteinsisoelectricpoints(pI). ThegreybarscorrespondtotheSAMD9deduced proteinspI,thebluebarstotheSAMD9LdeducedproteinspIandtheyellowbartotheopossum(Modo)SAMD9LdeducedproteinpI.The abbreviationscorrespondtothefollowingspeciescommonnames :Hosa-Human;Patr-Commonchimpanzee;Gogo-Westerngorilla; Poab-Sumatranorangutan;Nole-Northernwhite-cheekedgibbon;Mamu-Rhesusmonkey;Bota-Cow;Susc-Pig;Eqca-Horse; Mylu-Littlebrownmyotis;Orcu-Europeanrabbit;Rano-Brownrat ;Crgr-Chinesehamster;Capo-DomesticGuineapig;Soar-Common shrew;Caja-Commonmarmoset;Loaf-Afric anbushelephant;Calu-Domesticdog;Aime-Giantpanda;Ereu-WestEuropeanhedgehog; Mumu-Housemouse;Modo-Greyshort-tailedopossum.Toaccessthespeci esscientificnames,thelistofabb reviationsshouldbeconsulted. LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page9of16 http://www.biomedcentral.com/1471-2148/13/121

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originatingeventhadoccurredafterthemammalian radiation.Oneofourgoalswastointensifytheidentificationof SAMD9 and SAMD9L withindifferentmammalian genomesandalsoverifywhetherthelossofmouse SAMD9 wasauniqueeventrestrictedtothistaxon. Despitethegreatnumberofmorphological,molecular andphylogeneticstudiesfortheorderRodentia,controversiesrelatingtothedivergencetimesbetweenitsmajor subordersstillpersist[23].Inarecentstudyonrodent evolution[24]someinternalrodentbrancheshavebeen 34 5 6 7 8 9 10 0100200300400500600700800900100011001200130014001500z -Score valuesAmino acid positions Isoelectric point Equilibrium constant (ionization of COOH) Power to be at the C-terminal Alpha-helical tendencies Figure6 SlidingwindowforSAMD9aminoacidpropertiesunderpositiveselection. SAMD9aminoacidpropertiesunderdestabilizing selectionwithsignificantz-Scorevalues(>3.09). 3 4 5 6 7 8 0100200300400500600700800900100011001200130014001500z-Score valuesAmino acid positions Alpha-helical tendencies Equilibrium constant (ionization of COOH) Isoelectric point Power to be at the C-terminal Power to be at the middle of alpha-helix Solvent accessible reduction ratio Surrounding hydrophobicity Figure7 SlidingwindowforSAMD9Laminoacidpropertiesunderpositiveselection. SAMD9Laminoacidpropertiesunderdestabilizing selectionwithsignificantz-Scorevalues(>3.09). LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page10of16 http://www.biomedcentral.com/1471-2148/13/121

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resolved,wherethreemaingroupsinthephylogenetictree weresupported:theMouse-relatedclade,Ctenohystrica cladeandtheSquirrel-relatedclade.Ascenariohasbeen proposedwherethepre-Squirrel-relatedcladediverged earlyfromthecommonancesto rfollowedbyalaterseparationofthepre-Mouse-relatedandpre-Ctenohystrica clade[24].Wegatheredsequencesforoneorboth SAMD9 and SAMD9L genesforspeciesrepresentativeofthethree clades.Thetwogeneswerepresentinthethirteen-lined groundsquirrel(Squirrel-re latedclade),thedomestic Guineapig(Ctenohystricaclade),theChinesehamsterand thebrownrat(Mouse-relatedclade).Togetherwiththeabsenceof SAMD9 inthehousemousegenome,theOrd ’ s kangaroorat(Mouse-relatedclade)alsodidnothavethis geneannotatedinEnsembl.Withtheapparentregion syntenyfortheOrd ’ skangarooratwhencomparedto theothermammals,thisabsencemightjustbethecase ofagenomestilltobecompletelyannotated,leaving thehousemouseastheonlyrodenttaxonthathaslost SAMD9 ,atleastfromthecurrentlyavailablegenomic sequencedatabase. Agreatnumberoftheavailablemammaliangenomes arestillnotcompletelyannotated.Therefore,wemade noassumptionsregarding SAMD9 and SAMD9L for thosespecies.Nevertheless,weobservedthatthefairly wellannotatedcowandpiggenomes(OrderArtiodactyla)hadnomatchesorannotationsfor SAMD9L .This informationtogetherwiththeabsenceof SAMD9 inthe housemouseandthealreadysuggestedoriginofboth genesfromacommonancestorbyancientgeneduplication[1]ledustothefollowinghypothesis:insomelineages thepresenceofbothgenesmightbecostlyforthegenome, resultinginthelossofoneofthegenesthatfunctionally wouldbeovercomebytheremainingparalogue.Although theseobservationssupportthepotentialexistenceofcertaingeneredundancybetween SAMD9 and SAMD9L ,we alsonotethealmostnonexistentrecombinationbetween them,despitetheproximityinthelocationofthesetwo genesinthegenomesofalltheannotatedmammalianspecies.Thisgeneticisolationofthetwoparaloguesdoesnot supporttheexistenceoffunctionalredundancybetween SAMD9 and SAMD9L .Theseapparentcontradictoryhypotheseshavetobeconfirmedwiththeconductionof functionalstudiesindifferentspecies. Withalltheavailablemammaliansequencescollected forboth SAMD9 and SAMD9L genes,theperformed phylogeneticstudyresultedinatreewithawell-defined monophyleticgroup per genegatheringsolelyplacental mammalsandasingleoutgroup,themarsupialgrey short-tailedopossum.Thissupportedthespeculativehypothesisof SAMD9 and SAMD9L resultingfromagene duplicationevent,moreprecisely,afterthedivergenceof MarsupialiafromPlacentalia147.7Mya[25].Despitethe commonancestor,whentestingfortheoccurrenceof potentialpositiveselectionactingatthegeneandproteinlevels,weconcludedthatSAMD9LisunderstrongerselectionthanSAMD9.Thisissupportedbythefact thatahighernumberofsitesatthegenelevelandof specificlineageswerepositivelyselectedinSAMD9L thanSAMD9.Besides,agreaternumberofaminoacid propertieswereunderselectionatthededucedprotein levelofSAMD9LthanSAMD9. Whenweexaminedtheaminoacidsubstitutionsand changesonphysicochemicalpropertiesforsitesunder selection,itwasclear,forbothproteins,thatmembers oftheRodentiaorderpresentedthehighestnumberof divergentalterationsforthesamecodonscomparedto othermammalianorders.Sinceitisknownthatinmany proteinstheaminoacidsubstitutionscausedbypositive selectionarenotrandom[21,26],forinstancesthePrimateAPOBEC3GresiduesinvolvedinHIV-1Vifinteraction[27],wehypothesizethatanyoccurringalteration inrodentsoreveninotherlineagesmaybetheresultof consistentarmsracebetweenthehostandapathogen stressor.Thiscouldbeasignificantobservation,given thatanti-viralpropertieshavebeenalreadyassignedto humanSAMD9inculturedhumancells.Specifically,a uniqueviralgeneproduct,M062ofmyxomavirus,was foundtoantagonizetheanti-viralpropertiesofSAMD9 proteininordertopermitthereplicationofthisvirusin culturedhumancells[8]. Consideringthemammalianspeciesincludedinthis study,selectionanalysesperformedonSAMD9 and/or SAMD9L genesforeachspeciesindividuallyonemay havedifferentresultsfromtheobtainedinourwork, sincerecombinationratesandeffectivepopulationsizes areexpectedtodifferamongspecies.Thesespeciesand populationspecificselectionanalysesshouldresultin theidentificationofsitesunderselectionin SAMD9 and/or SAMD9L genesthatcanbeusedingeneticpopulationstudiesbydeterminingparameterslikealleleand genotypefrequencies,andFSTandnucleotidediversity values.Thiscontributestothedefinitionofgenotypes thatmightbefavorableornot,forexample,tothedefenceagainstcertainpathogens. HumanSAMD9andSAMD9Lhavesolelyonedefined domain,theSterileAlphaMotif(SAM),amoduleof about70aminoacidresidueslong[28],specifically65 aminoacidsand66inSAMD9andSAMD9L,respectively.SAMdomains,oneofthemostcommonprotein domainsfoundineukaryoticcells,areprotein-protein interactionmodulesthatperformalargenumberofdifferentfunctions[29,30]andarenoteasilycategorized. Indeed,differentSAMdomainscanself-associate,bind tootherSAMdomainsand/ortonon-SAMproteins, andeveninteractwithRNA,DNAorlipids[30].Becauseofthegreatvarietyofknownfunctions,thepresenceofaSAMdomaindoesnotnecessarilyinvolveaLemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page11of16 http://www.biomedcentral.com/1471-2148/13/121

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specificfunctionorpathway,butanarrayofpossible functions.ForbothhumanSAMD9andSAMD9L,no functionhasyetbeenassignedtotheirSAMdomains, butforSAMD9theabilitytoformSAMpolymershas beensuggested[31].Fromourevolutionarystudyon bothproteins,noneoftheidentifiedsitesoraminoacid propertiesunderpositiveselectionoverlappedwiththe deducedSAMdomains,demonstratingahighlevelof conservationamongthemammalianspecies.ConclusionsSincetheoriginandevolutionofthe SAMD9 and SAMD9L geneswerefirstreported,agreatnumberof mammaliangenomeshavebeensequenced,allowingnow amoredetailedviewintotheevolutionaryhistoryofboth genes.Ourstudysupportsthepreviouslysuggestedorigin of SAMD9 and SAMD9L fromamammalianancestralduplicationevent.Specifically,accordingtotheresultsfrom ourstudy,thiseventoccurredafterthedivergenceof MarsupialiafromPlacentalia.Whenconsideringthemostly completemammaliangenomescollectedforthisstudy,the apparentlossof SAMD9 or SAMD9L insomespeciesled ustoproposethatsomeoverlappingfunctionalredundancyexistsbetweenthetwoproteins,despitethealmost nonexistentrecombinationbetweenthetwocloselylocated genesfromotherspecies.Fromthepositiveselectionanalysesperformed,bothatgeneandproteinlevels,wedemonstratethatSAMD9andSAMD9Lcontinuetobeunder longtermselectivepressure,withevenstrongerevidence forpositiveselectioninSAMD9L. Both SAMD9 and SAMD9L genesareupregulatedby typeIinterferon,aclassicfeatureassociatedwithmanyinnatepathogen-responsegenescalledinterferon-stimulated genes(ISGs).Indeed,humanSAMD9hasalreadybeen showntobeafunctionalinhibitorforatleastoneviral pathogen,apoxviruscalledmyxomavirus,thatexpressesa specificviralinhibitor(M062)thatcounteractstheantiviralpropertiesofSAMD9[8].Ourresultssuggestthatat leasttheSAMD9genesmayhavebeenundersustainedselectionpressureexertedbyviralpathogens. Ourworkisthefirstcompletestudytoinvestigatethe evolutionaryhistoryofmammalianSAMD9andSAMD9L.MethodsSAMD9andSAMD9LnucleotideandproteinsequencesAlltheavailablemammalian SAMD9 and SAMD9L genescodingsequencesusedinthephylogeneticand positiveselectionanalyseswereretrievedfromNCBI (http://www.ncbi.nlm.nih.gov)andEnsembl(http://www. ensembl.org/index.html)databases.Next,sequenceswere alignedwithClustalW[32]implementedinBioEditv7.0.9 [33],followedbyvisualinspection.Nucleotidesequences translationintoproteinsequenceswasperformedusing alsoBioEdit. SAMD9 and SAMD9L genescodingsequenceswerecollectedforfifteenandnineteenspecies,respectively.Based ontheMammalSpeciesoftheWorlddatabaseclassification(http://www.buckn ell.edu/msw3/),representativespeciesofmammalianinfraclassesMetatheria (OrderDidelphimorphia)andEutheria(OrderArtiodactyla,Carnivora,Chiroptera,Erinaceomorpha,Lagomorpha,Perissodactyla,Primates,Proboscidea,Rodentia andSoricomorpha)wereincludedinthisstudy.Table1 summarizesthespeciescollectedforeachgeneandtheir respectiveaccessionnumbers. Theisoeletricpoint(pI)ofSAMD9andSAMD9L deducedproteinsfordifferentspecieswasestimated usingDAMBE(DataAnalysisandMolecularBiology andEvolution)[34].RecombinationandphylogeneticanalysesRecombinationcanmisleadphylogeneticandpositive selectionanalyses[35],andparticularlyfor SAMD9 and SAMD9L ,thegenescloselocation(~12kbinhuman genome,forexample)mightincreasetheprobabilityof recombinationtooccur.Therefore,wefirstperformedrecombinationtestingonplacental SAMD9 and SAMD9L nucleotidesequencesalignments,andalsoonthealignmentofbothgenestogether( SAMD9 + SAMD9L ).The softwareGARD(GeneticAlgorithmforRecombination Detection)[13,14],implementedintheDatamonkey webserver[36],wasusedtodetectpossiblerecombination breakpoints. For SAMD9 and SAMD9L genesalignmentsnosignificantbreakpointsweredetectedwhileusingGARD,thus thecompletealignmentswereusedtoestablisheach genephylogeny.AsindicatedbytheAkaikeInformation Criterion(AIC)implementedinjModelTestv0.1.1[37], thenucleotidesubstitutionmodelTVM+Gwasusedfor SAMD9 treeestimation,whiletheGTR+Gmodelwas theconsensusmodelselectedfor SAMD9L phylogenetic treeconstruction.Ontheotherhand,asignificant breakpointwasdetectedwhenrunningGARDforthe SAMD9 + SAMD9L alignmentandaphylogenetictree wasestimatedforeachsegment.Fortheleftsegment, theAICinjModelTestindicatedGTR+I+Gasthebestfitnucleotidesubstitutionmodel,whereasfortheright segmenttheTPM2uf+Gmodelwasindicatedasthebest forthetreeestimation.Also,forthe SAMD9 + SAMD9L alignment,aphylogenetictreewasestimatedwithout testingrecombination.Inthiscase,thejModelTestAIC estimatedGTR+I+Gmodelasthebest-fitnucleotide substitutionmodel. Toestablishmammalianphylogenyfor SAMD9 SAMD9L and SAMD9 + SAMD9L ,basedonnucleotide sequences,theMaximumLikelihood(ML)methodimplementedonGARLIv2.0(GeneticAlgorithmforRapid LikelihoodInference)wasused[38].TheanalyseswereLemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page12of16 http://www.biomedcentral.com/1471-2148/13/121

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performedwith1,000,000generationsand1,000bootstrap searches.MLtreesweredisplayedusingFigTreev1.3.1 (http://tree.bio.ed.ac.uk/).Codon-basedanalysesofpositiveselectionAusefulmeasurementforidentifyingadaptiveprotein evolutionisthenonsynonymous( dN)/synonymoussubstitution( dS)rate( = dN/ dS),wherevaluesof =1,<1, and>1indicateneutralselection,negativeselection,and positiveselection,respectively[39,40].Naturally,and duetoproteinstructuralandfunctionalconstraints, is expectedtobecloseto0andfullproteinanalysisrarely detectspositiveselection[41].Asaresult,several methods,basedonmodelsofcodonsubstitution,have beendevelopedtodetectadaptiveevolution(positiveselection)atindividualsitesinabackgroundofnegative selection[42,43].Weemployedsixdifferentmethodsto detectsitesunderselection,andbasedonthemethodologyadoptedbyseveralauthors[19,20]onlycodons identifiedbyatleastthreeofthesixusedmethodswere consideredtobeunderpositiveselection. Todetectselectionbasedontheratio andatthe gene-level,forboth SAMD9 and SAMD9L ,PAMLv4.4 (PhylogeneticAnalysisbyMaximumLikelihood)[16,17] wasusedandthecodonfrequencymodelF3x4was fittedtobothalignments.Inthesite-specificmodels thatallowtheratio tovaryamongcodons,we performedLikelihoodRatioTests(LRTs)with2degreesoffreedomtocomparethefollowingmodels ( NSsites ):M1(nearlyneutral)withM2(selection) andM7(neutral, distributionof <1)withM8 (selection, distributionof >1).AsignificantLRT demonstratesthattheselectionmodelfitsbetterthan theneutralmodel[42,43].FormodelM8,aBayes empiricalBayes(BEB)approachwasemployedtodetectcodonswithaposteriorprobability>90%ofbeing underselection[44].Alsot hebranch-sitemodelAwas performedfortestingpositiveselectiononindividual sitesalongaspecificlineage,calledforegroundbranch, wheretheotherlineagesarebackgroundbranches.In branch-sitemodelA,three ratiosareassumedfor foreground(0< 0< 1, 1=1, 2>1)andtwo ratiosfor background(0< 0< 1, 1=1).Thenullmodelisthesame asmodelA,but 2=1isfixed.WealsousedBEBapproachtocalculatetheposteriorprobabilityofaspecific codonsiteandtoidentifythosemostlikelytobeunder positiveselection(posteriorprobability>90%)[44]. Both SAMD9 and SAMD9L geneswerealsoanalyzed usingHyPhysoftwareimplementedintheDatamonkey webserver[36].DatamonkeyincludesthreeclassicML methodstodetectsitesunderselection:theSingleLikelihoodAncestorCounting(SLAC)model,theFixed EffectLikelihood(FEL)modelandtheRandomEffect Likelihood(REL)model[45].Besidesthesethreemethods, twootherrecentlydevelopedandimplementedinthe Datamonkeywebserverwereappliedtoourdataset:the MixedEffectsModelofEvolution(MEME)thatallowsthe distributionof tovaryfromsitetositeandalsofrom branchtobranchatasite,beingcapableofidentifyingboth episodicandpervasivepositiveselection[46],andtheFast UnbiasedBayesianAppRoximation(FUBAR)methodthat candetectpositiveselectionunderamodelfasterthan theexistingfixedeffectslikelihoodmodelsthroughthe introductionofanultra-fastMarkovchainMonteCarlo (MCMC)routineandthatallowstovisualizeBayesian inferenceforeachsite[47].Allthesemethodswere runusingthebestmodelchosenbyAIConadefined Neighbor-Joining(NJ)phylogenetictreeafterrunning GARDtodetectrecombination.Toavoidahighfalsepositiverate,duetothereducednumberofsequences [45],siteswith pvalues<0.1forSLAC,FELandMEME models,BayesFactor>50forRELmodelandaposterior probability>0.90forFUBARwereacceptedascandidatesforselection. FromtheHyPhysoftwareavailableontheDatamonkey webserver,wealsorunthePARRISmethodusedtodetect ifaproportionofsitesinthealignmentevolvewith dN/dS>1andthataccountsforsynonymousratevariation andrecombination[18].Aminoacid-basedanalysesofpositiveselectionByusingTreeSAAPv3.2(SelectionofAminoAcidPropertiesbasedonPhylogeneticTrees)[22]itwaspossible todetectselectionsignaturesattheaminoacidlevel, morespecifically,positivelyselectedaminoacidpropertiesthatresultinradicalstructuralandfunctional changesinlocalregionsoftheprotein(destabilization). Propertiesthatfellintocategories6through8(themost radicalvaluesdenotingpositivedestabilizingselection), presentedz-scorevaluesof3.09andhigher,andwitha probabilityvalueof0.001wereplottedinaslidingwindow (length=20). Thirty-oneaminoacidpropertieswereevaluated acrossSAMD9andSAMD9Lphylogenetictreestoidentifyproteinregionsthatpresentedevidenceofpositive destabilizationforeachproperty.Thethirty-oneamino acidpropertiesarethefollowing:alpha-helicaltendencies, averagenumberofsurroundingresidues,beta-structure tendencies,bulkiness,buriedness,chromatographicindex, coiltendencies,composition,c ompressibility,equilibrium constant(ionizationofCOOH),helicalcontactarea,hydropathy,isoelectricpoint,long-rangenon-bondedenergy, meanr.m.s.fluctuationdisplacement,molecularvolume, molecularweight,normalizedconsensushydrophobicity, partialspecificvolume,polarrequirement,polarity,power tobeattheC-terminal,powertobeatthemiddleof alpha-helix,powertobeattheNterminal,refractiveindex, shortandmediumrangenon-bondedenergy,solventLemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page13of16 http://www.biomedcentral.com/1471-2148/13/121

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accessiblereductionratio,surroundinghydrophobicity, thermodynamictransferhydrophobicity,totalnon-bonded energyandturntendencies.AdditionalfilesAdditionalfile1:FigureS1. MammalianSAMD9andSAMD9Ldeduced proteinsequencesalignment. SAMD9 and SAMD9L genescodingsequences werecollectedforfifteenandnineteenspecies,respectively.Sequences werealignedwithClustalWimplementedinBioEdit.Theabbreviations correspondtothefollowingspeciescommonnames:Hosa-Human; Patr-Commonchimpanzee;Gogo-Westerngorilla;Poab-Sumatran orangutan;Nole-Northernwhite-cheekedgibbon;Mamu-Rhesus monkey;Bota-Cow;Susc-Pig;Eqca-Horse;Mylu-Littlebrown myotis;Orcu-Europeanrabbit;Rano -Brownrat;Crgr-Chinesehamster; Capo-DomesticGuineapig;Soar-Commonshrew;Caja-Common marmoset;Loaf-Africanbushelephant;Calu-Domesticdog;Aime-Giant panda;Ereu-WestEuropeanhedgehog;Mumu-Housemouse;Modo-Grey short-tailedopossum.Toaccessthespeciesscientificnames,thelistof abbreviationsshouldbeconsulted.Codonsarenumberedaccordingto humanSAMD9protein. “ ? ” representsundeterminedcodons; “ ” represents identitywiththereferencesequenceofhumanSAMD9protein. Additionalfile2:TableS1. Detectionofrecombinationbreakpoints from SAMD9 and SAMD9L genesalignmentusingGARDanalysis. SAMD9 and SAMD9L completecodingsequenceswerealignedtogetherandthe softwareGARDwasusedtolookforanyevidenceofrecombination. Threebreakpointswereidentified,butonlyonewasstronglysupported bytheKishino-Hasegawa(KH)test. Additionalfile3:FigureS2. Mammalian SAMD9 and SAMD9L genes estimatedMaximumLikelihoodtreewithouttestingrecombination.A phylogenetictreewasestimatedforthemammalian SAMD9 and SAMD9L genesalignmentusingtheMaximumLikelihood(ML)methodandunder theGTR+I+Gnucleotidesubstitutionmodel.Theanalyseswereperformed with1,000,000generationsand1,000bootstrapsearches.Thebootstrap valuesareindicatedonthebranches.Theabbreviationscorrespondto thefollowingspeciescommonnam es:Aime-Giantpanda;Bota-Cow; Caja-Commonmarmoset;Calu-Domesticdog;Capo-Domestic Guineapig;Crgr-Chinesehamster;Eqca-Horse;Ereu-WestEuropean hedgehog;Gogo-Westerngorilla;Hosa-Human;Loaf-Africanbush elephant;Mamu-Rhesusmonkey;Modo-Greyshort-tailedopossum; Mumu-Housemouse;Mylu-Littlebrownmyotis;Nole-Northern white-cheekedgibbon;Orcu-Europeanrabbit;Patr-Commonchimpanzee; Poab-Sumatranorangutan;Rano-Brownrat;Soar-Common shrew;Susc-Pig.Toaccessthespeciesscientificnames,thelistof abbreviationsshouldbeconsulted. Additionalfile4:FigureS3. Evolutionaryrelationshipsofeutherian mammals.Placentalmammals ’ evolutionaryrelationshipstreeretrieved andadaptedfromSong etal. [15]. Additionalfile5:FigureS4. MammalianSAMD9deducedprotein sequencesalignment.SAMD9deducedproteinsequencesfromfifteen specieswerealignedwithClustalWimplementedinBioEdit.The abbreviationscorrespondtothefollowingspeciescommonnames: Hosa-Human;Patr-Commonchimpanzee;Gogo-Westerngorilla; Poab-Sumatranorangutan;Nole-Northernwhite-cheekedgibbon; Mamu-Rhesusmonkey;Bota-Cow;Susc-Pig;Eqca-Horse;Mylu-Little brownmyotis;Orcu-Europeanrabbit;Rano-Brownrat;Crgr-Chinese hamster;Capo-DomesticGuineapig;Soar-Commonshrew.Toaccessthe speciesscientificnames,thelistofabbreviationsshouldbeconsulted. CodonsarenumberedaccordingtohumanSAMD9protein. “ ? ” represents undeterminedcodons; “ ” representsidentitywiththereferencesequenceof humanSAMD9protein. Additionalfile6:FigureS5. MammalianSAMD9Ldeducedprotein sequencesalignment.SAMD9Ldeducedproteinsequencesfrom eighteenspecieswerealignedwithClustalWimplementedinBioEdit. Theabbreviationscorrespondtothefollowingspeciescommonnames: Hosa-Human;Patr-Commonchimpanzee;Gogo-Westerngorilla; Poab-Sumatranorangutan;Nole-Northernwhite-cheekedgibbon; Caja-Commonmarmoset;Mamu-Rhesusmonkey;Loaf-African bushelephant;Eqca-Horse;Calu-Domesticdog;Aime-Giant panda;Ereu-WestEuropeanhedgehog;Orcu-Europeanrabbit; Mumu-Housemouse;Crgr-Chinesehamster;Rano-Brownrat; Capo-DomesticGuineapig;Soar-Commonshrew.Toaccessthe speciesscientificnames,thelistofabbreviationsshouldbe consulted.CodonsarenumberedaccordingtohumanSAMD9L protein. “ ? ” representsundeterminedcodons; “ ” representsidentity withthereferencesequenceofhumanSAMD9Lprotein.Additionalfile7:FigureS6. Mammalian SAMD9 geneestimated MaximumLikelihoodtree.Thephylogenetictreeofmammalian SAMD9 genealignmentwasestimatedusingtheMaximumLikelihoodmethod andthenucleotidesubstitutionmodelTVM+G.Theanalyseswere performedwith1,000,000generationsand1,000bootstrapsearches.The bootstrapvaluesareindicatedonthebranches.Theabbreviations correspondtothefollowingspeciescommonnames:Hosa-Human; Patr-Commonchimpanzee;Gogo-Westerngorilla;Poab-Sumatran orangutan;Nole-Northernwhite-cheekedgibbon;Mamu-Rhesus monkey;Bota-Cow;Susc-Pig;Eqca-Horse;Mylu-Littlebrown myotis;Orcu-Europeanrabbit;Rano -Brownrat;Crgr-Chinesehamster; Capo-DomesticGuineapig;Soar-Commonshrew.Toaccessthespecies scientificnames,thelistofabbreviationsshouldbeconsulted. Additionalfile8:FigureS7. Mammalian SAMD9L geneestimated maximumlikelihoodtree.Thephylogenetictreeofmammalian SAMD9L genealignmentwasestimatedusingtheMaximumLikelihoodmethod andthenucleotidesubstitutionmodelGTR+G.Theanalyseswere performedwith1,000,000generati onsand1,000bootstrapsearches. Thebootstrapvaluesareindicatedonthebranches.Theabbreviations correspondtothefollowingspeciescommonnames:Hosa-Human; Patr-Commonchimpanzee;Gogo-Westerngorilla;Poab-Sumatran orangutan;Nole-Northernwhite-cheekedgibbon;Caja-Common marmoset;Mamu-Rhesusmonkey;Loaf-Africanbushelephant; Eqca-Horse;Calu-Domesticdog;Aime-Giantpanda;Ereu-West Europeanhedgehog;Orcu-Europeanrabbit;Mumu-Housemouse; Crgr-Chinesehamster;Rano-Brownrat;Capo-DomesticGuineapig; Soar-Commonshrew.Toaccessthespeciesscientificnames,thelist ofabbreviationsshouldbeconsulted. Additionalfile9:TableS2. SAMD9 and SAMD9L likelihoodratiotest (LRT)forPARRISanalysisfromHyPhysoftware.Only SAMD9L wasfound tobeunderselectionwhenusingthisspecificmethod. Additionalfile10:TableS3. Positively-selectedcodonpositionsin SAMD9 and SAMD9L determinedbysixdifferentMaximumLikelihood methods.ThesixmethodscorrespondtoPAMLM8,SLAC,FEL,REL, MEMEandFUBAR.Codonspositionsarenumberedaccordingto humanSAMD9andSAMD9Lproteins(Additionalfile5FigureS4and Additionalfile6FigureS5). Additionalfile11:TableS4. SAMD9andSAMD9Laminoacid propertiesunderpositiveselectiondeterminedinTreeSAAP.SAMD9 exhibitedthreeandSAMD9Levidencedsevenaminoacidproperties underpositiveselection. Abbreviations Aime: GiantpandaAiluropodamelanoleuca ;Bota:CowBostaurus ; Caja:CommonmarmosetCallithrixjacchus ;Calu:DomesticdogCanis lupusfamiliaris ;Capo:DomesticGuineapigCaviaporcellus ; Chho:Hoffmann ’ stwo-toedslothCholoepushoffmanni ;Crgr:Chinese hamsterCricetulusgriseus ;Dano:Nine-bandedarmadilloDasypus novemcinctus ;Dior:Ord ’ skangarooratDipodomysordii ;Ecte:Lesser hedgehogtenrecEchinopstelfairi ;Eqca:HorseEquuscaballus ;Ereu:West EuropeanhedgehogErinaceuseuropaeus ;Feca:DomesticcatFeliscatus ; Gogo:WesterngorillaGorillagorilla ;Hosa:HumanHomosapiens ; Ictr:Thirteen-linedgroundsquirrelIctidomystridecemlineatus ;Loaf:African bushelephantLoxodontaafricana ;Mamu:RhesusmonkeyMacaca mulatta ;Modo:Greyshort-tailedopossum-Monodelphisdomestica ; Mumu:HousemouseMusmusculus ;Mylu:LittlebrownmyotisMyotis lucifugus ;Nole:Northernwhite-cheekedgibbonNomascusleucogenys ; Ocpr:AmericanpikaOchotonaprinceps ;Orcu:EuropeanrabbitOryctolagus cuniculus ;Otga:NortherngreatergalagoOtolemurgarnettii ;Patr:Common chimpanzeePantroglodytes ;Poab:SumatranorangutanPongoabelii ;LemosdeMatos etal.BMCEvolutionaryBiology 2013, 13 :121Page14of16 http://www.biomedcentral.com/1471-2148/13/121

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Prca:RockhyraxProcaviacapensis ;Ptva:LargeflyingfoxPteropus vampyrus ;Rano:BrownratRattusnorvegicus ;Soar:CommonshrewSorex araneus ;Susc:PigSusscrofa ;Tasy:PhilippinetarsierTarsiussyrichta ; Vipa:AlpacaVicugnapacos Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions ALMparticipatedinthedesignoftheresearch,performedthedataanalyses anddraftedthemanuscript.JL,GMandPJEconceivedthestudy,designed theresearchanddraftedthemanuscript.Allauthorsreadandapprovedthe finalmanuscript. Acknowledgements ThePortugueseFoundationforScienceandTechnologysupportedthe doctoralfellowshipofALM(SFRH/BD/48566/2008).Aresearchprojectfrom thePortugueseFoundationforScienceandTechnology(PTDC/BIA-BEC/ 103158/2008)alsosupportedthestudy.Thisworkwasalsosupportedby grantR01AI080607fromtheNationalInstituteofHealthtoGM.This researchhasalsobeenassistedbytheBITCoreoftheUniversityof California,SanDiego,CenterforAIDSResearch(NIHP30AI036214). Authordetails1CIBIO-CentrodeInvestigaoemBiodiversidadeeRecursosGenticos/ InBioLaboratrioAssociado,UniversidadedoPorto,4485-661Vairo, Portugal.2DepartamentodeBiologia,FaculdadedeCincias,Universidade doPorto,4169-007Porto,Portugal.3DepartmentofMolecularGeneticsand Microbiology,UniversityofFloridaCollegeofMedicine,Gainesville,Florida 32610USA.4CentrodeInvestigaoemTecnologiasdaSade,IPSN,CESPU, 4585-116Gandra,Portugal. Received:6February2013Accepted:6June2013 Published:12June2013 References1.LiCF,MacDonaldJR,WeiRY,RayJ,LauK,KandelC,KoffmanR,BellS, SchererSW,AlmanBA: Humansterilealphamotifdomain9,anovelgene identifiedasdown-regulatedinaggressivefibromatosis,isabsentinthe mouse. BMCGenomics 2007, 8: 92. 2.AsouH,MatsuiH,OzakiY,NagamachiA,NakamuraM,AkiD,InabaT: Identificationofacommonmicrodeletionclusterin7q21.3subband amongpatientswithmyeloidleukemiaandmyelodysplasticsyndrome. 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Table S3 : Positively selected codon positions in SAMD9 and SAMD9L determined by six different Maximum Likelihood methods a Codons with posterior probabilities >90% in the BEB analyses (*: P>95%; **: P>99%) b C odons with significance level < 0.1 (*: p<0.05; **: p< 0.01) c Codons with Bayes F actor >50 SAMD9 SAMD9L PAML M8 a 21*, 48 *, 52, 88 491 736, 1006 *, 1085**, 1088, 1116 *, 1120*, 1258 *, 1348, 1380, 1398 *, 1440 71**, 126**, 146*, 156 260 267 *, 329*, 340 **, 357 *, 368*, 452 **, 586 *, 639, 653 *, 1145*, 1183, 1276 *, 1295**, 1318*, 1339, 1380*, 1408* SLAC b 352 383 491 *, 513 731 1006 *, 1116 1320 *, 1329 1398 156 267 *, 340 **, 362 *, 586 *, 606 776 *, 978 *, 1018, 1186 1229 1429 1474 FEL b 38, 45*, 75*, 88 *, 151, 170*, 176, 279 *, 331 352 383 395 491 513 596 610, 618 731 735, 783 872 875 993 1006 ** 1095, 1116 ** 1127, 1258 1320 1329 1333, 1351, 1376, 1393, 1398 **, 1424, 1503, 1528 5, 39 56, 76, 83, 88, 92, 156 260 267 ** 282, 340 ** 357 362 ** 452 ** 519, 540, 585, 586 602, 606 653 776 783, 978 1006, 1154 1186 1189 1229 1255, 1276 1308 1360 1364, 1374, 1413, 1429 1474 REL c 48 52, 69, 106, 116, 193, 279 312, 331 372, 376, 397, 491 594, 611, 731 736, 827, 872 956, 993 1006 1017, 1085, 1088, 1092, 1116 1120, 1167, 1177, 1258 1314, 1320 1327, 1329 1348, 1380, 1398 15 19, 1576 39 126, 156 260 267 279, 325, 328, 340 357 362 452 586 601, 639, 653 721, 776 822, 953, 978 996, 1018, 1078, 1106, 1109, 1183, 1229 1231, 1276 1295, 1308 1316, 1335, 1367, 1380, 1413, 1429 1535 MEME b 21, 38**, 45*, 48 61*, 75*, 77*, 79**, 85**, 88 *, 96, 109, 121, 143, 145, 170, 173, 176, 187, 191, 193, 201**, 218*, 279 331 *, 352 **, 353, 365, 369, 383 387, 395, 491 *, 513 *, 596, 610, 618, 623*, 642*, 731 735**, 744, 783, 859, 872 875*, 969**, 993 *, 1003*, 1006 **, 1007, 1041, 1063, 1116 *, 1174*, 1199, 1252, 1258 *, 1320 *, 1329 *, 1341*, 1347*, 1351*, 1376*, 1393, 1398 **, 1415, 1422, 1444**, 1467*, 1528, 1556**, 1566* 5, 24 39 76 92, 142 149 156 170, 181 212 213 267 ** 282 299, 318, 323 340 ** 341 *, 350, 351, 357 359, 362 366, 373 377 426 452 ** 468 509, 519 540, 555 585, 586 602, 606 632 ** 653 723, 725 754, 776 789 791 848, 856, 859, 861 888 ** 889 945 963, 978 1004 1006 1109 1150 1154, 1170, 1186 1189, 1229 1250, 1252 1276 1284, 1287, 1308 1335 ** 1339 1343, 1346, 1360 1374 1385, 1390, 1429 1474 1535 FUBAR d 1006 1398 156 267 340 ** 357 452

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d Codons with posterior probabilities > 0. 90 (*: P>0.95 ; **: P> 0.99 ) Codons identified by three or more than three methods are underlined and in bold.



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10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa MAKQLNLPENTDDWTKEDV NQWLESHKIDQKHREILTEQDVNGAVLKWLKKEHLVDMGITHGPAIQIEELFKELRKTAIEDSIQTSKMGKPSKNAPKDQT SAMD9_Patr .................................................................................................... SAMD9_Gogo .....................................G......... ...........................Q...T..................... SAMD9_Poab .....................................G..............................................M............... SAMD9_Nole .................E...................G..................................... ...................V..... SAMD9_Mamu ........Q...........R................G....................................Q.............R........... SAMD9_Bota ..A.........................E....A...A...S..I..Y.T.D...A....F.......Q.....LE.SSG.PF..C.S..G..SV..T. S AMD9_Susc ..A.....K...........R............D...A......N..Y.T....I.I...F.......H.....ME.ST..PS.IC.RE.G..DV..K. SAMD9_Eqca ..A.................R...............IA...S..I....T.NN.IE............N.....Q..SS.G.L..N.KK.G...V..T.. SAMD9_Mylu ..A.............D ...R..D.........K.............F.T.N..IE............Q...K.Q.ESS..P...CQKK.G..KV..T.. SAMD9_Orcu ..E.PE..............R.......H....D..VA...S.....F...SD.IE........V...G..R..Q..SSK.P.R..EQK.G...VSNK.P SAMD9_Rano ..ET....K...........L.....R.........MA...S.V. ....N.KN..E..................Q..PK.NLTK.C.KT.GR..I..T. SAMD9_Crgr ..EK.......N........L...G.........T.ML...........D.N...E............A.....Q.SSSKQPT....RKRG...I..T. SAMD9_Capo ..E.PH....I.........R...............IA...S..T....N.SD......P.......VG..N. .Q..SS..PV..P.AK.S.T.T.GK.H SAMD9_Soar ..A.P..SK.P.........I...E.N..K.Y....VA.....D...L.T.K......V......L..N.V.K.Q..SS.N....REKKTNN..I.EV.. 110 120 130 140 150 160 170 180 190 20 0 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa VSQKERRETSKQKQKGKENPDMANPSAMS ----------TTAKGSKSLKVELI -EDKIDYTKERQPSIDLTCVSYPFDEFSNPYRYKLDFSLQPETGP SAMD9_Patr ...... ......R................ ----------.............. -...........L................................ SAMD9_Gogo ............................. ----------.............. -............................................ SAMD9_Poab .....H.........D..........TI. ---------.I..S........M -..Q.............N...................I....... SAMD9_Nole ....A.....................T.. ----------.I............ -..E...........................H............. SAMD9_Mamu .....S.........D..........T.. ----------.I..V.E....... -...... ..............................I....... SAMD9_Bota -.. DGG... -..N.KKS.KV.D.TV. ----------.VT.......N.FM -..E..D..KK...TEP..MA.......D......N.K....... SAMD9_Susc --EKN.........D..TS.RVTD.TI. ----------.VTE..M..NN.FM -..E.ND.QKK.S.VE.M..P.......D...... N......... SAMD9_Eqca .M.E.NG........N..KS....APT.. ----------.VT.D.....N.FK -VNE..D...K...VE............D......N......... SAMD9_Mylu LM.E.NE......H....KS....A.T.. ----------.V --.....S.F. -G.E..D...K.................D........C....... SAMD9_Orcu LV ...K.D.......N...S.P.DG..A. ----------PGE.EP..P.T..TDN..ERGD...KL.AKEPS.R.H..NK.DEQW....H.I..... -SAMD9_Rano --T.S....N.N.RAE..SCK.DT..V.PEGNSQRTAASPE......E.ND.V -...AVQ QDK.A.PGP..IA...N..........H.I....... SAMD9_Crgr --T.I...P..N.R.E..T.K.DA..LR ---------PE...AE.A.N..M -...TEL QGK.SPPGP..IA...N..........H.I....... SAMD9_Capo LM...G......R.SD..ISHK. -------------------.E...PDS..M -.NQP.D -----VKQP..LP...NT.........H.I....... SAMD9_Soar L..S.NG.K..K...D..KS.TVDT.TTH ----------.V..S...VEN.F. -.. D. --Q.K.L.TEQ..IP.......D.....EN.V....... 210 220 230 240 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. ...|....|....|....|....| SAMD9_Hosa GNLIDPIHEFKAFTNTATATEEDVKMKFSNEVFRFASACMNSRTNGTIHFGVKDKPHGKIVGIKVTNDTKEALINHFNLMINKYFEDHQVQQAKKCIREP SAMD9_Patr .................................................................................................... SAM D9_Gogo ..................................................................S................................. SAMD9_Poab ....................................................I.............S................................. SAMD9_Nole ................... ...............................................S................................. SAMD9_Mamu ................E.............................................V...S................................. SAMD9_Bota L...............E.G....A....................... ...........T...VEF.TI......D......HQ........K..N..... SAMD9_Susc L...............E...K.........................................VE..TV....F.D...M..HQ........K..N..... SAMD9_Eqca L...............ER.R...I......................................V..STV...T..D ......QQ........K........ SAMD9_Mylu L..........E....KN............................................VN..SV......D......HQ........K........ SAMD9_Orcu -----------------------------------.............L...... .Q...V.LASI..N...D..DE..P....E.......S...V. S AMD9_Rano L.....V.....L...E......I............................Q...Q.R...MEL.TV..D...D......VQ...V............. SAMD9_Crgr L.....V.........EK.S...I.......I........................Q.R...VDLSTV..DT.........DQ...A............. SAMD9_Capo Q...........L.D.. ....K.I.......T..............................M...SV......E...S..PQ...A............. SAMD9_Soar L............K.IEKG.K..I.I.......K..................E.......I.VRFIAI...V..D...QI.HQ...EG...K..N.....

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310 320 330 340 350 360 370 380 390 400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa RFVEVLLPNSTLSDRFVIEVDIIPQFSECQYDYFQIKMQNY NNKIWEQSKKFSLFVRDGTSSKDITKNKVDFRAFKADFKTLAESRKAAEEKFRAKTN SAMD9_Patr ......................................... -...T.................R................................... SAMD9_Gogo ......................................... -...T.....N................. .............................. SAMD9_Poab ......................................... -...T............E.......M.......T..T..................... SAMD9_Nole ...........A............................. -...T..............P.....M.............................. .. SAMD9_Mamu .....................................I... -...T........V...........M...A...T........................ SAMD9_Bota .....................VV.KY...EV.........C -S.T..K..PN..V.....A.....M.SNM..K...L.L.R......E....C.V... SAMD9_Susc ..........IP.........VV.KY...EQE......... -S..T.K..P...V.M...A....VM..N.E.KT..L.L.K...A..E....C.L... SAMD9_Eqca ...........P.........V..KY...ED...R..I..C -D.NT.K..S...I.....A.....M..NA..KE..L.L.....A..E....Y.L..S SAMD9_Mylu ...........P.........V..KY.. .EH.........C -...V...R.H..V.....A.....M......KI.QLGLEA...H..E....C.I... SAMD9_Orcu ........D..V.........V..HY.V.GH......K.I.DN...K....S...VL....ARTVN.V.T.T..KM..LNL........E..DNCT.... SAMD9_Rano ...........P.NK..........Y...KE.F.....HWH -KDET.Q.IP.Y.V .....PK....IG..A..K...L.L.A..D...E..K.SKV.SD SAMD9_Crgr ...........P.NK......VL..H...KE........HL -KSQT.Q..S.H.V.....PQ....IG..T..K...LGL.EV.D...E..T..KE.PE SAMD9_Capo ...........V.....V...VV..Y...KH...R.T.... -.HET.QP.S.Y.V.I...PNT.N.I......E..QL.L.SV .T...E....CKL... SAMD9_Soar .......T.GIP...Y........KY...EH.........F -...T.NK.SR..V.....A.C..LM.TPA..K.Y.S.L........E...N..L..S 410 420 430 440 450 460 470 480 490 500 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa KKEREGPKLVKLLTGNQDLLDNSYYEQYILVTNKCHPDQTKHLDFLKEIKWFAVLEFDPESNINGVVKAYKESRVANLHFPSVYVEQKTTPNETISTLNL SAMD9_Patr ................. ................................................................................... SAMD9_Gogo .................................................................................................... SAMD9_Poab ............................................. .........................................E......M...... SAMD9_Nole ...............................................................K......................E...S..M...... SAMD9_Mamu ...................................................................QD..A. .........I...G......K...... SAMD9_Bota ...S.....................DW....I.....T.I.....................VSK........T..V......L...G... T.K.TS... SAMD9_Susc ...S............K........DW....I.....T.I.....................VSE...........V......L...G... ..K.IS... SAMD9_Eqca ...N.....................DW..........N.I.....................VS...........IT......L.M.G...S..K.TS... SAMD9_Mylu N.NS.....IE.....K.........W..........N.I...............D.....VSK...N......IV...L..L...G......K..S... SAMD9_Orcu NRH ..FR..E.... ..........DW..........N.IQY...................ES....RSF....L.......Q.S.G...T..M..N... SAMD9_Rano GSNSQ.Q..ID.....K......H..W..................................ES......F.G..........LFI.E.P.VS.K..S... SAMD9_Crgr GNKS..Q..ID...............W...I......N.NS.. E.................ES......F.Q..........QF..E...L..K..S... SAMD9_Capo .NNS..S...T..............DW............II............I.......KTK..L..F....A....S..QF..E...TE.K..S... SAMD9_Soar ...S......RM.............SL.........QN.VN.........L..........ASK....... ...........Q...E...TD.K..S... 510 520 530 540 550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa YHQPSWIFCNGRLDLDSEKYKPFDPSSWQRERASDVRKLISFLTHEDIMPRGKFLVVFLLLSSVDDPRDPLIETFCAFYQDLKGMENILCICVHPHIFQG SAMD9_Patr ................. ................................................................................... SAMD9_Gogo .................................................................................................... SAMD9_Poab ...............N............................R ....................................................... SAMD9_Nole ......................L......................................................................Q...... SAMD9_Mamu ............S.......................................................................I........Q...... SAMD9_Bota .Q.L..............E...L.A......K..E.........Q................................................DSR.C.. SAMD9_Susc .Q.L..................L..... ...K..E.......I.............................................M...A.SR.C.. SAMD9_Eqca .Q..........I.........L........K..E...........................................................S..C.R SAMD9_Mylu .Q....................L......K.K..E............V........ ............................I.....V...S..C.R SAMD9_Orcu .Q..........S..GI.....L..I........E............V..K...........P................................Q.C.. SAMD9_Rano .Q.............E.D..Q.L..I........E..R......RD..................E.................N. .............Y.V SAMD9_Crgr .Q..........S..E.D....L..I........E..R....F.RD....K.R.........P.............G..............S.FSS.Y.V SAMD9_Capo .QK.........S.......E.V..V...K....E.........R......................K........T.....S..........Q.N.C.. SAMD9_Soar HQ..... .........D....L.. ???????????????????????? ... -... ....P.... .. Y............L....V. LNSQLC.I

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610 620 630 640 650 660 670 680 690 700 ....| ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa WKDLLEARLIKHQDEISSQCISALSLEEINGTILKLKSVTQSSKRLLPSIGLSTVLLKKEEDIMTALEIICENECEGTLLEKDKNKFLEFKASKEEDFYR SAMD9_Patr .................................. .................................................................. SAMD9_Gogo .................................................................................................... SAMD9_Poab .................................................................................................... SAMD9_Nole .................................................................................................... SAMD9_Mamu ............................ .....................................S.................................. SAMD9_Bota .........TTQ...L.N..V.S......................F.....S.................L......D.I.....K.L.....L....... SAMD9_Susc .........A.QE..L.............................F...V.S.... ..R..........L.............EK.......L....... SAMD9_Eqca .........T.Q...L.N........................L..F.....S.............S...L..............K...D..T........ SAMD9_Mylu .........T. ...L......S.................K..Q.F.....S.....EM..........L.....K.....N.. ........L....... SAMD9_Orcu .........T.E...L.N......N.Q..................F.....S.......Q..T......L......D...KD..K..........KA... SAMD9_Rano ......V..SSQ...L...S.FS.N.A...........L......F...V.S......R..E.......L.............RE.LF...T...K.... SAMD9_Crgr .........AGQ...L.NRS....N.A...........L......F.....ST........ .......L...........N..K.LF...TA..Q.... SAMD9_Capo .........TNE...L.DK.....N..Q......N...I....E......CS.......... ......L.V........H...T.LQ...T........ SAMD9_Soar .........KIQ...LAN......N. ................V.K......S.................L......D.D.........A..TL....... 710 720 730 740 750 760 770 780 790 800 ....|....|....|....|....|....|....|....|.... |....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa GGKVSWWNFYFSSESYSSPFVKRDKYERLEAMIQNCADSSKPTSTKIIHLYHHPGCGGTTLAMHILWELRKKFRCAVLKNKTVDFSEIGEQVTSLITYGA SAMD9_Patr ......................................................................... ........................... SAMD9_Gogo ..................................................................................A................. SAMD9_Poab .................................................................................................... SAMD9_Nole ..................................................................................A................. SAMD9_Mamu ...........................................C.......................D.........................N...... SAMD9_Bota ....T.........N ............K..E...RG.....S.CV................................................N.....T SAMD9_Susc ..............N........ER..K..T...E..A.....CV...................V................M...C.......N.....T SAMD9_Eqca .............K...L.........I..E....W....... CV.....................................M.........AT...... SAMD9_Mylu ..............N............K..R..H.......S.CS............................................K...N.....T SAMD9_Orcu ..............NH..A................W......ICA.......................... ......................N.....T SAMD9_Rano ..............N...S.......VK..KK..EW....Q.VCA.....................................E..........N...... SAMD9_Crgr ..............N...S........K..KK..EW.. ..Q.VCA......................D..............E..........N...... SAMD9_Capo ........... ..Y...S........K.KE....Y..P..LMCV...............................................V....... SAMD9_Soar .............KN...A........K.KD....W ....S.CV...Q................. .N.....................K.I.D.....T 810 820 830 840 850 860 870 880 890 900 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.... |....|....|....| SAMD9_Hosa MNRQEYVPVLLLVDDFEEQDNVYLLQYSIQTAIAKKYIRYEKPLVIILNCMRSQNPEKSAR IPDSIAVIQQLSPKEQRAFELKLKEIKEQHKNFEDFYS SAMD9_Patr ........................................................K...K ......L............................... SAMD9_Gogo ............................................................. ...................................... SAMD9_Poab .....................................V....................... ...................................... SAMD9_Nole ......I...................S .............................K.... ...................................... SAMD9_Mamu ....D........................................................ ...................................... SAMD9_Bota T.N...L.I.................A..H..V.NR..............R.... .....K .S....L...................ED.....Q.... SAMD9_Susc ASS...L................F..A....T..N...............R.........K N.N...L..N..S...........K.E......K.... SAMD9_Eqca T.Q...L...........E.......S.....V.N.......................C.K NL.G..L..N..........F .....E...E..K.... SAMD9_Mylu A.H...L............GDI....A......TS...............V.......C.K MS....L.....S.............E........... SAMD9_Orcu T.H...L...............F...S.....V.E.H.......................K ....V.LLHH.............EK..KEYD....... SAMD9_Ran o T.H...L...................T.....V...H.............T.........K N...V.LV....D..........T........V..... SAMD9_Crgr TS....L...................A.....V.N.H.............T.........K ....V.LVH...D..............KH.E....... SAMD9_Capo T.H...L.................. .AA....V.Q...............A.........K ......LR.K..R..KE...........H......... SAMD9_Soar TSH...L............E......S..H..VSS.H.................D.R.C.KNF.N...L......S............EK....V.....

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910 920 930 940 950 960 970 980 990 1000 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa FMIMKTNFNKEYIENVVRNILKGQNIFTKEAKLFSFLALLNSYVPDTTISLSQCEKFLGIGNKKAFWG TEKFEDKMGTYSTILIKTEVIECGNYCGVRII SAMD9_Patr .................................................................................................... SAMD9_Gogo ................................................................................................ .... SAMD9_Poab .....................................T.............................................................. SAMD9_Nole ............M....................................................................................... SAMD9_Mamu .......... .............................................R............................................ SAMD9_Bota ..........K...D.......E...S.................................TS..........................V........... SAMD9_Susc ........D.K...............C........... ......................T..........L............................ SAMD9_Eqca .........EK...............S............................N....T.......V...............R...V...K....... SAMD9_Mylu ........D.K...............S.................................TT...Y .....L................V...K....... SAMD9_Orcu ........................DPS............................N....A..........L.E..........R...L...M....... SAMD9_Rano .........................VT.......A......AF............L....S....Y.. .................S......S....... SAMD9_Crgr ........DRK..............VA.......A......T.............YY...S........................S......S.W..... SAMD9_Capo .............KK..........KS.................SG..........Y...A..........L............Q.K.E...T... .... SAMD9_Soar ........D.M......K........C..................E.........Q....T......EL..L..............D.V...C....... 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa HSLIAEFSLEELKKSYHLNKSQIMLDMLTENLFFDTGMGKSKFLQDMHTLLLTRHRDEHEGETGNWFSPFIEALHKDEGNEAVEAVLLESIHRFNPNAFI SAMD9_Patr ............................. ....................................................................... SAMD9_Gogo .........................H.......................................................................... SAMD9_Poab .....K................................................... ...S............................G.R........ SAMD9_Nole .....K.....................................................D.............................G....K..... SAMD9_Mamu .....K................................. .................N................................G.......... SAMD9_Bota .P...IR......I..D.D....T.........Y...I.R....E.VQ......Q.N.N...M.TL..........E...V..KN..RGG.R........ SAMD9_Susc .P...TR......I..N.D....I.........Y...I..D...E..Q......Q.ND......T.. ........R....V..KN....G.RQ..S.... SAMD9_Eqca .P...DR.....RT....D..............YE..L...R.SEHIQ......Q.N.......TL..............V..KE....G.R...S.... SAMD9_Mylu .P...IR......I..D.D.....W......V.Y...I.R...F...Q......Q.I.......T..........E....K...K....G..... ..... SAMD9_Orcu .P...SL......N..G.GRC.M..........YE..I........VQ........N...D...................D..KM..EKG.N...K.... SAMD9_Rano .H...TL......R..N.S..K.VM........Y...I.....Y...Q...I..Q.N...R...................D..K...H.ATR..D..... SAMD9_Crgr .HE..SA.. ....R..N.S..E.VMN.......Y.M.I.....F...Q......Q.N.......................D..KD....ATR........ SAMD9_Capo .P...IL.........D.D..............Y...L.R...F...Q......E.S.Q.....T..................KQ..CQG.E..K..... SAMD9_Soar .P...LR......L..Q...........R....Y.R....T..I..IQ....I.Q.N......ET..................KE..I.GVD..KQ.... 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 ....|....|....|... .|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa CQALARHFYIKKKDFGNALNWAKQAKIIEPDNSYISDTLGQVYKSKIRWWIEENGGNGNISVDDLIALLDLAEHASSAFKESQQQSEDREYEVKERLYPK SAMD9_Patr ............................................... ..................................................... SAMD9_Gogo .........................................................RS..........N.............................. SAMD9_Poab ...............D.......E.................................R...........N..... .......................L. SAMD9_Nole ..............................................M.......ER.............N.............................. SAMD9_Mamu .......................................................R.R....N......N...Q.............Y..........Q. S AMD9_Bota ....T......E...DS..H..N...K...............F.......MDD.ER.RS...GE.SD.....VQ..N...............G...F.Q. SAMD9_Susc ....S......ER..NS..H..NE..K......................... D.R.W...A...AD.....V...N......R............F.Q. SAMD9_Eqca .........L.E...SS ..K..N...K...H.....................D..K.R.....N.TD..E..VE.TN............Q..G...F.Q. SAMD9_Mylu ...........ER..TS..H..N...N...N...................T.N.ER.R....A..TE.....V...E..........YK.D.AM..FNQ. SAMD9_Orcu R....... --------------------------.......... .L.....D..RGNT..AN..TD..Q..V..AE......R........G.....Q. SAMD9_Rano .........L.E...ES..L.....RK.A.N...............L...V.D.IK..I....E.TE.....K...E...D....T......G..SV.Q. SAMD9_Crgr .........L.E...K...F.....RT...N....................DD.IR..S..AE..TD...S.V ...D...D..R.T......G....NQ. SAMD9_Capo ..V....Y..RE...N...L...E..K...N.....................DTER.RD......ST.........N......R........G....GQ. SAMD9_Soar ...........E...ER..E..NK..N...Y....................DD..R.R....A..TM..E..V...N......R...YK.R.A.GKFHQ.

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1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa SKR RYDTYNIAGYQGEIEVGLYTIQILQLIPFFDNKNELSKRYMVNFVSGSSDIPGDPNNEYKLALKNYIPYLTKLKFSLKKSFDFFDEYFVLLKPRNNI SAMD9_Patr .................................................................................................... SAMD9_Gogo ..........................................................D......................................... SAMD9_Poab ..........................................D....I.................................................... SAMD9_Nole ............................ ......K.......D....I.................................................... SAMD9_Mamu ..........................................D........G.....Q.D..Q..................................... SAMD9_Bota .........T...L..............F.............D.I..I...K.... EA...F..V...F.S...N......................... SAMD9_Susc ..........................................D.I..I..TT.........F..V.N.F...................D........... SAMD9_Eqca ..................A...............S.......D.I..I.........SS..F......F.....N..C...RC. ....D........... SAMD9_Mylu ......................A...................D.I..I..N..........F......F.......RS..........D........S.. SAMD9_Orcu .........................................KD.......T......S...F.SV...F.....N.R...........D........... SAMD9_Rano ...........................K.L...NS.....RQV.I..I..NC.....L ..F.SV...F.S...N.QS..........D........... SAMD9_Crgr ........................K...........D.....D.I..I..NR.....L ..F.I....F.....N.QS...RA.....D........... SAMD9_Capo ..........S..H........A... ........K.....R.D.I..I..N......L ..F......F.....T..S...DA.....D........... SAMD9_Soar ..................................S....F.KD.I..I..T......S...F....S.F.S...N.R....N......D..........M 1310 1320 1330 1340 1350 1360 1370 1380 1390 1400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa KQNEEAKTRRKVAGYFKKYVDIFCLLEESQNNTGLGSKFSEPLQVERCRRNLVALKADKFSGLLEYLIKSQED AISTMKCIVNEYTFLLEQCTVKIQSKE SAMD9_Patr .................................................................................................... SAMD9_Gogo .................................D.R..............S...........................EY..........R......... SAMD9_Poab ..............................K .DP...........................................E...K.......R.A....... SAMD9_Nole ...................I....VS..... KD...............SS....................A..V...EN..DK....S........... SAMD9_Mamu .............V. ....T......G...D I...........I....KS...........................ED...K.A..........E... SAMD9_Bota ...................A...GPS..L.. KDF...L.V.....LY..S.EV...............A....VH..ED.MDK....F.......LI.. SAMD9_Susc .........K...V.....A...GPS.D..S KY....L.L.....Q...S.EV...............N.K..A...ED.M.K.S..F.....RML... SAMD9_Eqca ......R.......H........GPSV.... KD..A.L.I.....LY.KS.EV...........N...N.....R..DD...K....F..G.....P.. SAMD9_Mylu ....QS.............A...GPF.. L.. RD...QL.L..R..LY..S.EV..................I..N..ED..KK....F...A.R..Q.. SAMD9_Orcu .....T.......TC..R.L...GPSA.ILS K.....I.LS...QE...S.E..............V....H.....EN..HK....F..S.E...L.. SAMD9_Rano E.Y....S.....M......E..GP.A.L.. QNFR..L.L......T..S.E... ..............PK.TV.S.ED...K........AI...Q.. SAMD9_Crgr ..Y..S.C.S..V....R.....S....... QNFR..LTL......I..T.E.................PK..V.S.EG...Q..S.....VI.T.Q.. SAMD9_Capo ..H.........VV.....I...GPSG..L. RD.EL.L.L......NQ.S.E................T. ..V.S.EY...T .I..FD..I.RMHL.. SAMD9_Soar ..SD -.............G...D.FNGT.S KDF..NI.LS.KI.LF.SK.EV.............V.N.NSP.E..ES..EQ.S.I..H.AAS -T.. 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa KLNFILANIILSCIQPTSRLVKPVEKLKDQLREVLQPIGLTYQFSEPYFLASLLFWPENQQLDQHSEQMKEYAQALKNSFKGQYKHMHRTKQPIAYFFLG SAMD9_Patr ..............K.. ................................................................................... SAMD9_Gogo ..............K...........................................Y......................................... SAMD9_Poab ..............K...........................R.. .....................K.....S...E....................... SAMD9_Nole ..............K...........................R.......................G.........E....................... SAMD9_Mamu ..............K...T......................HR.......................Q...A.. ...E....................... SAMD9_Bota .Q.........Y..K...KI...IK........L..QT.MS.RYP...................D.RL.EK..SS.E...R......Y............ SAMD9_Susc .Q.........Y..K...KI.M.IK...E.......QAETICRQ....................D.K..EK..RS.E...R......C............ SAMD9_Eqca .Q.........Y..K...KI...IR...........Q.....R.................H...D.K..EK..RS.E...R......Y............ SAMD9_Mylu .Q.........Y..K...KI.M.TK........I..Q..PS...................K...D.K..EKH.RS.Q...R......Y............ SAMD9_Orcu .Q............K ...KS..TIK......Q.I..QV..NCR.....................D.K..EK...S.E...R......Y............ SAMD9_Rano .Q.....S......N.I.KF...IK...E....I..............................D.K..EK.....EK..R....RLY............ SAMD9_Crgr .Q............N.A.K.....K...E....I..............................D.K..EK..W..ET..R....R.Y...........E SAMD9_Capo .Q............K.A.K....IK...EH..DI..QV.P.HR.....................D.KE.EK.V.S.EK...................... SAMD9_Soar .Q.......V.Y..K...KF.S..K...E....... V.TN.R.P.............T.N...D.K..EK..RL.ET..L.K.R..Y............

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1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa KGKRLERLVHKGKIDQCFKKTP DINSLWQSGDVWKEEKVQELLLRLQGRAENN CLYIEYGINEKITIPITPAFLGQLRSGRSIEKVSFYLGFSIGGPL SAMD9_Patr ..................E... .... ........................... ............................................. SAMD9_Gogo ...................... ............................... ............................................. SAMD9_Poab ..E..K............E... ............................... ............................................. SAMD9_Nole .....K............E..L ............................... ............................................. SAMD9_Mamu .....K............E... ..............A................ ..................T......... ................. SAMD9_Bota ..NSVN............E..A ...F..........K...K.....K..VG.. ..H.......................................... SAMD9_Susc ..NNMN..I.R......C...AA..K.F.........KE..K..F..K...... ..............................D.........T.... SAMD9_Eqc a ..NNMT............RN.S ..............RN........K...... .......V.D..........F........................ SAMD9_Mylu ..NNMN...........YG..L ...IW....E....K.........K...... ..........R.........W........................ SAMD9_Orcu ..NNMN............E..L .. ............R................ .......VSD...L......W.........D.............. SAMD9_Rano ..NSRN..I......EN.ENLS ....W.HT.K..E.K...D..V..P...... ................K.THF......................L. SAMD9_Crgr ..NNRN..I......EN.E.VS ....W.H..K..E.K...D........... N.............Q...TH.......................L. SAMD9_Capo ..TNMN.........E..GEMS ...F.....K..E.K...D............EK.F.................F........................ SAMD9_Soar .A.NIN............G.. -.......N....R.K...D.....N...... .V..D...K...I.....T.F..... ................... ....|.. SAMD9_Hosa AYDIEIV SAMD9_Patr ....... SAMD9_Gogo ....... SAMD9_Poab ....... SAMD9_Nole ....... SAMD9_Mamu ....... SAMD9_Bota ......I SAMD9_Susc .....VI SAMD9_Eqca ......I SAMD9_Mylu ......I SAMD9_Orcu ......I SAMD9_Rano ....K.. SAMD9_Crgr ....K.. SAMD9_Capo ....Q.L SAMD9_Soar ....QVI



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10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa MAKQLNLPENTDDWTKED VNQWL ESHKIDQKHREILTEQDVNGAVLKWLKKEHLVDMGITHGPAIQIEELFKELRKTAIEDSIQTSKMGKP -SKNAPK SAMD9_Patr ....................... .................................................................... -...... SAMD9_Gogo ....................... ..............G..... ...............................Q...T............ -...... SAMD9_Poab ....................... ..............G..............................................M...... -...... SAMD9_Nole .................E..... ..............G............................... ...................... -...V.. SAMD9_Mamu ........Q...........R.. ..............G....................................Q.............R.. -...... SAMD9_Bota ..A.................... .....E....A...A...S..I..Y.T.D...A....F.......Q.....LE.SSG.PF..C.S..G -.. SV.. SAMD9_Susc ..A.....K...........R.. ..........D...A......N..Y.T....I.I...F.......H.....ME.ST..PS.IC.RE.G -..DV.. SAMD9_Eqca ..A.................R.. .............IA...S..I....T.NN.IE............N.....Q..SS.G.L..N.KK.G -...V.. SAMD9_Mylu ..A.............D...R.. D.........K.............F.T.N..IE............Q...K.Q.ESS..P...CQKK.G -..KV.. SAMD9_Orcu ..E.PE..............R.. .....H....D..VA...S.....F...SD.IE........V...G..R..Q..SSK.P.R..EQK.G -...VSN SAMD9_Rano ..ET....K...........L.. .. .R.........MA...S.V.....N.KN..E..................Q..PK.NLTK.C.KT.G -R..I.. SAMD9_Crgr ..EK.......N........L.. .G.........T.ML...........D.N...E............A.....Q.SSSKQPT....RKRG -...I.. SAMD9_Capo ..E.PH....I.........R.. .............IA...S..T....N. SD......P.......VG..N..Q..SS..PV..P.AK.S -.T.T.G SAMD9_Soar ..A.P..SK.P.........I.. .E.N..K.Y....VA.....D...L.T.K......V......L..N.V.K.Q..SS.N....REKKTN -N..I.E SAMD9L_Hosa .S..VS...MIK.....H.KK.VN.DL..NEQYGQ..LSEE.T.L..QE.TEKD..E..LPW...LL.KRSYNK.NSK SP.SDNHDPGQLDN -.. -.S SAMD9L_Patr .S..VS...MIK.....H.KK.VT.DL..NEQYGQ..LSGE.T.L..QE.TEKD.IE..LPW...LL.KRSYNK.NSKSP.SDNHDPGQLDN -.. -.S SAMD9L_Gogo .S..VS...MIK.....H.KK.VN.DL..SEQYGQ..FSEE.T.L..QE.TEKD.IE..LPW...LL.KRSYNK.NSKSP.SDNHDPGQLDN -.. -.S SAMD9L_Poab .S..VS...MIE.....H.KK.VT.DL..NEQYGQ..LSEE.T.L..QE.TEKD.IE..LPR...LL.KRSYNKFYSKSP.SDNHDPGQLDN -.. -.S SAMD9L_Nole .S..VS...MIK.....H.KK.VT.DL..NEQYGQ..LSEE.T.L..QE.TEKD.IE..LPR...LL.KRSYNK.NSKSP.SDNRDPGQLDN -.. -.S SAMD9L_Caja .S..VT...MIK. ....H.KK.VT.DL...E.YGQ..LNEE.T.L..QE.TEKD.IE..LPR...LL.KRSYNK.NN.SA.SDNHDPGQLSH -.. -SS SAMD9L_Mamu .S..VS...IIK.....H.KK.VTDDL..NEQYGQ..LSEE.T.L..QE.TEKD.IE..LPR...LL.KRAYNK.NSKSP.SDNHDPGQLDH -.. -.S SAMD9L_Loaf .SE.VT...M.Q.....H.KK.VT.DL...E.YGQ..LN EE.T.L..QE.TEKD.T...LPR...LL.KRGYNK.NNSSP.SHN.DFEQLDH -T. -TS SAMD9L_Eqca .NE.A....M.K.....H.K..VTKDL..GE.YGQ..LNEE.T.L..QE.TEKD.IE..LPR....L.KRAYNR.NNSSP.SNN.D.GQLDH -T. -SS SAMD9L_Calu .NEEV....VV......H.K..VTKHLNV.E.YGQ..LREE.T.L..QE.TEKD.RE..LPW.S. LL.KRKYNK.NNSSS.SNN.D.GQLDH -T. -.S SAMD9L_Aime .SE.V....IIN.....H.K..VTKDL...E.YGQ..LTEE.T.L..QE.TEND.RE..LPR...LL.KRAYNR.NNSSS.SNN.D.GQVDH -T. -.S SAMD9L_Ereu .DE.V....M.Q.....H.K..VTNDLQ..E.YGQ..LCEE.T.R..QV.TEKD.IE..LPR...LL.KRK..S.NNLSSKN.N.N.GQLD H -AE -.S SAMD9L_Orcu .NE.VT...LVK.....H.KK.VT.DL.V.E.YGQ..LNEE.T.L...E.TEDD.KE..LPR...LL.KRACNK.LNSSP.SDN.D.GKLDN -I. -.S SAMD9L_Mumu .SG.VTQ.KLIK.....H.RK.VT.DLN.VE.YAQ..FKEE.T.M..QE.TE.D.RE..LPR...LL.KRMYNK.IS SP.SHN.D.RELND -K. -LS SAMD9L_Crgr .NE.VTA.KLVK.....Q.KK.IT.DLN..E.YA...FNEE.T.M..QE.TEKD.RE..LPR...LL.KRAYNK.SN .T.SDN.D..QLHN -K. -LS SAMD9L_Rano .DRHVTQ.KLIK.....H.RK.IT.DL...E.YAQ.VF.EE.T.M..QE.TEKD.RE..LPR...LL.KRMYNK.IS SP.GHN.D.RQLNN -KT -LS SAMD9L_Capo .NE.GT...MIK....DH.KK.IT.E L...E.YGQ..FNEE.T.L..QE.TEKD.KE..LPR...LL.KRMYNK.NTSFP.SDNPN.RQVHD -T. -SS SAMD9L_Soar .SEHT....M.KA......K..VT.VLQ..EEYGQ..LNEK.S.LA.QEITE.D.RE..LPR...LL.KRTYNK.NNISP.SNSPN.RPLDS -TN -VS SAMD9L_Modo ..E.T.....P....V...C..IT.KL..NS.YT...KKEE...KS..VSD. ND......K....L..INS....N. SS.CPK...EEQ.DKQTE -VQ

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110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa DQTVSQKERRETSKQKQKGKENPDMANPSAMS -------------TTAKGSKSLKVELI -EDKIDYTKERQPSIDLTCVSYPFDEFSNPYRYKLDFSL SAMD9_Patr .............. .R................ -------------.............. -...........L.......................... SAMD9_Gogo ................................ -------------.............. -...................................... SAMD9_Poab ........H.........D..........TI. ------------.I..S........M -..Q.............N...................I. SAMD9_Nole .......A.....................T.. -------------.I............ -..E...........................H....... SAMD9_Mamu ........S.........D..........T.. -------------.I..V.E....... -.... ................................I. SAMD9_Bota T. ---QKDGG... -..N.KKS.KV.D.TV. -------------.VT.......N.FM -..E..D..KK...TEP..MA.......D......N.K. SAMD9_Susc K. ---EKN.........D..TS.RVTD.TI. -------------.VTE..M..NN.FM -..E.ND.QKK.S.VE.M..P.......D.. ....N... SAMD9_Eqca T...M.E.NG........N..KS....APT.. -------------.VT.D.....N.FK -VNE..D...K...VE............D......N... SAMD9_Mylu T..LM.E.NE......H....KS....A.T.. -------------.V --.....S.F. -G.E..D...K.................D........C. SAMD9_Orcu K.PLV...K.D.......N...S.P.DG..A. -------------PGE.EP..P.T..TDN..ERGD...KL.AKEPS.R.H..NK.DEQW....H.I. SAMD9_Rano T. ---T.S....N.N.RAE..SCK.DT..V.P --EGNSQRTAASPE......E.ND.V -...AVQ QDK.A.PGP..IA...N..........H.I. SAMD9_Crgr T. ---T.I...P..N.R.E..T.K. DA..LR -------------PE...AE.A.N..M -...TEL QGK.SPPGP..IA...N..........H.I. SAMD9_Capo K.HLM...G......R.SD..ISHK. ----------------------.E...PDS..M -.NQP.D -----VKQP..LP...NT.........H.I. SAMD9_Soar V..L..S.NG.K..K...D..KS.TVDT.TTH -------------.V..S. ..VEN.F. -..D. --Q.K.L.TEQ..IP.......D.....EN.V. SAMD9L_Hosa KT ----.HQKNP.HTK.EE..SMSS.I DYDP --REIRDIKQEESIL ---MKENV.DEVANAKHKK.GKLKPEQ...MP....Q.HDSH..IEHYT. SAMD9L_Patr KT ----.HQKNP.HTK.EE..SMSS.I DYDP --REIRDIKQEESIL ---MKENV.DEVANAK.KK.GKLKPEQ. ..MP....Q.HDSH..IEHYT. SAMD9L_Gogo KT ----.HQKNP.HTK.EE..STSS.I DYDP --REIRDIKQEESIL ---MKENV.DEVANAK.KK.GKLKPEQ...MP....Q.HDSH..IEHYT. SAMD9L_Poab KT ----.HQKNP..TK.EE..SMSS.I DYDP --REIRDIKEQESIL ---MKENV.DEVANAK.KK.GKLKPEQ...MP....Q.HDSH..IEHYT. SA MD9L_Nole KT ----.HQKNP..TK.EE..SKSS.I DYDP --REIRDVKQQESIL ---MKENV.DEVANAK.KK.GKLKPEQ...MP....Q.HDSH..IEHYT. SAMD9L_Caja KT ----.HHKKP..TK.KEK.SMSSSI DYDP --REVRDIKEQKSIL ---MKENV.DEA.NAK.KK.DKLKPER...MP....Q.HDGQ..IEHYT. SAMD9L_Mamu KR ----.HQKDP..TK.EE..STSS.I DYDP --REVRDIKERESIL ---MKENV.EEVANAK.KK.GELKPEQ...MP....Q.HDSH..IEHYT. SAMD9L_Loaf KK ----.Q KKA. ----..KSISSSI DHDL --TEIGDIKERESIL ---MKENATNEVAATK.KK.NKVKTEQ...MP....Q.HDSQ..TEHYI. SAMD9L_Eqca KE ----.HK.KPQ.TK.E..KSTSS .I DHNL --RETRDTKEQESIL ---MKENA.NEVV TK.KQ.NKLQAEQ...MP....Q.HDSQ..IEHYI. SAMD9L_Calu KK ----.HPKKPQ.MK.EE.KSVLS.I DHDL --REARDTKEQESIL ---MKEDA.NEGATAE.QNEDKLGIKQ...MP....Q.H.SHC.IENSV. SAMD9L_Aime KK ----.HPKKPQKMK.EE.KSVLS.N DHDL --REMRDTKEQESIL --VKEDA.NEEVTTE.QNED.TETEQ...MP....Q.H.SQ..IEHSI. SAMD9L_Ereu KK ----.HQRK -H --KT..T LS.N DHDV --REIQNAKAQELA. ---TG.NAQDEVGITEEK .KKRKIVQS..MP....Q.HASHH.TEHYI. SAMD9L_Orcu KK ----KQQKKP...S.EEGTSMSL.I DHDL --RETTEIEVQESIP ---LKEKA.DETVNAA.K ENAIQTER. ..MP....Q.HDSQ..IEHYI. SAMD9L_Mumu TK ----.QQTK --TKNEE..SVSS.S DHGL --RETGQNEEQEPSL ---TKENM.GDVV TK.MEDNKPKPEQMS.TP....S.CDVKQ.IEHSI. SAMD9L_Crgr IK ----.HPKK --TNNEE.KLISS.S GHDL --REMGLNTEQEPSL ---LKEKA.SDVL TK.MEGNTAKPEQMS.MP....S.HDDR..IERYI. SA MD9L_Rano IN ----.QPKK --SNSEE..SISS.S DFGL --RETGQNEEQEPSI ---MKVNT.GDV. TK.MKDNMPK.EQMS.MPH..NFAHDAK..IEHSI. SAMD9L_Capo KK ----.HQKKP ---NLEKELMPSSI DQDL --SESRNIKDQDSIP ---MEENAANEVSNT..KK.NKLKTEN...PP....N.HDGQ..IEHYI. SAMD9L_Soar KK ----KSKKN.Q -NTKE.KLVSS.S DHDL --KESVNTKEQESVP ---IE.DS.DYQGIPE.Q .GKLKKEQS...P....Q.HDSR..VEHQI. SAMD9L_Modo -W.T.EKN.KR -----RR --HHTD. KNDTTTQDEEVYESPKTNPS ---KGTESNAEET -----.DNL.FRGQ..QT....K.HKSF..REQTII

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210 220 230 240 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa Q PET GPGNLIDPIHEFKAFTNTATATEEDVKMKFSNEVFRFASACMNSRTNGTIHFGVKDKPHGKIVGIKVTNDTKEALINHFNLMINKYFEDHQVQQA SAMD9_Patr .................................................................................................. SAMD9_Gogo ............................. ..........................................S.......................... SAMD9_Poab .........................................................I.............S.......................... SAMD9_Nole ....................................................... ................S.......................... SAMD9_Mamu .....................E.............................................V...S.......................... SAMD9_Bota .....L...............E.G....A..................................T...VEF.TI......D. .....HQ........K. SAMD9_Susc .....L...............E...K.........................................VE..TV....F.D...M..HQ........K. SAMD9_Eqca .....L...............ER.R...I......................................V..STV...T..D......QQ........K. SAMD9_M ylu .....L..........E....KN............................................VN..SV......D......HQ........K. SAMD9_Orcu ... ------------------------------------? .............L...... .Q...V.LASI..N...D..DE..P....E...... SAMD9_Rano .....L.....V.....L...E......I............................Q...Q.R...MEL.TV..D...D......VQ...V...... SAMD9_Crgr .....L.....V.........EK.S...I.......I........................Q.R...VDLSTV..DT.........DQ...A...... SAMD9_Capo .....Q...........L.D.... ..K.I.......T..............................M...SV......E...S..PQ...A...... SAMD9_Soar .....L............K.IEKG.K..I.I.......K..................E.......I.VRFIAI...V..D...QI.HQ...EG...K. SAMD9L_Hosa ....AL...........L...E....V.I..................... .............E...V.I.SKA -.F.D...V..K....ESEINE. SAMD9L_Patr ....AL...........L...E....V.I..................................E...V.I.SKA -.F.D...V..K....ESEINE. SAMD9L_Gogo ....AL...........L...E....V.I..................................E...V.I.SKA -.F.D...V..K....ESEINE. SAMD9L_Poab ....AL...........L...E....A.IN.................................E...V.I..KA -SF.D...V..R....ESEINE. SAMD9L_Nole ....AL...........L...E....A.I..............................N...E...V.I.SKA -.F.D...V..K....ESEINE. SA MD9L_Caja ....SL...........L...ER.....I........L.........................E...VNI.SKD -VF.D......K....ESDINE. SAMD9L_Mamu ....AL...........L...E....A.I..................................E...V.I.SKD -.F.D......K....ESEIN.. SAMD9L_Loaf .....L...........L...D....K.IH.................................E...V..ASKD -DF........KQ....SDIKE. SAMD9L_Eqca .....L...........L...E....K.I.......A.....................N....E...V...SKD -SF.D...V..KQ...ESEIKV. SAMD9L_Calu .....L...........L...EA. ..K.I.............................NR...Q...V..A.KD -.F.D...I..RQ...ESEINEV SAMD9L_Aime .....L...........L...EA...K.I.............................N....Q...V...SKD -.F.D...V..RQ...GNEINE. SAMD9L_Ereu .....L...........L...E....K.I..................... .......L.....E...V..RSKD -.F.D......KQ...ESEIKE. SAMD9L_Orcu .....L...........L....A.....I..................................E...V.ISSKD -V......V..K.....SDINE. SAMD9L_Mumu RVA....L...............KK... ..I.......T....A.....................E...VQ..SKD -IFV....T..T.....SEISE. SAMD9L_Crgr .VA....L.....V.....L...E......I.......I..........H...............E...V.IPSKD -VFVD...K..K.....SDIKE. SAMD9L_Rano RVA....L.....V.........E...Q.QM.......T....A....T..... ...........E...VQ..SKD -IF.....V..T.....SDIHE. SAMD9L_Capo .....L...........L...EN.....L.......T..........................E...V...SKD -.F.D......K....ESEISE. SAMD9L_Soar .....R...........L...ESVE.K.I............................L...Q.E...V...RKD -SF .D...K..KH....SEIKE. SAMD9L_Modo ....SF...........L...ER.....IL................C............S...E...L.L.EED..KFVD..YKK.EV...KDD.S..

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310 320 330 340 3 50 360 370 380 390 400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa KKCIREPRFVEVLLPNSTLSDRFVIEVDIIPQFSECQYDYFQIKMQNY -NNKIWEQSKKFSLFVRD GTSSKDITKN ---KVDFRAFKADFKTLAESRK SAMD9_Patr ................................................ -...T.................R..... ---................... SAMD9_Gogo ................................................ -...T.....N................. ---............ ....... SAMD9_Poab ................................................ -...T............E.......M.. ---.....T..T.......... SAMD9_Nole ..................A............................. -...T..............P.....M.. ---................... SAMD9_Mamu .... ........................................I... -...T........V...........M.. ---.A...T............. SAMD9_Bota .N..........................VV.KY...EV.........C -S.T..K..PN..V.....A.....M.S ---NM..K...L.L.R...... SAMD9_Susc .N...............IP.........VV .KY...EQE......... -S..T.K..P...V.M...A....VM.. ---N.E.KT..L.L.K...A.. SAMD9_Eqca ..................P.........V..KY...ED...R..I..C -D.NT.K..S...I.....A.....M.. ---NA..KE..L.L.....A.. SAMD9_Mylu ..................P.........V..KY...EH.........C -...V...R.H..V.....A.....M.. ---....KI.QLGLEA...H.. SAMD9_Orcu .S...V.........D..V.........V..HY.V.GH......K.I.DN...K....S...VL....ARTVN.V.T ---.T..KM..LNL........ SAMD9_Rano ..................P.NK.... ......Y...KE.F.....HWH -KDET.Q.IP.Y.V.....PK....IG. ---.A..K...L.L.A..D... SAMD9_Crgr ..................P.NK......VL..H...KE........HL -KSQT.Q..S.H.V.....PQ....IG. ---.T..K...LGL.EV.D... SAMD9_Capo ..................V.....V...VV..Y...KH...R.T.... -.H ET.QP.S.Y.V.I...PNT.N.I.. ---....E..QL.L.SV.T... SAMD9_Soar .N............T.GIP...Y........KY...EH.........F -...T.NK.SR..V.....A.C..LM.T ---PA..K.Y.S.L........ SAMD9L_Hosa ..............Q.N.P.........T..KH.I.NDK..Y.Q..IC -KD...K.NQNL.....E.A..R..LA.S KQRD...K..LQNL.S.VA... SAMD9L_Patr ..............Q.N.P.........T..KH.I.NDK..Y.Q..IC -KD...K.NQNL.....E.A..R..LA.SKQRD...K..LQNL.S.VA... SAMD9L_Gogo ..............Q.N.P.........T..KH.I.NDK..Y.Q..IC -KD...K.NQNL...I.E.A..R..LA.SKQRD...K..LQNL.S.VA... SA MD9L_Poab ..............Q.N.P............KH.I.KDK..Y.Q..IC -KD.T.K.NQNL.....E.A..R..LA.SKQRD...K..LQNL.S.VA... SAMD9L_Nole ..............Q.N.P..........N.KH.I.KDK..Y.Q..IC -KD...K.NQNT.....E.A..R..LA.SKQRD...K..LQNL.S.VA... SAMD9L_Caja ..............Q .N.P.......I....KH.V.KDM..C.R..S. -TD.K.K.N.NS.....E.A..R..LADPNKRD...K..LQNLES.VA... SAMD9L_Mamu ..............Q.NMP............KY.I.KDK..Y.Q..IF -KD...K.NQNS.....E.A..R..LA.SKQRD...K..LQNL.S.VA... SAMD9L_Loaf E....Q.......QQ.N.P..K......V..R H.V.KDK..Y.N..IC -KDGS.R.D.DY.....Y.A.....LA.VRQRDA..K..TLN.ESVVA... SAMD9L_Eqca ..............Q.N.P.........V..KH.I.KEK..Y.....C -K.EK.K.NEDH.......A.....LA.GKQRDT..K..TQNL.S.VT... SAMD9L_Calu ..............Q.N...........V..KH.V.EKK..L.R...C -KSET.KPN QDL.......P.....LA.VKQRELNLK..LQNLTSVVA... SAMD9L_Aime ..............Q.N.P.........V..KY.V.GEK..L.R..SC -K.ET.KPNQDL.......P.....LA.VKQRDIAYKE.SQN..SV.T... SAMD9L_Ereu ..............Q.Y.P.........V..KH.I.KEK..YTN..S. -K.DT.K.NQES.......A..V..LA.GKQRDKN .KT.LQNL.S...D.. SAMD9L_Orcu ..............Q..MP.........V..KH.V.KEK..Y....SC -KDNV.K.NQEY...I.E.A..R..LA.AKQRDP..K..TQNL.S.VA... SAMD9L_Mumu RA............Q.N.Q.N.......V..RH.I..EK..Y.M..SS -TG.T.K...DT.....E.A...N.LG.PNQRDRE.KK.LE.L.MWTA... SAMD9L_C rgr RE............Q.N.P.........V..KH.I.KEK..Y.I..S. -TD.T.K...ENC....E.....N.LA.AKQRDRE..K.LENL.AWTA... SAMD9L_Rano RA............L.N.Q.N.......V..KH.I..EK..YVML.TC -TGTT.K...DT.....E.A..R..LG.PKQRDRE.KK.LENL.MSIA... SAMD9L_Capo Q....Q...ID...Q.N.... ..........KY.V.KEK..Y..L.SL -TD.T.K.NQNL.......A.....LA.VKRREIE.K..YENL.SC.AA.. SAMD9L_Soar R.............Q.N.S.........V..KH.I..EK..YT...EL -KDG..K.TENT.....E.A.T...LA.ERTRDAG.K..LQ.L.S.ITM.. SAMD9L_Modo .Q...P........K.NIV.......I..V.KH.I.ENK...V...I F -KENK.GKNAEL..Y........N.YA.PKQKDGNIK..L.ELEKCVKL..

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410 420 430 440 450 460 470 480 490 500 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa AAEEKFRAKTNKKEREGPKLVKLLTGNQDLLDNSYYEQYILVTNKCHPDQTKHLDFLKEIKWFAVLEFDPESNINGVVKAYKESRVANLHFPSVYVEQKT SAMD9_Patr .............. ...................................................................................... SAMD9_Gogo .................................................................................................... SAMD9_Poab ........................................ .........................................................E.. SAMD9_Nole ..........................................................................K......................E.. SAMD9_Mamu .................................................................. ............QD..A..........I...G.. SAMD9_Bota E....C.V......S.....................DW....I.....T.I.....................VSK........T..V......L...G.. SAMD9_Susc E....C.L......S............K........DW....I.....T.I.....................VSE...........V..... .L...G.. SAMD9_Eqca E....Y.L..S...N.....................DW..........N.I.....................VS...........IT......L.M.G.. SAMD9_Mylu E....C.I...N.NS.....IE.....K.........W..........N.I...............D.....VSK...N......IV...L..L...G.. SAMD9_Orcu E.. DNCT....NRH ..FR..E..............DW..........N.IQY...................ES....RSF....L.......Q.S.G.. SAMD9_Rano E..K.SKV.SDGSNSQ.Q..ID.....K......H..W..................................ES......F.G..........LFI.E.P SAMD9_Crgr E..T..KE.PEGNKS..Q..ID....... ........W...I......N.NS..E.................ES......F.Q..........QF..E.. SAMD9_Capo E....CKL....NNS..S...T..............DW............II............I.......KTK..L..F....A....S..QF..E.. SAMD9_Soar E...N..L..S...S......RM.............SL.........QN.VN... ......L..........ASK..................Q...E.. SAMD9L_Hosa E...EYGM.AM...S..L......I..R.S......DW..........N.I.....................M...................NQ.EDKT. SAMD9L_Patr E...EYGM.AM...S..L......I..R.S......DW..........N.I........M............M........ ...........NQ.EDKT. SAMD9L_Gogo E...EYGM.AM...S..L......I..R.S......DW..........N.I.....................M...................NQ.EDKT. SAMD9L_Poab E...EYGM.AI...S..L......I..R.S......DW..........N.I.....................M...................NQ.EDKT. SAMD9 L_Nole E...EYGM.AM...S..L......I..R.S......DW.V........N.I.....................M.........Q.........NQ.EDKT. SAMD9L_Caja E...EY.M.AK...S..L.........R.S......DW....I.....N.I.....................QS.......R..........NQ.EDKTM SAMD9L_Mamu E...EYGM..M...S..L ......I..R.S......DW..........N.I.....................V...................NQ.EDKT. SAMD9L_Loaf E...EYGV.A....S..L..A...I.SR.S..D...DW.......S..N.......................VSK.................DQ.EKKAA SAMD9L_Eqca E...EYEV.A....S..Q......I..R.S......NW....I. ....N...N...................QSK..A..F.K..........Q.E.KS. SAMD9L_Calu E...EYEM.ADR..S..Q......I..R.S......NW..........N...N......M.L..........VSK.......K..I......NQFE.KTN SAMD9L_Aime V...EYEV.A....S..Q......I..R.S......SW..........N...N...... M............VSK.......K.........NQFE.KSN SAMD9L_Ereu D..KEHEM.A....C..Q..I...I....S......NW........N.S.R -...................ESK..A.T.R........ .NQ.A.KT. SAMD9L_Orcu E...EYGR.AT...S..L......I..R.S......SW..........N.......................G............ ........Q.EDKTS SAMD9L_Mumu ....EL.M VT...S..L..S....RH.GS..E...DW......T.A.T.LE..E.I..M.L....D...Y.H.K......R...I....L..H.E.K SAMD9L_Crgr E...QQ.M VT...S..L..A....RH.GS......DW......N.A.N.IE.........L....D...Y.Q.K......R...I....L..Q.E.K S SAMD9L_Ra no .. .ECMV VS..DS..L..S....RH.GS..K...DW......S.V.T.ME....I....L....D...Y.H.K......R........L..H.E.K SAMD9L_Capo D...EHQM..T.R.S..L......I..R.S......NS..........N.MQ....................R....A...QK.....M...NQ.E.K.. SAMD9L_Soar E....DEV.V..Q.S..Q..I. ..I..R.S..S...NW..........N...Y.......S.....D.....VSK....VFQ...........Q...G.S SAMD9L_Modo E. .DHQP.EI.T.N..T...H....... ......D.....V..S..S.IE........E...........LNT..A...R.T.A......NQ.QDN..

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510 520 530 540 550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa TPNETISTLNLYHQPSWIFCNGRLDLDSEKYKPFDPSSWQRERASDVRKLI SFLTHEDIMPRGKFLVVFLLLSSVDDPRDPLIETFCAFYQDLKGMENIL SAMD9_Patr .................................................................................................... SAMD9_Gogo ............................................................................. ....................... SAMD9_Poab ....M.....................N............................R............................................ SAMD9_Nole .S..M............................L.................................................................. S AMD9_Mamu ....K..................S.......................................................................I.... SAMD9_Bota T.K.TS....Q.L..............E...L.A......K..E.........Q............................................ SAMD9_Susc ..K.IS....Q. L..................L........K..E.......I.............................................M SAMD9_Eqca .S..K.TS....Q..........I.........L........K..E...................................................... SAMD9_Mylu ....K..S....Q....................L...... K.K..E............V....................................I.... SAMD9_Orcu .T..M..N....Q..........S..GI.....L..I........E............V..K...........P.......................... SAMD9_Rano .VS.K..S....Q.............E.D..Q.L..I........E..R......RD......... .........E.................N...... SAMD9_Crgr .L..K..S....Q..........S..E.D....L..I........E..R....F.RD....K.R.........P.............G............ SAMD9_Capo .TE.K..S....QK.........S.......E.V..V...K....E.........R......................K........T.... .S...... SAMD9_Soar .TD.K..S...HQ..... .........D....L.. ???????????????????????? ... -... ....P.... .. Y............L.... SAMD9L_Hosa NMW.K.......Q..........S..K..T...LE.HL.......E.....L...D.N..T..............ES.G.......W....A......M. SAMD9L_Patr NMR .K.......Q..........S..K..T...LE.HL.......E.....L...D.N..T..............ES.G.......W....A......M. SAMD9L_Gogo NMR.K.......Q..........S..K..T...LE.HL.H.....E.....L...D.N..T..............ES.G.......W....A......M. SAMD9L_Poab NMR.K.......Q..........S..K.. TH..LE.HL.......E.....L...D.N..T..............ES.G.......W....A......M. SAMD9L_Nole NMR.K.......Q..........S..K..T...LE.YL.......E.....L...D.N..T..............ES.G.......W....A......M. SAMD9L_Caja NMR.K..A....Q..........S..K..T...LEAHL.......E.....L... D.N..T.R.........FT.ES.V.......W....A......M. SAMD9L_Mamu NMR.K..A....Q..........S..K..T...LE.HL.......E.....L...D.N..T..............ES.G.......W....A......M. SAMD9L_Loaf .IR.M.F.....E..........S..NN.T...L..HL.......E.....L...D.N..TK.........F...ES.G.. ..........A........ SAMD9L_Eqca PMR.K..S....Q..........T..KD.RH..LE.HL.......EI....Q...D.NV.T..R.........A.ES.G.....S......A......M. SAMD9L_Calu NIR.K..S....Q.T........S..KN.S...LE.HL.H.....E.....L...D.N..T............P.ES.G............A........ SAMD9 L_Aime NIR.K..S....E..........S..KN.S...LE.HL.H.....E.....L...D.N..T..............ES.G............A........ SAMD9L_Ereu .MR.K..S ? S.W ---------------------E.RQ..E....GRA..NY...D.N..T............P.ES.G.....P..T.FKV.N..Q..M SAMD9L_Orcu SLR.K.......Q.... V.....S..T..T....E.HL.......E.....F...D.N..A..............ES.G............A......M. SAMD9L_Mumu .IA.K....K..E..........V.. --SCQ.LE.HL...D...G..R......D.N.IVK..V........PIENQK............VFN..D.M. SAMD9L_Crgr .IE.K....K.FE.............KNDSCQ.LE.HL...D. ..G..R......D.N..V...V........P.NNQK............VFN..D.M. SAMD9L_Rano .IE.K....K..E..........V.. --SCQ.LE.HL...D...G.........DGN.IA...V........P.ENQK...L........VFN..D.M. SAMD9L_Capo .MRQK..S....E.T........S..QN.T...LEAHL...D..CE.........D.NL.AK....... ......ESLG..FT........A.R....M. SAMD9L_Soar .Q...VCS....Q.T........S..KGAV...LE.HL.NKN...E.T...L...D.S..A............P.ES.G........V..KA.N...... SAMD9L_Modo SIS.K.ESF..FK.T........A.FNT.E.Q.LEL...IKK..TK.........C..V.QP.R.........P.E..K..F....ST...E.G. ..D..

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610 620 630 640 650 660 670 680 690 700 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.. ..| SAMD9_Hosa CICVHPHIFQGWKDLLEARLIKHQDEISSQCISALSLEEINGTILKLKSVTQSSKRLLPSIGLSTVLL KKEEDIMTALEIICENECEGTLLEKDKNKFL SAMD9_Patr .................................................................... ............................... SAMD9_Gogo .................................................................... ............................... SAMD9_Poab .................................................................... ............................... SAMD9_Nole ....Q..................... .......................................... ............................... SAMD9_Mamu ....Q............................................................... ........S...................... SAMD9_Bota ....DSR.C...........TTQ...L.N..V.S.................. ....F.....S..... ............L......D.I.....K.L. SAMD9_Susc ...A.SR.C...........A.QE..L.............................F...V.S..... .R..........L.............EK... SAMD9_Eqca .....S..C.R.........T.Q...L.N........................L..F.....S..... ........S ...L..............K... SAMD9_Mylu .V...S..C.R.........T. ...L......S.................K..Q.F.....S..... EM..........L.....K.....N...... SAMD9_Orcu ......Q.C...........T.E...L.N......N.Q..................F.....S..... ..Q..T......L......D...KD..K... SA MD9_Rano ........Y.V......V..SSQ...L...S.FS.N.A...........L......F...V.S..... .R..E.......L.............RE.LF SAMD9_Crgr ..S.FSS.Y.V.........AGQ...L.NRS....N.A...........L......F.....ST.... .... .......L...........N..K.LF SAMD9_Capo ....Q.N.C...... .....TNE...L.DK.....N..Q......N...I....E......CS..... ..... ......L.V........H...T.LQ SAMD9_Soar V. LNSQLC.I.........KIQ...LAN......N.................V.K......S..... ............L......D.D......... SAMD9L_Hosa ..S.NS..Y.R.....QT.MKM E..LTNHS..T.NI.LV. S.........R..R.F..AR.S.S.I.E..K..VL.....L.....TE.DI....S... SAMD9L_Patr ..S.NS..Y.R.....QT.MKM E..LTNHS..T.NI.LV.S.........R..R.F..AR.S.S.I.E..K..VL.....L.....TE.DI....S... SAMD9L_Gogo ..S.NS..Y.R.....QT.MKM E..LTNHS..T.NI.LV.S.........R..R.F..AR.S.S.I .E..K..VL.....L.....TE.DI....S... SAMD9L_Poab ..S.NS..Y.R.....QT.MKM E..LTNHS..T.NI.LV.S............R.F..AH.S.S.I.E..K..VL.....L.....KD.DI...ES... SAMD9L_Nole ..S.NS..Y.R.....QT.MKM E..LTNHS..T.NI.LV.S...........PR.F..AH.S.S.I.E..K.. VL.....L.....RD.DI...ES... SAMD9L_Caja ..S.SS..Y.R.....QT.MNI E..LTNHS..T.NI.LV.SS...........R.F..AH.S.S.I.K..K..AL.....L.....RD.DI...ES... SAMD9L_Mamu ..S.NS..Y.R.....QT.MKM E..LTNHS..T.NI.LV.S............R.F..AR.S.S.I.E..K..VL.....L.....RD.DI...ES... SAMD9L_Loaf ....KSQ.Y.R.....RT..TI A..LTNHS..T.N..Q..S........I...R.F...R.S.S.I.E.....LF.....L.....KD.DI....S... SAMD9L_Eqca ....NSQ.Y.R.....QT..TV A..LANHS..T.N..L..S......P..E..R.F...S...S.I.E......L.....L.....RD.DI...EF..Q SAMD9L_Calu ....NS..Y.R .....QT..TI A..LTNHS..T.N..L..S......P....LR.F...H.F.SII.E......L.....L.....KD.DI....S..Q SAMD9L_Aime ....NSQ.Y.R.....QT..TA A..LTNHS..T.N..L..S......P.....R.F...H.FAS.I.E.....TL.....L.....KD.DI...ES..Q SAMD9L_Ereu ....T.Q.Y.R.....QT.FVM S..LTNHS..T... .LL.S..............F...R.S.S.I.E.....FL.....L.....GD.DI...EC..E SAMD9L_Orcu ....NSQ.Y.R.....KT..TV AH.L.NHS..T.N..LV.S.........R..R.F...R.S.S.I.E..K..TL.....L.....RD.DI...ES... SAMD9L_Mumu ....NSA.Y.Q.S...QV..EI K.DLAKHS..T.NI.LV.N........I...R. F...C.S.S.I.E.MD....S....L.....KD.DI...ESQ.. SAMD9L_Crgr ....N...Y.R.S...QV..EI KG.LAEHS..T.NIQLV.N........I...R.F...C.S.S.I.E.I...TL.....L.....RD.DI..NESE.. SAMD9L_Rano ....N.S.Y.Q.S...QV..EI K.DLAKHS..T.NI.LV.S........I...R.F...C.S.S.I.E.MD....S....L .....RD.DI...ESQ.. SAMD9L_Capo ....NSK.Y.R.....QT..TI G..L.KHSV.T.NI.L..S.......L....RKF...Y.S.S.I.E.....LL...Q.LS....KE.DI....S... SAMD9L_Soar ....NLD.YKQ.....K....E ES.LEDHT..T.N..FL.S.........R....F...K.S.S.T.E.....TF.T...L.....KD.DI..NAS..Q SAMD9L _Modo S.S.DQQ.GSQ..E...GKFSS I. LCNR..TT....QL........PQ.E.PI.F...N.S. II.Q..D..S.S....L.....KD.AI...E ..T

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710 720 730 740 750 760 770 780 790 800 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa EFKASKEEDFYRGGKVSWWNFYFSSESYSSPFVKRDKYERLEAMIQNCADSSKPTSTKIIHLYHHPGCGGTTLAMHILWELRKKFRCAVLKNKTVDFSEI SAMD9_Patr ...................... .............................................................................. SAMD9_Gogo ..............................................................................................A..... SAMD9_Poab ................................................ .................................................... SAMD9_Nole ..............................................................................................A..... SAMD9_Mamu ....................................................... C.......................D.................... SAMD9_Bota ....L...........T.........N............K..E...RG.....S.CV........................................... SAMD9_Susc ....L.....................N........ER..K..T...E..A.....CV...................V.... ............M...C.. SAMD9_Eqca D..T.....................K...L.........I..E....W.......CV.....................................M..... SAMD9_Mylu ....L.....................N............K..R..H.......S.CS........................................... SAMD9 _Orcu .......KA.................NH..A................W......ICA........................................... SAMD9_Rano ...T...K..................N...S.......VK..KK..EW....Q.VCA.....................................E..... SAMD9_Crgr ...TA..Q.......... ........N...S........K..KK..EW....Q.VCA......................D..............E..... SAMD9_Capo ...T................... ..Y...S........K.KE....Y..P..LMCV........................................... SAMD9_Soar A..TL....................KN...A........K.KD. ...W ....S.CV...Q..................N.................... SAMD9L_Hosa ...K....H.................N...D.....S..K.KDL.HCW.E.P..IFA...N...............V..D.K.N..........T..A.. SAMD9L_Patr ...K....H.................N...D.....S..K.KDL.HCW.E.P..IFA...N......... ......V..D.K.N..........T..A.. SAMD9L_Gogo ...K....H.................N...D.....S..K.KDLVHCW.E.P..IFA...N...............V..D.K.N..........T..A.. SAMD9L_Poab ...K....H.......C.........N...D.....S..K.KDL.RCW.E.P..IFA...N...............V..D.K.N..........T. .A.. SAMD9L_Nole ...K....H.................N...D.....S..K.KDL.HCW.E.P..IFA...N...............V..D.K.N..........T..A.. SAMD9L_Caja ..RK....H.................N.P.D.....S..K.KDL..CL.E.P..IFA...N...............V..D.KE...........T..V.. SAMD9L_Mamu ...K... .H.................N...D.....S..K.KDL..CW.E.P..IFA...N...............V..D.K.N..........TG.A.. SAMD9L_Loaf ..RK....H.....R...........N...A.....S..H.KDL..CW.E.P..VFA...N...............V..N.K.N.........AT..T.. SAMD9L_Eqca ...K...KY.................N...A.. ...S..K.KDL.LCR.Q.P..PFA..VN..............NV..D.K.N.........AT..A.. SAMD9L_Calu ...KL...H.................N...A.....S..K.KVL.KCW.E.P..EFA...N..............NV..D.K.N..........T..E.. SAMD9L_Aime ...T....H.................N...A.....S..K.KDL..CW...P..VFA.. .N..............NV..D.K.S..........T..G.. SAMD9L_Ereu ..RK....H.................N... .I...R..E.RSL.EDW.E.P..VFA...N..............NV..DF............AT..A.. SAMD9L_Orcu ...K....H.................N...A.....S.KE.KNL..CW.E.PR.VFA...N...............V..D.K.N. ........AT..A.. SAMD9L_Mumu ...K.R..H.....R...........N...A.....SF.E.TTL..Q....P..VFV.V.N...............V..D.KQ..........AT..V.. SAMD9L_Crgr K..KLR..H.....RA..........N...A.....G..E.TTL..Q....PE.VFA.V.N...............V..D.K...........AT..L.. SAMD9L_Ra no ...KLR..H.....R...........N...A.....NF.E.TTL..Q....P..VFA.V.N...............V..D.KQ..........AT..I.. SAMD9L_Capo ..RK.R..H.................N...A.....M.KN.KDL.ER..ECP..EFA...N..................D.KQ.Y........AI..E.V SAMD9L_Soar ..IK.N..Q.... S............NHT.A.....R..N.KKL.EDW.E.P..LFA...N..............NV...............I.A..A.. SAMD9L_Modo ..QMN..KH..L...........Y..NHT.D.IR..S..I..K...SL.N.PRESGAEV.N....................K.N.......S..FASED.

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810 820 830 840 850 860 870 880 890 900 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa GEQVTSLITYGAMNRQEYVPVLLLVDDFEEQDNVYLLQYSIQ TAIAKKYIRYEKPLVIILNCMRSQNPEKSAR IPDSIAVIQQLSPKEQRAFELKLKEI SAMD9_Patr ....................................................................K...K ......L................... SAMD9_Gogo .................................................................... ..... .......................... SAMD9_Poab .................................................V....................... .......................... SAMD9_Nole ..................I...................S.............................K.... .................... ...... SAMD9_Mamu .....N..........D........................................................ .......................... SAMD9_Bota .....N.....TT.N...L.I.................A..H..V.NR..............R.........K .S....L................... SAMD9_Susc ..... N.....TASS...L................F..A....T..N...............R.........K N.N...L..N..S...........K. SAMD9_Eqca ....AT......T.Q...L...........E.......S.....V.N.......................C.K NL.G..L..N..........F..... SAMD9_Mylu .K...N.....TA.H...L............ GDI....A......TS...............V.......C.K MS....L.....S............. SAMD9_Orcu .....N.....TT.H...L...............F...S.....V.E.H.......................K ....V.LLHH.............EK. SAMD9_Rano .....N......T.H...L...................T.....V...H........ .....T.........K N...V.LV....D..........T.. SAMD9_Crgr .....N......TS....L...................A.....V.N.H.............T.........K ....V.LVH...D............. SAMD9_Capo ....V.......T.H...L...................AA....V.Q...............A.........K ......LR. K..R..KE......... SAMD9_Soar .K.I.D.....TTSH...L............E......S..H..VSS.H.................D.R.C.KNF.N...L......S............ SAMD9L_Hosa A...IN.V..R.KSH.D.I............E...F..NA.HSVL.E.DL....T..........R..DE..K LA....LNY...S......GA..... SAMD9L_ Patr A...IN.V..R.KSH.D.I............E...F..NA.HSVL.E.DL....T..........R..DE..K LA....LNY...S......GA..... SAMD9L_Gogo A...IN.V..R.KSH.D.I............E...F..NA.HSVL.E.DL....T..........R..DE..K LA....LNY...S......GA..... SAMD9L_Poab A...IN.V..R.KSH.D.I. ...........E...F..NA.HSLL.E.DL....T..........R..DE..K LA....LNY...S......GA..... SAMD9L_Nole A...IN.V..K.KSH.D.I............E...F..NA.H.VL.E.DL....T..........R..DE..K LA....LNY...S......GA..... SAMD9L_Caja .......V..K.KSH.D.I............E...I..NV.HSVL. E.DL...NT.............DE..K SA....LNY...S......GA..... SAMD9L_Mamu ....IN.V..K.KSH.D.I............E...F..NA.HSVL.E.DL....T..........R..DE..K LA....LNY...S......GA..... SAMD9L_Loaf .D...N....K.TSH...I...........LE.ICI..DA.NSSL...GL....T.............DR.. K SA..V.LKY...........A..E.. SAMD9L_Eqca ..........K.TSH.D.I...........PE..CV..NA...IF.E.DL....T.............DE..K SAY...LMHR.........GD..... SAMD9L_Calu V....K....K.TSHED.F..........D.E..CV..NA.DSILGE.GL....T.............DET.K LA..V.LTY...S.......A..E .. SAMD9L_Aime V....K....K.TSPED.F..........D.E...V..NA.DSIL.E.GL....T.............DET.K LA..V.LTY...........A..E.. SAMD9L_Ereu .....N..A.K.SSY.D.I............E..FV..NA.HSIL.E.DL....T.............DE..K LD...VLKY......K...ST..... SAMD9L_Orcu .....N... .K.TCH.D.I............E...I..NA.NSIL.E.DL....T.............DE..K LI....LKY..........GA..E.. SAMD9L_Mumu ....SK.MS.K.TSHEDFI............E.A.I..NA.NAF..E.GL....T.............DE..K LAN..SLKY...........A..Q.. SAMD9L_Crgr ....SK..S.K.TSH.D.I............E.T. I..NT.NSF..E.GL....T.............DA..K LA...SLKY...........A..Q.. SAMD9L_Rano ....SK..S.K.SSH.D.I............Q.T.I..NA.NSF..E.GV....T.............DE..K LA...SLKY......K....A..Q.. SAMD9L_Capo ...IIH.V..K.TSH.D.I............E.I.I..NA.HSVL..RDLQ...T..V... .......DQ..K LA...SLKYA..A.......A..E.. SAMD9L_Soar .....K....K.SSH.D.I...........LE...N..DA.HSSL.E.DV....T..............E..K LA....LTN...L..R...GA..... SAMD9L_Modo .A..RT.V...SEDQHS.M..........D.EA.SD.RNH.IS.LEEA..Q..T................D.K N...V...NK..S ......VK.F...

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910 920 930 940 950 960 970 980 990 1000 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa KEQHKNFEDFYSFMIMKTNFNKEYIENVVRNILKGQNIFTKEAKLFSFLALLNSYVPDTTISLSQCEKFLGIGNKKAFWGTEKFEDKMGTYSTILIKTEV SAMD9_Patr .............. ...................................................................................... SAMD9_Gogo .................................................................................................... SAMD9_Poab ........................................ .........T.................................................. SAMD9_Nole ........................M........................................................................... SAMD9_Mamu .................................................................. .R................................ SAMD9_Bota ED.....Q..............K...D.......E...S.................................TS.......................... SAMD9_Susc E......K............D.K...............C.................................T..........L........ ........ SAMD9_Eqca E...E..K.............EK...............S............................N....T.......V...............R... SAMD9_Mylu E...................D.K...............S.................................TT...Y.....L................ SAMD9_Orcu .KEYD...............................DPS............................N....A..........L.E..........R... SAMD9_Rano ......V..............................VT.......A......AF............L....S....Y...................S.. SAMD9_Crgr .KH.E...............DRK... ...........VA.......A......T.............YY...S........................S.. SAMD9_Capo ..H......................KK..........KS.................SG..........Y...A..........L............Q.K. SAMD9_Soar EK....V.............D.M......K........C............. .....E.........Q....T......EL..L..............D. SAMD9L_Hosa EK....C.N........S..DET.............DVDS...Q.I......S...T.S...V....I....IYTSTP.EP.SL.........L...... SAMD9L_Patr EK....C.N........S..DET.............DVDS...Q.I......S...T.S...V....I....IYTSTP .EP.SL.........L...... SAMD9L_Gogo EK....C.N........S..DET.............DVDS...Q.I......S...T.S...V....I....IYTSTP.EP.SL.........L...... SAMD9L_Poab EK....C.K.IP.....S..DET.............DVDR...Q.I..........T.S...V....I....IYTSTP.EP.SL.........L...... SA MD9L_Nole EK....C.N........S..DET.............DVDS...Q.I..........T.S...V....I....IYTSTP.EP.NL.........L...... SAMD9L_Caja EK....C.N........S..DET.............DVDS..VQ.I..........T.S...V....I....IYTSTP.EP.SL.........L...... SAMD9L_Mamu EK ....C.N........S..DET.............DVDS...Q.I..........T.S...V....I....IYTSTP.EP.SL.........L...... SAMD9L_Loaf EK....C.N........S..DDM..............VDS...Q.I..........INS...V....F....TSARTP.EP.TL.........L...... SAMD9L_Eqca EK....C.N........S..DEI....L ........DADS..TQ.I..........I.S...V....I....MCTSIP.VP.SL.N...A...L..N... SAMD9L_Calu EK....C.N........S....M...K..........VDS..GQ.I..........TES...V....I....IYTSMP.EP.SL.....A...L..N... SAMD9L_Aime EK....C.N......L.S...EI..............VDS..GQ.I........ ..T.S...V....I....IYTSTP.EP.SLD....A...L..N... SAMD9L_Ereu EK......N........S..DEM......G......DGDS..TQ.I..........T.S...VP...I....TYI.TP.KQ.SL.........L..NADI SAMD9L_Orcu EK...DC.N......L.R..DET..K..........DVYS...Q.I.......T..T.S...V....I....IYTSTP.K P.SL.........L..N... SAMD9L_Mumu EKE...C.N......L.G..DTT..K...K.T..DLDAKSRR.Q.I.Y........T.S...V....I....TYT.KYGKP.TV.KN......L..R... SAMD9L_Crgr EKE...C.N.....ML.GS.ETT......K.T..DLD.HSRR.Q.I.Y........T.S........I....IYTNKHGKP.SV..N......LV.R... SAMD 9L_Rano EKEY.DC.N......L.D..DTT..K...K.T..DLDAHSRK.QFI.Y.....T..I.S...V....I....TYTIKRGKP.TV..N......L..R... SAMD9L_Capo EK....C.N........G..DEK...........E...NS...Q.I..........T.S........I....IYTSVP.KP.SL..R....Y.L..S... SAMD9L_Soar EKE.....N......V. S..DDT.........F....ADS..TQ.I.Y........T.S...VP...I....RHTPSP.VP.SI.........LI..... SAMD9L_Modo EKK.E.CR.........E...RT..Q...KH....L.TDS..GQ.I...S.....IS.SDF.VQ......E.T.QR.N.KRGSL.N...N......C.KA

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1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa IECGN YCGVRIIHSLIAEFSLEELKKSYHLNKSQIMLDMLTENLFFDTGMGKSKFLQDMHTLLLTRH --RDEH --EGETGNWFSPFIEALHKDEGNEA SAMD9_Patr ................................................................... --.... --....................... SAMD9_Gogo ............................... ......H............................. --.... --....................... SAMD9_Poab .................K................................................. --.... --.S..................... SAMD9_Nole .................K....................................... .......... --.... --D...................... SAMD9_Mamu .................K................................................. --.N.. --....................... SAMD9_Bota V............P...IR......I..D.D....T.........Y...I.R....E.VQ......Q --.N.N --...M.T L..........E...V. SAMD9_Susc .............P...TR......I..N.D....I.........Y...I..D...E..Q......Q --.ND. --.....T..........R....V. SAMD9_Eqca V...K........P...DR.....RT....D..............YE..L...R.SEHIQ......Q --.N.. --.....TL..............V. SAMD9_M ylu V...K........P...IR......I..D.D.....W......V.Y...I.R...F...Q......Q --.I.. --.....T..........E....K. SAMD9_Orcu L...M........P...SL......N..G.GRC.M..........YE..I........VQ....... --.N.. --.D...................D. SAMD9_Rano ....S........H...TL. .....R..N.S..K.VM........Y...I.....Y...Q...I..Q --.N.. --.R...................D. SAMD9_Crgr ....S.W......HE..SA......R..N.S..E.VMN.......Y.M.I.....F...Q......Q --.N.. --.....................D. SAMD9_Capo E...T........P...IL.........D.D.............. Y...L.R...F...Q......E --.S.Q --.....T................. SAMD9_Soar V...C........P...LR......L..Q...........R....Y.R....T..I..IQ....I.Q --.N.. --....ET................. SAMD9L_Hosa A.Y.R.T......P...LYC.K..ER....D.C..A.NI.E....Y.S.I.RD..QH.VQ......Q --. KVY --GD..DTL...LM...QNKD ---SAMD9L_Patr A.Y.R.T......P...LYC.K..ER....D.C..A.NI.E....Y.S.I.RD..QH.VQ......Q --.K.Y --GD..DTL...LM...QNKD ---SAMD9L_Gogo A.Y.R.T......P...LYC.K..ER....D.C..A.NI.E....Y.S.I.RD..QH.VQ......Q --.K.Y --GD..DTL...LM...QNKD --SAMD9L_Poab A.Y.R.T......P...VYC.K..ER....D.C..A.NI.E....Y.S.I.RD..QH.VQ......Q --.K.Y --GD..DTL...LM...QNKD ---SAMD9L_Nole A.Y.R.T......P...LYC.K..ER....D.C..A.NI.E....Y.S.I.RD..QH.VQ......Q --.K.Y --GD..DTL...LM...QNKD ---SAMD9L_Caja S.Y.R.T. .....P...VYC.K..ER....D.C..A.KI.E....Y.S.I.RD..QH.VQ...V..Q --.R.Y --GD..DTL...LM...QNKD ---SAMD9L_Mamu A.Y.R.T...V..P...LYC.K..ER....D.C..A.NI.D....Y.S.I.RD..QH.VQ......Q --.K.Y --GD..DTL...LM...QNKD ---SAMD9L_Loaf A.Y.R.T......P...IAC.K..E.....D... .A.NL.R.K..YVS.I.RE...H.VQ......Q --.R.Y --GD..DTL...L....PNSK ---SAMD9L_Eqca T.YRR.T......P...ISC.K..E...D.D.CK.A.KI.K....Y.S.I.RD..Q..VQ......Q --.K.. --GD..DTL...L....QNE. ---SAMD9L_Calu T.Y.R.T......P...ISC.K..EE..D...C..A.KI.N....YVS.I.RE..QH.VQ ......Q --.R.Y --GD..DTL.A.L....QNED ---SAMD9L_Aime A.F.R.T......P...I.C.K..EN..N...C..A.KI.K.D..YVS.I.RE..QH.VQ......Q --.K.Y --GD..DTL.A.L....ENE. ---SAMD9L_Ereu A.Y. FP......P...I.C.K........D..R.A.KL.S....YNS.I..D..QH.VQ......Q --.KGY --G...D M.. .L.D .Q ??? ---SAMD9L_Orcu S.Y.R.T......P...IHC.K..EEKH....HH.A.KL.N....Y.F.I.RD..QH.VQ......R --.K.Y --GD..DTL...L..D.QNK. ---SAMD9L_Mumu SDY.R.T.I....P...THC.K..EM..RMD.C..A.N..E..VLY.S.L.RD..KY.VQ......Q --.K.. --GA..DTL...L..E.QNE. ---SAMD9L_Crg r LDY.R.T.I....P...IHC.K..EM..GMG.C..T.NI.E.KV.Y.S.I.RD..KH.VQ......Q --.K.. --GA..DTR...L..E.KNE. ---SAMD9L_Rano SDY.R.A.I....P...IHC.K..EM.HGMD.C..A.N..E..I.Y.S.I.RD..KH.VQ......Q --.K.. --GA.IDTL......E.QNEQ ---SAMD9L_Capo A.Y.R.T......P...IHC.K. .ER...MD.C..A.NI.N.TV.Y.S.I.RD..QH.VQ......Q --.K.Y --GA..DTL...L..T.KNE. ---SAMD9L_Soar K.Y. .K..........LCC.K..E...Q.DM.R.A.N..R.K..Y.S.I..D..QH.VQ....... --.K.. --GD..DTL...L..D.KNED --I SAMD9L_Modo .D.KR.DR...V.P...SCC.....ITSEMKR.D.AVNL.K.DTLYSP. IRRD..VRIVQ....K.QKEYSK.TLISGKDMNTP....LMDIK.....DQ

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1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa VEAVLLESIHRFNPNAFICQALARHFYIKKKDFGNALNWAKQAKIIEPDNSYISDTLGQVYKSKIRWWIEENGGNGNISVDDLIALLDLAEHASSAFKES SAMD9_Patr .............. ...................................................................................... SAMD9_Gogo ...........................................................................RS..........N............ SAMD9_Poab .......G.R.......................D...... .E.................................R...........N............ SAMD9_Nole .......G....K...................................................M.......ER.............N............ SAMD9_Mamu .......G.......................................................... .......R.R....N......N...Q........ SAMD9_Bota .KN..RGG.R............T......E...DS..H..N...K...............F.......MDD.ER.RS...GE.SD.....VQ..N..... SAMD9_Susc .KN....G.RQ..S........S......ER..NS..H..NE..K......................... D.R.W...A...AD.....V. ..N..... SAMD9_Eqca .KE....G.R...S.............L.E...SS..K..N...K...H.....................D..K.R.....N.TD..E..VE.TN..... SAMD9_Mylu ..K....G.....................ER..TS..H..N...N...N...................T.N.ER.R....A..TE.....V...E..... SAMD9_Orcu .KM ..EKG.N...K....R....... --------------------------...........L.....D..RGNT..AN..TD..Q..V..AE..... SAMD9_Rano .K...H.ATR..D..............L.E...ES..L.....RK.A.N...............L...V.D.IK..I....E.TE.....K...E...D. SAMD9_Crgr .KD....ATR.................L. E...K...F.....RT...N....................DD.IR..S..AE..TD...S.V...D...D. SAMD9_Capo .KQ..CQG.E..K.......V....Y..RE...N...L...E..K...N.....................DTER.RD......ST.........N..... SAMD9_Soar .KE..I.GVD..KQ...............E...ER..E..NK..N...Y...... ..............DD..R.R....A..TM..E..V...N..... SAMD9L_Hosa I.K..SAGSR..PQ...............E...NT..D..R...MKA.K..............E.K..LDG.KNCRS.T.N..TH..EA..K..R..... SAMD9L_Patr I.K..SAGSR..PQ...............E...NT..D..R...MKA.K..............E.K..LDG.KNCRS.T. N..TH..EA..K..R..... SAMD9L_Gogo I.K..SAGSR..PQ...............E...NT..D..R...MKA.K..............E.K..LDG.KNCRS.T.N..TH..EA..K..R..... SAMD9L_Poab I.K..SAGSR..PQ...............E...NT..D..R...MKA.K..............E.K..LDG.KNCRS.T.N..TH..EA..K..R..... SAMD 9L_Nole I.K..SAGSR..PQ...............E...NT..D..H...MKA.K..............E.K..LDG.KNCRS.T.N..TH..EA..K..R..... SAMD9L_Caja I.M..SAGSR..PQ...............E...NT..D..C...MKA.K..............E.K..LD..KNCR..T.SQ.TH..EA..K..R..... SAMD9L_Mamu I.K..SAGSR..PQ... ............E...NT..D..R...MKA.K..............E.K..LDG.KNCRS.T.N..TH..EA..K..R..... SAMD9L_Loaf I.N..TAGSIQ.PQ..........Y....E...NT..H..N...K.A.K.......V........K..LD..KTCREVT.K..THF.ET..K..G..... SAMD9L_Eqca I.K..V.GSI..PQ...............E.K.NI..E..E.. QKRA.K...........H....K..LG..KNCKD.T.N..THF.EA..K..R...K. SAMD9L_Calu I.K..IAGAT..PQ..........Y....E.N..T..E..N...KKA.K..............Q.K..LD..KNSTD.T.N...C..EA..N..K...K. SAMD9L_Aime I.K..MAGTL..PQ..........Y....E.N.ST..D..NE..KKA.K..............R.K.RL D.MKNAKD.T.N...C..EA..N..K...K. SAMD9L_Ereu ???????? SN..PQ.......... L..HERN.NT..D..Y...RKA.K.............NEVK..LD..RSCKE.TI...MHC..A..K..R...T. SAMD9L_Orcu I.E..VAGCS..PQ...............E.N.ET..E..NL..TRA.K................K..LD..KNSKV.T.K..TH..EA..K..G ..... SAMD9L_Mumu T.K..IAGSD..PQ...............E.N.ST..V..NL..RKA.K..............ELKQ.LGK.KTC........AYF.EV..K..K..... SAMD9L_Crgr T.KI.TVGSD..PQ...............E.N.ST..V..NL..RKA.K..............E.KQ.LGK.ENCRL......TH..EI..K.TK..... SAMD9L_Rano T.K..T AGSD..PQ.............L.E.N.ST..V..N...RKA.K..............E..Q.L -----------------KV..K..K..... SAMD9L_Capo .KK..TRGSS..PE...............E.N.ND..T..NL..ERA.K..............E.KC. ------------------ET.GK.AR..R.. SAMD9L_Soar ..N..TAGSN..PK.................N .DT..F..T...TKA.N..............E.K..L.K.KNCKI..AS..TYF.EA..K..K...M. SAMD9L_Modo ..R..T.GTI..KDH.L.Y.........NENN.EA.IFY.EE..KRSVR..FV..........QLIFCNNKISQGKS.PCQE.EY..NI..K..N...D.

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121 0 1220 1230 1240 1250 1260 1270 1280 1290 1300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa QQQSEDREYEV -KERLYPKSKRRYDTYN IAGYQGEIEVGLYTIQILQLIPFFDNKNELSKRYMVNFVSGSSDIPGDP --NNEYKLALKNYIPYLTKLK SAMD9_Patr ........... -................................................................. --................... SAMD9_Gogo ........... -.......................................... ....................... --D.................. SAMD9_Poab ........... -.....L...........................................D....I.......... --................... SAMD9_Nole ........... -.........................................K.......D....I.......... --................... SAMD9_Mamu .....Y..... -.....Q...........................................D........G.....Q --.D..Q.............. SAMD9_Bota ..........G -...F.Q..........T...L..............F.............D.I..I...K....EA --...F..V...F.S...N.. SAMD9 _Susc .R......... -...F.Q...........................................D.I..I..TT...... --...F..V.N.F........ SAMD9_Eqca .......Q..G -...F.Q...................A...............S.......D.I..I.........S --S..F......F.....N.. SAMD9_Mylu .....YK.D.A -M..FN Q.......................A...................D.I..I..N....... --...F......F.......R SAMD9_Orcu .R........G -.....Q..........................................KD.......T......S --...F.SV...F.....N.R SAMD9_Rano ...T......G -..SV.Q......................... ...K.L...NS.....RQV.I..I..NC.....L ---..F.SV...F.S...N.Q SAMD9_Crgr .R.T......G -....NQ.........................K...........D.....D.I..I..NR.....L ---..F.I....F.....N.Q SAMD9_Capo .R........G -....GQ...........S..H........A...........K.....R.D.I..I.. N......L ---..F......F.....T.. SAMD9_Soar .R...YK.R.A -.GKFHQ...................................S....F.KD.I..I..T......S --...F....S.F.S...N.R SAMD9L_Hosa .R.TDSKN..T -ENWSPQ..Q....M..T.CFL...............T...HKE.....KH..Q.L..KWT..P.. --R..CY...SKFTSH. KN.Q SAMD9L_Patr .R.TDSKN..T -ENWSPQ..Q....M..T.CFL...............T...HKE.....KH..Q.L..KWT..P.. --R..CY...SKFTSH.KN.Q SAMD9L_Gogo .R.TDSKN..T -ENWSPQ..Q....M..T.CFL...............T...HKE.....KH..Q.L..KWT..P.. --R..CY...SKFTSH.KN.Q SAMD9L_Poab .R.TDSK N..T -ENWSPQ.YQ....M..T.CFL...............T...HKE.....KH..Q.L..KWT..P.. --R..CY...SKFTSH.KN.Q SAMD9L_Nole .R.TDSKN..T -ENWSPQ..Q....M..T.CFL...............T...HKE.....KH..Q.L..KWT..P.. --R..CY...SKFTSH.KN.Q SAMD9L_Caja .R.TDSKNC.T -ETWSPQ..Q...G...T.CF L...............T...QKE....ENP..Q.L..KWT..P.. --K...Y...SKFTSH.KH.Q SAMD9L_Mamu .R.TDSKN..T -E.WSPQ..Q....M..T.CFL...............T...HKE....IKT..Q.L..KWT..P.. --R...Y...SKFTSH.KT.Q SAMD9L_Loaf .R.NDSKD..P -EVWPLQ.YQ.K..M..T..FL........S.E....T.L.HKE... AQKPL.Q.L..IG..LA.. --E...H.T...FTS..QN.R SAMD9L_Eqca .E.TDRKN..T -EAW.PQ..Q.K.VM..T..FL........A......T.C.HKE.....KS..E.L..KGN.LTNS --KS..Y.V.RKFTS..QN.Q SAMD9L_Calu .E.T.RKG..TWADTWAKQTLQ.K.....T..FF........A......T.C.LKQ.....KA..E.L..KGM..TN. --KC.. Y...NKFTS..EN.Q SAMD9L_Aime .E.A.RKAC.T -EPWGPQ.LQ.K.....T..FV........A......T.C.LKE..F..KA.AD.L..KGMT.TN. --KC..Y...SKFTS..EN.Q SAMD9L_Ereu .E.TDKKDS.T -EAWSTQ..LK..EM..T..FL........A......T.C.HKE.K..Q.HT.E.L..KGN..EHS --KGK.Y..ISKFTSH.RN.Q SAMD9L_Or cu ...TDSKN..T -EAWSAQ..Q.......T..FF...............T...HKD.VSFLKP..Q.L..KG...F.A --R...Y...SKFTSL.QN.Q SAMD9L_Mumu .N..DSKD.GT -EAWSPQN.Q.....F.T..FF.......D...L...T.L.HKE..I..ES.AE.L..KGT.LS.. --KG..CVV.SKFTSL.QN.H SAMD9L_Crgr .E.TDNKD..T -EAWSPR..Q .......T..FF.......D.......T.P.HKES.IAIEA..Q.L..KGT.S .. --KD..GAV.SKFASH.QN.Q SAMD9L_Rano .E.TDSKD..I -EVWSPQ..Q.......T..FF.......D...L...T.L.HKE..M..ES.AQ.L..KGT..P.. --KG..YVV.NKFKSF.QN.Q SAMD9L_Capo ...TDSKD..T -EAWSPQ.PQ.......T..FL.....S........ .T...HKED.S.LES..Q.L..KEN.SP.. --K...Y...SKFTAH.QS.Q SAMD9L_Soar .E.TDKKDC.R -GVW.QQRFR.K..M..T..FL.......FA........C.HKE.D..EKR..K.LL.KVNV.T.. --K.D.Y...RQFTS..QN.Q SAMD9L_Modo ..YN.NKDDKT -G -Q.HRY..S ....TSC.F......IQI..L.EKVFL..ER..SP.GL.GQLLR.N.GV LLN.LIQR.M.YPVI.KH.D..NA.Q

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1310 1320 1330 1340 1350 1360 1370 1380 1390 1400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa FSLKKSFDFFDEYFVLLKPRNNIKQNEEAKTRRKVAGYFKKYVDIFCLLEESQNNTGLGSKFSEPLQVERCRRNLVALKADKFSGLLEYLIKSQEDAIST SAMD9_Patr .............. ...................................................................................... SAMD9_Gogo ........................................................D.R..............S.......................... SAMD9_Poab ........................................ .............K .DP.......................................... SAMD9_Nole ..........................................I....VS..... KD...............SS....................A..V.. SAMD9_Mamu ....................................V.....T......G...D I.......... .I....KS.......................... SAMD9_Bota ..........................................A...GPS..L.. KDF...L.V.....LY..S.EV...............A....VH. SAMD9_Susc ...........D....................K...V.....A...GPS.D..S KY....L.L.....Q...S.EV............... N.K..A.. SAMD9_Eqca C...RC.....D.................R.......H........GPSV.... KD..A.L.I.....LY.KS.EV...........N...N.....R. SAMD9_Mylu S..........D........S......QS.............A...GPF..L.. RD...QL.L..R..LY..S.EV..................I..N. SAMD9_Orcu ... ........D................T.......TC..R.L...GPSA.ILS K.....I.LS...QE...S.E..............V....H.... SAMD9_Rano S..........D...........E.Y....S.....M......E..GP.A.L.. QNFR..L.L......T..S.E.................PK.TV.S SAMD9_Crgr S...RA.....D.............Y..S .C.S..V....R.....S....... QNFR..LTL......I..T.E.................PK..V.S SAMD9_Capo S...DA.....D.............H.........VV.....I...GPSG..L. RD.EL.L.L......NQ.S.E................T. ..V.S SAMD9_Soar ....N......D..........M..SD -.............G...D.FNGT.S KDF..NI.LS.KI.LF.SK.EV.............V.N.NSP.E. SAMD9L_Hosa SD..RC....ID.M....M.YTQ.EIA.IMLSK..SRC.R..TEL..H.D ---PCL.Q..E.QL..E.N..KK.E..R..R.A......NPNYK.. T. SAMD9L_Patr SD..RC....ID.M....M.YTQ.EIA.IMLSK..SRC.R..TEL..H.D ---PCL.Q..E.HL..E.N..KK.E..R.. R.A......NPNYK.. T. SAMD9L_Gogo SD..RC....ID.M....M.YTQ.EIA.IMLSK..SRC.R..TEL..H.D ---PCL.Q..E.QL..E.N..KK.E..R..R.A......NPNYK.. T. SAMD9L_Poab SD..RC....ID.M....M.YTQ.EIA.ITLGK..SRC.R..TEL..H.D ---PCL.Q..E.QL..E.N..KK.E..R..R.A......NPNYK.. A. SAMD9 L_Nole SD..RC....ID.M....M.YTQ.EIT.IMLSK..SRC.R..TEL..H.. ---PCL.Q..E.QL..E.N..KK.E..R..R.A......NPNYK.. A. SAMD9L_Caja SD..RC....ID.M....M.YAQ.EIV.VMLSK..SRC.R..TEL..H.D ---SDQ.QN.G.QL..E.N..KK.E..R..R.A......NPKYK.SATI SAMD9L_Mamu SD..RC....ID.M.... M.YTQ.EIV.ITLSK..SRC.R..TEL..H.D ---PCL.R..E.QLF.E.N..KK.E..R..R.A......NPNFKN.AT. SAMD9L_Loaf LD...C....VD.M....T..TH.EIV.SILSK..SR.YR..TE...H.G ---LDP.Q..E -L..E.NY.KS.E..R..R........NPNHK..AN. SAMD9L_Eqca SD..RC.E..AD.M...EMK.TQ.ETA.LSLNK.ISR C.R..MEL..H.D ---LGV.Q.RE.QL.KE.N..KA.E..R..R........NSNHKEVAT. SAMD9L_Calu SD..RC.....D.L....MK.TQ.ETG.ISLSK.ITRC.....EL..H.D ---SGP.HR E.QL..E.N..KA.E..R..R........NPNHRE.ATN SAMD9L_Aime SD..RC....ND.I...QM..TQ.ETV.ISLNK.ITRC.R..GEL..C.D ---LGL. Q..E.QFF.E.SF.KG.E..R..R........NPNHRE.ATN SAMD9L_Ereu SD..RC....AD.M.F..T..TQ.ETA.IILSK..SR..RQ.IEL..HSD ---VGI.QN.G.QL..E.YY.KS.E.KR..R........NP.HQETGNI SAMD9L_Orcu SN..RC....ID.M....M..TQ.EMA.ILLSK..SRCY...R.L..I.D ---PSP.H..E.QL.LE.N..KK.E.WR..R.. ......SLNHKE.T.. SAMD9L_Mumu SD.ERC.H..GD.MGF.....TP.ELT.LLLSK..SRC.....EL..H.D ---TNLVQG.EDLL..K.N..KRIQ.WR..T........NPNHKE. NN SAMD9L_Crgr SE..RC....LD.IG.....ITP.ETT.LSLIK...R.....AGL..RMN ---TNL.QG.ENVL..E.N..KRI..WR..T........NPNHKEV DN SAMD9L_R ano SD..RC....LD.MG......TP.ELT.LSLSK..SRC....A.L..Q ------.QG.EDLL..E.N..KRIK.WR..T........NPNHKE. NN SAMD9L_Capo SD.I.C.H..SD.M....T..FQ.EMT.L.LSK..IFCY.T.TKL..H.D ---S.P.QG.E.QL..E.N..KR.E.WR..R........NSNHK..ATI SAMD9L_Soar PD..RC....AD.M.F..T.T VH.EIT.ISL.K.IGHC.MS.IS..NH.. ---LGTTQAQENLL..E.N..KR.EKCR..R..L.....NPNYK..AT. SAMD9L_Modo KR.ETI.NNLQD..SF..LKD.E.EIV.E.NQI.MEKL..Q.LS..SNS. ------IRPSM ---VTSV.W.E.EISN.AR.....K..T.TH.N.EME

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1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa MKCIVNEYTF LLEQCTVKI ---QSKEKLNFILANIILSCIQPTSRLVKPVEKLKDQLREVLQPIGLTYQFSEPYFLASLLFWPENQQLDQHSEQMKEYA SAMD9_Patr ................... ---..................K.......................................................... SAMD9_Gogo .EY..........R..... ---............. .....K...........................................Y.............. SAMD9_Poab .E...K.......R.A... ---..................K...........................R.......................K.....S SAMD9_Nole .EN..DK....S....... ---..................K.................... .......R.......................G...... SAMD9_Mamu .ED...K.A.......... ---E.................K...T......................HR.......................Q...A.. SAMD9_Bota .ED.MDK....F....... ---LI...Q.........Y..K...KI...IK........L..QT.MS.RYP................ ...D.RL.EK.. SAMD9_Susc .ED.M.K.S..F.....RM ---L....Q.........Y..K...KI.M.IK...E.......QAETICRQ....................D.K..EK.. SAMD9_Eqca .DD...K....F..G.... ---.P...Q.........Y..K...KI...IR...........Q.....R.................H...D.K..EK.. SAMD9_Mylu .ED..KK....F...A.R. ---.Q...Q.........Y..K...KI.M.TK........I..Q..PS...................K...D.K..EKH. SAMD9_Orcu .EN..HK....F..S.E.. ---.L...Q............K...KS..TIK......Q.I..QV..NCR.....................D.K..EK.. SAMD9_Rano .ED...K........AI.. --.Q...Q.....S......N.I.KF...IK...E....I..............................D.K..EK.. SAMD9_Crgr .EG...Q..S.....VI.T ---.Q...Q............N.A.K.....K...E....I..............................D.K..EK.. SAMD9_Capo .EY...T.I..FD..I.RM ---HL...Q............K.A.K ....IK...EH..DI..QV.P.HR.....................D.KE.EK.V SAMD9_Soar .ES..EQ.S.I..H.AAS -----T...Q.......V.Y..K...KF.S..K...E....... V.TN.R.P.............T.N...D.K..EK.. SAMD9L_Hosa .ES.....A...Q.NSK.P ---MTN..Q.S.........LK.N.K.IQ.LTT..K.......FV..SH.YP G.....C........E...D.KLIEK.V SAMD9L_Patr .ES.....A...Q.NSK.P ---MTN..Q.S.........LK.N.K.IQ.LTT..K.......FV..SH.YPG.....C........E...D.KLIEK.V SAMD9L_Gogo .ES.....A...Q.NSK.P ---MTN..Q.S.........LK.N.K.IQ.LTT..K.......FV..SH.YPG.....C........E...D.KLIEK .V SAMD9L_Poab .ES.........Q.NSK.H ---MTN..Q.S.........LK.N.KFIQ.LTT..K.......FV.PSH.YPG.....C........E...D.KLIEK.V SAMD9L_Nole .ES.....A...Q.NSK.R ---MTN..Q.S.........LK.K.K.IQ.LTT..K.......CV..SH.YPG.....C........E...D.KLIEK.V SAMD9L_Caja .ES.....A ...H.NSK.C ---MTN..Q.S.........LK.N.KSIQ.LNT..K.......FV..SH.YPN.....C........E...D.KLIEK.V SAMD9L_Mamu .EI..D..A...K.NSN.R ---MTN..Q.S.......N.LK.S..FIQ.LTM..K.......FV..SH.YPD.....C........E...D.KLIEK.V SAMD9L_Loaf VER...Q.AL.FQ.NLN.K ---LI...Q...... .....LK.S.KYIR.FNI..K......ELVEP.H.YPD.....C........E...D.KL.EK.V SAMD9L_Eqca .ENV..K.....Q.NPN.Q ---LTR..Q.........N.LK...KSIQ.LS...K..Q...HIV.PRH.YPD.....C....L...E..ED.QL.EK.V SAMD9L_Calu .EN...K.N...Q.NPK.Q ---MT...Q.........N.LK.H.KFIQ.LPI..K..... .LS..PSH.YPN.....C.....Q..E..ED.KF.EK.V SAMD9L_Aime .EN...K.S...R.NPN.Q ---LT.............N.LK.H.KSIQ.LSI..KL...A.LS..PS..YPD.....C........E..ED.KL.EK.V SAMD9L_Ereu .EN..E..A...Q.NPN.R ---MI...Q...........LK.N.KFIQ.LDT..K.......LVEINR.YPD.....C..... D..E...D.KL.EK.V SAMD9L_Orcu .EN..D..A...K.NPN.R ---LT...Q...........LK...KSIQ.LNM..K.......LV..HH.Y.D.....C........E...D.KL.EK.V SAMD9L_Mumu IEN..GN.....QDILN.QLSKVLT.DIQ...........LK.S.KYIL.FST..KK......IV...HSYPD.....C.......KE..ED.TLIEK.V SAMD9L_C rgr .ES..KD.....QHSLS.RVTKGLT..TQ...........LK.S.KYIL.FNT..TK.....ELV..GHPYPD.....C.......KE...D.TLIEK.V SAMD9L_Rano .EN..EH.....QHTLN.QLSKALI.DTQ...........LK.S.KHIL.FST..KK......IV...HSYPD.....C.......KE...D.SLIEK.V SAMD9L_Capo .EN.....A...Q.NPSIR --SI...Q...........VK.N.KFIQ.LSI..Q.......LV..N..YPD.....C........K...D.KL.EK.V SAMD9L_Soar IES..KD.....Q.NLQNP ---MR...Q...........LN.N.VFIE.LDT..EL......LAK.DHSYPD.F...C.......EV...D.QLLEK.V SAMD9L_Modo .ES..DR.ML.VQ.ASSM ----KTRA.Q.........N.LK.N.KI R..LDE..AM.Q...LQV..HSSYPD.....T........H.N.N.TEIID.V

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1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 ....|....|....|....|....|....|....|....|....|.... |....|....|....|....|....|....|....|....|....|....| SAMD9_Hosa QALKNSFKGQYKHMHRTKQPIAYFFLGKGKRLERLVHKGKIDQCFKKTP DINSLWQSGDVWKEEKVQELLLRLQGRAENN CLYIEYGINEKITIPITP SAMD9_Patr ............................................. E... ............................... .................. SAMD9_Gogo ................................................. ............................... .................. SAMD9_Poab ...E.........................E..K............E... ..................... .......... .................. SAMD9_Nole ...E............................K............E..L ............................... .................. SAMD9_Mamu ...E............................K............E... ..............A................ ............... ... SAMD9_Bota SS.E...R......Y..............NSVN............E..A ...F..........K...K.....K..VG.. ..H............... SAMD9_Susc RS.E...R......C..............NNMN..I.R......C...AA..K.F.........KE..K..F..K...... .................. SAMD9_Eqca RS.E...R ......Y..............NNMT............RN.S ..............RN........K...... .......V.D........ SAMD9_Mylu RS.Q...R......Y..............NNMN...........YG..L ...IW....E....K.........K...... ..........R....... SAMD9_Orcu .S.E...R......Y..............NNMN. ...........E..L ..............R................ .......VSD...L.... SAMD9_Rano ...EK..R....RLY..............NSRN..I......EN.ENLS ....W.HT.K..E.K...D..V..P...... ................K. SAMD9_Crgr W..ET..R....R.Y...........E..NNRN..I......EN.E.VS ....W.H..K ..E.K...D............ N.............Q... SAMD9_Capo .S.EK........................TNMN.........E..GEMS ...F.....K..E.K...D............EK.F............... SAMD9_Soar RL.ET..L.K.R..Y.............A.NIN............G.. -.......N....R.K...D.....N...... .V.. D...K...I..... SAMD9L_Hosa SS.NR..R....R.C.S..ASTL.Y...R.G.NSI...A..E.Y.D.AQ NT....H......KNE.KD..R..T.Q..GK LISV...TE...K..VIS SAMD9L_Patr SS.NR..R....R.C.S..ASTL.Y...R.G.NSI...AE.E.Y.D.AQ NT....H......KNE.KD..R..T.Q..GK LIS....TE...K..VIS SAMD9L_Gog o SS.NR..R....R.C.S..ASTL.Y...R.G.NSI...A..E.Y.D.AQ NT....H......KNE.KD..R..T.Q..GK LIS....TE...K..VIS SAMD9L_Poab SS.NR..R....R.C.S..ASTL.Y...R.G.NSI...A..E.Y.D.AQ NT....H......KNE.KD..R..T.Q..GK LIS....TE...K..VIS SAMD9L_Nole SS.NR..R....R.C.S..ASTL .Y...R.G.NSI...AE.E.Y.D.AQ NT....H......KNE.KD..R..T.Q..GK LIS....TE...K..V.S SAMD9L_Caja SS.NR..R......C.S..ASTL.....R.G.NSI...AE.E.Y.G.VH NT...........KNE.KD..R..I.Q..GK LIS....TK...K..VIS SAMD9L_Mamu SS.NR..R....R.C.S..ASTL.Y...RRG.NSI...AE.E.Y.D.VQ NT....H......KHE.KH..C..I.Q..GK LIS....TE...K..VIS SAMD9L_Loaf SS.NR..GR..R..C.S..ASTL.Y...S.G.HSI...AE.E.Y.S.EQ NT...........KNE.KD..R..T.Q.VGK LIS....TE...R..VIS SAMD9L_Eqca SS.NR...R..SN.C.SR.ASTV.Y...K.G.HS....A..E.Y.G.VQ NT.....N....EKKE.KD..C..T .Q..GK RIS....TK...K..VIS SAMD9L_Calu SS.NRN..R..RS.C.S..ASTL.Y...Q.G.HS..C.AE.E.Y.N.AQ NTS..S..A..C.K ..KD..C..T.Q..GK LISM...TEK.VK..VI. SAMD9L_Aime SS.NRT..R..RS.C.S..ASTL.Y...K.G.HS..C.AE.E.Y.N.AQ NV...SP......KKE.KD..C..T.Q..GK LISM...TEK..K..VI. SAMD9L_Ereu SS..K..NRH.RL.C.S..ASTL.Y..NR.G.NS....AE.E.YIS.VK NT..F....H...K.E.KG..R..N....GK LILM...TE...K..VIS SAMD9L_Orcu SS.NRT.R....R.C.S..ASTF.Y...R.GFNG..L.AE.E.Y.S.AQ N............KKE.KD..H..V.Q..GK LIS....TE...K..V.S SAMD9L_Mumu SS.NR.. RR.....C.SR..STL.Y..QK.G.NS....AE.ERY.SEVQ .S..F.H..V..EKRE.KD..RL.D.Q..GK LISL...TEA..K..V.S SAMD9L_Crgr SS.NR..RR.....C.S.K.STL.Y..QK.G.HS....AE.ERYVSEVQ NS..F....V..EKRE.KD..RL.D.Q..GK LISV...TEA..K..V.S SAMD9L_Rano SS.NR..RR.....C.SR..STL.Y..QK.G.N S....AE.ERY.SEVQ .S..F....V..EKGE.KDI.RL.D.Q..GK LISL...TEA..K..V.S SAMD9L_Capo VS.NR..R....R.C.S..ASTF.Y..TK.G.NS....AELEKHIS..K NT..F.....I..T E.KH..H..T.Q..GK LIS............... SAMD9L_Soar SS..K..NR..RR.C.S..ASTL.Y..N..G.KG....TE.E.Y.S.V. NTSI...N. ....KKE.KS..C..T.Q..GK LIS.Q..TE...N..VIS SAMD9L_Modo T..RKT.WD..GQ.C.G...FTH.Y..NDRG.N....R...E...QDAS .VSI...R....R....KD.....S..V..D LI..DC.SK.NAE..VR.

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1610 1620 1630 ... .|....|....|.. ..|....|....|.... SAMD9_Hosa AFLGQLRSGRSIEKVSFYLGFSIGGPLAYDIEIV SAMD9_Patr .................................. SAMD9_Gogo .................................. SAMD9_Poab .................................. SAMD9_Nole .................................. SAMD9_Mamu T................................. SAMD9_Bota .................................I SAMD9_Susc ............D.........T.........VI SAMD9_Eqca ..F..............................I SAMD9_Mylu ..W.... ..........................I SAMD9_Orcu ..W.........D....................I SAMD9_Rano THF......................L.....K.. SAMD9_Crgr TH.......................L.....K.. SAMD9_Capo ..F............................Q.L SAMD9_Soar T.F............... .............QVI SAMD9L_Hosa VYS.P.....N..R.........E........VI SAMD9L_Patr VYS.P.....N..R.........E........VI SAMD9L_Gogo VYS.P.....N..R.........E........VI SAMD9L_Poab VYS.P.....N..R.........E........VI SAMD9L_Nole VYS.P.....N..R.........E..... ..KVI SAMD9L_Caja VYS.P.....N..R.........E.......QVI SAMD9L_Mamu VYS.P.....N..R.........E........VI SAMD9L_Loaf VYS.P....GN..R.........E........VI SAMD9L_Eqca VYS.P.Q...N..R........ME........VI SAMD9L_Calu VYS.P....GN..R........ME.LQ..E...I SAMD 9L_Aime VYS.P....GN..R.....A...E..Q......I SAMD9L_Ereu VYS.P.....N.QR........ME........VI SAMD9L_Orcu VYS.P.....N............E........V. SAMD9L_Mumu VYSAP.....N..R.........E.....G.KVI SAMD9L_Crgr VYS.P.....N..R.V.......E.....G.KVI SAMD9L_Rano VY S.P.....N..R.........E.....G.KVI SAMD9L_Capo ..F............................Q.L SAMD9L_Soar VYS.P.....H.QS........ME........LI SAMD9L_Modo .V.....R.Q.M.R........TA..V....KNI