Tag Archives: Phylogenetics

BLAST from the past – a bit of history behind Craig Pikaard’s discovery in 2000 of RNA Pol IV in Arabidopsis

I saw this post by Craig Pikaard on Facebook and it brought back some memories:

New paper from my lab in which we identified the RNAs made by RNA Polymerase IV, an enzyme we discovered ~15 years ago. Took us more than ten years to find the little buggers, but we finally got ’em. The paper is “open access”, meaning that anyone can read it without paying a download fee or subscription. So have at it if you need a nap. 

 

And the post included a link to a new paper in Elife.  This brought back memories because I had a small part in the discovery (or more accurately, some post discovery analysis).  So – let’s step into a time machine here provided by, well, me keeping all my email forever I guess.

It was September 2000.  I was working as a faculty member at TIGR (The Institute for Genomic Research) and I was doing some evolutionary analysis of the Arabidopsis thaliana genome, for what would become my most highly cited paper: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana.  And then on Sept 6 day I got an email from someone who I had gotten to know a little bit who was also analyzing the genome:

———————————-
9/6

Dear Jonathan, 

In helping Mike Bevan search for the general transcription machinery, I’ve
stumbled across something odd that might also interest you given its
evolutionary implications. 

There should be three related genes in the Arabidopsis genome (or more if
any of the genes are duplicated) encoding ~135 kd (2nd largest)
DNA-dependent RNA polymerase subunits – one each for pol I, II and III.
These subunits are similar and are clearly related to one another (also to
the B subunit of the single bacterial RNA polymerase) yet they have
distinct motifs that allow them to be placed in each class (pol I, II, or
III) based on clustal analysis with orthologs from other species. Anyway,
there ARE three distinct ~135 kd subunit genes in the thaliana genome and
based on multiple alignments vs. mouse, yeast, drosophila etc genes, and
clustal analysis to draw phylogenetic trees, one is clearly for pol II, and
one is clearly for pol III. The third paralog is strange- it does not group
with other pol I 135 kd subunits (from yeast, Drosophila, Euplotes, mouse,
C. elegans), nor with pol II or III subunits. In fact, it appears as an
outgroup even when archael subunits (e.g. Sulfolobus) are included in the
analysis: archael subunits are more closely related to the pol II second
largest subunit than the mystery subunit is to other pol I, II, or III
subunits. By BLAST searching Genbank, the mystery subunit does not match
anything better than eukaryotic 135 kd subunits and it doesn’t look like a
chloroplast or mitochondrial subunit. I’m wondering if a plant Pol I can
really be that weird. 

Is this something you would be interested in looking at if I send you the
protein sequences for clustal analysis? 

Cheers
Craig 

Craig S. Pikaard
Associate Professor
Biology Department, Washington University
Campus Box 1137, One Brookings Drive
St. Louis, MO 63130

Now this certainly seemed interesting and as I was doing a variety of analyses of RNA polymerase homologs for some studies of the evolution of microbes, it was something I actually knew a little bit about. So I wrote back immediately:

Craig 

This sounds quite interesting. I have found that for many of the DNA repair genes I have been looking at, the A. thaliana genes do show quite long branches, so long branches might be a possibility. A good phylogenetic analysis should be able to detemrine if that is the case. If you send me the sequences and/or an alignment, I would be happy to put them through a more deailed phylogenetic analysis.

Jonathan

Then, a few minutes later I got another email:

Hi Jonathan,

I’m pasting below the sequences I used for the multiple alignments (using
DNAStar), starting with the mystery gene and then known second subunits of
pol I, II, III, and archae.
Thanks for having a look at this.
Craig
———–

Arabidopsis mystery gene (from chromosome 3):
DEFINITION DNA-dependent RNA polymerase II [Arabidopsis thaliana].
ACCESSION BAB02021
A. thal chromosome III sequence. Does not group with pol I, II or III
despite its description. Two chromosome 3 P1 clones and two partial cDNAs
(that are the same)from developing seeds match it (see accessions below,
with match scores)
GSDB:S:3264005|AB020749|AB020749|Arabidopsis thaliana genomic D… 598 0.0
GSDB:S:4681131|AP000377|AP000377|Arabidopsis thaliana genomic D… 566 0.0
GSDB:S:1038672|Z19120|ATRNAPIIM|A.thaliana mRNA for RNA polymer… 504 e-142
GSDB:S:8430488|BE522782|BE522782|M28H12STM Arabidopsis developi… 171
1e-046
GSDB:S:8430529|BE522823|BE522823|M29C3STM Arabidopsis developin… 171
2e-041

mdvdeiesagqiniselgesflqtfckkaatsffeefglishqlnsynffiehglqnvfesfgdilvepsfdvikkkdgd
wryatvfkkivikhdkfktgqdeyvekeildvkkqdiligsipvmvksvlcktsekgkenckkgncafdqggyfvikgae
kvfiaqeqmctkrlwisnspwtvsfrsetkrnrfivrlsenekaedykimekvltvyflsteipvwllffalgvssdkea
mdliafdgddasitnsliasiheadavceafrcgnnaltyvehqikstkfppaesvddclrlylfpclqglkkkarflgy
mvkcllsayagkrkcenrdsfrnkrielagellereirvhlaharrkmtramqkqlsgdgdlkpiehyldasvitnglnr
afstgawshpfrkmervsgvvanlgranplqtlidlrrtrqqvlytgkvgdarhphpshwgrvcflstpdgencglvknm
sllglvstqglesvvemlftcgmeelmndtstplcgkhkvllngdwvglcadsesfvgelksrrrqselplemeikrdkd
dnevriftdagrllrpllvvenlhklkqdkptqypfkhlldqgileligieeeedcttawgikqllkepknythceldls
fllgvscaivpfanhdhgkrvlyqsqkhcqqaigfsstnpnircdtlsqqlfypqkplfktlaseclekevlfngqnaiv
avnvhlgynqedsivmnkaslergmfrseqirsykaevdtkdsekrkkmdelvqfgktyskigkvdsleddgfpfiganm
stgdivigrctesgadhsiklkhtergivqkvvlssndegknfaavslrqvrspclgdkfssmhgqkgvlgyleeqqnfp
ftiqgivpdivinphafpsrqtpgqlleaalskgiacpiqkkegssaaytkltrhatpfstpgvteiteqlhragfsrwg
nervyngrsgemmrslifmgptfyqrlvhmsenkvkfrntgpvhpltrqpvadrkrfggirfgemerdcliahgasanlh
erlftlsdssqmhicrkcktyanviertpssgrkirgpycrvcassdhvvrvyvpygakllcqelfsmgitlnfdtklc
——————————— 

Known Pol I ~135 kd subunits: 

Yeast (S. cerevisae)
MSKVIKPPGQARTADFRTLERESRFINPPKDKSAFPLLQEAVQPHIGSFNALTEGPDGGLLNLGVKDIGEKVIFDGKPLN
SEDEISNSGYLGNKLSVSVEQVSIAKPMSNDGVSSAVERKVYPSESRQRLTSYRGKLLLKLKWSVNNGEENLFEVRDCGG
LPVMLQSNRCHLNKMSPYELVQHKEESDEIGGYFIVNGIEKLIRMLIVQRRNHPMAIIRPSFANRGASYSHYGIQIRSVR
PDQTSQTNVLHYLNDGQVTFRFSWRKNEYLVPVVMILKALCHTSDREIFDGIIGNDVKDSFLTDRLELLLRGFKKRYPHL
QNRTQVLQYLGDKFRVVFQASPDQSDLEVGQEVLDRIVLVHLGKDGSQDKFRMLLFMIRKLYSLVAGECSPDNPDATQHQ
EVLLGGFLYGMILKEKIDEYLQNIIAQVRMDINRGMAINFKDKRYMSRVLMRVNENIGSKMQYFLSTGNLVSQSGLDLQQ
VSGYTVVAEKINFYRFISHFRMVHRGSFFAQLKTTTVRKLLPESWGFLCPVHTPDGSPCGLLNHFAHKCRISTQQSDVSR
IPSILYSLGVAPASHTFAAGPSLCCVQIDGKIIGWVSHEQGKIIADTLRYWKVEGKTPGLPIDLEIGYVPPSTRGQYPGL
YLFGGHSRMLRPVRYLPLDKEDIVGPFEQVYMNIAVTPQEIQNNVHTHVEFTPTNILSILANLTPFSDFNQSPRNMYQCQ
MGKQTMGTPGVALCHRSDNKLYRLQTGQTPIVKANLYDDYGMDNFPNGFNAVVAVISYTGYDMDDAMIINKSADERGFGY
GTMYKTEKVDLALNRNRGDPITQHFGFGNDEWPKEWLEKLDEDGLPYIGTYVEEGDPICAYFDDTLNKTKIKTYHSSEPA
YIEEVNLIGDESNKFQELQTVSIKYRIRRTPQIGDKFSSRHGQKGVCSRKWPTIDMPFSETGIQPDIIINPHAFPSRMTI
GMFVESLAGKAGALHGIAQDSTPWIFNEDDTPADYFGEQLAKAGYNYHGNEPMYSGATGEELRADIYVGVVYYQRLRHMV
NDKFQVRSTGPVNSLTMQPVKGRKRHGGIRVGEMERDALIGHGTSFLLQDRLLNSSDYTQASVCRECGSILTTQQSVPRI
GSISTVCCRRCSMRFEDAKKLLTKSEDGEKIFIDDSQIWEDGQGNKFVGGNETTTVAIPFVLKYLDSELSAMGIRLRYNV
EPK
 

C. elegans
MDCDIASYHVDSFDFLVSKGCQFAAQAVPAEKFRLKNGDAVTMKFTSAQLHKPTLDTGAKLTSDTLPLLPAECRQRGLTY
AGNLKVGIDVHVNGSRLDIIEIILGKVPIMLRSEGCHLRGMSRKELVVAGEEPIEKGGYFIVNGSEKVIRLLIANRRNFP
IAIIRKTFKEKGKLFSEFGVMMRSVKENHTAVMMTLHYLDTGTMQLALQFRREIFYVPLMYIVKALTDKNDAVISAGFKR
GRNQDQFYSSCILNMLAQCQEEEILNQEAAIRAIGSRFRVAVSDRVAPWEDDLEAGRFIIRECVLIHLDSDEEKFHTLAY
MTQKLIALVKGECAPETPDNPQFQEASVSGHILLLILRERMENIIGMVRRKLEYMSSRKDFILTSAAILKALGNHTGGEI
TRGMAYFLATGNLVTRVGLALQQESGFSVIAERINQLRFVSHFRAIHRGAFFMEMRTTDVRKLRPEAWGFICPVHTPDGA
PCGLLNHVTASCRIVTDLSDNSNVPSLLAELGMYTHKTVALAPPGEELYPVLMNGRFLGYVPITKAASIERYLRCAKVAK
DARIPYTSEIALVRRSTDIKNIQTQYPGIYILSDAGRLIRPVRNLAMDAVEHIGTFEQVYLSVVLDPEEAEPGVTMHQEL
HPSCLFSFAGNLIPFPDHNQSPRNVYQCQMGKQTMGTAVHAWHSRADNKMYRLQFPQQPMLKLEAYEKYEMDEYPLGTNA
CVAVISYTGYDMEDAMTINKASYQRGFAHGTVIKVERINLVTERERKTIFYRNPREEIKTVGPDGLPIPGRRYFLDEVYY
VTFNMETGDFRTHKFHYAEPAYCGLVRIVEQGEGDSGAKHALIQWRIERNPIIGDKFASRHGQKGINSFLWPVESLPFSE
TGMVPDIIFNPHGFPSRMTIGMMIESMAGKAAATHGENYDASPFVFNEDNTAINHFGELLTKAGYNYYGNETFYSGVDGR
QMEMQIFFGIVYYQRLRHMIADKFQVRATGPIDPITHQPVKGRKKGGGIRFGEMERDAIIAHGTSFVLQDRLLNCSDRDV
AYACRRCGSLLSVLMSSRAGSHLLKKKRKDDEPLDYTETQRCRTCDKDDQVFLLQVPRVFRYLTAELAAMNVKIKLGIEH
PSKVTGS
 

D. melanogaster
MLEEMQQMKTIPVLTNSRPEFKQIPKKLSRHLANLGGPHVDSFDEMLTVGLDNSAKHMIPNHWLSPAGEKISMKVESIWI
AKPKVPQDVIDVRTREIYPTDSRQLHVSYSGMCSVRLGWSVNGVQKTPINMDLGEVPIMLRSKACNLGQATPEEMVKHGE
HDSEWGGIFVIRGNEKIVRMLIMTRRNHPICVKRSSWKDRGQNFSDLGMLVQTVREDESSLSNVVHYLNNGTAKFMFSHV
KRLSYVPVCLILKCLMDYTDEEIYNRLVQGYESDQYYVSCVQAMLREVQNENVYTHAQCKSFIGNLFRARFPEVPEWQPD
DDVTDFILRERVMIHLDTYEDKFQLIVFMIQKLFQCAQGKYKVENVDSSMMQEVLLPGHLYQKYLSERVESWVSQVRRCL
QKKLTSPDALVTSAVMTQCMRQAGGVGRAIESFLATGNIASRTGLGLMQNSGLVIMAENINRMRYMSHFRAIHRGSYFTT
MRTTEARQLLPDAWGFICPVHTPDGTPCGLLNHLTLTCEISMRPDPKLVKAIPKHLIDMGMMPLSNRRYLGEKLYVVFLD
GKHLGHIHQSEAEKIVDELRYGKIFGTLPQMMEIGFIPFKKNGQFPGLYIATGPARLMRPVWNLKWKRVEYIGTLEQLYM
EIAIDAKEMYPDFTTHLELAKTHFMSNLANLIPMPDYNQSPRNMYQCQMGKQTMGTPCLNWPKQAANKLYRLQTPGTPLF
RPVHYDIIQLDDFAMGTNAIVAVISYTGYDMEDAMIINKAAYERGFAYGSIYKTKFLTLDKKSSYFARHPHMPELIKHLD
TDGLPHPGSKLSYGSPLYCYFDGEVATYKVVKMDEKEDCIVESIRQLGSFDLSPTKMVAITLRVPRPATIGDKFASRAGQ
KGICSQKYPAEDLPFTESGLIPDIVFNPHGFPSRMTIAMMIETMAGKGAAIHGNVYDATPFRFSEENTAIDYFGKMLEAG
GYNYYGTERLYSGVDGREMTADIFFGVVHYQRLRHMVFDKWQVRSTGAVEARTHQPIKGRKRGGGVRFGEMERDALISHG
AAFLLQDRLFHNSDKTHTLVCHKCGSILAPLQRIVKRNETGGLSSQPDTCRLCGDNSSVSMIEIPFSFKYLVTELSSVNI
NARFKLNEI
 

mouse
MDVDGRWRNLPSGPSLKHLTDPSYGIPPEQQKAALQDLTRAHVDSFNYAALEGLSHAVQAIPPFEFAFKDERISLTIVDA
VISPPSVPKGTICKDLNVYPAECRGRKSTYRGRLTADISWAVNGVPKGIIKQFLGYVPIMVKSKLCNLYNLPPRVLIEHH
EEAEEMGGYFIINGIEKVIRMLIEPRRNFPVAMVRPKWKSRGLGYTQFGVSMRCVREEHSAVNMNLHYVENGTVMLNFIY
RKELFFLPLGFALKALVSFSDYQIFQELIKGKEEDSFFRNSVSQMLRIVIEEGCHSQKQVLNYLGECFRVKLSLPDWYPN
VEAAEFLLNQGICIHLQSNTDKFYLRCLMTRKLFALARGECMDDNPDSLVNQEVLSPGQLFLMFLKEKMENWLVSIKIVL
DKRAQKANVSINNENLMKIFSMGTELTRPFEYLLATGNLRSKTGLGFLEDSGLCVVADKLNFLRYLSHFRCVHRGAAFAK
MRTTTVRRLLPESWGFLCPVHTPDGAPCGLLNHLTAVCEVVTKFGDTASIPALLCGLGVTGADTAPCRPYSDCYPVLLDG
VMVGWVDKDLAPEVADTLRRFKVLREKRIPPWMEVALIPMTGKPSLYPGLFLFTTPCRLVRPVQNLELGREELIGTMEQL
FMNVAIFEDEVFGGISTHQELFPHSLLSVIANFIPFSDHNQSPRNMYQCQMGKQTMGFPLLTYQNRSDNKLYRLQTPQSP
LVRPCMYDFYDMDNYPIGTNAIVAVISYTGYDMEDAMIVNKASWERGFAHGSVYKSEFIDLSEKFKQGEDNLVFGVKPGD
PRVMQKLDDDGLPSIGAKLEYGDPYYSYLNLNTGEGFVVYYKSKENCVVDNIKVCSNDMGSGKFKCICITVRIPRNPTIG
DKFASRHGQKGILSRLWPAEDMPFTESGMMPDILFNPHGFPSRMTIGMLIESMAGKSAALHGLCHDATPFIFSEENSALE
YFGEMLKAAGYNFYGTERLYSGISGMELEADIFIGVVYYQRLRHMVSDKFQVRTTGARDKVTNQPLGGRNVQGGIRFGEM
ERDALLAHGTSFLLHDRLFNCSDRSVAHVCVECGSLLSPLLEKPPPSWSAMRNRKYNCTVCGRSDTIDTVSVPYVFRYFV
AELAAMNIKVKLDVI
 

Euplotes
MKTNAKFDRKEISKIYKNIARHHIDSFDFAMSTCLNRACEHMLPFDYIVPEESASCGFKKLTLWYDSFELGQPSLGEIDY
DSHILYPSECRQRKMTYTIPLFATIFKKFDDEMVDNFKVKLGDIPTMGRKKFCNLKGLTKKELAKRGEDMLEFGGYFIVN
GNEKVIRMLIVPKRNFPIAFKRSKFLERGKDFTDYGVQMRCVRDDFTAQTITLTYLSDGSVSLRLIYQKQEFLIPIILIL
KALKNCTDRQIYERIVKGNFNQRQISDRVEAILAVGKDLNIYDSDQSKALIGSRFRIVLAGITSETSDIDAGDLFLSKHI
CIHTDSYEAKFDTLILMIDKLYASVANEVELDNLDSVAMQDVLLGGHLYLQILSEKLFDCLHINLRARLNKELKRHNFDP
MKFRDVLTNQKINCGIGLIGKRMENFLATGNLISRTNLDLMQTSGFCIIGDKLNNIRFLSHFRSIHRGQYFAEQKTTSVR
KLLPESWGFICPVHTPDGAPCGLLNHISMSCVPIGSEEKQIDIDKFRNILGELGMNSISSDLCLNYHTGYYPVIFDGIHL
GYVEKDIGESFVEGLRYLKCTQSQPDYAIPRTLEIAFIPFSGYSRNLQWPGIFLASTPARFTRPVKNLHYNCIEWISPLE
QMNLSIACTDEDITPETTHQELDPINILSIVASVGVFAEYNQSPRNMYQCQMAKQTMGTPYHNHQFRTDNKIYRLLFPHR
PIVKTRTQVDFDIEEYPSGTNAVVAVISYTGYDLEDAMIINKSSYERGFGHGVVYKSYTHDLNESNSQSTRGIKSSVRYK
FLNNVSQKDKSKIKLENIDPDGLPKIGSQLTKGKPELCIFDTLKRGAKLSKFKDSEKARIETVRVCGNDDKNPDNLSIGY
TIRYSRIPVIGDKFSSRHGQKGVLSVLWPQVDMPFTENGITPDLIINPHAFPSRMTMGMLIQSMAAKSGSLRGEFKTVET
FQRYDDNDIVGHFGKELLDKGFNYHGNELMYSGIFGTPLKADIFIGVVYYQRLRHMVSDKSQARGTGPIDILTHQPVKGR
KKGGGIRFGEMERDSLLAHGAAYCLNDRLFRSSDYSEGFVCQNCGSILSCYVNRAIMKTQTFIPPSLDESNKDTEDKEIH
MNEKVICKVCKKNSNCKKVALPFVLRFLANELASMGIKLKFTVNDF
——————–
 

Pol II second largest subunits 

S. cerevisae
msdlansekyydedpygfedesapitaedswavisaffrekglvsqqldsfnqfvdytlqdiicedstlileqlaqhtte
sdnisrkyeisfgkiyvtkpmvnesdgvthalypqearlrnltyssglfvdvkkrtyeaidvpgrelkyeliaeesedds
esgkvfigrlpimlrskncylseatesdlyklkecpfdmggyfiingsekvliaqersagnivqvfkkaapspishvaei
rsalekgsrfistlqvklygregssartikatlpyikqdipiviifralgiipdgeilehicydvndwqmlemlkpcved
gfviqdretaldfigrrgtalgikkekriqyakdilqkeflphitqlegfesrkafflgyminrlllcaldrkdqddrdh
fgkkrldlagpllaqlfktlfkkltkdifrymqrtveeahdfnmklainaktitsglkyalatgnwgeqkkamssragvs
qvlnrytysstlshlrrtntpigrdgklakprqlhnthwglvcpaetpegqacglvknlslmscisvgtdpmpiitflse
wgmepledyvphqspdatrvfvngvwhgvhrnparlmetlrtlrrkgdinpevsmirdirekelkiftdagrvyrplfiv
eddeslghkelkvrkghiaklmateyqdieggfedveeytwssllneglveyidaeeeesiliamqpedlepaeaneend
ldvdpakrirvshhattfthceihpsmilgvaasiipfpdhnqsprntyqsamgkqamgvfltnynvrmdtmanilyypq
kplgttrameylkfrelpagqnaivaiacysgynqedsmimnqssidrglfrslffrsymdqekkygmsitetfekpqrt
ntlrmkhgtydkldddgliapgvrvsgedviigkttpispdeeelgqrtayhskrdastplrstengivdqvlvttnqdg
lkfvkvrvrttkipqigdkfasrhgqkgtigityrredmpftaegivpdliinphaipsrmtvahliecllskvaalsgn
egdaspftditvegiskllrehgyqsrgfevmynghtgkklmaqiffgptyyqrlrhmvddkiharargpmqvltrqpve
grsrdgglrfgemerdcmiahgaasflkerlmeasdafrvhicgicglmtviaklnhnqfeckgcdnkidiyqihipyaa
kllfqelmamnitprlytdrsrdf
 

C. elegans
myddedemvndpmdgdyiddsdeisaeawqeacwvvisayfdekglvrqqldsfdefvqmnvqrivedsppvelqsenqh
lgtdmenpakfslkfnqiylskpthwekdgapmpmmpnearlrnltyasplyvditkvvtrddsatekvydkvfvgkvpv
mlrssycmlsnmtdrdltelnecpldpggyfvingsekvliaqekmatntvyvfsmkdgkyafktecrsclenssrptst
mwvnmlargggggkktamgqriigilpyikqeipimivfralgfvsdrdilghiiydfndpemmemvkpsldeafviqeq
nvalnfigargakpgvtreqrikyareilqkellphvgvsehcetkkaffigymvhrlllaalgrrelddrdhignkrld
lagpllaflfrslfrnllkemrmtaqkyinknddfaldvcvktstitrgltyslatgnwgdqkkahqsragvsqvlnrlt
ytatlshlrranspigregklakprqlhntqwgmvcpaetpegqavglvknlalmayisvgslpepilefleewsmenle
evspsaiadatkifvngawvgihrepdqlmttlkklrrqmdiivsevsmvrdirdreiriytdagrvcrpllivenqkla
lkkrhidqlkeaadeankytwsdlvgggvvelidsmeeetsmiammpedlrsggycdththceihpamilgvcasiipfp
dhnqsprntyqsamgkqamgvyttnfhvrmdtlahvlyypqkplvttrsmeylrfnelpaginaivailsysgynqedsv
imnnsaidrglfrsvfyrsyrdneanldnaneeliekptrekcsgmrhslydkldedgiispgmrvsgddviigktvalp
didddldasgkkypkrdastflrssetgivdqvmlslnsdgnkfvkirmrsvrlpqigdkfasrhgqkgtmgimyrqedm
pftaegltpdiiinphavpsrmtighlieclqgklsankgeigdatpfndtvnvqkisgllceygyhlrgnevmynghtg
kklttqiffgptyyqrlkhmvddkihsrargpiqmmnrqpmegrardgglrfgemerdcqishgatqflrerlfevsdpy
hvyvcnncglivvanlrtnsfeckacrnktqvsavripyackllfqelmsmsiaprlmvkprqskrskhqsea
 

Drosophila
msvqrivedspaielqaerqhtsgevetpprfslkfeqiylskpthwekdgspspmmpnearlrnltysaplyvditktk
nvegldpvetqhqktfigkipimlrstycllsqltdrdltelnecpldpggyfiingsekvliaqekmatntvyvfsmkd
gkyafkteirsclehssrptstlwvnmmargsqnikksaigqriiailpyikqeipimivfralgfvadrdilehiiydf
ddpemmemvkpsldeafvvqeqnvalnfigargarpgvtkdkrikyakeilqkemlphvgvsdfcetkkayflgymvhrl
llaslgrrelddrdhygnkrldlagpllaflfrglfknlmkevrmytqkfidrgkdfnlelaiktniitdglryslatgn
wgdqkkahqaragvsqvlnrltfastlshlrrvnspigrdgklakprqlhntlwgmlcpaetpegaavglvknlalmayi
svgsqpspilefleewsmenleeiapsaiadatkifvngcwvgihrdpeqlmatlrklrrqmdiivsevsmirdirdrei
riytdagricrpllivengslllkkthvemlkerdyknyswqvlvasgvveymytleeetvmiamspydlkqdkdyayct
tythceihpamilgvcasiipfpdhnqsprntyqsamgkqamgvyitnfhvrmdtlahvlyypmkplvttrsmeylrfre
lpaginsivailcytgynqedsvilnasavergffrsvfyrsykdsenkrvgdqeenfekphrgtcqgmrnahydklddd
giiapgirvsgddvvigktitlpenddeldsntkrfskrdastflrnsetgivdqvmltlnsegykfckirvrsvripqi
gdkfasrhgqkgtcgiqyrqedmaftceglapdiiinphaipsrmtighlieclqgklgsnkgeigdatpfndavnvqki
stflqeygyhlrgnevmynghtgrkinaqvflgptyyqrlkhmvddkihsrargpvqilvrqpmegrardgglrfgemer
dcqishgaaqflrerlfevsdpyrvhicnfcgliaianlrnntfeckgcknktqisqvrlpyaakllfqelmsmniaprl
mvt
 

Human
mydadedmqydedddeitpdlwqeacwivissyfdekglvrqqldsfdefiqmsvqrivedappidlqaeaqhasgevee
ppryllkfeqiylskpthwerdgapspmmpnearlrnltysaplyvditktvikegeeqlqtqhqktfigkipimlrsty
cllngltdrdlcelnecpldpggyfiingsekvliaqekmatntvyvfakkdskyaytgecrsclenssrptstiwvsml
arggqgakksaigqrivatlpyikqevpiiivfralgfvsdrdilehiiydfedpemmemvkpsldeafviqeqnvalnf
igsrgakpgvtkekrikyakevlqkemlphvgvsdfcetkkayflgymvhrlllaalgrrelddrdhygnkrldlagpll
aflfrgmfknllkevriyaqkfidrgkdfnlelaiktriisdglkyslatgnwgdqkkahqaragvsqvlnrltfastls
hlrrlnspigrdgklakprqlhntlwgmvcpaetpeghavglvknlalmayisvgsqpspilefleewsmenleeispaa
iadatkifvngcwvgihkdpeqlmntlrklrrqmdiivsevsmirdirereiriytdagricrpllivekqklllkkrhi
dqlkereynnyswqdlvasgvveyidtleeetvmlamtpddlqekevaycstythceihpsmilgvcasiipfpdhnqsp
rntyqsamgkqamgvyitnfhvrmdtlahvlyypqkplvttrsmeylrfrelpaginsivaiasytgynqedsvimnrsa
vdrgffrsvfyrsykeqeskkgfdqeevfekptretcqgmrhaiydkldddgliapgvrvsgddviigktvtlpenedel
estnrrytkrdcstflrtsetgivdqvmvtlnqegykfckirvrsvripqigdkfasrhgqkgtcgiqyrqedmpftceg
itpdiiinphaipsrmtighlieclqgkvsankgeigdatpfndavnvqkisnllsdygyhlrgnevlyngftgrkitsq
ifigptyyqrlkhmvddkihsrargpiqilnrqpmegrsrdgglrfgemerdcqiahgaaqflrerlfeasdpyqvhvcn
lcgimaiantrthtyecrgcrnktqislvrmpyackllfqelmsmsiaprmmsv
Peperomia (Plant)
wgmmcpaetpegqacglvknlalmvyitvgsaanpilefleewstenfeeispavipqatkifvngcwvgihrnpdllvk
tlrqlrrqidvntevgvirdirlkelrlytdygrcsrplfivenqkllikkrdiqalqqretqeegwhflvskgfieyvd
teeeettmismtindlvqarrskdaysttythceihpslilgvcasiipfpdhnqsprntyqsamgkqamgiyvtnyqlr
mdtlayvlyypqkplvttramehlhfrqlpaginaivaiacysgynqedsvimnqssidrgffrslffrsyrdeekkmgt
lvkedfgrpnrentmgmrhgsydkldddglappgtrvsgedviigktspiaqdesqgqasrynrrdhstslrhsesgmvd
qvllttnadglrfvkvrmrsvripqigdkfssrhgqkgtvgmtytqedmpwtaegitpdiivnqhaipsrmtigqlieci
mgkvaahmgkegdatpftdvtvdniskalhkcgyqmrgfetmynghtgrrlsamiflgptyyqrlkhmvddkih
 

Arabidopsis
Columbia; BAC clone F17L22.
Essentially identical to Larkin and Guilfoyle sequence for pol II 2nd
 

largest subunit
MEYNEYEPEPQYVEDDDDEEITQEDAWAVISAYFEEKGLVRQQLDSFDEFIQNTMQEIVDESADIEIRPESQHNPGHQSD
FAETIYKISFGQIYLSKPMMTESDGETATLFPKAARLRNLTYSAPLYVDVTKRVIKKGHDGEEVTETQDFTKVFIGKVPI
MLRSSYCTLFQNSEKDLTELGECPYDQGGYFIINGSEKVLIAQEKMSTNHVYVFKKRQPNKYAYVGEVRSMAENQNRPPS
TMFVRMLARASAKGGSSGQYIRCTLPYIRTEIPIIIVFRALGFVADKDILEHICYDFADTQMMELLRPSLEEAFVIQNQL
VALDYIGKRGATVGVTKEKRIKYARDILQKEMLPHVGIGEHCETKKAYYFGYIIHRLLLCALGRRPEDDRDHYGNKRLDL
AGPLLGGLFRMLFRKLTRDVRSYVQKCVDNGKEVNLQFAIKAKTITSGLKYSLATGNWGQANAAGTRAGVSQVLNRLTYA
STLSHLRRLNSPIGREGKLAKPRQLHNSQWGMMCPAETPEGQACGLVKNLALMVYITVGSAAYPILEFLEEWGTENFEEI
SPSVIPQATKIFVNGMWVGVHRDPDMLVKTLRRLRRRVDVNTEVGVVRDIRLKELRIYTDYGRCSRPLFIVDNQKLLIKK
RDIYALQQRESAEEDGWHHLVAKGFIEYIDTEEEETTMISMTISDLVQARLRPEEAYTENYTHCEIHPSLILGVCASIIP
FPDHNQSPRNTYQSAMGKQAMGIYVTNYQFRMDTLAYVLYYPQKPLVTTRAMEHLHFRQLPAGINAIVAISCYSGYNQED
SVIMNQSSIDRGFFRSLFFRSYRDEEKKMGTLVKEDFGRPDRGSTMGMRHGSYDKLDDDGLAPPGTRVSGEDVIIGKTTP
ISQDEAQGQSSRYTRRDHSISLRHSETGMVDQVLLTTNADGLRFVKVRVRSVRIPQIGDKFSSRHGQKGTVGMTYTQEDM
PWTIEGVTPDIIVNPHAIPSRMTIGQLIECIMGKVAAHMGKEGDATPFTDVTVDNISKALHKCGYQMRGFERMYNGHTGR
PLTAMIFLGPTYYQRLKHMVDDKIHSRGRGPVQILTRQPAEGRSRDGGLRFGEMERDCMIAHGAAHFLKERLFDQSDAYR
VHVCEVCGLIAIANLKKNSFECRGCKNKTDIVQVYIPYACKLLFQELMSMAIAPRMLTKHLKSAKGRQ
—————–
 

RNA polymerase III 

Yeast (cerevisae)
mvaatkrrkthihkhvkdeafddllkpvykgkkltdeintaqdkwhllpaflkvkglvkqhldsfnyfvdtdlkkiikan
qlilsdvdpefylkyvdirvgkksssstkdyltpphecrlrdmtysapiyvdieytrgrniimhkdveigrmpimlrsnk
cilydadeskmaklnecpldpggyfivngtekvilvqeqlsknriiveadekkgivqasvtsstherksktyvitkngki
ylkhnsiaeeipiaivlkacgilsdleimqlvcgndssyqdifavnleesskldiytqqqaleyigakvktmrrqkltil
qegieaiattviahltvealdfrekalyiammtrrvvmamynpkmiddrdyvgnkrlelagqlisllfedlfkkfnndfk
lsidkvlkkpnrameydallsinvhsnnitsglnraistgnwslkrfkmeragvthvlsrlsyisalgmmtrissqfeks
rkvsgpralqpsqfgmlctadtpegeacglvknlalmthittddeeepikklcyvlgveditlidsaslhlnygvylngt
ligsirfptkfvtqfrhlrrtgkvsefisiysnshqmavhiatdggricrpliivsdgqsrvkdihlrklldgeldfddf
lklglveyldvneendsyialyekdivpsmthleiepftilgavaglipyphhnqsprntyqcamgkqaigaiaynqfkr
idtllylmtypqqpmvktktielidydklpagqnatvavmsysgydiedalvlnkssidrgfgrcetrrktttvlkryan
htqdiiggmrvdengdpiwqhqslgpdglgevgmkvqsgqiyinksvptnsadapnpnnvnvqtqyreapviyrgpepsh
idqvmmsvsdndqalikvllrqnrrpelgdkfssrhgqkgvcgiivkqedmpfndqgivpdiimnphgfpsrmtvgkmie
lisgkagvlngtleygtcfggskledmskilvdqgfnysgkdmlysgitgeclqayiffgpiyyqklkhmvldkmharar
gpravltrqptegrsrdgglrlgemerdcviaygasqlllerlmissdafevdvcdkcglmgysgwcttcksaeniikmt
ipyaakllfqellsmniaprlrledifqq
S. pombe
mgvntagdpqksqpkinkggigkdesfgalfkpvykgkkladpvptiedkwqllpaflkvkglvkqhldsynyfvdvdlk
kivqanekvtsdvepwfylkyldirvgapvrtdadaiqasisphecrlrdltyganiyvdieytrgkqvvrrrnvpigrm
pvmlrsnkcvlsgknememaalnecpldpggyfivkgtekvilvqeqlsknriiveaepkkglwqasvtsstherkskty
vitkngklylkhnsvaddipivvvlkamglqsdqeifelvagaeasyqdlfapsieecaklniytaqqaleyigarvkvn
rraganrlppheealevlaavvlahinvfnlefrpkavyigimarrvlmamvdplqvddrdyvgnkrlelagqllallfe
dlfkkfnsdlklnidkvlkkphrtqefdaynqltvhsdhitqgmvralstgnwslkrfkmeragvthvlsrlsyisalgm
mtritsqfektrkvsgprslqasqfgmlctsdtpegeacglvknlalmthittdeeeepiiklayafgiedihvisgrel
hshgtylvylngailgisrypslfvasfrklrrsgkispfigifinthqravfistdggricrpliivqnglpkveskhi
rllkegkwgfedflkqglveyvdvneendslisvyerditpdtthleiepftilgavaglipyphhnqsprntyqcamgk
qaigaiaynqlqridtllylmvypqqpmvktktieligydklpagqnatvaimsysgydiedalvlnkssidrgfgrcqv
fhkhsvivrkypngthdrigdpqrdpetgevvwkhgvveddglagvgcrvqpgqiyvnkqtptnaldnsitlghtqtves
gykatpmtykapepgyidkvmltttdsdqtlikvlmrqtrrpelgdkfssrhgqkgvcgvivqqedmpfndqgicpdiim
nphgfpsrmtvgkmiellsgkvgvlrgtleygtcfggtkvedasrilvehgynysgkdmltsgitgetleayifmgpiyy
qklkhmvmdkmharargpravltrqptegrsrdgglrlgemerdcliaygasqlllerlmissdacdvdvcgqcgllgyk
gwcnscqstrevvkmtipyaakllfqellsmnivprlaledefky
 

Drosophila
mvelkmgdhnveattwdpgdskdwsvpikpltekwklvpaflqvkglvkqhidsfnhfinvdikkivkanelvtsgadpl
fylkyldvrvgkpdiddgfnitkattphecrlrdttysapitvdieytrgtqrikrnnlligrmplmlrsncaltgksef
elsklnecpldpggyfvvrgqekviliqeqlswnkmltedfngvvqcqvtssthekksrtlvlskhgkyylkhnsmtddi
pivvifkalgvvsdqeiqsligidsksqnrfgaslidaynlkvftqqraleymgsklvvkrfqsattktpseearelllt
tilahvpvdnfnlqmkaiyvsmmvrrvmaaeldktlfddrdyygnkrlelagsllsmmfedlfkrmnwelktiadknipk
vkaaqfdvvkhmraaqitaglesaissgnwtikrfkmeragvtqvlsrlsyisalgmmtrvnsqfektrkvsgprslqps
qwgmlcpsytpegeacglvknlalmthitteveerpvmivafnagvedirevsgnpinnpnvflvfingnvlgltlnhkh
lvrnlrymrrkgrmgsyvsvhtsytqrciyihtdggrlcrpyvivenrrplvkqhhldelnrgirkfddflldglieyld
vneendsfiawnedqiedrtthleietftllgvcaglvpyphhnqsprntyqcamgkqamgmigynhnnridslmynlvy
phapmvksktieltnfdklpagqnatvavmsysgydiedalilnkasidrgygrclvyknskctvkryanqtfdrimgpm
kdaltnkvifkhdvldtdgivapgeqvqnkqiminkempavtsmnplqgqsaqvpytavpisykgpepsyiervmvsana
eedflikillrqtriprgdkfssrhgqkgvtgliveqedmpfndfgicpdmimnphgfpsrmtvgktlellggkaglleg
kfhygtafggskvediqaelerhgfnyvgkdffysgitgtpleayiysgpvyyqklkhmvqdkmharargpkavltrqpt
qgrsregglrlgemerdclisygasmlimerlmissdafevdvcrtcgrmaycswchfcqssanvskismpyackllfqe
ltsmnvvpkmileny
 

A. thaliana (chromosome 5)
DEFINITION DNA-directed RNA polymerase subunit [Arabidopsis thaliana].
 

ACCESSION BAB11387
mliiflhgfqitdsliaklramgldqedldltnddhfidkeklsapikstadkfqlvpeflkvrglvkqhldsfnyfinv
gihkivkansritstvdpsiylrfkkvrvgepsiinvntveninphmcrladmtyaapifvnieyvhgshgnkaksakdn
viigrmpimlrscrcvlhgkdeeelarlgecpldpggyfiikgtekvlliqeqlsknriiidsdkkgninasvtsstemt
ksktviqmekekiylflhrfvkkipiiivlkamgmesdqeivqmvgrdprfsasllpsieecvsegvntqkqaldyleak
vkkisygtppekdgralsilrdlflahvpvpdnnfrqkcfyvgvmlrrmieamlnkdamddkdyvgnkrlelsgqlisll
fedlfktmlseaiknvdhilnkpirasrfdfsqclnkdsrysislglertlstgnfdikrfrmhrkgmtqvltrlsfigs
mgfitkispqfeksrkvsgprslqpsqwgmlcpcdtpegescglvknlalmthvttdeeegplvamcyklgvtdlevlsa
eelhtpdsflvilnglilgkhsrpqyfanslrrlrragkigefvsvftnekqhcvyvasdvgrvcrplviadkgisrvkq
hhmkelqdgvrtfddfirdglieyldvneennalvclraeaakadtthieiepftilgvvaglipyphhnqsprntyqca
mgkqamgniaynqlnrmdtllyllvypqrpllttrtielvgydklgagqnatvavmsfsgydiedaivmnkssldrgfgr
civmkkivamsqkydnctadrilipqrtgpdaekmqildddglatpgeiirpndiyinkqvpvdtvtkftsalsdsqyrp
areyfkgpegetqvvdrvalcsdkkgqlcikyiirhtrrpelgdkfssrhgqkgvcgiiiqqedfpfselgicpdlimnp
hgfpsrmtvgkmiellgskagvscgrfhygsafgersghadkvetisatlvekgfsysgkdllysgisgepveayifmgp
iyyqklkhmvldkmhargsgprvmmtrqptegkskngglrvgemerdcliaygasmliyerlmissdpfevqvcracgll
gyynyklkkavcttckngdniatmklpyackllfqvktiglffklklstsshlendkiilisgykflpkisknh

——————— 

Archae second subunit (only one polymerase, though multi-subunit similar to
eukaryotes)
 

Sulfolobus
DEFINITION DNA-DIRECTED RNA POLYMERASE SUBUNIT B.
ACCESSION P11513
PID g133422
mldtesrwaiaesffktrglvrqhldsfndflrnklqqviyeqgeivtevpglkiklgkiryekpsiretdkgpmreitp
mearlrnltysspiflsmipvenniegepieiyigdlpimlksvadptsnlpidklieigedpkdpggyfivngsekmii
aqedlatnrvlvdygksgsnithvakvtssaagyrvqvmierlkdstiqisfatvpgripfaiimralgfvtdrdivyav
sldpqiqnellpsleqassitsaeealdfignrvaigqkrenriqkaeqvidkyflphlgtspedrkkkgyylasavnki
lelylgrrepddkdhyankrvrlagdlftslfrvafkafvkdlvyqlekskvrgrrlsltalvradiiterirhalatgn
wvggrtgvsqlldrtnwlsmlshlrrvvsslargqpnfeardlhgtqwgrmcpfetpegpnsglvknlallaqvsvgine
svvervayelgvvsvedvirriseqnedvekymswskvylngrllgyyedgkelakkiresrrqgklsdevnvayiatdy
lnevhincdagrvrrpliivnngtplvdtedikklkngeitfddlvkqgkiefidaeeeenayvalnpqdltpdhthlei
wpsailgiiasiipypehnqsprntyqsamakqslglyasnyqirtdtrahllhypqmplvqtrmlgvigyndrpagana
ilaimsytgynmedsiimnkssiergmyrstffrlysteevkypggqedkivtpeagvkgykgkdyyrlledngvvspev
evkggdvligkvspprflqefkelspeqakrdtsivtrhgengivdlvlitetlegnklvkvrvrdlripeigdkfatrh
gqkgvvgilidqvdmpytakgivpdiilnphalpsrmtigqimeaiggkyaalsgkpvdatpfletpklqemqkeilklg
hlpdstevvydgrtgqklksrilfgivyyqklhhmvadkmharargpvqiltrqptegraregglrfgemerdcligfgt
amlikdrlldnsykavvyicdqcgyvgwydrsknryvcpvhgdksvlhpvtvsyafklliqelmsmvisprlilgekvnl
ggasne

Well, this was helpful. Sequences and useful notes about them. So I played around with the sequences and searched for some other homologs and built a few alignments, build some masks to filter out poorly aligned regions, and then fed the data into PAUP and built a tree. (I note – I know about this because amazingly I still have all the files)

And I wrote back to Mike Bevan and Craig on Sept 8:

Mike and Craig 

Attached is a phylogenetic tree of RNA polymerase subunits (Craig suggested I look at these because of an unusual protein in the A. thaliana genome). A. thaliana has representatives in five different subfamilies – Pol-I, Pol-II, Pol-III and RpoB (for the chloroplast) as would be expected and then this novel Pol which I have called Pol-IV. 

I do not know much about RNA polymerase, but it seems like this is a pretty big deal and I think should be emphasized in the paper. What do you think? I could try to make a pretty tree figure to show the different families. 

Jonathan

I got an email back:

Dear Jonathan (and Mike), 

Many thanks for the detailed phylogenetic tree of the mystery pol subunit.
I think a figure is the only way to show clearly that this protein defines
a new clade. Is there room for such a figure, Mike? 

In the lab we have also been calling it a putative pol IV subunit just for
the shock value of saying the words (a radical idea in the transcription
field), though in the absence of knowing what other subunits associate with
it, I’m not sure what to call it in the annotation or figure. Maybe
“oddpol” or “atypical polymerase 2nd subunit”. It takes more than a dozen
subunits to make a eukaryotic polymerase, so it is not clear that one
unusual subunit is enough to confer new properties-i.e. a true pol IV.
Obviously, that will require quite a bit of work. 

Cheers,
Craig

Me to Craig on 9/11/00:

Yes 

I agree that it is too early to call it a true polIV, and I was doing it for the shock value too 

Jonathan 

PS. Do you mind if I present this at the TIGR GSAC meeting later this week 

Jonathan

Craig to me 9/11:

Hi Jonathan, 

Feel free to show the data. In thinking more about this, it is worth also
making a phylogenetic tree for the largest pol subunit (the equivalent of
eubacterial B’) just to see if there might be a fourth class out there for
the largest subunit, too. If there is, pol IV may not be such a wild idea. 

In case you are interested in giving this a try, I’m including some
sequences below. In the meantime, is there a good web site for performing
the types of extensive phylogenetic trees you’ve done for the mystery
subunit? I should do this for many of the general transcription factors
just to be sure they really group with the correct homologs, as you
suggested. 

Anyway, here are some largest subunit sequences for pol I, II and III.
Vive la difference! 

Craig

Pol I:
rat
mlaskhtpwrrlqgisfgmysaeelkklsvksitnpryvdslgnpsadglydlalgpadskevcstcvqdfnncsghlgh
idlpltvynpllfdklylllrgsclnchmltcpraaihllvcqlkvldvgalqavyelerilsrfleetsdpsafeiqee
leeytskilqnnllgsqgahvknvcesrsklvahfwkthmaakrcphcktgrsvvrkehnskltitypamvhkksgqkda
elpegapaapgideaqmgkrgyltpssaqehlfaiwknegfflnylfsglddigpessfnpsmffldfivvppsryrpin
rlgdqmftngqtvnlqavmkdavlirkllavmaqeqklpcemteitidkendssgaidrsflsllpgqsltdklyniwir
lqshvnivfdsdmdklmlekypgirqilekkeglfrkhmmgkrvdyaarsvicpdmyintneigipmvfatkltypqpvt
pwnvqelrqavingpnvhpgasmvinedgsrtalsavdatqreavakqlltpstgipkpqgakvvcrhvkngdilllnrq
ptlhrpsiqahrahilpeekvlrlhyanckaynadfdgdemnahfpqselgraeayvlactdqqylvpkdgqplagliqd
hmvsganmtirgcfftreqymelvyrgltdkvgrvklfppailkpfplwtgkqvvstlliniipedytplnltgkakigs
kawvkekprpvpdfdpdsmcesqviiregellcgvldkahygssayglvhccyeiyggetsgrvltclarlftaylqlyr
gftlgvedilvkpnadvmrqriieestqcgpravraalnlpeaascdeiqgkwqdaiwrkdqrdfnmidmkfkeevnhys
neinkacmpfglhrqfpennlqmmvqsgakgstvntmqiscllgqielegrrpplmasgkslpcfepyeftpraggfvtg
rfltgirppefffhcmagreglvdtavktsrsgylqrciikhleglviqydltvrdsdgsvvqflygedgldipktqflq
pkqfpflasnyevimkskhlhevlsradpqkvlrhfraikkwhhrhssallrkgaflsfsqkiqaavkalnlegktqngr
spetqqmlqmwheldeqsrrkyqkraapcpdpslsvwrpdihfasvsetfekkiddysqewaaqaekshnrselsldrlr
tllqlkwqrslcdpgeavgllaaqsigepstqmtlntfhfagrgemnvtlgiprlreilmvasaniktpmmsvpvfntkk
alrrvkslkkqltrvclgevlqkvdiqesfcmgekqnkfrvyelrfqflphayyqqekclrpedilhfmetrffkllmea
ikkknskasafrsvntrratqkdlddtedsgrnrreeerdeeeegnivdaeaeegdadasdtkrkekqeeevdyeseeeg
eeeeeedvqeeenikgegahqthepdeeegsgleeessqnppcrhsrpqgaeamerriqavreshsfiedyqydteeslw
cqvtvklplmkinfdmsslvvslahnaivyttkgitrcllnetinsknekefvlnteginlpelfkysevldlrrlysnd
ihavantygieaalrviekeikdvfavygiavdprhlslvadymcfegvykplnrfgiqssssplqqmtfetsfqflkqa
tmmgshdelkspsaclvvgkvvkggtglfelkqplr
 

Drosophila
mgskramdvhmfpsdlefavftdqeirklsvvkvitgitfdalghaipgglydirmgsygrcmdpcgtclklqdcpghmg
hielgtpvynpffikfvqrllcifclhcyklqmkdheceiimlqlrlidagyiieaqelelfkseivcqntenlvaikng
dmvhphiaamykllekneknssnstktscslrtaithsalqrlgkkcrhcnksmrfvrymhrrlvfyvtladikervgtg
aetggqnkvifadecrrylrqiyanypellkllvpvlglsntdltqgdrspvdlffmdtlpvtpprarplnmvgdmlkgn
pqtdiyiniiennhvlnvvlkymkggqeklteeakaayqtlkgetaheklytawlalqmsvdvlldvnmsremksgeglk
qiiekkcglirshmmgkrvnyaartvitpaypninvdeigipdifakklsypvpvtewnvtdvrkmvmngpdvhpganyi
qdkngfttyipadnaskreslaklllsnpkdgikivhrhvlngdvlllnrqpslhkpsimghkarilhgektfrlhysnc
kaynadfdgdemnahypqsevaraeaynlvnvasnylvpkdgtplggliqdhvisgvklsirgrffnredyqqlvfqgls
qlkkdikllpptilkpavlwsgkqilstiiiniipegyerinldsfakiagknwnvsrprppicgtnpegndlsesqvqi
rngellvgvldkqqygattyglihcmyelyggdvstllvtaftkvftfflqlegftlgvkdilvtdvadrkrrkiirecr
nvgnsavaaaleledepphdelvekmeaayvkdskfrvlldrkykslldgytndinstclprglitkfpsnnlqlmvlsg
akgsmvntmqiscllgqielegkrpplmisgkslpsftsfetspksggfidgrfmtgiqpqdfffhcmagreglidtavk
tsrsgylqrclikhleglsvhydltvrdsdnsvvqflygedgldilkskffndkfcadfltqnatailrpaqlqlmkdee
qlvkvqrhekhirswekkkpaklraafthfseelreevevkrpnevnsktgrrrfdegllklwkkadaedkalyrkkyar
cpdptvavykqdlyygsvsertrklitdyakrkpalketiadimrvktikslaapgepvgliaaqsigepstqmtlntfh
fagrgemnvtlgiprlreilmlassniktpsmdipikpgqqhqaeklrinlnsvtlanlleyvhvstgltldpersyeyd
mrfqflprevykedygvrpkhiikymhqtffkqlipppilkvsnasrttkivviddkkdadkdddndldngdevgrskak
andddssddnddddatgvklkqrktdekdyddpddveelhdanddddeaededdeekgqdgndndgddkaverllsndmv
kaytydkenhlwcqvklnlsvryqkpdltsiirelagksvvhqvqhikraiiykgndddqllktdginigemfqhnkild
lnrlysndihaiartygieaasqvivkevsnvfkvygitvdrrhlsliadymtfdgtfqplsrkgmehsssplqqmsfes
slqflksaagfgradelsspssrlmvglpvrngtgafelltkic
 

yeast (S.c.)
mdiskpvgseitsvdfgiltakeirnlsakqitnptvldnlghpvsgglydlalgaflrnlcstcgldekfcpghqghie
lpvpcynplffnqlyiylrasclfchhfrlksvevhryacklrllqyglidesykldeitlgslnssmytddeaiedted
emdgegskqskdisstllnelkskrseyvdmaiakalsdgrttergsftatvnderkklvhefhkkllsrgkcdncgmfs
pkfrkdgftkifetalnekqitnnrvkgfirqdmikkqkqakkldgsneasandeesfdvgrnpttrpktgstyilstev
knildtvfrkeqcvlqyvfhsrpnlsrklvkadsffmdvlvvpptrfrlpsklgeevhensqnqllskvlttsllirdln
ddlsklqkdkvsledrrvifsrlmnafvtiqndvnafidstkaqgrtsgkvpipgvkqalekkeglfrkhmmgkrvnyaa
rsvispdpnietneigvppvfavkltypepvtayniaelrqavingpdkwpgatqiqnedgslvsligmsveqrkalanq
lltpssnvsthtlnkkvyrhiknrdvvlmnrqptlhkasmmghkvrvlpnektlrlhyantgaynadfdgdemnmhfpqn
enaraealnlantdsqyltptsgspvrgliqdhisagvwltskdsfftreqyqqyiygcirpedghttrskivtlpptif
kpyplwtgkqiittvllnvtppdmpginlisknkikneywgkgslenevlfkdgallcgildksqygaskygivhslhev
ygpevaakvlsvlgrlftnyitataftcgmddlrltaegnkwrtdilktsvdtgreaaaevtnldkdtpaddpellkrlq
eilrdnnksgildavtsskvnaitsqvvskcvpdgtmkkfpcnsmqamalsgakgsnvnvsqimcllgqqalegrrvpvm
vsgktlpsfkpyetdamaggyvkgrfysgikpqeyyfhcmagreglidtavktsrsgylqrcltkqlegvhvsydnsird
adgtlvqfmyggdaiditkeshmtqfefcldnyyallkkynpsaliehldvesalkyskktlkyrkkhskephykqsvky
dpvlakynpakylgsvsenfqdklesfldknsklfkssdgvnekkfralmqlkymrslinpgeavgiiasqsvgepstqm
tlntfhfaghgaanvtlgiprlreivmtasaaiktpqmtlpiwndvsdeqadtfcksiskvllsevidkvivtettgtsn
taggnaarsyvihmrffdnneyseeydvskeelqnvisnqfihlleaaivkeikkqkrttgpdigvavprlqtdvansss
nskrleedndeeqshkktkqavsydepdedeietmreaekssdeegidsdkesdsdsededvdmneqinksiveannnmn
kvqrdrqsaiishhrfitkynfddesgkwcefklelaadtekllmvniveeicrksiirqiphidrcvhpepengkrvlv
tegvnfqamwdqeafidvdgitsndvaavlktygveaarntivneinnvfsryaisvsfrhldliadmmtrqgtylafnr
qgmetstssfmkmsyettcqfltkavldnereqldspsarivvgklnnvgtgsfdvlakvpnaa
 

Arabidopsis
MAHAQTTEVCLSFHRSLLFPMGASQVVESVRFSFMTEQDVRKHSFLKVTSPILHDNVGNPFPGGLYDLKLGPKDDKQACN
SCGQLKLACPGHCGHIELVFPIYHPLLFNLLFNFLQRACFFCHHFMAKPEDVERAVSQLKLIIKGDIVSAKQLESNTPTK
SKSSDESCESVVTTDSSEECEDSDVEDQRWTSLQFAEVTAVLKNFMRLSSKSCSRCKGINPKLEKPMFGWVRMRAMKDSD
VGANVIRGLKLKKSTSSVENPDGFDDSGIDALSEVEDGDKETREKSTEVAAEFEEHNSKRDLLPSEVRNILKHLWQNEHE
FCSFIGDLWQSGSEKIDYSMFFLESVLVPPTKFRPPTTGGDSVMEHPQTVGLNKVIESNNILGNACTNKLDQSKVIFRWR
NLQESVNVLFDSKTATVQSQRDSSGICQLLEKKEGLFRQKMMGKRVNHACRSVISPDPYIAVNDIGIPPCFALKLTYPER
VTPWNVEKLREAIINGPDIHPGATHYSDKSSTMKLPSTEKARRAIARKLLSSRGATTELGKTCDINFEGKTVHRHMRDGD
IVLVNRQPTLHKPSLMAHKVRVLKGEKTLRLHYANCSTYNADFDGDEMNVHFPQDEISRAEAYNIVNANNQYARPSNGEP
LRALIQDHIVSSVLLTKRDTFLDKDHFNQLLFSSGVTDMVLSTFSGRSGKKVMVSASDAELLTVTPAILKPVPLWTGKQV
ITAVLNQITKGHPPFTVEKATKLPVDFFKCRSREVKPNSGDLTKKKEIDESWKQNLNEDKLHIRKNEFVCGVIDKAQFAD
YGLVHTVHELYGSNAAGNLLSVFSRLFTVFLQTHGFTCGVDDLIILKDMDEERTKQLQECENVGERVLRKTFGIDVDVQI
DPQDMRSRIERILYEDGESALASLDRSIVNYLNQCSSKGVMNDLLSDGLLKTPGRNCISLMTISGAKGSKVNFQQISSHL
GQQDLEGKRVPRMVSGKTLPCFHPWDWSPRAGGFISDRFLSGLRPQEYYFHCMAGREGLVDTAVKTSRSGYLQRCLMKNL
ESLKVNYDCTVRDADGSIIQFQYGEDGVDVHRSSFIEKFKELTINQDMVLQKCSEDMLSGASSYISDLPISLKKGAEKFV
EAMPMNERIASKFVRQEELLKLVKSKFFASLAQPGEPVGVLAAQSVGEPSTQMTLNTFHLAGRGEMNVTLGIPRLQEILM
TAAANIKTPIMTCPLLKGKTKEDANDITDRLRKITVADIIKSMELSVVPYTVYENEVCSIHKLKINLYKPEHYPKHTDIT
EEDWEETMRAVFLRKLEDAIETHMKMLHRIRGIHNDVTGPIAGNETDNDDSVSGKQNEDDGDDDGEGTEVDDLGSDAQKQ
KKQETDEMDYEENSEDETNEPSSISGVEDPEMDSENEDTEVSKEDTPEPQEESMEPQKEVKGVKNVKEQSKKKRRKFVRA
KSDRHIFVKGEGEKFEVHFKFATDDPHILLAQIAQQTAQKVYIQNSGKIERCTVANCGDPQVIYHGDNPKERREISNDEK
KASPALHASGVDFPALWEFQDKLDVRYLYSNSIHDMLNIFGVEAARETIIREINHVFKSYGISVSIRHLNLIADYMTFSG
GYRPMSRMGGIAESTSPFCRMTFETATKFIVQAATYGEKDTLETPSARICLGLPALSGTGCFDLMQRVEL
 

Pol II:
Arabidopsis
mdtrfpfspaevskvrvvqfgilspdeirqmsvihvehsettekgkpkvgglsdtrlgtidrkvkcetcmanmaecpghf
gylelakpmyhvgfmktvlsimrcvcfncskiladeamkiknpknrlkkildacknktkcdggddiddvqshstdepvkk
srggcgaqqpkltiegmkmiaeyknskeendepdqlpepaerkqtlgadrvlsvlkrisdadcqllgfnpkfarpdwmil
evlpippppvrpsvmmdatsrseddlthqlamiirhnenlkrqekngaprhiisrftqllqfhiatyfdnelpgqpratq
ksgrpiksicsrlkakegrirgnlmgkrvdfsartvitpdptinidelgvpwsialnltypetvtpynierlkelvdygp
hpppgktgakyiirddgqrldlrylkkssdqhlelgyryvllsysihsthkrlflevvifmlswsqverhlqdgdfvlfn
rqpslhkmsimghririmpystfrlnlsvtspynadfdgdemnmhvpqsfetraevlelmmvpkcivspqanrpvmgivq
dtllgcrkitkrdtfiekdvfmntlmwwedfdgkvpapailkprplwtgkqvfnliipkqinllrysawhadtetgfitp
gdtqvriergellagtlckktlgtsngslvhviweevgpdaarkflghtqwlvnywllqngftigigdtiadsstmekin
etisnaktavkdlirqfqgkeldpepgrtmrdtfenrvnqvlnkarddagssaqkslaetnnlkamvtagskgsfinisq
mtacvgqqnvegkripfgfdgrtlphftkddygpesrgfvensylrgltpqefffhamggreglidtavktsetgyiqrr
lvkamedimvkydgtvrnslgdviqflygedgmdavwiesqkldslkmkksefdrtfkyeiddenwnptylsdehledlk
girelrdvfdaeyskletdrfqlgteiatngdstwplpvnikrhiwnaqktfkidlrkisdmhpveivdavdklqerllv
vpgddalsveaqknatlffnillrstlaskrvleeyklsreafewvigeiesrflqslvapgemigcvpaqsigepatqm
tlntfhyagvsaknvtlgvprlreiinvakriktpslsvyltpeaskskegaktvqcaleyttlrsvtqatevwydpdpm
stiieedfefvrsyyempdedvspdkispwllrielnremmvdkklsmadiaekinlefdddltcifnddnaqklilrir
imndegpkgelqdesaeddvflkkiesnmltemalrgipdinkvfikqvrksrfdeeggfktseewmldtegvnllavmc
hedvdpkrttsnhlieiievlgieavrralldelrvvisfdgsyvnyrhlailcdtmtyrghlmaitrhginrndtgplm
rcsfeetvdilldaaayaetdclrgvtenimlgqlapigtgdcelylndemlknaielqlpsymdglefgmtparspvsg
tpyhegmmspnyllspnmrlspmsdaqfspyvggmafspssspgyspsspgysptspgysptspgysptspgysptspty
spsspgysptspaysptspsysptspsysptspsysptspsysptspsysptspsysptspaysptspaysptspayspt
spsysptspsysptspsysptspsysptspsysptspaysptspgysptspsysptspsygptspsynpqsakyspsiay
spsnarlspaspysptspnysptspsysptspsyspssptyspsspyssgaspdyspsagysptlpgyspsstgqytphe
gdkkdktgkkdaskddkgnp
 

Drosophila
mstptdskaplrqvkrvqfgilspdeirrmsvteggvqfaetmeggrpklgglmdprqgvidrtsrcqtcagnmtecpgh
fghidlakpvfhigfitktikilrcvcfycskmlvsphnpkikeivmksrgqprkrlayvydlckgkticeggedmdltk
enqqpdpnkkpghggcghyqpsirrtgldltaewkhqnedsqekkivvsaervweilkhitdeecfilgmdpkyarpdwm
ivtvlpvpplavrpavvmfgaaknqddlthklsdiikannelrkneasgaaahviqenikmlqfhvatlvdndmpgmpra
mqksgkplkaikarlkgkegrirgnlmgkrvdfsartvitpdpnlridqvgvprsiaqnltfpelvtpfnidrmqelvrr
gnsqypgakyivrdngeridlrfhpkssdlhlqcgykverhlrdddlvifnrqptlhkmsmmghrvkvlpwstfrmnlsc
tspynadfdgdemnlhvpqsmetraevenihitprqiitpqankpvmgivqdtltavrkmtkrdvfitreqvmnllmflp
twdakmpqpcilkprplwtgkqifsliipgnvnmirthsthpdeedegpykwispgdtkvmvehgelimgilckkslgts
agsllhicflelghdiagrfygniqtvinnwllfeghsigigdtiadpqtyneiqqaikkakddvinviqkahnmelept
pgntlrqtfenkvnrilndahdktggsakkslteynnlkamvvsgskgsninisqviacvgqqnvegkripygfrkrtlp
hfikddygpesrgfvensylagltpsefyfhamggreglidtavktaetgyiqrrlikamesvmvnydgtvrnsvgqliq
lrygedglcgelvefqnmptvklsnksfekrfkfdwsnerlmkkvftddvikemtdsseaiqeleaewdrlvsdrdslrq
ifpngeskvvlpcnlqrmiwnvqkifhinkrlptdlspirvikgvktllercvivtgndriskqanenatllfqclirst
lctkyvseefrlsteafewlvgeietrfqqaqanpgemvgalaaqslgepatqmtlntfhfagvssknvtlgvprlkeii
niskkpkapsltvfltggaardaekaknvlcrlehttlrkvtantaiyydpdpqrtvisedqefvnvyyempdfdptris
pwllrieldrkrmtdkkltmeqiaekinvgfgedlncifnddnadklvlririmnneenkfqdedeavdkmeddmflrci
eanmlsdmtlqgieaigkvymhlpqtdskkrivitetgefkaigewlletdgtsmmkvlserdvdpirtssndiceifqv
lgieavrksvekemnavlqfyglyvnyrhlallcdvmtakghlmaitrhginrqdtgalmrcsfeetvdvlmdaaahaet
dpmrgvseniimgqlpkmgtgcfdllldaekcrfgieipntlgnsmlggaamfigggstpsmtppeldsawancntpryf
sppghvsamtpggpsfspsaasdasgmspswspahpgsspsspgpsmspyfpaspsvspsysptspnytasspggaspny
spsspnysptsplyaspryasttpnfnpqstgyspsssgysptspvysptvqfqsspsfagsgsniyspgnayspsssny
spnspsysptspsyspsspsysptspcysptspsysptspnytpvtpsysptspnysaspqyspaspaysqtgvkyspts
ptysppspsydgspgspqytpgspqyspaspkysptsplyspsspqhspsnqysptgstysatspryspnmsiyspsstk
ysptsptytptarnysptspmysptapshysptspayspssptfeesedvrkggrg
 

human
mhgggppsgdsacplrtikrvqfgvlspdelkrmsvteggikypetteggrpklgglmdprqgviertgrcqtcagnmte
cpghfghielakpvfhvgflvktmkvlrcvcffcskllvdsnnpkikdilakskgqpkkrlthvydlckgkniceggeem
dnkfgveqpegdedltkekghggcgryqprirrsglelyaewkhvnedsqekkillspervheifkrisdeecfvlgmep
ryarpewmivtvlpvpplsvrpavvmqgsarnqddlthkladivkinnqlrrneqngaaahviaedvkllqfhvatmvdn
elpglpramqksgrplkslkqrlkgkegrvrgnlmgkrvdfsartvitpdpnlsidqvgvprsiaanmtfaeivtpfnid
rlqelvrrgnsqypgakyiirdngdridlrfhpkpsdlhlqtgykverhmcdgdivifnrqptlhkmsmmghrvrilpws
tfrlnlsvttpynadfdgdemnlhlpqsletraeiqelamvprmivtpqsnrpvmgivqdtltavrkftkrdvflergev
mnllmflstwdgkvpqpailkprplwtgkqifsliipghincirthsthpddedsgpykhispgdtkvvvengelimgil
ckkslgtsagslvhisylemghditrlfysniqtvinnwllieghtigigdsiadsktyqdiqntikkakqdvievieka
hnneleptpgntlrqtfenqvnrilndardktgssaqkslseynnfksmvvsgakgskinisqviavvgqqnvegkripf
gfkhrtlphfikddygpesrgfvensylagltptefffhamggreglidtavktaetgyiqrrliksmesvmvkydatvr
nsinqvvqlrygedglagesvefqnlatlkpsnkafekkfrfdytneralrrtlqedlvkdvlsnahiqnelerefermr
edrevlrvifptgdskvvlpcnllrmiwnaqkifhinprlpsdlhpikvvegvkelskklvivngddplsrqaqenatll
fnihlrstlcsrrmaeefrlsgeafdwllgeieskfnqaiahpgemvgalaaqslgepatqmtlntfhyagvsaknvtlg
vprlkeliniskkpktpsltvfllgqsardaerakdilcrlehttlrkvtantaiyydpnpqstvvaedqewvnvyyemp
dfdvarispwllrveldrkhmtdrkltmeqiaekinagfgddlncifnddnaeklvlririmnsdenkmqeeeevvdkmd
ddvflrciesnmltdmtlqgieqiskvymhlpqtdnkkkiiitedgefkalqewiletdgvslmrvlsekdvdpvrttsn
diveiftvlgieavrkalerelyhvisfdgsyvnyrhlallcdtmtcrghlmaitrhgvnrqdtgplmkcsfeetvdvlm
eaaahgesdpmkgvsenimlgqlapagtgcfdllldaekckygmeiptnipglgaagptgmffgsapspmggispamtpw
nqgatpaygawspsvgsgmtpgaagfspsaasdasgfspgyspawsptpgspgspgpsspyipspggamspsysptspay
eprspggytpqspsysptspsysptspsysptspnysptspsysptspsysptspsysptspsysptspsysptspsysp
tspsysptspsysptspsysptspsysptspsysptspsysptspsysptspsysptspsysptspnysptspnytptsp
sysptspsysptspnytptspnysptspsysptspsysptspsyspssprytpqsptytpsspsyspsspsysptspkyt
ptspsyspsspeytpaspkysptspkysptspkysptsptyspttpkysptsptysptspvytptspkysptsptyspts
pkysptsptysptspkgstysptspgysptsptysltspaispddsdeen
 

yeast (S.c)
mvgqqyssaplrtvkevqfglfspeevraisvakirfpetmdetqtrakigglndprlgsidrnlkcqtcqegmnecpgh
fghidlakpvfhvgfiakikkvcecvcmhcgkllldehnelmrqalaikdskkrfaaiwtlcktkmvcetdvpseddptq
lvsrggcgntqptirkdglklvgswkkdratgdadepelrvlsteeilnifkhisvkdftslgfnevfsrpewmiltclp
vppppvrpsisfnesqrgeddltfkladilkanisletlehngaphhaieeaesllqfhvatymdndiagqpqalqksgr
pvksirarlkgkegrirgnlmgkrvdfsartvisgdpnleldqvgvpksiaktltypevvtpynidrltqlvrngpnehp
gakyvirdsgdridlryskragdiqlqygwkverhimdndpvlfnrqpslhkmsmmahrvkvipystfrlnlsvtspyna
dfdgdemnlhvpqseetraelsqlcavplqivspqsnkpcmgivqdtlcgirkltlrdtfieldqvlnmlywvpdwdgvi
ptpaiikpkplwsgkqilsvaipngihlqrfdegttllspkdngmliidgqiifgvvekktvgssngglihvvtrekgpq
vcaklfgniqkvvnfwllhngfstgigdtiadgptmreitetiaeakkkvldvtkeaqanlltakhgmtlresfednvvr
flneardkagrlaevnlkdlnnvkqmvmagskgsfiniaqmsacvgqqsvegkriafgfvdrtlphfskddyspeskgfv
ensylrgltpqefffhamggreglidtavktaetgyiqrrlvkaledimvhydnttrnslgnviqfiygedgmdaahiek
qsldtiggsdaafekryrvdllntdhtldpsllesgseilgdlklqvlldeeykqlvkdrkflrevfvdgeanwplpvni
rriiqnaqqtfhidhtkpsdltikdivlgvkdlqenllvlrgkneiiqnaqrdavtlfccllrsrlatrrvlqeyrltkq
afdwvlsnieaqflrsvvhpgemvgvlaaqsigepatqmtlntfhfagvaskkvtsgvprlkeilnvaknmktpsltvyl
epghaadqeqaklirsaiehttlksvtiaseiyydpdprstvipedeeiiqlhfslldeeaeqsfdqqspwllrleldra
amndkdltmgqvgerikqtfkndlfviwsedndekliircrvvrpksldaeteaeedhmlkkientmlenitlrgvenie
rvvmmkydrkvpsptgeyvkepewvletdgvnlsevmtvpgidptriytnsfidimevlgieagraalykevynviasdg
syvnyrhmallvdvmttqggltsvtrhgfnrsntgalmrcsfeetveilfeagasaelddcrgvsenvilgqmapigtga
fdvmideeslvkympeqkiteiedgqdggvtpysnesglvnadldvkdelmfsplvdsgsndamaggftayggadygeat
spfgaygeaptspgfgvsspgfsptsptysptspaysptspsysptspsysptspsysptspsysptspsysptspsysp
tspsysptspsysptspsysptspsysptspsysptspsysptspsysptspsysptspaysptspsysptspsysptsp
sysptspsysptspnysptspsysptspgyspgspayspkqdeqkhnenensr

pol III:
human
mvkeqfretdvakktshicfgmkspeemrqqahiqvvsknlysqdnqhapllygvldhrmgtsekdrpcetcgknladcl
ghygyidlelpcfhvgyfravigilqmicktcchimlsqeekkqfldylkrpgltylqkrglkkkisdkcrkknichhcg
afngtvkkcgllkiihekyktnkkvvdpivsnflqsfetaiehnkevepllgraqenlnplvvlnlfkripaedvplllm
npeagkpsdliltrllvpplcfrpsvvsdlksgtneddltmklteiiflndvikkhrisgaktqmimedwdflqlqcaly
inselsgiplnmapkkwtrgfvqrlkgkqgrfrgnlsgkrvdfsgrtvispdpnlridevavpvhvakiltfpekvnkan
inflrklvqngpevhpganfiqqrhtqmkrflkygnrekmaqelkygdiverhlidgdvvlfnrqpslhklsimahlarv
kphrtfrfnecvctpynadfdgdemnlhlpqteeakaealvlmgtkanlvtprngepliaaiqdfltgaylltlkdtffd
rakacqiiasilvgkdekikvrlppptilkpvtlwtgkqifsvilrpsddnpvranlrtkgkqycgkgedlcandsyvti
qnselmsgsmdkgtlgsgsknnifyillrdwgqneaadamsrlarlapvylsnrgfsigigdvtpgqgllkakyellnag
ykkcdeyiealntgklqqqpgctaeetlealilkelsvirdhagsaclreldksnspltmalcgskgsfinisqmiacvg
qqaisgsrvpdgfenrslphfekhsklpaakgfvansfysgltptefffhtmagreglvdtavktaetgymqrrlvksle
dlcsqydltvrsstgdiiqfiyggdgldpaamegkdeplefkrvldnikavfpcpsepalsknelilttesimkkseflc
cqdsflqeikkfikgvsekikktrdkygindngtteprvlyqldritptqvekfletcrdkymraqmepgsavgalcaqs
igepgtqmtlktfhfggvasmnitlgvprikeiinaskaistpiitaqldkdddadyarlvkgriektllgeiseyieev
flpddcfilvklslerirllrlevnaetvrysictsklrvkpgdvavhgeavvcvtprenskssmyyvlqflkedlpkvv
vqgipevsravihideqsgkekykllvegdnlravmathgvkgtrttsnntyevektlgieaarttiineiqytmvvnhg
msidrrhvmllsdlmtykgevlgitrfglakmkesvlmlasfektadhlfdaayfgqkdsvcgvseciimgipmnigtgl
fkllhkadrdpnppkrplifdtnefhiplvt

trypanosome
mlkgssstsfllpqqfveplphapveisalhygllsrndvhrlsvlpcrrvvgdvkeygvndarlgvcdrlsicetcgln
siecvghpghidleapvfhlgffttvlricrtickrcshvllddteidyykrrlssssleplqrtmliktiqtdayktrv
clkcgglngvvrrvrpmrlvhekyhveprrgegprenpggffdaelrtacaynkvvgecrefvhdfldpvrvrqlflavp
pgevillglapgvsptdllmttllvppvpvrprgcagtttvrdddltaqyndilvstdtmqdgsldatrytetwemlqmr
aarlldsslpgfppnvrtsdlksyaqrlkskhgrfrcnlsgkrvdysgrsvispdpnldvdelavplhvarvltypqrvf
kanhelmrrlvrngphvhpgattvylaqegskkslknerdrhrlaarlavgdiverhvmngdlvlfnrqpslhrvsmmah
rarvlpfrtfrfnecccapynadfdgdemnvhfvqtekaraealqlmstarniisakngepiiactqdflaaaylvtsrd
vffdrgefsqmvshwlgpvtqfrlpipailkpvelwtgkqlfelivrpspevdvllsfeaptkfytrkgkhdcaeegyva
fldscfisgrldkkllgggakdglfarlhtiagggytarvmsriaqftsryltnygfslglgdvaptpelnkqkaavlar
svevcdgliksaktgrmiplpgltvkqslearlntelskvrdecgtaavqtlsihnntplimvqsgskgsalniaqmmac
vgqqtvsgkrildafqdrslphfhrfeeapaargfvansfysglsptefffhtmagreglvdtavktaetgyiyrrlmka
menlsvrydgtvrntkgdviqlrfgedgldpqlmegnsgtplnleqewlsvraayarwvvgllagsktasdgnairdnen
yfnefismlptegpsfveaclngdqealkvceeqesredalhnsngktndresrprtgrlrravlishlvkvcsrkfkdd
iqdffvkkvreqqrirnllnlpntsrertegggdnsgpiankrtkkrapslkvkdskeggrvselrdlemlqtellpltr
gmvtrfiaqcaskylrkacepgtpcgaiaaqsvgepstqmtlrtfhfagvasmsitqgvprlvevinanrniatpvvtap
vllmegeenhceifrkrarfvkaqiervllrevvseivevcsdtefylrvhlnmsvitklhlpinaitvrqrilaaaght
msplrmlnedcievfsldtlavyphfqdarwvhfslrrilgllpdvvvggigginramissngtevlaegaelravmnlw
gvdstrvvcnhvavvervlgieaarrvivdeiqnilkayslsidvrhvylladlmtqrgvvlgitrygiqkmnfnvltma
sferttdhlynaaatqrvdrdlsvsdsiivgkpvplgttsfdllldgsisndilppqrcvkrgmgpnfhtakrhhlvpla
aegvfrldlf 

yeast (S.c)
mkevvvsetpkrikglefsalsaadivaqsevevstrdlfdlekdrapkangaldpkmgvsssslecatchgnlaschgh
fghlklalpvfhigyfkatiqilqgickncsaillsetdkrqflhelrrpgvdnlrrmgilkkildqckkqrrclhcgal
ngvvkkaaagagsaalkiihdtfrwvgkksapekdiwvgewkevlahnpeleryvkrcmddlnplktlnlfkqiksadce
llgidatvpsgrpetyiwrylpappvcirpsvmmqdspasneddltvklteivwtsslikagldkgisinnmmehwdylq
ltvamyinsdsvnpamlpgssngggkvkpirgfcqrlkgkqgrfrgnlsgkrvdfsgrtvispdpnlsidevavpdrvak
vltypekvtrynrhklqelivngpnvhpganyllkrnedarrnlrygdrmklaknlqigdvverhledgdvvlfnrqpsl
hrlsilshyakirpwrtfrlnecvctpynadfdgdemnlhvpqteearaeainlmgvknnlltpksgepiiaatqdfitg
sylishkdsfydratltqllsmmsdgiehfdipppaimkpyylwtgkqvfsllikpnhnspvvinldaknkvfvppksks
lpnemsqndgfviirgsqilsgvmdksvlgdgkkhsvfytilrdygpqeaanamnrmaklcarflgnrgfsigindvtpa
ddlkqkkeelveiayhkcdelitlfnkgeletqpgcneeqtleakiggllskvreevgdvcineldnwnaplimatcgsk
gstlnvsqmvavvgqqiisgnrvpdgfqdrslphfpknsktpqskgfvrnsffsglsppeflfhaisgreglvdtavkta
etgymsrrlmksledlscqydntvrtsangivqftyggdgldplemegnaqpvnfnrswdhaynitfnnqdkgllpyaim
etaneilgpleerlvrydnsgclvkredlnkaeyvdqydaerdfyhslreyingkatalanlrksrgmlglleppakelq
gidpdetvpdnvktsvsqlyriseksvrkfleialfkyrkarlepgtaigaigaqsigepgtqmtlktfhfagvasmnvt
lgvprikeiinaskvistpiinavlvndnderaarvvkgrvektllsdvafyvqdvykdnlsfiqvridlgtidklqlel
tiediavaitrasklkiqasdvniigkdriainvfpegykaksistsakepsendvfyrmqqlrralpdvvvkglpdisr
avinirddgkrellvegyglrdvmctdgvigsrtttnhvlevfsvlgieaarysiireinytmsnhgmsvdprhiqllgd
vmtykgevlgitrfglskmrdsvlqlasfekttdhlfdaafymkkdavegvseciilgqtmsigtgsfkvvkgtnisekd
lvpkrclfeslsneaalkan

9/11 me to Craig

sorry .. no useful sites out there for doing phylogenetic analysis … I am working on such a type of thing right now. I tis tricky becuase to do it correctly you need to filter out parts of a multiple sequence alignment to remove badly aligned regions as well as hypervariable regions.

9/12 Craig to Me

Dear Mike, 

Yes, I can do this for the atypical RNA polymerase 2nd subunit. I have
already done multiple alignments with it against pol I, II, III subunits
and it is clear that the atypical subunit has amino acid differences that
set it apart, rather than large indels that skew the data. So I think
Jonathan is safe to go ahead and make a figure while I examine the gene
sequences and gene models more carefully. 

Any comments on the tone/amount of detail in the section I wrote on the
general transcription machinery? Either way, I will add some references
and send you an updated version as soon as I can. 

cheers
Craig

———————
>Speaking on behalf of the editorial committee whom I have not consulted, I
>would be delighted to have this in our section. But we need to check out the
>gene structure in detail (dodgy gene prediction, missing exons etc. Craig,
>could you so this as you know most about these enzymes
>
>All the best
>
>Mike

Me to Craig

Craig 

I am still working on a slightly better figure … but I have attached the latest version … I think it is sufficient for submission 

I have attached it in a few different formats. 

I will be out of town for a few days but checking email. 

Jonathan

Craig to Me:

Hi Jonathan,

The phytlogenetic tree figure for the atypical pol subunit looks good
though the font size may need to be reduced to fit “Fungal Plasmids”
between the dividing lines for the adjacent categories. Have you sent a
copy to Mike?

Craig

Craig again

Hi Jonathan, 

I forwarded a copy to Mike. Did you ever have a chance to do a tree for the
largest subunit to further test the hypothesis of a pol IV? 

Hope you are having fun in LA 

Craig

> I am not sure if I sent a copy to mike
>
>I am in LA right now and it would be easier if you could send mike a copy to
>make sure he has one. I will try and edit the figure and send one with a
>smaller font.
>
>J

10/3 Me to Craig:

Criag 

Attached is a new version of the rna pol tree with fonts corrected. I am going to add a few more sequences a rerun it and make a new tree tomorrow. 

Jonathan 

PS Also … here is a potential figure legend 

Figure. Phylogenetic tree of RNA polymerase homologs. Homologs of RNA polymerase were identified by searching sequence databases with representatives of the major known RNA polymerase subfamilies. These proteins, as well as six DNA polymerase homologs from A. thaliana, were aligned using clustalx using default settings. Phylogenetic trees were generated from the alignment (with ambiguously aligned regions and hypervariable regions excluded) using the PAUP* program. The tree shows was generated using the neighbor-joining algorithm with pairwise distances between sequences calculated with a PAM-like matrix. Numbers on the branches are bootstrap values indicating the percentage of 100 trees in which the proteins to the right of the node grouped together to the exclusion of all other proteins.

Craig 10/3

Hi Jonathan, 

I will look forward to seeing the final tree, as will Mike, I’m sure. For
the legend, the fact that this is an alignment of second-largest subunits
should be made clear. Here is a stab at a minor revision:

Figure—–. Phylogenetic tree for the second-largest subunit of
DNA-dependent RNA polymerases. Homologs of RNA polymerase second-largest
subunits were identified by searching sequence databases with
representatives of the major known subfamilies (e.g. pol I, II, III and
eubacterial beta subunits). Identified proteins, including six homologs
from A. thaliana, were
aligned using clustalx using default settings. Phylogenetic trees were
generated from the alignment (with ambiguously aligned regions and
hypervariable regions excluded) using the PAUP* program. The treewas generated using the neighbor-joining algorithm with pairwise distances
between sequences calculated with a PAM-like matrix. Numbers on the
branches are bootstrap values indicating the percentage of 100 trees inwhich the proteins to the right of the node group together to the
exclusion of all other proteins.

Thanks,
Craig

Me:

much better figure legend 

j

Anyway – and so it went. Alas, for a variety of reasons not much made it into the final paper. What was there was this:

Unexpectedly, Arabidopsis has two genes encoding a fourth class of largest subunit and second-largest subunit (Supplementary Information Fig. 5). It will be interesting to determine whether the atypical subunits comprise a polymerase that has a plant-specific function.

And of course, this Supplemental Information is not exactly easy to find and does not actually work correctly anymore:

Downloading the Zip file and opening first page.htm gets one to this

And then clicking on the Figure 5 you get a broken page w/o the Figure.

But there, hidden in the folder with the Supplemental Information is the figure

So that is the beginning of the story on RNA Pol IV in Arabidopsis.

Go read the E-life paper and some of what it cites for the last 15 years of the story.


Horizontal gene transfer into humans? I am not convinced. Full text of my comments to reporters here

Some news stories about a new paper claiming evidence for horizontal gene transfer into humans and other chordates. I got asked by many reporters about it and some used some of my email comments in their articles.

See for example

 Here is the full text of my responses:


“got asked by another reporter to comment on this

so – have seen the paper 

it is interesting .. but I am not overwhelmed by what they present in the paper itself. For example, the HAS story seems really incomplete as presented (e.g., the Figure showing the tree does not have all the HAS1, HAS2, HAS3 genes even though they imply they studied that). “


I have been looking through the supplemental information. I find it impossible to judge the quality of this paper without being able to see the alignments they used for each phylogenetic tree. I cannot find alignments for the trees even after going to their Figshare site with the trees. I therefore think there is not much to say about the paper until being able to see those. 

Without seeing the alignments I offer multiple alternative hypothesis for their findings

  1. They have identified genes for which they are unable to produce reasonable alingnments. Alignments are central to phylogenetic analysis and if their alignments are poor quality then the trees will show all sorts of anomalies that have nothing to do with phylogenetic history. By scanning through 1000s of genes and flagging those with unusual patterns they may be selectively identifying genes for which producing good alignments between species is tough. I note – clustalw is a bit notorious for not producing idea alignments in some cases.
  2. I do not buy their arguments for why gene loss is not a possible explanation. They need to present more detail on how many gene losses would be required for each gene family under consideration and then present some evidence for why that # of gene losses is less likely than HGT.
  3. They have not even considered as far as I can tell, the possibility of divergent evolution (as opposed to gene loss) in many taxa which could lead to them being unable to identify homologs in some species
  4. I am not convinced by the arguments against long branch attraction as an explanation for some of the tree patterns.
  5. Related to alignments they need to show which regions of alignments they excluded from phylo
  6. Convergent evolution could also explain some of the patterns they observe.
  7. I could go on. I am NOT saying that HGT into chordates is impossible. It seems plausible. But it is up to them to exclude other MORE plausible alternatives and I just do not think they have done that.

Reporter: asking if it was OK to quote me

Yes it is OK to quote from me. I would like to reiterate – I am not saying they are wrong. Just that I would like to see (1) all the data (e.g., alignments) that unreels their conclusions and (2) them do more to exclude other possibilities.


Reporter asking what other analyses could they do

So – I don’t want to be difficult, but it is their job to figure out how to do such tests before claiming they have strong evidence for HGT. 

In general, this is pretty typical of claims of HGT. Many researchers show evidence that is consistent with the occurence of HGT (which they did here) but few actually explicitly test alternative hypotheses such as gene loss, bad alignments, convergence, divergence, contamination, random noise, and more. I think their work is certainly interesting, but they just have not tested all of these alternatives. And I personally have grown a bit tired of pointing out how people can do better controls for their papers.


Reporter asking about initial impressions:

I see little here that is particularly convincing evidence for HGT …


My follow up email

Note – I am not saying that this is a bad paper — just that I am not overwhelmed by their evidence and especially by what they put in the paper. 

For example, the HAS1 gene story seems incomplete.  Figure 3 seems to show just HAS1 but in the text the say they show the same thing for all HAS genes.  And the tree they show shows a tiny subset of all the available sequences (e.g., HAS1 HAS2 HAS3 and fungal and bacterial homologs).  They claim that they now have proof that HAS1 was transferred near the base of chordates but I just don’t see how they tested alternative hypotheses …


Some related links:

Also here are some presentations from many years ago with some discussion of HGT

APRIL WORKSHOP: Phylogenetically correct stats for evolutionary ecologists (RSVP needed)

From CPB Postdoc Luke Mahler:

Dear Davis Ecologists and Evolutionary Biologists,

This Spring Quarter, I’ll be delivering my CPB Postdoctoral Fellowship Workshop, which will be entitled:

“Phylogenetically correct analysis of species data in ecology and evolution”.

This workshop will consist of 3 sessions, to take place over three days:

April 2: 4:10PM-5:30PM (lecture; note this will be the CPB seminar for this week)
April 3: 11:00AM-1:00PM (tutorial 1 – feel free to bring your lunch)
April 4: 1:00PM-3:00PM (tutorial 2)

The format of this workshop will be an introductory lecture followed by two hands-on R tutorials.

In the lecture, which will take place during that week’s CPB seminar slot, my goal is to (1) review why it’s important to account for phylogeny during data analysis, (2) briefly introduce a suite of current methods for doing so, and (3) critically discuss the inherent assumptions and limitations of phylogenetic comparative methods, outlining best practices for conducting basic analyses.

In tutorials, I will lead participants in a series of computer exercises (all in R) designed to provide hands-on experience implementing the most commonly used phylogenetic statistical analyses. In the first tutorial, participants will learn how to prepare and import phylogenetic trees and species data set for analysis, how to visually explore these data, and how to measure phylogenetic signal and select an appropriate model of trait evolution. In the second tutorial, we’ll conduct a number of phylogenetic statistical analyses with a focus on linear models and principal component analysis. We’ll discuss parameter estimation and interpretation under these models, and will cover techniques for assessing sensitivity of results to phylogenetic uncertainty.

The target audience for this workshop are scientists at any stage who are familiar with phylogenetic trees but don’t necessarily have specialized knowledge about how to work with trees when analyzing ecological or other comparative data. No prior experience using R is necessary, although attendees will need to bring their own laptops, and will need to install R in advance of the workshop (it’s free). Attendees do not need to bring data files – we’ll analyze example data sets which I will provide. Faculty, postdocs, and students are all welcome to attend.

If you are interested in attending this workshop, PLEASE CONTACT ME BY EMAILING lmahler@ucdavis.edu. We haven’t decided yet where to hold the workshop – this decision will be based on anticipated attendance. We’ll do our best to secure a room that can accommodate everyone who interested in attending, but can’t guarantee there will be room for you if you don’t let me know in advance.

Thanks, and I hope you can make it.

Best,
Luke Mahler

Center for Population Biology Postdoctoral Fellow
2320 Storer Hall
University of California, Davis
One Shields Avenue
Davis, CA 95616, USA

PLoSOne paper: Parallel polymorphisms for pepper population phylogenetics, from #UCDavis, not #Pepperspray

Interesting new paper from colleagues at UC Davis: PLOS ONE: Characterization of Capsicum annuum Genetic Diversity and Population Structure Based on Parallel Polymorphism Discovery with a 30K Unigene Pepper GeneChip.

Press release is here: http://www.news.ucdavis.edu/search/news_detail.lasso?id=10497

Good to see something on peppers from UC Davis not about spraying.

Online phylogenetics seminar 2/5 9 AM PST: Fiona Jordan "Testing hypotheses about cultural evolution"

From phyloseminar.org home. Online phylogenetics seminar 2/5: Fiona Jordan “Testing hypotheses about cultural evolution”

From the website:
Anthropologists had a name for the non-independence-of-species-problem way back in the 1880s. Solving “Galton’s Problem”, and the promise of comparative methods for testing hypotheses about cultural adaptation and correlated evolution was a major catalyst for the field of cultural phylogenetics. In this talk I will show how linguistic, cultural, and archaeological data is used in comparative phylogenetic analyses. The “treasure trove of anthropology” – our vast ethnographic record of cultures – is now being put to good use answering questions about cross-cultural similarities and differences in human social and cultural norms in a rigorous evolutionary framework.

West Coast USA:
09:00 (09:00 AM) on Tuesday, February 05

East Coast USA:
12:00 (12:00 PM) on Tuesday, February 05

UK:
17:00 (05:00 PM) on Tuesday, February 05

France:
18:00 (06:00 PM) on Tuesday, February 05

Japan:
02:00 (02:00 AM) on Wednesday, February 06

New Zealand:
06:00 (06:00 AM) on Wednesday, February 06

From iSEEM project: Phylogenetic Diversity Theory Sheds Light on the Structure of Microbial Communities

Quick post.  Another paper is out based on the Gordon and Betty Moore Foundation funded iSEEM project I co-ran with Jessica Green and Katie Pollard.

PLOS Computational Biology: Phylogenetic Diversity Theory Sheds Light on the Structure of Microbial Communities.

O’Dwyer JP, Kembel SW, Green JL (2012) Phylogenetic Diversity Theory Sheds Light on the Structure of Microbial Communities. PLoS Comput Biol 8(12): e1002832. doi:10.1371/journal.pcbi.1002832.

It has one of my favorite paper figures ever.

Figure 1. The local community and metacommunity framework casts local biodiversity of coexisting species in terms of a sampling process from a larger reference pool, or metacommunity. 

And the paper is definitely worth checking out.

Interesting new #PLOS One paper on study design in rRNA surveys

Interesting new paper in PLoS One:  PLOS ONE: Taxonomic Classification of Bacterial 16S rRNA Genes Using Short Sequencing Reads: Evaluation of Effective Study Designs

Abstract: Massively parallel high throughput sequencing technologies allow us to interrogate the microbial composition of biological samples at unprecedented resolution. The typical approach is to perform high-throughout sequencing of 16S rRNA genes, which are then taxonomically classified based on similarity to known sequences in existing databases. Current technologies cause a predicament though, because although they enable deep coverage of samples, they are limited in the length of sequence they can produce. As a result, high-throughout studies of microbial communities often do not sequence the entire 16S rRNA gene. The challenge is to obtain reliable representation of bacterial communities through taxonomic classification of short 16S rRNA gene sequences. In this study we explored properties of different study designs and developed specific recommendations for effective use of short-read sequencing technologies for the purpose of interrogating bacterial communities, with a focus on classification using naïve Bayesian classifiers. To assess precision and coverage of each design, we used a collection of ~8,500 manually curated 16S rRNA gene sequences from cultured bacteria and a set of over one million bacterial 16S rRNA gene sequences retrieved from environmental samples, respectively. We also tested different configurations of taxonomic classification approaches using short read sequencing data, and provide recommendations for optimal choice of the relevant parameters. We conclude that with a judicious selection of the sequenced region and the corresponding choice of a suitable training set for taxonomic classification, it is possible to explore bacterial communities at great depth using current technologies, with only a minimal loss of taxonomic resolution.

Not sure I like everything in the paper.  For example, they focus on naive Bayesian classification methods … when (of course) I prefer phylogenetic methods.  But that is a small issue.  Overall there is a lot of useful detail in here about rRNA based taxonomic studies.  I note – some of this probably applies to metagenomic studies as well … perhaps this group will do a comparable analysis of metagenomics next?

Mizrahi-Man O, Davenport ER, Gilad Y (2013) Taxonomic Classification of Bacterial 16S rRNA Genes Using Short Sequencing Reads: Evaluation of Effective Study Designs. PLoS ONE 8(1): e53608. doi:10.1371/journal.pone.0053608

I note – if you want to catch up / learn / research metagenomics and phylogeny or classification check out the Mendeley group I started on the topic:

http://www.mendeley.com/groups/1152921/phylogenetic-and-related-analyses-of-metagenomic-data/widget/29/3/

Some arguments for why Carl Woese (and probably Norm Pace) deserves a Nobel Prize

Compiling posts and articles discussing why Carl Woese deserves a Nobel Prize.  Will be writing a new article on this but felt like I should share the articles in case I don’t get done in time

I note I do not think Woese should win a Nobel for discovering the archaea.  That was a groundbreaking finding but it does not fit well with the Nobel Prize categories.  I think he should win it for the concept of molecular classification of microorganisms and applying this in general to the microbial world around us.  This concept (expanded by Norm Pace and colleagues to uncultured microbes) revolutionized our approach to studying single microbes in the environment, to studying single microbes infecting people and to studying communities of microbes in and on people.  And thus Woese and Pace in my opinion deserve the Nobel Prize for Medicine.  I will be expanding on this in a future post …

Three talks, 1.5 days at #ISMB … phylogeny, phylogenomics, open science and more

Gave three talks in 1.5 days here in Long Beach as part of the satellite meetings associated with the “Intelligent Systems for Molecular Biology” (ISMB) 2012 Conference. I will write more about the meeting and the craziness of giving three very different talks in 1.5 days. But for now I wanted to at least get my talks posted here since I posted the slides to slideshare and recorded the audio in synch with the slides and posted these “slideshows” to YouTube. Here are the talks below:



Talk 1 for the “Bioinformatics Open Source Conference” BOSC2012.  Was asked to talk about Open Science … so … I did …

Slideshow with audio:
 



Talk 2 for the Student Council Symposium SCS2012. Sort of supposed to be a career guidance discussion so I geared my talk on the lines of “lessons learned” …

Slideshow with audio:



Talk 3 for the “Automated function prediction” AFP2012 satellite meeting.  I decided to talk about phylogenetic and phylogenomics approaches to functional prediction …

Slideshow with audio:


http://storify.com/phylogenomics/jonathan-eisen-talks-at-ismb-satellite-meetings.js[<a href=”http://storify.com/phylogenomics/jonathan-eisen-talks-at-ismb-satellite-meetings” target=”_blank”>View the story “Jonathan Eisen @phylogenomics talks at #ISMB Satellite Meetings” on Storify</a>]

Useful comparative analysis of sequence classification systems w/ a few questionable bits

There is a useful new publication just out: BMC Bioinformatics | Abstract | A comparative evaluation of sequence classification programs by Adam L Bazinet and Michael P Cummings.  In the paper the authors attempt to do a systematic comparison of tools for classifying DNA sequences according to the taxonomy of the organism from which they come.

I have been interested in such activities since, well, since 1989 when I started working in Colleen Cavanaugh’s lab at Harvard sequencing rRNA genes to do classification.  And I have known one of the authors, Michael Cummings for almost as long.

Their abstract does a decent job of summing up what they did

Background
A fundamental problem in modern genomics is to taxonomically or functionally classify DNA sequence fragments derived from environmental sampling (i.e., metagenomics). Several different methods have been proposed for doing this effectively and efficiently, and many have been implemented in software. In addition to varying their basic algorithmic approach to classification, some methods screen sequence reads for ’barcoding genes’ like 16S rRNA, or various types of protein-coding genes. Due to the sheer number and complexity of methods, it can be difficult for a researcher to choose one that is well-suited for a particular analysis. 

Results
We divided the very large number of programs that have been released in recent years for solving the sequence classification problem into three main categories based on the general algorithm they use to compare a query sequence against a database of sequences. We also evaluated the performance of the leading programs in each category on data sets whose taxonomic and functional composition is known. 

Conclusions
We found significant variability in classification accuracy, precision, and resource consumption of sequence classification programs when used to analyze various metagenomics data sets. However, we observe some general trends and patterns that will be useful to researchers who use sequence classification programs.

The three main categories of methods they identified are

  • Programs that primarily utilize sequence similarity search
  • Programs that primarily utilize sequence composition models (like CompostBin from my lab)
  • Programs that primarily utilize phylogenetic methods (like AMPHORA & STAP from my lab)
The paper has some detailed discussion and comparison of some of the methods in each category.  They even made a tree of the methods
Figure 1. Program clustering. A neighbor-joining tree
 that clusters the classification programs based on their similar attributes. From here.
In some ways – I love this figure.  Since, well, I love trees.  But in other ways I really really really do not like it.  I don’t like it because they use an explicitly phylogenetic method (neighbor joining, which is designed to infer phylogenetic trees and not to simply cluster entities by their similarity) to cluster entities that do not have a phylogenetic history.  Why use neighbor-joining here?  What is the basis for using this method to cluster methods?  It is cute, sure.  But I don’t get it.  What do deep branches represent in this case?  It drives me a bit crazy when people throw a method designed to represent branching history at a situation where clustering by similarity is needed.  Similarly it drives me crazy when similarity based clustering methods are used when history is needed.
Not to take away from the paper too much since this is definitely worth a read for those working on methods to classify sequences as well as for those using such methods.  They even go so far as to test various web served (e.g., MGRAST) and discuss time to get results.  They also test the methods for their precision and sensitivity.  Very useful bits of information here.
So – overall I like the paper.  But one other thing in here sits in my craw in the wrong way.  The discussion of “marker genes.”  Below is some of the introductory text on the topic.  I have labelled some bits I do not like too much:

It is important to note that some supervised learning methods will only classify sequences that contain “marker genes”. Marker genes are ideally present in all organisms, and have a relatively high mutation rate that produces significant variation between species. The use of marker genes to classify organisms is commonly known as DNA barcoding. The 16S rRNA gene has been used to greatest effect for this purpose in the microbial world (green genes [6], RDP [7]). For animals, the mitochondrial COI gene is popular [8], and for plants the chloroplast genes rbcL and matK have been used [9]. Other strategies have been proposed, such as the use of protein-coding genes that are universal, occur only once per genome (as opposed to 16S rRNA genes that can vary in copy number), and are rarely horizontally transferred [10]. Marker gene databases and their constitutive multiple alignments and phylogenies are usually carefully curated, so taxonomic and functional assignments based on marker genes are likely to show gains in both accuracy and speed over methods that analyze input sequences less discriminately. However, if the sequencing was not specially targeted [11], reads that contain marker genes may only account for a small percentage of a metagenomic sample.  

I think I will just leave these highlighted sections uncommented upon and leave it to people to imagine what I don’t like about them .. for now.

Anyway – again – the paper is worth checking out.  And if you want to know more about methods used for classifying sequences see this Mendeley collection which focuses on metagenomic analysis but has many additional paper on top of the ones discussed in this paper.