Evolutionary Genome Scanning - talk by J. Eisen in 2000 at MBL Molecular Evolution Course.

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Topics of Discussion
• Introduction to phylogenomics
• Phylogenomics Examples
– Functional prediction
– Identifying “unusual” genes in genomes
– Gene duplication
– Genetic exchange within genomes
– Gene loss
– Horizontal gene transfer
– Specialization
– Comparing close relatives
– Species evolution

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“Nothing in biology makes sense
except in the light of evolution.”
T. H. Dobzhansky (1973)

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Uses of Evolutionary Analysis in
Molecular Biology
• Identification of mutation patterns (e.g., ts/tv ratio)
• Amino-acid/nucleotide substitution patterns useful in
structural studies (e.g., rRNA)
• Sequence searching matrices (e.g., PAM, Blosum)
• Motif analysis (e.g., Blocks)
• Functional predictions
• Classifying multigene families
• Evolutionary history puts other information into
perspective (e.g., duplications, gene loss)
TIGRTIGR

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Phylogenomic Analysis
Phylogenomics involves combining evolutionary
reconstructions of genes, proteins, pathways, and
species with analysis of complete genome
sequences.

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Why use Phylogenomics?
Evolutionary information improves genome analysis
-Classification of multigene families
-Predicting functions
-Origins of genes and pathways
Genomics information improves evolutionary
reconstructions
-More sequences of genes
-Unbiased sampling
-Presence/absence needed to infer certain events
Feedback loop between two types of analysis
TIGRTIGR

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Uses of Phylogenomics I:
Functional Predictions

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Predicting Function
• Identification of motifs
• Homology/similarity based methods
– Highest hit
– Top hits
– Clusters of orthologous groups
– HMM models
– Structural threading and modeling
– Evolutionary reconstructions
TIGRTIGR

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Types of Molecular Homology
• Homologs: genes that are descended from a common
ancestor (e.g., all globins)
• Orthologs: homologs that have diverged after speciation
events (e.g., human and chimp β-globins)
• Paralogs: homologs that have diverged after gene
duplication events (e.g., α and β globin).
• Xenologs: homologs that have diverged after lateral
transfer events
• Positional homology: common ancestry of specific amino
acid or nucleotide positions in different genes

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Blast Search of H. pylori “MutS”
Score E
Sequences producing significant alignments: (bits) Value
sp|P73625|MUTS_SYNY3 DNA MISMATCH REPAIR PROTEIN 117 3e-25
sp|P74926|MUTS_THEMA DNA MISMATCH REPAIR PROTEIN 69 1e-10
sp|P44834|MUTS_HAEIN DNA MISMATCH REPAIR PROTEIN 64 3e-09
sp|P10339|MUTS_SALTY DNA MISMATCH REPAIR PROTEIN 62 2e-08
sp|O66652|MUTS_AQUAE DNA MISMATCH REPAIR PROTEIN 57 4e-07
sp|P23909|MUTS_ECOLI DNA MISMATCH REPAIR PROTEIN 57 4e-07
• Blast search pulls up Syn. sp MutS#2 with
much higher p value than other MutS
homologs

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H. pylori and MutS
• Prior to this genome, all species that
encoded a MutS homolog also encoded a
MutL homolog
• Experimental studies have shown MutS and
MutL always work together in mismatch
repair
• Problem: what do we conclude about H.
pylori mismatch repair

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Table 3. Presence of MutS Homologs in Complete Genomes Sequences
Species # of MutS
Homologs
Bacteria
Escherichia coli K12 1
Haemophilus influenzae Rd KW20 1
Neisseria gonorrhoeae 1
Helicobacter pylori 26695 1
Mycoplasma genitalium G-37 0
Mycoplasma pneumoniae M129 0
Bacillus subtilis 169 2
Streptococcus pyogenes 2
Synechocystis sp. PCC6803 2
Treponema pallidum Nichols 1
Borrelia burgdorferi B31 2
Aquifex aeolicus 2
Deinococcus radiodurans R1 2
Archaea
Archaeoglobus fulgidus VC-16, DSM4304 0
Methanococcus janasscii DSM 2661 0
Methanobacterium thermoautotrophicum ∆H 1
Eukaryotes
Saccharomyces cerevisiae 6
Homo sapiens 5

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MutS Alignment
EEDLKNRLCQKF . DA . HYNT IWMPT IQA I SN IDCLLA I TRTSEYLGAPSC
DTSLKDCMRRLFCNFDKNHKDWQSAVEC IAVLDVLLCLANYSQGGDGPMC
CSAEWLDFLEK . FS . . EHYHSLCKAVHHLATVDC I FSLAKV . . AKQGDYC
SELQYKEFLNK . I T . . AEYTELRK I TLNLAQYDC I LSLAAT . . SCNVNYV
EYELYKELRER . VV . . KELDKVGNNASAVAEVDF IQSLAQ I . . AYEKDWA
EYELFTELREK . VK . . QY I PRLQQLAKQMSELDALQCFAT I . . SENRHYT
EYE I FTEVRAT . VA . . EKAQP IRDVAKAVAA IDVLAGLAEV . . AVYQGYC
EQRVLKS I TDE . IV . . SHHKTLRSLANALDELD I STSLATL . . AQEQDFV
EAN I IDLFKRK . F I . . DRSNVVRQVATTLGYLDTLSSFAVL . . ANERNLV
QDA IVKE IVN I . SS . . GYVEPMQTLNDVLAQLDAVVSFAHVSNGAPVPYV
QSALVRE I IN I . TL . . TYTPVFEKLSLVLAHLDV IASFAHTSSYAP I PY I
EEER I LRQLSDQVL . . EVLLDLEHLLA IATRLDLATARVRY . . . SFWLGA
EVRKVLQR I TEY IG . . DYAKELLESFEACVEVDFQQCKYRF . . SKLVEGS
E I ER I LRVLTEKTA . . EYTEELFLDLQVLQTLDF I FAKARY . . AKAVKAT
TYMIVCKLLSE . IY . . EH IHCLYKLSDTVSMLDMLLSFAHA . . CTLSDYV
SEETVDELLDK . IA . . TH I SELFMIAEAVA I LDLVCSFTYN . . LKENNYT
ETLLMYQLQCQ . VL . . ARAAVLTRVLDLASRLDVLLALASA . . ARDYGYS
E I E I LFSLQEQ . I L . . RRKTQLTAYN I LLSELE I LLSFAQV . . SAERNYA
RPT IVDEVDSKTNTQLNGFLKFKSLRHPCFNLGA . . . TTA . KDF I PND I E
RPE IVLP . . GEDTHP . . . FLEFKGSRHPC I TKTF . . . FG . . DDF I PND I L
RPTVQEE . . . . . . R . . . . K IV IKNGRHPV IDVLL . . GEQ . . DQYVPNNTD
RPTFVNG . . . . . QQ . . . . A I IAKNARNP I I ESLD . . . . . . . VHYVPND IM
KPQ IHE . . . . . . GY . . . . EL I I EEGRHPV I EEF . . . . . V . . ENYVPNDTK
KPEFSK . . . . . . D . . . . . EVEV I EGRHPVVEKVM. . . DS . . QEYVPNNCM
RP IMQM. . . . . EPG . . . . L ID I EAGRHPVVEQSL . . . GA . . GFFVANDTQ
RPVVDD . . . . . SH . . . . . AHTV IQGRHP IVEKGL . . SHKL . I PFTPNDCF
CPKVDE . . . . . SN . . . . . KLEVVNGRHLMVEEGL . . SARSLETFTANNCE
RPA I LEK . . . . GQG . . . . R I I LKASR . . . VEVQD . . . . E . . IAF I PNDVY
RPKLHPM. . . DSER . . . . RTHL I SSRHPVLEMQD . . . . D . . I SF I SNDVT
HPPQWL . . . TPGDEK . . . P I TLRQLRHPLLHWQA . . EKEGGPAVVP I TLT
FPDFGE . . . . .WVE . . . . . . . LYEARHPVLVLVKED . . . . . VVPVG I LLK
KP IMND . . . . . TG . . . . . F IRLKKARHPLLPP . . . . . . . . . DQVVAND I E
RPEFTD . . . . . . . . . . . . TLA IKQGWHP I LEK I S . . . . A . . EKP IANNTY
I P I FTN . . . . . . . . . . . . NLL IRDSRHPLLEKVL . . . . . . . KNFVPNT I S
RPRYSPQ . . . . VL . . . . . GVR IQNGRHPLMELCA . . . . . . . RTFVPNSTE
EPQLVE . . . . . DEC . . . . I LE I INGRHALYETFL . . . . . . . DNY I PNSTM
LGKE . . . . . . QPR . . . . . .
IGCE . . . EEAEEHGKAY . .
LSED . . . . . . SER . . . . . .
MSPE . . . . . . NGK . . . . . .
LDRD . . . . . . SF . . . . . . .
MGDN . . . . . . RQ . . . . . . .
LGHD . . . . . . HWHPD . . . .
VGNGNV . . . . N . . . . . . . .
LAKD . . . . . . N . . . . . . . .
FEKD . . . . . . KQM. . . . . .
LESG . . . . . . KGD . . . . . .
IDSQ . . . . . . IR . . . . . . .
EKKG . . . . . . . . . . . . . . .
LGRD . . . . . . FS . . . . . . .
VTE . . . . . . . GSN . . . . . .
STKH . . . . . . SSS . . . . . .
CGGD . . . . . . KGR . . . . . .
IDGG . . LFSELSWCEQNKG
. LGLLTGANAAGKST I LRMAC IAV IMAQMGC
. CVLVTGPNMGGKSTL IRQAGLLAVMAQLGC
. VMI I TGPNMGGKSSY IKQVAL I T IMAQ IGS
. IN I I TGPNMGGKSSY IRQVALLT IMAQ IGS
. IHV I TGPNMAGKSSY IRQVGVLTLLSH IGS
.MLL I TGPNMSGKSTYMRQ IAL I S IMAQ IGC
. LV I LTGPNASGKSCYLRQVGL IQLMAQTGS
. IWL I TGPNMAGKSTFLRQNA I I S I LAQ IGS
. LWV I TGPNMGGKSTFLRQNA I IV I LAQ IGC
. FH I I TGPNMGGKSTY IRQTGV IVLMAQ IGC
. FL I I TGPNMGGKSTY IRQVGV I SLMAQ IGC
. V IA I TGPNTGGKTVTLKTLGLVALMAKVGL
. . L I LTGPNTGGKTVALKTLGLSVLMFQSA I
. T IV I TGPNTGGKTVTLKTLGLLTLMAQSGL
. FL I I TGPNMSGKSTYLKQ IALCQ IMAQ IGS
. LQ I I TGCNMSGKSVYLKQVAL IC IMAQMGS
. VKV I TGPNSSGKS IYLKQVGL I TFMALVGS
R I IVVTGANASGKSVYLTQNGL IVYLAQ IGC
YVPCESA . VLTP IDR IMTRLGANDN IMQGKSTFFVELAETKK I LD . . . . .
YVPAEKC . RLTPVDRVFTRLGASDR IMSGESTFFVELSETAS I LR . . . . .
YVPAEEA . T IG IVDG I FTRMGAADN IYKGRSTFMEELTDTAE I IR . . . . .
FVPAEE I . RLS I FENVLTR IGAHDD I INGDSTFKVEMLD I LH I LK . . . . .
F I PARRA . K I PVVDALFTR IGSGDVLALGVSTFMNEMLEVSN I LN . . . . .
FVPAKKA . VLP I FDQ I FTR IGAADDL I SGQSTFMVEMLEAKNA IV . . . . .
F I PAKTA . TLS ICDR I FTRVGAVDDLATGQSTFMVEMNETAN I LN . . . . .
FVPASNA . R IG IVDQ I FSR IGSADNLYQQKSTFMVEMMETSF I LK . . . . .
FVPCSKA . RVG IVDKLFSRVGSADDLYNEMSTFMVEMI ETSF I LQ . . . . .
FVPCESA . EVS IVDC I LARVGAGDSQLKGVSTFMAEMLETAS I LR . . . . .
FVPCEEA . E IA IVDA I LCRVGAGDSQLKGVSTFMVE I LETAS I LK . . . . .
Y I PAKETVEMPWFAQ I LAD IGDEQSLQQNLSTFSGH ICR I IR I LQALPSG
PVPASPNSKLPLFEKVFTD IGDEQS I EQNLSTFSAHVKNMAEFLP . . . . .
H I PADEGSEAAVFEHVFAD IGDEQS I EQSLSTFSSHMVN IVG I LE . . . . .
YVPAEYS . SFR IAKQ I FTR I STDDD I ETNSSTFMKEMKE IAY I LH . . . . .
G I PALYG . SFPVFKRLHARV . CNDSMELTSSNFGFEMKEMAYFLD . . . . .
FVPAEEA . E IGAVDA I FTR IHSCES I SLGLSTFMIDLNQVAKAVN . . . . .
FVPAERA . R IG IADK I LTR IRTQETVYKTQSSFLLDSQQMAKSLS . . . . .
C
S
A
A
A
S
A
A
A
A
A
A
A
G
A
S
L
. . . . . . . . . . . . .MATNRSLLVVDELGRGGSSSDGFA I
. . . . . . . . . . . . . HATAHSLVLVDELGRGTATFDGTA I
. . . . . . . . . . . . . KATSQSLV I LDELGRGTSTHDG IA I
. . . . . . . . . . . . . NCNKRSLLLLDEVGRGTGTHDG IA I
. . . . . . . . . . . . . NATEKSLV I LDEVGRGTSTYDG IA I
. . . . . . . . . . . . . NATKNSL I LFDE IGRGTSTYDGMAL
. . . . . . . . . . . . . HATAKSLVLLDE IGRGTATFDGLA I
. . . . . . . . . . . . . NATRRSFV IMDE IGRGTTASDG IA I
. . . . . . . . . . . . . GATERSLA I LDE IGRGTSGKEG I S I
. . . . . . . . . . . . . SATKDSL I I IDELGRGTSTYDGFGL
. . . . . . . . . . . . . NASKNSL I IVDELGRGTSTYDGFGL
VQDVLDPE IDSPNHP I FPSLVLLDEVGAGTDPTEGSAL
. . . . . . . . . . . . . KSDENTLVL IDELGAGTDP I EGSAL
. . . . . . . . . . . . . QVNENSLVLFDELGAGTDPQEGAAL
. . . . . . . . . . . . . NANDKSL I L IDELGRGTNTEEG IG I
. . . . . . . . . . . . . D INTETLL I LDELGRGSS IADGFCV
. . . . . . . . . . . . . NATAQSLVL IDEFGKGTNTVDGLAL
. . . . . . . . . . . . . LATEKSL I L IDEYGKGTD I LDGPSLF
Y
ESVLHHVATH I
SAVVKELAET I
YATLEYF IRDV
AL IKYFSELS
KA IVKY I SEKL
QA I I EYVHDH I
WSVAEYLAGE I
YGCLKYLST IN
YATLKYLLENN
WA I SEY IATK I
WA IAEH IASK I
IALLRHLADQP
IG I LEYLKKKK
MS I LDDVHRTN
YAVCEYLLSLK
LAVTEHLLRTE
AAVLRHWLARG
GS IMLNMSKSE
. QSLGF . FATHYGTLASSFKHHPQ . VRPLKMS I L . . . VDE . . . . . A . . . .
. KCRTL . FSTHYHSLVEDYSKSVC . VRLGHMACM. . . VENECEDPS . . . .
. KSLTL . FVTHYPPVCELEKNYSHQVGNYHMGFL . . . VSEDESKLDPGAA
. DCPL I LFTTHFPMLGE IKSPL . . . IRNYHMDYV . . . . EEQKTGED . . . .
. KAKTL . LATHFLE I TELEGK I EG . VKNYHMEVE . . . . . . . . . . . KT . . .
. GAKTL . FSTHYHELTVLEDKLPQ . LKNVHVRAE . . . . . . . . . . . EY . . .
. QART I . FATHYHELNELASLLEN . VANFQVTVK . . . . . . . . . . . EL . . .
. HSRTL . FATHAHQLTNLTKSFKN . VECYCTNLS . . . . . IDRD . . . . . . .
. QCRTL . FATHFGQELKQ I IDNKC . SKGMSEKVK . . . . . . FYQSG I TDLG
. GAFCM. FATHFHELTALANQ I PT . VNNLHVTALT . . . . . . . . . . . . . . .
. GCFAL . FATHFHELTELSEKLPN . VKNMHVVAH I . . . . . EKNLKEQKH .
. . CLTV . ATTHYGELKALKYQDAR . FENASVEFD . . . . . . . . . . . . . . . .
. . AWVF . VTTHHTP IKLYSTNSDY . YTPASVLFD . . . . . . . . . . . . . . . .
. . ARVL . ATTHYPELKAYGYNREG . VMNASVEFD . . . . . . . . . . . . . . . .
. . AFTL . FATHFLELCH IDALYPN . VENMHFEVQ . . . . . . . . HVK . . . NT
. . ATVF . LSTHFQD I PK IMSKKPA . VSHLHMDAV . . . . . . . . LLN . . . . .
PTCPH I FVATNFLSLVQLQLLPQGPLVQYLTMET . . . . . . . . . . . . . . . .
. KCPR I IACTHFHELFNENVLTEN IKG IKHYCTD I L I SQKYNLLETAHVG
. . . . TRNVTFLYKMLEGQSEGSFGMHVASMCG I SKE I IDNAQ IAAD
. . . . QET I TFLYKF IKGACPKSYGFNAARLANLPEEV IQKGHRKAR
EQV . PDFVTFLYQ I TRG IAARSYGLNVAKLADVPGE I LKKAAHKSK
. . . .WMSV I FLYKLKKGLTYNSYGMNVAKLARLDKD I INRAFS I SE
. . . . PEG IRFLY I LKEGKAEGSFG I EVAKLAGLPEEVVEEARK I LR
. . . . NGTVVFLHQ IKEGAADKSYG IHVAQLAELPGDL IARAQD I LK
. . . . PEE I I FLHQVTPGGADKSYG I EAGRLAGLPSSV I TRARQVMA
. . . . DHTFSFDYKLKKGVNYQSHGLKVAEMAG I PKNVLLAAEEVLT
. . . . GNNFCYNHKLKPG ICTKSDA IRVAELAGFPMEALKEARE I LG
. . . TEETLTMLYQVKKGVCDQSFG IHVAELANFPKHV I ECAKQKAL
. . . DDED I TLLYKVEPG I SDQSFG IHVAEVVQFPEK IVKMAKRKAN
. . . . DQSLSPTYRLLWG I PGRSNALA IAQRLGLPLA IVEQAKDKLG
. . . . RETLKPLYK IAYNTVGESMAFY IAQKYG I PSEV I E IAKRHVG
. . . . I ETLSPTYKLL IGVPGRSNAFE I SKRLGLPDH I IGQAKSEMT
SRNKEA I LYTYKLSKGLTEEKNYGLKAAEVSSLPPS IVLDAKE I TT
. . . . DNSVKMNYQLTQKSVA I ENSG IRVVKK I FNPD I IAEAYNMDS
. . . CEDGNDLVFFYQVCEGVAKASHASHTAAQAGLPDKLVARGKEV
EDHESEG I TFLFKVKEG I SKQSFG IYCAKVCGLSRD IVERAEELSR
----------------I------------------ -----------II------------ ------------III------------
------IV------
MSH6__Yeast
MSH6__Mouse
MSH3__Human
MSH3__Yeast
MutS__Aquae
MutS__Bacsu
MutS__Synsp
MSH1__Pombe
MSH1__Yeast
MSH2__Human
MSH2__Yeast
MutS2_Synsp
MutS2_Aquae
MutS2_Bacsu
MSH4__Human
MSH4__Yeast
MSH5__Human
MSH5__Yeast
MSH6__Yeast
MSH6__Mouse
MSH3__Human
MSH3__Yeast
MutS__Aquae
MutS__Bacsu
MutS__Synsp
MSH1__Pombe
MSH1__Yeast
MSH2__Human
MSH2__Yeast
MutS2_Synsp
MutS2_Aquae
MutS2_Bacsu
MSH4__Human
MSH4__Yeast
MSH5__Human
MSH5__Yeast
MSH6__Yeast
MSH6__Mouse
MSH3__Human
MSH3__Yeast
MutS__Aquae
MutS__Bacsu
MutS__Synsp
MSH1__Pombe
MSH1__Yeast
MSH2__Human
MSH2__Yeast
MutS2_Synsp
MutS2_Aquae
MutS2_Bacsu
MSH4__Human
MSH4__Yeast
MSH5__Human
MSH5__Yeast

16. TIGRTIGR
Phylogenetic Tree of MutS Family
Aquae Trepa
Fly
Xenla
Rat
Mouse
Human
Yeast
Neucr
Arath
Borbu
Strpy
Bacsu
Synsp
Ecoli
Neigo
Thema
TheaqDeira
Chltr
Spombe
Yeast
Yeast
Spombe
Mouse
Human
Arath
Yeast
Human
Mouse
Arath
StrpyBacsu
Celeg
Human
Yeast
MetthBorbu
Aquae
Synsp
Deira Helpy
mSaco
Yeast
Celeg
Human

17. TIGRTIGR
MutS Subfamilies
Aquae Trepa
Fly
Xenla
Rat
Mouse
Human
Yeast
Neucr
Arath
Borbu
Strpy
Bacsu
Synsp
Ecoli
Neigo
Thema
TheaqDeira
Chltr
Spombe
Yeast
Yeast
Spombe
Mouse
Human
Arath
Yeast
Human
Mouse
Arath
StrpyBacsu
Celeg
Human
Yeast
MetthBorbu
Aquae
Synsp
Deira Helpy
mSaco
Yeast
Celeg
Human
MSH4
MSH5
MutS2
MutS1
MSH1
MSH3
MSH6
MSH2

18. TIGRTIGR
MutS Subfamilies
• MutS1 Bacterial MMR
• MSH1 Euk - mitochondrial MMR
• MSH2 Euk - all MMR in nucleus
• MSH3 Euk - loop MMR in nucleus
• MSH6 Euk - base:base MMR in nucleus
• MutS2 Bacterial - function unknown
• MSH4 Euk - meiotic crossing-over
• MSH5 Euk - meiotic crossing-over

19. TIGRTIGR
Table 3. Presence of MutS Homologs in Complete Genomes Sequences
Species # of MutS
Homologs
Which
Subfamilies?
Bacteria
Escherichia coli K12 1 MutS1
Haemophilus influenzae Rd KW20 1 MutS1
Neisseria gonorrhoeae 1 MutS1
Helicobacter pylori 26695 1 MutS2
Mycoplasma genitalium G-37 0 -
Mycoplasma pneumoniae M129 0 -
Bacillus subtilis 169 2 MutS1,MutS2
Streptococcus pyogenes 2 MutS1,MutS2
Synechocystis sp. PCC6803 2 MutS1,MutS2
Treponema pallidum Nichols 1 MutS1
Borrelia burgdorferi B31 2 MutS1,MutS2
Aquifex aeolicus 2 MutS1,MutS2
Deinococcus radiodurans R1 2 MutS1,MutS2
Archaea
Archaeoglobus fulgidus VC-16, DSM4304 0 -
Methanococcus janasscii DSM 2661 0 -
Methanobacterium thermoautotrophicum ∆H 1 MutS2
Eukaryotes
Saccharomyces cerevisiae 6 MSH1-6
Homo sapiens 5 MSH2-6

20. TIGRTIGR
Overlaying Functions onto Tree
Aquae Trepa
Rat
Fly
Xenla
Mouse
Human
Yeast
Neucr
Arath
Borbu
Synsp
Neigo
Thema
Strpy
Bacsu
Ecoli
TheaqDeira
Chltr
Spombe
Yeast
Yeast
Spombe
Mouse
Human
Arath
Yeast
Human
Mouse
Arath
StrpyBacsu
Human
Celeg
Yeast
MetthBorbu
Aquae
Synsp
Deira Helpy
mSaco
Yeast
Celeg
Human
MSH4
MSH5
MutS2
MutS1
MSH1
MSH3
MSH6
MSH2

21. TIGRTIGR
Functional Prediction Using Tree
Aquae Trepa
Fly
Xenla
Rat
Mouse
Human
Yeast
Neucr
Arath
Borbu
Strpy
Bacsu
Synsp
Ecoli
Neigo
Thema
TheaqDeira
Chltr
Spombe
Yeast
Yeast
Spombe
Mouse
Human
Arath
Yeast
Human
Mouse
Arath
MSH1
Repair
in Mictochondria
MSH3
Repair of Loops
in Nucleus
MSH6
Repair of Mismatches
in Nucleus
MutS1
Repair of Loops and Mismatches
StrpyBacsu
Celeg
Human
Yeast
MetthBorbu
Aquae
Synsp
Deira Helpy
mSaco
Yeast
Celeg
Human
MSH4
Meiotic Crossing-Over
MSH5
Meiotic Crossing-Over
MutS2 Unknown Functions
MSH2
Repair of Loops and Mismatches
in Nucleus

22. TIGRTIGR
Table 3. Presence of MutS Homologs in Complete Genomes Sequences
Species # of MutS
Homologs
Which
Subfamilies?
MutL
Homologs
Bacteria
Escherichia coli K12 1 MutS1 1
Haemophilus influenzae Rd KW20 1 MutS1 1
Neisseria gonorrhoeae 1 MutS1 1
Helicobacter pylori 26695 1 MutS2 -
Mycoplasma genitalium G-37 - - -
Mycoplasma pneumoniae M129 - - -
Bacillus subtilis 169 2 MutS1,MutS2 1
Streptococcus pyogenes 2 MutS1,MutS2 1
Mycobacterium tuberculosis - - -
Synechocystis sp. PCC6803 2 MutS1,MutS2 1
Treponema pallidum Nichols 1 MutS1 1
Borrelia burgdorferi B31 2 MutS1,MutS2 1
Aquifex aeolicus 2 MutS1,MutS2 1
Deinococcus radiodurans R1 2 MutS1,MutS2 1
Archaea
Archaeoglobus fulgidus VC-16, DSM4304 - - -
Methanococcus janasscii DSM 2661 - - -
Methanobacterium thermoautotrophicum ∆H 1 MutS2 -
Eukaryotes
Saccharomyces cerevisiae 6 MSH1-6 3+
Homo sapiens 5 MSH2-6 3+

23. TIGRTIGR
Why was the MutS2 Family Missed?
Blast Search of Syn. sp. MutS#2
Sequences producing significant alignments: (bits) Value
sp|Q56239|MUTS_THETH DNA MISMATCH REPAIR PROTEIN MUT 91 3e-17
sp|P26359|SWI4_SCHPO MATING-TYPE SWITCHING PROTEIN 87 4e-16
sp|P27345|MUTS_AZOVI DNA MISMATCH REPAIR PROTEIN MUTS 83 1e-14
sp|P74926|MUTS_THEMA DNA MISMATCH REPAIR PROTEIN MUTS 81 3e-14
sp|Q56215|MUTS_THEAQ DNA MISMATCH REPAIR PROTEIN MUTS 81 4e-14
sp|P10564|HEXA_STRPN DNA MISMATCH REPAIR PROTEIN HEXA 80 5e-14
• Blast search pulls up standard MutS genes
but with only a moderate p value (10-17
)

24. TIGRTIGR
Problems with Similarity Based
Functional Prediction
• Prone to database error propagation.
• Cannot identify orthologous groups reliably.
• Perform poorly in cases of evolutionary rate
variation and non-hierarchical trees (similarity will
not reflect evolutionary relationships in these cases)
• May be misled by modular proteins or large
insertion/deletion events.
• Are not set up to deal with expanding data sets.
TIGRTIGR

25. TIGRTIGR
Non-hierarchical Tree
2 31 4 5 6

26. TIGRTIGR
Evolutionary Rate Variation
2
3
1
4
5
6

27. TIGRTIGR
Rate Variation and Duplication
Species 3
Species 1
Species 2
1A
2A
3A
1B
2B
3B
Duplication

28. TIGRTIGR
AlkA Domain (O6-Me-Gglycosylase)
Ogt Domain (O6-Me-Galkyltransferase)
Ada Domain (transcriptions regulator)
Ada E. coli
Ada H. infl
Ogt E. coli
Ogt H. infl
Ogt Gram+
Ogt D. radio
AlkA Gram+
AlkAE. coli
MGMTEuks
Alkylation Repair Genes

29. TIGRTIGR
Evolutionary
Method
P H Y L O G E N E N E T IC P R E D IC T IO N O F G E N E F U N C T IO N
ID E N T IFY H O M O L O G S
O V E R L A Y K N O W N
FU N C T IO N S O N T O T R E E
IN FE R L IK E L Y FU N C T IO N
O F G E N E (S) O F IN T E R E ST
1 2 3 4 5 6
3 5
3
1A 2A 3A 1B 2B 3B
2A 1B
1A
3A
1B
2B
3B
A L IG N SE Q U E N C E S
C A L C U L A T E G E N E T R E E
1
2
4
6
C H O O S E G E N E (S) O F IN T E R E ST
2A
2A
5
3
S pecies 3S pecies 1 S pecies 2
1
1 2
2
2 31
1A 3A
1A 2A 3A
1A 2A 3A
4 6
4 5 6
4 5 6
2B 3B
1B 2B 3B
1B 2B 3B
A C T U A L E V O L U T IO N
(A SSU M E D T O B E U N K N O W N )
Duplication?
E X A M P L E A E X A M P L E B
D uplication?
D uplication?
D uplication
5
M E T H O D
A m biguous

30. TIGRTIGR
MutS.Aquae
orf.Trepa
SPE1.Drome
MSH2.Xenla
MSH2.Rat
MSH2.Mouse
MSH2.Human
MSH2.Yeast
MSH2.Neucr
atMSH2.Arath
MutS.Borbu
orf.Strpy
MutS.Bacsu
MutS
SynspMutS
Ecoli orf
Neigo
MutS
Thema
MutS
Theaq
orf.Deira
orf.Chltr
MSH1.Spombe
MSH1.Yeast
MSH3.Yeast
Swi4.Spombe
Rep3.Mouse
hMSH3.Human
orf.Arath
MSH6.Yeast
GTBP.Human
GTBP.Mouse
MSH6.Arath
orf
Strpy
yshD
Bacsu
MSH5
Caeel
hMHS5
human
MSH5
Yeast
MutS.Metth
orf
Borbu
MutS2
Aquae MutS
Synsporf
Deira
MutS.Helpy
sgMutS.Saugl
MSH4.Yeast
MSH4.Caeel
hMSH4.Human
A.
Aquae Trepa
Fly
Xenla
Rat
Mouse
Human
Yeast
Neucr
Arath
Borbu
Strpy
Bacsu
Synsp
Ecoli
Neigo
Thema
TheaqDeira
Chltr
Spombe
Yeast
Yeast
Spombe
Mouse
Human
Arath
Yeast
Human
Mouse
Arath
MutS2.Metth
MutS2.Saugl
StrpyBacsu
Caeel
Human
Yeast
Borbu
Aquae
Synsp
Deira Helpy
Yeast
Caeel
Human
MSH4
MSH5
MutS2
MutS1
MSH1
MSH3
MSH6
MSH2
B.
Aquae Trepa
Xenla
Neucr
Arath
Borbu
Synsp
Neigo
Thema
Deira
Chltr
Spombe
Spombe
Arath
Mouse
Mouse
Fly
Rat
Mouse
Human
Yeast
Strpy
Bacsu
Ecoli
Theaq
Yeast
Yeast
Human
Yeast
Human
Arath
StrpyBacsu
Human
MutS2-MetthBorbu
Aquae
Synsp
Deira Helpy
MutS2-Saugl
Caeel
Yeast
Yeast
Caeel
Human
MSH4
MSH5
MutS2
MutS1
MSH1
MSH3
MSH6
MSH2
C. MutS2StrpyBacsu
MutS2.MetthBorbu
Aquae
Synsp
Deira Helpy
MutS2.Saugl
Caeel
Yeast
Yeast
Caeel
Human
Human
MSH4
Segregation &
Crossover
MSH5
Segregation &
Crossover
Fly
Mouse
Human
Yeast
Aquae Trepa
Xenla
Neucr
Arath
Borbu
Synsp
Neigo
Thema
Deira
Chltr
Spombe
Spombe
Arath
Arath
MutS1
All MMR
(Bacteria)
Rat
Strpy
Bacsu
Ecoli
Theaq
Yeast
Yeast
Mouse
Human
Yeast
Human
Mouse
MSH1
MMR in
Mitochondria
MSH3
MMR of
Large Loops
in Nucleus
MSH6
MMR of
Mismatches and
Small Loops
in Nucleus
MSH2
All MMR
in Nucleus
D.

31. TIGRTIGR
Clustering vs. Neighbor-joining
MutS2.Syns
MutS2.Bacs
MutS2.Help
MutS2.Deir
Mutsl.Mett
MSH4.Celeg
MSH4.Yeast
MSH4.human
mMutS.Saco
MSH3.yeast
C23C11.Spo
MSH1.Yeast
MSH3.Human
REP1.Mouse
GTBP.Mouse
GTBP.Human
MSH6.Yeast
MSH5.Human
MSH5.Celeg
MSH5.Yeast
MSH2.Human
MSH2.Mouse
MSH2.Yeast
MutS.Ecoli
MutS.Synsp
MutS.Deira
MutS.Bacsu
M utS.Ecoli
M utS.Synsp
M utS.B acsu
M utS.Deira
M SH 2.H uman
M SH 2.M ouse
M SH 2.Yeast
M SH 3.H uman
R EP1.M ouse
G TB P.M ouse
G TB P.H uman
M SH 6.Yeast
C 23C 11.Sp o
M SH 1.Yeast
M SH 3.yeast
M SH 4.C eleg
M SH 4.human
M SH 5.C eleg
M SH 5.Yeast
mM utS.Saco
M SH 5.H uman
M SH 4.Yeast
M utS2.Syns
M utS2.B acs
M utS2.Deir
M utS2.H elp
M utsl.M ett
UPGMANeighbor-Joining

32. TIGRTIGR
Deinococcus radiodurans

33. TIGRTIGR
UvrA Gene Family
• UvrA has conserved role in nucleotide excision repair in
bacteria (part of UvrABCD complex)
• UvrA homologs found in all complete bacterial genomes
• Some UvrA homologs have been found to be involved in
resistance to DNA damaging antibiotics
• UvrA accumulates at membrane after DNA damage
• All UvrAs are members of the ABC transporter family
• Possible role in DNA damage export?

34. TIGRTIGR
UvrAs in D. radiodurans
• UvrA homolog in D. radiodurans shown to be
part of UV endonuclease α complex
• D. radiodurans genome sequence reveals a second
UvrA gene - on the large megaplasmid
• D. radiodurans known to export DNA repair
products (e.g., damaged bases) out of cell after
damage
• Export may be important for radiation resistance
(Battista 1997)

35. TIGRTIGR
UvrA Evolution
• Originated by gene duplication of an ABC transporter
• Subsequently, there was a tandem duplication of the
ABC transporter motif within UvrA
• Ancient duplication into UvrA1 and UvrA2
subfamilies
• UvrA1s - conserved role in NER
• UvrA2s - transport of DNA damage?
• UvrA2 in D. radiodurans may be from lateral transfer

36. TIGRTIGR
Evolution of UvrA Family
UvrA2
UvrA2 S. coelicolor
DrrC S. peuceteus
UvrA2 D. radiodurans
Duplication
in UvrA
family
UvrA1
UvrA H. influenzae
UvrA E. coli
UvrA N. gonorrhoaea
UvrA R. prowazekii
UvrA S. mutans
UvrA S. pyogenes
UvrA S. pneumoniae
UvrA B. subtilis
UvrA M. luteus
UvrA M. tuberculosis
UvrA M. hermoautotrophicum
UvrA H. pylori
UvrA C. jejuni
UvrA P. gingivalis
UvrA C. tepidum
uvra1 D. radiodurans
UvrA T. thermophilus
UvrA T. pallidum
UvrA B. burgdorefi
UvrA T. maritima
UvrA A. aeolicus
UvrA Synechocystis sp.
UvrA1
UvrA2
OppDF
UUP
NodI
LivF
XylG
NrtDC
PstB
MDR
HlyB
TAP1
CFTR, SUR
A. ABC Transporters B. UvrA Subfamily

37. TIGRTIGR
UvrA Evolution
Diversification of ABC family
UvrA
UvrAC UvrAN
UvrA1C UvrA1N UvrA2C UvrA2N
ABC1ABC2
ABC
Tandem Duplication
Gene Duplication

38. TIGRTIGR
Three V. cholerae Photolyases
Phr.S thyp
PHR E. coli
ORFA00965* * * * * * * * *
phr.neucr
Phr.Tricho
Phr.Yeast
Phr.B firm
phr.strpy
phr.haloba
PHR STRGR
pCRY1.huma
phr.mouse
phr2.human
phr2.mouse
phr.drosop
phr3.Synsp
O RF02295.V ibch* * * * * * * *
phr.neigo
ORF01792.V ibch* * * * * * *
Phr.Adiant
Phr2.Adian
Phr3.Adian
phr.tomato
CRY1 ARATH
phr.phycom
CRY2 ARATH
PHH1.arath
PHR1 SINAL
phr.chlamy
PHR ANANI
phr.Synsp
PHR SYNY3
phr.Theth
Rh.caps
MTHF type
Class I CPD
Photolyases
6-4
Photolyases
Blue
Light
Receptors
8-HDF type
CPD
Photolyases
Three Photolyase Homologs inV. cholerae

39. TIGRTIGR
MFS phylogenetic tree
Bmr Bsu
TetB Eco
Vmt1 Rno
Mmr Sco
EmrB Eco
QacA Sau
Sge1 Sce
TetK Sau
NarK Bsu
NasA Bsu
CrnA Eni
NapO Ocu
Ykh4 Cel
Hup1 Cke
AraE Eco
Itr1 Sce
Gtr1 Hsa
ProP EcoKgtP Eco
CitA Sty
HI1104 Hin
NanT Eco
YjhB Eco
Ycy8 Sce
YaeC Spo
Pho84 Sce
UhpT Eco
PgtP Sty
UhpC Eco
GlpT Bsu
NupG Eco
XapB Eco
LacY Eco
LacY Kpn
RafB Eco
CscB Eco
YhjX Eco
Y38K Tte
OxlT Ofo
T02G5 Cel
XpcT Hsa
Mct2 Rno
Gal Bab
FucP Eco
Yhe7 Sce
Yhe0 Sce
YK86 Sce
OFA
OHS
NHS
OPA
PHS
SHS
MHS
SPACS
NNP
DHA14
DHA12
UMF
FGHS
MCP

40. TIGRTIGR
Uses of Phylogenomics II:
Knowing when to Not Predict
Functions

41. TIGRTIGR
DNA Repair Genes in D.
radiodurans Complete Genome
Process Genes in D. radiodurans
Nucleotide Excision Repair UvrABCD, UvrA2
Base Excision Repair AlkA, Ung, Ung2, GT, MutM, MutY-Nths,
MPG
AP Endonuclease Xth
Mismatch Excision Repair MutS, MutL
Recombination
Initiation
Recombinase
Migration and resolution
RecFJNRQ, SbcCD, RecD
RecA
RuvABC, RecG
Replication PolA, PolC, PolX, phage Pol
Ligation DnlJ
dNTP pools, cleanup MutTs, RRase
Other LexA, RadA, HepA, UVDE, MutS2

42. TIGRTIGR
Recombination Genes in Genomes
Pathway |------------------------------Bacteria---------------------------| |---Archaea---| Euks
Protein Name(s)
Initiation
RecBCD pathway
RecB + + - - - - - - + + - + - - - - - - - -
RecC + + - - - - - - + ±+ - ± - - - - - - - -
RecD + + - - ± - - - + ±+ - ++ - ± ±+ - - - - -
RecF pathway
RecF + + + - + - - + + - + ± - - + - - ± ± ±
RecJ + + + + + - - + - + + + + + + - - - - -
RecO + + - - + - - + + - - - - - ± - - - - -
RecR + + + ±+ + - - + + - + + - + + - - - - -
RecN + + + + + - - + + - + - ± + + - - ± ± -
RecQ + + - - + - - + - - + - - - + - - - - + ++
RecE pathway
RecE/ExoVIII + - - - - - - - - - - - - - - - - - - -
RecT + - - - + - - - - - - - - - - - - - - -
SbcBCD pathway
SbcB/ExoI + + - - - - - - - - - - - - - - - - - -
SbcC + - - - + - - + - + + - + + + ± ± ± ± ± ±
SbcD + - - - + - - + - + + - + + + ± ± ± ± ± ±
AddAB Pathway
AddA/RexA - - + - + - - - - - + + - ± - - - - - -
AddB/RexB - - - - + - - - - - - - - - - - - - - -
Rad52 pathway
Rad52, Rad59 - - - - - - - - - - - - - - - - - - - ++ +
Mre11/Rad32 ± - - - ± - - ± - ± ± - ± ± ± + + + + + +
Rad50 ± - - - ± - - ± - ± ± - ± ± ± + + + ± + +
Recombinase
RecA, Rad51 + + + + + + + + + + + + + + + + + + + ++ ++
Branch migration
RuvA + + + + + + + + + + + + + - + - - - - -
RuvB + + + + + + + + + + + + + - + - - - - -
RecG + + + + + - - + + + + - + + + - - - - -
Resolvases
RuvC + + + + - - - + + - + + + - + - - - - -
RecG + + + + + - - + + + + - + + + - - - - -
Rus + - - - - - - - ±+ - - - - ±+ - - - - - -
CCE1 - - - - - - - - - - - - - - - - - - - +
Other recombination proteins
Rad54 - - - - - - - - - - - - - - - - - - - + +
Rad55 - - - - - - - - - - - - - - - - - - - + +
Rad57 - - - - - - - - - - - - - - - - - - - + +
Xrs2 - - - - - - - - - - - - - - - - - - - +

43. TIGRTIGR
Unusual Features of D. radiodurans
DNA Repair Genes
Process Genes
Nucleotide excision repair Two UvrAs
Base excision repair Four MutY-Nths
Recombination RecD but not RecBC
Replication Four Pol genes
dNTP pools Many MutTs, two RRases
Other UVDE

44. TIGRTIGR
Problem:
List of DNA repair gene homologs
in D. radiodurans genome is not
significantly different from other
bacterial genomes of the similar size

45. TIGRTIGR
Repair Studies in Different Species
(determined by Medline searches as of 1998)
Humans 7028
E. coli 3926
S. cerevisiae 988
Drosophila 387
B. subtilits 284
S. pombe 116
Xenopus 56
C. elegans 25
A. thaliana 20
Methanogens 16
Haloferax 5
Giardia 0

46. TIGRTIGR
-Ogt
-RecFRQN
-RuvC
-Dut
-SMS
-PhrI
-AlkA
-Nfo
-Vsr
-SbcCD
-LexA
-UmuC
-PhrI
-PhrII
-AlkA
-Fpg
-Nfo
-MutLS
-RecFORQ
-SbcCD
-LexA
-UmuC
-TagI
-PhrI
-Ogt
-AlkA
-Xth
-MutLS
-RecFJORQN
-Mfd
-SbcCD
-RecG
-Dut
-PriA
-LexA
-SMS
-MutT
-PhrI
-PhrII?
-AlkA
-Fpg
-Nfo
-RecO
-LexA
-UmuC
-PhrI
-Ung?
-MutLS
-RecQ?
-Dut
-UmuC
-PhrII
-Ogg
-Ogt
-AlkA
-TagI
-Nfo
-Rec
-SbcCD
-LexA
-Ogt
-AlkA
-Nfo
-RecQ
-SbcD?
-Lon
-LexA
-AlkA
-Xth
-Rad25?
-AlkA
-Rad25
-Nfo
-Ogt
-Ung
-Nfo
-Dut
-Lon
-Ung
-PhrII
-PhrI
Ecoli
Haein
Neigo
Helpy
Bacsu
Strpy
Mycge
Mycpn
Borbu
Trepa
Synsp
Metjn
Arcfu
Metth
Human
Yeast
BACTERIA ARCHAEA EUKARYOTES
from mitochondria
+Ada
+MutH
+SbcB
dPhr
+TagI?
+Fpg
+UvrABCD
+Mfd
+RecFJNOR
+RuvABC
+RecG
+LigI
+LexA
+SSB
+PriA
+Dut?
+Rus
+UmuD
+Nei?
+RecE
tRecT?
+Vsr
+RecBCD?
+RFAs
+TFIIH
+Rad4,10,14,16,23,26
+CSA
+Rad52,53,54
+DNA-PK, Ku
dSNF2
dMutS
dMutL
dRecA
+Rad1
+Rad2
+Rad25?
+Ogg
+LigII
+Ung?
+SSB,
+Dut?
+PhrI, PhrII
+Ogt
+Ung, AlkA, MutY-Nth
+AlkA
+Xth, Nfo?
+MutLS?
+SbcCD
+RecA
+UmuC
+MutT
+Lon
dMutSI/MutSII
dRecA/SMS
dPhrI/PhrII
+Spr
t3MG
+Rad7
+CCE1
+P53
dRecQ
dRad23
+MAG?
-PhrII
-RuvC
tRad25
+TagI?
+RecT
tUvrABCD
tTagI ?
Gain and Loss of Repair Genes
TIGRTIGR

47. TIGRTIGR
Evolution of Uracil Glycosylase
• Ung activity has evolve many times (many non-
homologous proteins have uracil-DNA glycosylase
activity)
• Therefore, absence of homologs of these genes
should not be used to infer likely absence of
activity
• However, presence of homologs of Ung and MUG
genes can be used to indicate presence of activity
because all homologs of these genes have this
activity

48. TIGRTIGR
Evolution of Photoreactivation
• All known enzymes that perform photoreactivation are part of
a single large photolyase gene family
• Some members of the family do not function as photolyases,
but instead work as blue-light receptors
• If a species does not encode a member of the photolyase gene
family, it likely does not have photoreactivation capability
• If a species encodes a photolyase, one cannot conclude it has
photolyase activity
• Position of photolyase homologs within photolyase tree helps
predict what activities they have

49. TIGRTIGR
Evolution of Alkyltransferases
• All known alkyltransferases share a conserved,
homologous alkyltransferase domain
• Therefore, if a species does not encode any
protein with this domain, it likely does not have
alkyltransferase activity
• If a species does encode an member of this gene
family, it likely has alkyltransferase activity

50. TIGRTIGR
Uses of Phylogenomics III:
Gene Duplication

51. TIGRTIGR
Why Duplications Are Useful to Identify
• Allows division into orthologs and paralogs
• Aids functional predictions
• Recent duplications may be indicative of species’
specific adaptations
• Helps identify mechanisms of duplication
• Can be used to study mutation processes in
different parts of genome

52. TIGRTIGR
MurA Homologs in A. thaliana
A RA TH I F5F19.7
A R A TH II F26C24.7
A RA TH IV F7N22.13
A R A TH IV T3H13.11
A R A TH II F23M 2.29
A RA TH II T13H18.11
A RA TH IV T24H24.6
A R A TH IV T3H13.13A R A TH I F22O13.23
A R A TH II F9B22.14
A RA TH II F27C21.3
A R A TH II T9F8.6A R A TH II T13P21.2A R A TH II F5O4.4
A R A TH II T13E11.2
A RA TH IV T24M 8.2
A R A TH IV F7N22.10A R A TH IV T3F12.3
A R A TH II T13E11.15
A R A TH IV T7M 24.1
A R A TH IV T3F12.8
A RA TH V T21B04.1
A R A TH II F27L4.10
A R A TH II F26B6.15
A RA TH II F23M 2.24
A R A TH I F1N21.16
A R A TH IV F9D12.2
A RA TH II F9B22.8
A R A TH IV F28J12.70
A RA TH IV T3F12.12
A RA TH II T13P21.20
A R A TH II T13E11.10
A RA TH V T21B04.16
A R A TH V T19K24.12
A R A TH V T19K24.13
A R A TH V T19K24.17 A R A TH V T21B04.11
A R A TH V T21B04.14
A R A TH V T21B04.10
A R A TH II T13P21.21
A R A TH V T21B04.13
A R A TH V T21B04.12
A RA TH II T13P21.3
A R A TH V T19K24.15
A RA TH V T19K24.16
A R A TH II T13E11.20
A RA TH V T19K24.11
A R A TH II T13E11.21
A R A TH II T13E11.9
A R A TH V T19K24.10
A RA TH V T21B04.15
A RA TH V T19K24.14
A R A TH II T11J7.3

53. TIGRTIGR
MurA Homologs in A. thaliana
colored by chromosome
A R A TH I F5F19.7
A RA TH I F22O13.23
A R A TH I F1N21.16
A RA TH V T21B04.1
A RA TH V T21B04.16
A R A TH V T19K24.12
A R A TH V T19K24.13
A RA TH V T19K24.17 A RA TH V T21B04.11
A R A TH V T21B04.14
A R A TH V T21B04.10
A RA TH V T21B04.13
A RA TH V T21B04.12
A RA TH V T19K24.15
A R A TH V T19K24.16
A R A TH V T19K24.11
A R A TH V T19K24.10
A RA TH V T21B04.15
A R A TH V T19K24.14
A R A TH IV F7N22.13
A RA TH IV T3H13.11
A RA TH IV T24H24.6
A R A TH IV T3H13.13
A R A TH IV T24M 8.2
A R A TH IV F7N22.10
A R A TH IV T3F12.3
A RA TH IV T7M 24.1
A R A TH IV T3F12.8
A RA TH IV F9D12.2
A R A TH IV F28J12.70
A R A TH IV T3F12.12
A R A TH II F26C24.7
A R A TH II F23M 2.29
A R A TH II T13H18.11
A R A TH II F9B22.14
A R A TH II F27C21.3
A RA TH II T9F8.6A R A TH II T13P21.2A R A TH II F5O4.4A R A TH II T13E11.2
A R A TH II T13E11.15
A R A TH II F27L4.10
A R A TH II F26B6.15
A RA TH II F23M 2.24
A R A TH II F9B22.8
A RA TH II T13P21.20
A R A TH II T13E11.10
A R A TH II T13P21.21
A R A TH II T13P21.3
A RA TH II T13E11.20
A RA TH II T13E11.21
A R A TH II T13E11.9
A RA TH II T11J7.3

54. TIGRTIGR
Recent Duplications
• Gene duplication is frequently accompanied
by functional divergence
• Evolutionary analysis can identify recent
duplications with no bias towards type of
gene
• Location of duplicates can help identify
mechanisms of duplication

55. TIGRTIGR
MutY-Nth
DEIRAORF00829
DEIRAORF02784
DEIRA
AQUAE
METJA
METTH
THEMA
CHLTR
HAEIN
MCYTU
THEMA
METTH
PYRHO
AQUAE
METJA
ARCFU
CELEG
VIBCH
ECOLI
HAEIN
TREPA
RICPR
AQUAE
BACSU
CAMJE
HELPY
MCYTU
SYNSP
CHLPN
CHLTR
BBUR

56. TIGRTIGR
Expansion of MCP Family in V. cholerae
E.coligi1787690
B.subtilisgi2633766
Synechocystissp. gi1001299
Synechocystissp. gi1001300
Synechocystissp. gi1652276
Synechocystissp.gi1652103
H.pylori gi2313716
H.pylori99 gi4155097
C.jejuniCj1190c
C.jejuniCj1110c
A.fulgidusgi2649560
A.fulgidusgi2649548
B.subtilisgi2634254
B.subtilisgi2632630
B.subtilisgi2635607
B.subtilisgi2635608
B.subtilisgi2635609
B.subtilisgi2635610
B.subtilisgi2635882
E.coligi1788195
E.coligi2367378
E.coligi1788194
E.coligi1789453
C.jejuniCj0144
C.jejuniCj0262c
H.pylori gi2313186
H.pylori99 gi4154603
C.jejuniCj1564
C.jejuniCj1506c
H.pylori gi2313163
H.pylori99 gi4154575
H.pylori gi2313179
H.pylori99 gi4154599
C.jejuniCj0019c
C.jejuniCj0951c
C.jejuniCj0246c
B.subtilisgi2633374
T.maritima TM0014
T.pallidumgi3322777
T.pallidumgi3322939
T.pallidumgi3322938
B.burgdorferi gi2688522
T.pallidumgi3322296
B.burgdorferi gi2688521
T.maritima TM0429
T.maritima TM0918
T.maritima TM0023
T.maritima TM1428
T.maritima TM1143
T.maritima TM1146
P.abyssiPAB1308
P.horikoshiigi3256846
P.abyssiPAB1336
P.horikoshiigi3256896
P.abyssiPAB2066
P.horikoshiigi3258290
P.abyssiPAB1026
P.horikoshiigi3256884
D.radiodurans DR A00354
D.radiodurans DRA0353
D.radiodurans DRA0352
P.abyssiPAB1189
P.horikoshiigi3258414
B.burgdorferi gi2688621
M.tuberculosisgi1666149
V .c hole ra eV C0 5 1 2
V . c hol e ra eV CA1 0 3 4
V .c hole ra eV CA 0 9 7 4
V .c hole raeV CA 0 06 8
V . chol e ra eV C0 8 2 5
V . c hol e ra eV C0 28 2
V .c hol e raeV CA 0 9 0 6
V . chol e ra eV CA0 9 7 9
V .c hol e raeV CA 1 0 5 6
V . c hol e ra eV C1 64 3
V . c hol e ra eV C2 1 6 1
V .c hole ra eV CA 09 2 3
V .c hole raeV C0 5 1 4
V . c hol e ra eV C1 8 6 8
V . c hol era eV CA0 7 7 3
V .c hole raeV C1 3 1 3
V . c hol era eV C1 8 5 9
V . c hole ra eV C14 1 3
V .c hol e raeV CA 0 2 6 8
V .c hol e raeV CA0 6 5 8
V . c hole ra eV C14 0 5
V . c hol e ra eV C1 2 9 8
V . c hol e ra eV C1 2 4 8
V . c hol era eV CA0 8 6 4
V . c hole ra eV CA0 1 7 6
V. c hol e ra eV CA0 2 2 0
V .c hole ra eV C1 2 8 9
V .c hole ra eV CA 10 6 9
V . c hol e ra eV C2 43 9
V . chol e ra eV C1 9 6 7
V . chol e ra eV CA0 0 3 1
V . c hole ra eV C18 9 8
V . chol e ra eV CA0 6 6 3
V .c hole ra eV CA 0 9 8 8
V . c hol era eV C0 2 1 6
V . c hol era eV C0 4 4 9
V .c hole ra eV CA 0 0 0 8
V . c hole ra eV C14 0 6
V . chol e ra eV C1 5 3 5
V .c hole ra eV C0 8 4 0
V . c hol e raeV C0 0 98
V .c hole ra eV CA 1 0 9 2
V .c hole ra eV C1 4 0 3
V .c hole ra eV CA1 0 8 8
V . c hol e ra eV C1 3 9 4
V .c hole ra eV C0 6 2 2
NJ
* *
* *
* *
*
* *
* *
* *
* *
* *
*
* *
* *
* *
* *
*
* *
* *
* *
* *
* *
* *
* *
* *
* *
* *
*
* *
* *
* *
* ** *
* *
*
*
*
*
* *
*
* *
* *
* *
*
* *
* *
*

57. TIGRTIGR
Phosphate Transporters
ARCFU
SYNSP
THEMA
AQUAE
METJA
MCYTU
MCYTU
VIBCH
ECOLI
DEIRA_ORF00198
DEIRA_ORFA00139
DEIRA_ORF00510

58. TIGRTIGR
Levels of Paralogy Within A Genome
• All
– All members of a gene family are linked together
• Top matches
– Only top matching pairs are linked together.
Therefore, if in a large gene family, only the pair
from the most recent duplication event is included
• Recent
– Operational definition based on comparison to other
species. Only pairs which are more similar to each
other than to selected other species are included.
TIGRTIGR

59. TIGRTIGR
C. pneumoniae Paralogs - All
0
250000
500000
750000
1000000
1250000
SubjectOrfPosition
0 250000 500000 750000 1000000 1250000
Query Orf Position
TIGRTIGR

60. TIGRTIGR
C. pneumoniae Paralogs - Top
0
250000
500000
750000
1000000
1250000
SubjectOrfPosition
0 250000 500000 750000 1000000 1250000
Query Orf Position
TIGRTIGR

61. TIGRTIGR
C. pneumoniae Paralogs – Recent
0
250000
500000
750000
1000000
1250000
SubjectOrfPosition
0 250000 500000 750000 1000000 1250000
Query Orf Position
TIGRTIGR

62. TIGRTIGR
E. coli Paralogs - All
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
SubjectCoordinates
0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000
Query Coordinates
TIGRTIGR

63. TIGRTIGR
E. coli Paralogs - Top
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
SubjectCoordinates
0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000
Query Coordinates
TIGRTIGR

64. TIGRTIGR
E. coli Paralogs - Recent
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
SubjectCoordinates
0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000
Query Coordinates
TIGRTIGR

65. TIGRTIGR
0
1000
2000
3000
4000
ChromosomePositionofRecentDuplicate
0 1000 2000 3000 4000
Chromosome Position ofQuery
Recent Duplications in
N. meningitidis
TIGRTIGR

66. TIGRTIGR
0
500000
1000000
1500000
Query ORF Chromosome Position
C. pneumoniae AR39
BestMatchChromosomePosiion
0
500000
1000000
C. trachomatis MoPn
Query ORF Chromosome Position
BestMatchChromosomePosiion
A. B.
0 500000 1000000 1500000 0 500000 1000000

67. TIGRTIGR
Uses of Phylogenomics IV:
Genetic Exchange within Genomes

68. TIGRTIGR
Circular Maps

69. TIGRTIGR
D. radiodurans Transposase Family
DEIRA_ORF01427_transposase__ps
DEIRA_ORF01431_transposase_{Sy
DEIRA_ORF03257_transposase_{Sy
DEIRA_ORFB01001_transposase__p
DEIRA_ORFB01020_transposase_{S
DEIRA_ORFB01025_transposase_{S
DEIRA_ORFB01012_transposase_{S
DEIRA_ORFB01035_transposase_{S
DEIRA_ORFC0021_transposase_{Sy
DEIRA_ORFC0025_hypothetical_pr
DEIRA_ORFC0018_transposase__ps
ORFB
ORF0
ORFC

70. TIGRTIGR

71. TIGRTIGR
Uses of Phylogenomics V:
Gene Loss

72. TIGRTIGR
Why Gene Loss is Useful to Identify
• Indicates that gene is not absolutely required for
survival
• Helps distinguish likelihood of gene transfers
• Correlated loss of same gene in different species
may indicate selective advantage of loss of that
gene
• Correlated loss of genes in a pathway indicates a
conserved association among those genes

73. TIGRTIGR
EuksArch Bacteria
Loss
Evolutionary O rigin of Gene
MT MJ SC HS AA DR TA BS MG MP BB TP HP HI EC SS MT
Presence ( ) or Absence of Gene
Species Abbreviation
Kingdom
Example of Tracing Gene Loss
TIGRTIGR

74. TIGRTIGR
5
1
2
3
4
E.coli
H.influenzae
N.gonorrhoeae
H.pylori
Syn.sp
B.subtilis
S.pyogenes
M.pneumoniae
M.genitalium
A.aeolicus
D.radiodurans
T.pallidum
B.burgdorferi
A.aeolicus
Spyogenes
B.subtilis
Syn.sp
D.radiodurans
B.burgdorferi
Syn.sp
B.subtilis
S.pyogenes
A.aeolicus
D.radiodurans
B.burgdorferi
MutS2
MutS1
A. B.
Gene
Duplication
Gene
Duplication
Ancient Duplication in MutS Family

75. TIGRTIGR
Need for Phylogenomics Example:
Gene Duplication and Loss
• Genome analysis required to determine number of
homologs in different species
• Evolutionary analysis required to divide into
orthology groups and identify gene duplications
• Genome analysis is then required to determine
presence and absence of orthologs
• Then loss of orthologs can be traced onto
evolutionary tree of species

76. TIGRTIGR
Uses of Phylogenomics VII:
Specialization

77. TIGRTIGR
Circular Maps

78. TIGRTIGR
Species Distribution of Homologs of
D. radiodurans Genes
0
10
20
30
40
50
60
0 5 10 15 20
0
50
100
150
0 5 10 15 20
NumberofSpeciesWithHighHits
0
50
100
150
200
250
Frequency
0 5 10 15 20
PapaBear MamaBear BabyBear
0
100
200
300
400
500
0 5 10 15 20
E.coli

79. TIGRTIGR
Megaplasmid I:
Iron Utilization/Iron Transport
ORFB040 Na+/H+ antiporterORFB040 Na+/H+ antiporter
ORFB042 iron ABC transporter, ATP-binding proteinORFB042 iron ABC transporter, ATP-binding protein
ORFB044 iron ABC transporter, permease proteinORFB044 iron ABC transporter, permease protein
ORFB045 iron ABC transporter, permease proteinORFB045 iron ABC transporter, permease protein
ORFB046 iron-chelator utilization proteinORFB046 iron-chelator utilization protein
ORFB047 iron ABC transporter, periplasmic substrate bpORFB047 iron ABC transporter, periplasmic substrate bp
ORFB067 putative metal binding proteinORFB067 putative metal binding protein
ORFB141 iron-chelator utilization proteinORFB141 iron-chelator utilization protein
ORFB074 hemin ABC transporter, periplasmic hemin bpORFB074 hemin ABC transporter, periplasmic hemin bp
ORFB075 hemin ABC transporter, permease proteinORFB075 hemin ABC transporter, permease protein
ORFB076 hemin ABC transporter, ATP-binding proteinORFB076 hemin ABC transporter, ATP-binding protein

80. TIGRTIGR
Specialized Genetic Elements
(Chromosome II and Megaplasmid)
• Many two component systems
• Nitrogen metabolism
• LexA
• Ribonucleotide reductase
• UvrA2
• Many transcription factors (e.g., HepA)
• Iron metabolism

81. TIGRTIGR
Uses of Phylogenomics VIII:
Comparison of Closely Related
Genomes

82. TIGRTIGR
V. cholerae vs. E. coli All Hits
0
1000000
2000000
3000000
4000000
5000000E.coliCoordinates
0 1000000 2000000 3000000
V. cholerae CoordinatesTIGRTIGR

83. TIGRTIGR
V. cholerae vs. E. coli Top Hits
0
1000000
2000000
3000000
4000000
5000000
E.coliCoordinates
0 1000000 2000000 3000000
V. cholerae CoordinatesTIGRTIGR

84. TIGRTIGR
V. cholerae vs. E. coli
Only if EC-Orf is Closest in All Genomes
0
1000000
2000000
3000000
4000000
5000000
E.coliCoordinates
0 1000000 2000000 3000000
V. cholerae Coordinates
TIGRTIGR

85. TIGRTIGR
V. cholerae vs. E. coli Proteins
Top
0
1000000
2000000
3000000
4000000
V. cholerae ORF Coordinates

86. TIGRTIGR
V. cholerae vs. E. coli F+R
0
1000000
2000000
3000000
4000000
5000000
Bert
Ecoli R
Ecoli

87. TIGRTIGR
S. pneumoniae vs. S. pyogenes DNA F+R
0
500000
1000000
1500000
2000000
BSP vs Spyo

88. TIGRTIGR
M. tuberculosis vs. M. leprae DNA
0
1000000
2000000
3000000
4000000
M1

89. TIGRTIGR C. trachomatis MoPn
C.pneumoniaeAR39
Origin
Termination
C. trachomatis vs C. pneumoniae Dot Plot

90. TIGRTIGR
Duplication and Gene Loss Model
A
B
CD
E
F
A
B
CD
E
F
A
B
C
D
E
F
A
B
C
D
E
F
A ’
B’
C’
D’
E’
F ’
A
B
C
D
E
F
A ’
B’
C’
D’
E’
F’
A
C
D
F
A ’
B’
E’
E. coli
E. coli
B
C
D
F
A ’
B’
D’
E’
V. cholerae
A
B
C
D
E
F
A ’
B’
C’
D’
E’
F’

91. TIGRTIGR
B1
A1
B2
A2
B3
A3
A2
A1 A2
A3
B2
B1
B3
B2
24
23
22
21
20
19
18171615
14
13
12
11
10
9
6
7
258
26
27
28
29
30
1 2 3
4
5
3132
B1
3132
6
7
8
9
10
11
12
13
14
15161718
19
20
21
22
23
24
25
26
27
28
29
30
1 2 3
4
5
3132
B3 24
23
22
21
20
19
18171615
14
13
12
11
10
9
6
7
258
26
27
28
29
3
3231 30
4
5
2 1
A1
3132
6
7
8
9
10
11
12
13
14
15161718
19
20
21
22
23
24
25
26
27
28
29
30
1 2 3
4
5
3132
A2
3132
6
7
8
9
10
11
12
13
19
18171615
14
20
21
22
23
24
25
26
27
28
29
30
1 2 3
4
5
3132
A3
2
6
7
8
9
10
11
12
13
19
18171615
14
20
21
22
23
24
25
26
27
5
4
3 31 30
29
28
1 32
B2
Inversion
A round
Terminus (*)
Inversion
A round
Terminus (*)
Inversion
A round
Origin (*)
Inversion
A round
Origin (*)
* *
* *
* *
* *
Figure 4
C ommon
Ancestor of
A and B
3132
6
7
8
9
10
11
12
13
14
15161718
19
20
21
22
23
24
25
26
27
28
29
30
1 2
3
4
5
3132

92. TIGRTIGR
M. tuberculosis strain phylogeny (Indels)

93. TIGRTIGR
Musser-Type Evolution (Indel Phylogeny)
98a
107a
43a
73a
105a
133a
114a
169a
218a
290a
160a
159a
13a
18a
26a
30a
32a
53a
58a
70a
96a
97a
100a
124a
204a
208a
236a
239a
249a
286a
99a
279a
205a
304a
54a
155a
165a
CDC1551a
223a
110a
122a
245a
313a
36a
40a
71a
79a
168a
254a
283a
312a
4a
12a
41a
42a
52a
77a
187a
214a
81a
129a
274a
220a
64a
48a
55a
60a
72a
80a
83a
85a
89a
91a
95a
111a
170a
171a
182a
212a
219a
225a
244a
278a
301a
195a
2a
123a
207a
306a
69a
94a
101a
102a
112a
113a
121a
132a
211a
222a
235a
250a
284a
285a
N1a
87a
117a
120a
136a
191a
237a
261a
37a
131a
269a
240a
63a
197a
206a
75a
108a
263a
128a
172a
162a
86a
38a
109a
119a
248a
6a
65a
68a
189a
66a
106a
227a
31a
78a
202a
213a
62a
163a
224a
256a
276a
287a
173a
291a
252a
281a
295a
310a
251a
151a
188a
292a
140a
141a
103a
174a
229a
259a
H37Rv
88a
44a
74a
76a
126a
282a
166a
210a
84a

94. TIGRTIGR
Consistency Indices (Indel Phylogeny)
Calculated over stored trees
CI
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
maximum
average
minimum
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 201
Character

95. TIGRTIGR
M. tuberculosis strain phylogeny
(Indels/SNPs)

96. TIGRTIGR
Musser-Type Evolution (Combined
Phylogeny)

97. TIGRTIGR
Consistency Indices (Combined Phylogeny)
Calculated over stored trees
CI
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
maximum
average
minimum
2
9
0
4
3
1
3
5
3
1
9
4
4
0
9
0
4
2
3
9
4
2
4
6
4
5
8
9
5
1
9
8
4
2
5
1
1
0
4
9
2 4C 6 6B 7 8C 9B 11B 14 15 15B 18C 4 8 12 16 18 M
U
S
S
E
R
S
m
e
a
r
Si
te
2
1
3
4
Character

98. TIGRTIGR
Uses of Phylogenomics VI:
Horizontal Gene Transfer and
Species Evolution

99. TIGRTIGR
Vertical Inheritance

100. TIGRTIGR
Examples of Horizontal Transfers
• Antibiotic resistance genes on plasmids
• Insertion sequences
• Pathogenicity islands
• Toxin resistance genes on plasmids
• Agrobacterium Ti plasmid
• Viruses and viroids
• Organelle to nucleus transfers

101. TIGRTIGR
Why Gene Transfers Are Useful to Identify
• Laterally transferred genes frequently involved in
environmental adaptations and/or pathogenicity
• Helps identify transposons, integrons, and other
vectors of gene transfer
• Helps identify species associations in the
environment
•

102. TIGRTIGR
Steps in Lateral Gene Transfer
1
2
3-5
6
A B C D

103. TIGRTIGR
How to Infer Gene Transfers
• Unusual distribution patterns
• Unusual nucleotide composition
• High sequence similarity to supposedly
distantly related species
• Unusual gene trees
• Observe transfer events

104. TIGRTIGR
Inferring Lateral Transfers
Observation Other Causes Always Occurs
Unusual Distribution Sampling bias Not if recipient already has gene.
Unusual GC/Codons Selection Not if donor/recipient similar.
Not if it occurred long ago.
High hit to "distant" species Selection
Rate variation
Gene loss
Usually.
Incongruent trees Bad trees
Missed paralogs
Usually.
Correlation of above with
neighbors
Selection Only if genes keep order after
transfer.

105. TIGRTIGR
E. coli and S. typhimurium Transfer
E. coli S. typhimurium
Old Model
E. coli S. typhimurium
New Model

106. TIGRTIGR
PGKPGK
Neighbor-joining;Neighbor-joining;
bootstrap;bootstrap;
50% majority rule50% majority rule
consensusconsensus
outgroup = Archaeaoutgroup = Archaea
T. maritima
M. genitalium
M. pneumoniae
A. aeolicus
B. burgdorferi
T. pallidum
B. subtilis
Synechocystis
E. coli
H. influenzae
H. pylori
M. tuburculosis
S. cerevisiae
A. fulgidus
M. jannascii
M. thermoauto
P. horikoshii
89
57
100
59
58
58 100
83
100
B. subtilis
S. cerevisiae
T. maritima
H. pylori
M. pneumoniae
M. genitalium
Synechocystis
B. burgdorferi
T. pallidum
P. horikoshii
M. jannascii
M. thermoautoA. fulgidus
A. aeolicus
H. influenzae
E. coli
M. tuburculosis

107. TIGRTIGR
Archaeal genes in bacterial genomesArchaeal genes in bacterial genomes**
Bacterial speciesBacterial species Best hits to ArchaealBest hits to Archaeal
Thermotoga maritimaThermotoga maritima 451 (24%)451 (24%)
Aquifex aeolicusAquifex aeolicus 246 (16%)246 (16%)
SynechocystisSynechocystis sp.sp. 126 (4%)126 (4%)
Borrelia burgdorferiBorrelia burgdorferi 45 (3.6%)45 (3.6%)
Escherichia coliEscherichia coli 99 (2.3%)99 (2.3%)
** 1010-5-5
over 60% of sequenceover 60% of sequence

108. TIGRTIGR
Evidence for lateral gene transfer inEvidence for lateral gene transfer in
ThermotogaThermotoga
1. 81 archaeal-like genes are clustered in 15 regions which
range in size from ~ 4 to 20 kb; many share conserved gene
order with their archaeal counterparts.
2. Many of the archaeal-like genes correspond to regions with
a significantly different base composition than the rest of
the chromosome.
3. Some of these regions are associated with a 30 bp repeat
structure found only in thermophiles.
4. Initial phylogenetic analyses of some of these genes lends
support to lateral gene transfer.

109. TIGRTIGR
0987 09900989ThermotogaThermotoga ORFORF
Archaea homologArchaea homolog
Bacterial homologBacterial homolog
Eukaryote homologEukaryote homolog
ThermotogaThermotoga ORFORF
Archaea homologArchaea homolog
Bacterial homologBacterial homolog
Eukaryote homologEukaryote homolog
0988 0991 0992 0993 0994
0995 0996 0997 0998 0999 1000 10021001 1003
Region TM00987 - TM1003 ( 21kb Archaea-like stretch)Region TM00987 - TM1003 ( 21kb Archaea-like stretch)
79% 69% 69% 72%
72% 69% 65%61% 78%
72%
TransposonTransposon
54%
48%
68% 51%
73%
73%
Regulatory proteinRegulatory protein

110. TIGRTIGR
Species Distribution of Top Hits:
A. thaliana Chr II
0
250
500
750
1000
TopHits
EAB
Syn. sp

111. TIGRTIGR
A. thaliana T1E2.8 is a
Chloroplast Derived HSP60
AR ATH -T1E2.8**********
ECOL
HAEIN
VIBCH
VIBCH
RICPR
YEAST
CHLPN
CHLTR
AQUAE
CAMJE
HELPY
BBUR
TREPA
THEMA
BACSU
DEIRA
MCYTU
MCYTU
SYNSP
SYNSP
ODONT CPST
MYCGE
MYCPN
CHLPN
CHLTR
CHLPN
CHLTR
ARCFU
ARCFU
METJA
PYRHO
METTH
METTH
YEAST
YEAST
YEAST
YEAST
CELEG
YEAST
YEAST
YEAST
CELEG
YEAST
YEAST
CELEG
YEAST
CELEG
CELEG
Eukarya
Archaea
Bacteria
Cyano/Cpst

112. TIGRTIGR
ParA Phylogeny
pOMB25.Bor
BBl32.Borb
Borbu3
Borbu.2
BBM32.Borb
CP32-6.Bor
BBA20.Borb
Cp18.Borbu
pOMB10.Bor
pLp7E.Borb
BBE19.Borb
BBB12.Borb
BBN32.Borb
BBF13.Borb
BBH28.Borb
BBK21.Borb
BBU05.Borb
BBJ17.Borb
BBQ08.Borb
BBF24.Borb
OrfC.Borbu
BBG08.Borb
Pyrab
Pyrho
YZ24 METJA
IncC1.Enta
IncC2.Enta
INC1 ECOLI
INC2 ECOLI
Orf.pRK2
IncC.pRK2
pM3.ParA
ORF3.Pseae
ORFB.Psepu
2603.Vibch*****
ParA.Strco
Strco2
Strco3
Myctu4
Mycle3
Deira.Chro
Soj.Trepa
SOJ BACSU
Ricpr
YGI1 PSEPU
ParA.Caucr
pAG1.Corgl
Mycle
Mycle2
Rv1708.Myc
Strco
Rv3213.Myc
Helpy99
Helpy26695
A00900.Vib*****
ParB.pR27.
ParA.pMT1.
parA.pMT1
parA.phage
ParA phage
ORFA00900
SOPA ECOLI
F-Plasmid
PhageN13
pCD1.Yerpe
pCD1#2.Yer
pYVe227.Ye
pNL1.Sphar
pQPH1.Coxb
p42d.Rhile
p42d.Rhiet
REPA AGRRA
pRiA4b.Agr
pTiB6S3.Ag
pTi-SAKURA
pRL8JI.Rhi
Y4CK Plasm
ParA.Raleu
pL6.5.Psef
Chr2.Deira
MP1#2.Deir
MP1.Deira
PX02.Bacan
ORF298.Clo
SojC.Halsp
Borbu4
sojD.Halsp
plasmid.St
SojB.Halsp
ParA.Rhoer
SOJ MYCPN
SOJ MYCGE
MinD2.Pyra
Pyrho2
pK214.Lacl
PatA.synsp
Deira.ParA
pCHL1.Chlt2
GP5D CHLTR
pCHL1.Chlt
Chltr
Chlps
Chlps2
Chlpn
Chltr2
Chlpn2
Chromosomal
Plasmid
and
Phage
BBQ08.Borb
Chlamydial
Inc
Borrelia
Plasmids
Archaea
Misc
Evolution of Chromosome Partitioning Proteins (ParA)

113. TIGRTIGR
0
0.1
0.2
0.3
0.4
C
B
A
0
Best Matches by Genetic Element
(D. radiodurans)

114. TIGRTIGR
N. meningitidis hits vs. genome size
0.0 250.0 500.0 750.0 1000.0 1250.0
Number ofN. meningitidis ORFs that have a significant hit
0.01000.02000.03000.04000.05000.0
TotalORFsinGenome Proteome Comparison ofN. meningitidis to other Complete Genomes
Archaea
H. influenzae
V. cholerae
E. coli

115. TIGRTIGR
Horizontal Gene Transfer II

116. TIGRTIGR
Reconciling a Tree of Life in the
Context of Lateral Gene Transfer

117. TIGRTIGR
rRNA Tree of Complete Genomes
Mycobacterium tuberculosis
Bacillus subtilis
Synechocystis sp.
Caenorhabditis elegans
Drosophila melanogaster
Saccharomyces cerevisiae
Methanobacterium thermoautotrophicum
Archaeoglobus fulgidus
Pyrococcus horikoshii
Methanococcus jannaschii
Aeropyrum pernix
Aquifex aeolicus
Thermotoga maritima
Deinococcus radiodurans
Treponema pallidum
Borrelia burgdorferi
Helicobacter pylori
Campylobacter jejuni
Neisseria meningitidis
Escherichia coli
Vibrio cholerae
Haemophilus influenzae
Rickettsia prowazekii
Mycoplasma pneumoniae
Mycoplasma genitalium
Chlamydia trachomatis
Chlamydia pneumoniae
0.05 changes
Archaea
Bacteria
Eukarya

118. TIGRTIGR
Whole Genome Phylogeny

119. TIGRTIGR
rRNA vs. Whole Genome Trees
Mycobacterium tuberculosis
Bacillus subtilis
Synechocystis sp.
Caenorhabditis elegans
Drosophila melanogaster
Saccharomyces cerevisiae
Methanobacterium thermoautotrophicum
Archaeoglobus fulgidus
Pyrococcus horikoshii
Methanococcus jannaschii
Aeropyrum pernix
Aquifex aeolicus
Thermotoga maritima
Deinococcus radiodurans
Treponema pallidum
Borrelia burgdorferi
Helicobacter pylori
Campylobacter jejuni
Neisseria meningitidis
Escherichia coli
Vibrio cholerae
Haemophilus influenzae
Rickettsia prowazekii
Mycoplasma pneumoniae
Mycoplasma genitalium
Chlamydia trachomatis
Chlamydia pneumoniae
0.05 changes
Archaea
Bacteria
Eukarya

120. TIGRTIGR
Gram Positives
Archaea
Eukaryotes
Syn. sp
Aquifex
Thermotoga
Proteobacteria
Top Hits of D. radiodurans Genes

121. TIGRTIGR
rRNA Suggested Deinococcus-Thermus Relationship
From
Embley et al.
Syst. Appl.
Microbiol. 16:
25-29
1993

122. TIGRTIGR
Serratia marcescens
Proteus mirabilis
Proteus vulgaris
Escherichia coli
Erwinia carotovora
Yersinia pestis
Enterobacter agglomerans
Vibrio
anguillarum
Vibrio cholerae
Haem
ophilus influenzae
Pseudomonasfluorescens
Pseudomonasputida
Pseudomonasaeruginosa
Azotobacter vinelandii
Acinotobactercalcoaceticus
Methylophilusmethylotrophus
Methylomonasclara
Methylobacillusflagellatum
Burkholderia
cepacia
Bordetella
pertussis
Xanthomonas oryzae
Legionella pneumophila
Acidiphilum facilis
Thiobacillus ferrooxidans
Neisseria gonorrhoeae
Rhizobium viciae
Myxococcus xanthus1
Myxococcus xanthus2
Campylobacter jejuni
StreptomycesviolaceusStreptomyceslividans
Streptomycesambofaciens
Mycobacteriumleprae
Mycobacteriumtuberculosis
Corynebacteriumglutamicum
Arabidopis thaliana
CPST
Synechococcussp.PCC7002
Synechococcussp.PCC7942
Anabaenavariabilis
Thermotoga maritima
Lactococcuslactis
Streptococcuspneumoniae
Staphylococcusaureus
Bacillussubtilis
Acholeplasm
a
laidlawii
Borrelia burgdorferi
Mycoplasma pulmonis
Mycoplasma mycoides
Bacteroides fragilis
Chlaymida trachomatis
Thermus thermophilus
Thermus aquaticus
Deinococcus radiodurans
Aquifex pyrophilus
0.10
α
γ1
γ2
β
Gram '+' High GC Cyanobacteria
Gram '+' Low GC
D/T
Magnetospirillum magnetotacticum
Helicobacter pylori
ε
δ
95
98
79
100
100
100
90
63
100
94
84
100
95
10088
93 91
75
100
100
100
100
100
8398
100
100
100
Rhizobium phaseoli
Agrobacterium tumefaciens
Rhizobium meliloti
Brucella abortus
Rhodobacter sphaeroides
Rhodobacter capsulatus
Rickettsia prowazekii
Acetobacter polyoxogenes
72 97
78
100
71
100
100 77
88
100
61
55
54
48
49
42
48
46
50
63
46
100
40

123. TIGRTIGR
Deinococcus-Thermus Comparison
• Took all available T. thermophilus proteins
• Searched against database of all available
complete genomes (including D. radiodurans)
• Identified gene with highest fasta p value
• Phylogenetic analysis of all genes with >4
homologs

124. TIGRTIGR
Other Bacteria
Archaea
D. radiodurans
Top Hits of T. thermophilus Proteins

125. TIGRTIGR
Significance of Deinococcus-
Thermus Relationship
• Mechanisms of extreme heat, radiation, and
desiccation resistance may be similar
• Complete genome of Thermus will be very
useful in identifying novel genes in
Deinococcus
• Shows utility of incomplete genome
sequences.

126. TIGRTIGR
Outline of Phylogenomics
Gene Evolution Events
Phenotype Predictions
Database
Species tree Presence/AbsenceGene trees
Congruence Evol. Distribution
F(x) Predictions
Pathway Evolution
TIGRTIGR

127. TIGRTIGR
Steps in Phylogenomic Analysis
• Create database of genes of interest
• Presence/absence of homologs in complete genomes
• Phylogenetic trees of each gene family
• Infer evolutionary events (gene origin, duplication, loss and
transfer)
• Refine presence/absence (orthologs, paralogs, subfamilies)
• Functional predictions and functional evolution
• Analysis of pathways

128. TIGRTIGR
Phylogenomics I:
Presence/Absence of Homologs
• Important to have complete genomes
• Similarity searches with high “homology
threshold” (to prevent false positives)
• Iterative searches (to prevent false negatives)
• Multiple sequence alignments to confirm
assignment of homology and to divide up
multi-domain proteins

129. TIGRTIGR
Phylogenomics II:
Phylogenetic Analysis of Homologs
• Multiple sequence alignment
• Mask alignment (exclude certain regions)
– ambiguous regions of alignment
– hypervariable regions and regions with large gaps
• Phylogenetic tree with method of choice
• Robustness checks
– bootstrapping
– compare trees with different alignments
– compare trees with different tree-building methods

130. TIGRTIGR
Phylogenomics III:
Inferring Evolutionary Events
• Infer evolutionary distribution patterns (overlay
presence/absence onto species tree)
• Compare gene tree vs. species tree
• Compare gene tree vs. evolutionary distribution
• Infer gene duplication and transfer events
• Combine gene transfer and duplication
information with evolutionary distribution
analysis to infer gene loss, gene origin, and
timing of gene duplications and transfers

131. TIGRTIGR
Phylogenomics IV:
Functional Predictions and Evolution
• Overlay experimentally determined functions
onto gene tree
• Infer changes in function
– many changes suggests caution should be used in
making new predictions
• Predict functions based on position in tree
relative to genes with known functions and
based on orthology groups

132. TIGRTIGR
Phylogenomics V:
Pathway Analysis
• Correlated presence/absence of all genes in pathway in
different species?
– If not, maybe non-orthologous gene displacement
– Alternatively, pathway may be different between species
• Correlated evolutionary events for genes in pathway
– loss of all genes at once
– correlated duplications?
• Compare evolution of function between pathways
– The number of times an activity has evolved helps in making
predictions of function/phenotype

133. TIGRTIGR
Evolution as a Screening
Method
• Gene duplications
• Gene loss
• Lateral gene transfers
• Organellar genes
• Structurally constrained genes
• Correlated evolutionary changes

134. TIGRTIGR
Evolutionary Genome Scanning
• Distribution patterns/phylogenetic profiles
• Patterns of evolution
– (ds/dn)
– Structurally constrained genes
– Correlated evolutionary changes
• Lateral gene transfers
– Organellar genes
– Pathogenicity islands
• Subdividing gene families
– Orthologs vs paralogs
– Functional predictions
– Subfamilies
– Motif identification
• Gene duplications
• Gene loss

135. TIGRTIGR
Genome Sequences Allow
“Hypothesisless Research”
• DNA microarrays
• Proteomics
• GC skew and other nucleotide composition
analyses
• Parallel genome wide genetic experiments
• Evolutionary genome scanning
• Phylogenetic profiles

136. TIGRTIGR
Evolutionary Diversity Still Poorly
Represented in Complete Genomes
Tmf-penden
R-rubrum3
Azs-brasi2
Rm-vanniel
Rhb-legum8
Bdr-japoni
Spg-capsul
Ric-prowaz
Ste-maltop
Spr-voluta
Rub-gelat2
Rcy-purpur
Nis-gonor1
Hrh-halch2
Alm-vinosm
Ps-aerugi3
E-coliMyx-xanthu
Bde-stolpiDsv-desulfDsb-postgaC-leptum
C-butyric4
C-pasteuri
Eub-barker
C-quercico
Hel-chlor2
Acp-laidla
M-capricol
C-ramosum
B-stearoth
Eco-faecal
Lis-monoc3
B-cereus4
B-subtilis
Stc-therm3
L-delbruck
L-casei
Fus-nuclea
Glb-violac
Olst-lut_CZeamaysC
Nost-muscr
Syn-6301
Tnm-lapsum
Flx-litora
Cy-lytica
Emb-brevi2
Bac-fragil
Prv-rumcol
Prb-difflu
Cy-hutchin
Flx-canada
Sap-grandi
Chl-limico
Wln-succi2
Hlb-pylor6
Cam-jejun5Stm-ambofa
Arb-globif
Cor-xerosi
Bif-bifidu
Cfx-aurant
Tmc-roseum
Aqu-pyroph
env-SBAR12
env-SBAR16
Msr-barker
Tpl-acidop
Msp-hungat
Hf-volcani
Mb-formici
Mt-fervid1
Tc-celer
Arg-fulgid
Mpy-kandl1
M
c-vanniel
Mc-jannasc
env-pJP27
Sul-acalda
Thp-tenax
env-pJP89
Tt-maritim
Fer-island
M
ei-ruber4
D-radiodur
Chd-psitta
Acbt-capsl
env-MC18
Pir-staley
Lpn-illini
Lps-interKSpi-stenos
Trp-pallid
Bor-burgdo
Spi-haloph
Brs-hyodys
Fib-sucS85
Tmf-penden
R-rubrum3
Azs-brasi2
Rm-vanniel
Rhb-legum8
Bdr-japoni
Spg-capsul
Ric-prowaz
Ste-maltop
Spr-voluta
Rub-gelat2
Rcy-purpur
Nis-gonor1
Hrh-halch2
Alm-vinosm
Ps-aerugi3
E-coliMyx-xanthu
Bde-stolpiDsv-desulfDsb-postgaC-leptum
C-butyric4
C-pasteuri
Eub-barker
C-quercico
Hel-chlor2
Acp-laidla
M-capricol
C-ramosum
B-stearoth
Eco-faecal
Lis-monoc3
B-cereus4
B-subtilis
Stc-therm
3
L-delbruck
L-casei
Fus-nuclea
Glb-violac
Olst-lut_CZeamaysC
Nost-muscr
Syn-6301
Tnm
-lapsum
Flx-litora
Cy-lytica
Emb-brevi2
Bac-fragil
Prv-rumcol
Prb-difflu
Cy-hutchin
Flx-canada
Sap-grandi
Chl-limico
Wln-succi2
Hlb-pylor6
Cam-jejun5Stm-ambofa
Arb-globif
Cor-xerosi
Bif-bifidu
Cfx-aurant
Tmc-roseum
Aqu-pyroph
env-SBAR12
env-SBAR16
Msr-barker
Tpl-acidop
Msp-hungat
Hf-volcani
Mb-formici
Mt-fervid1
Tc-celer
Arg-fulgid
Mpy-kandl1
M
c-vanniel
Mc-jannasc
env-pJP27
Sul-acalda
Thp-tenax
env-pJP89
Tt-maritim
Fer-island
M
ei-ruber4
D-radiodur
Chd-psitta
Acbt-capsl
env-MC18
Pir-staley
Lpn-illini
Lps-interKSpi-stenos
Trp-pallid
Bor-burgdo
Spi-haloph
Brs-hyodys
Fib-sucS85
Bacteria Archaea Bacteria Archaea
A.rRNAtreeofBacterialandArchaealMajorGroups B.GroupswithCompletedGenomesHighlighted

137. TIGRTIGR
Acknowledgements
• Genome duplications: S. Salzberg, J. Heidelberg, O.
White, A. Stoltzfus, J. Peterson
• Genome sequences and analysis: J. Heidelberg, T.
Read, H. Tettelin, K. Nelson, J. Peterson, R.
Fleischmann
• Horizontal transfers: K. Nelson, W. F. Doolittle
• TIGR: C. Fraser, J. Venter, M-I. Benito, S. Kaul,
Seqcore
• $$$: DOE, NSH, NIH, ONR
TIGRTIGR

138. TIGRTIGR
TIGTIG
RR
OtherOther
peoplepeople
Mom and DadMom and Dad
S. KarlinS. Karlin
M. FeldmanM. Feldman
A. M. CampbellA. M. Campbell
R. FernaldR. Fernald
R. ShaferR. Shafer
D. AckerlyD. Ackerly
D. GoldsteinD. Goldstein
M. EisenM. Eisen
J. CourcelleJ. Courcelle
R. MyersR. Myers
C. M. CavanaughC. M. Cavanaugh
P. HanawaltP. Hanawalt
NSFNSF
J. HeidelberJ. Heidelber
T.ReadT.Read
S. KaulS. Kaul
M-I BenitoM-I Benito
J. C. VenterJ. C. VenterC. FraseC. Fraser
S. SalzbergS. Salzberg
O. WhiteO. White
K. NelsonK. Nelson
$$$$$$
ONRONR
DOEDOE
NIHNIH
H. TettelinH. Tettelin

139. TIGRTIGR
Using Evolutionary Analysis To
Help Identify Novel Features in D.
radiodurans

140. TIGRTIGR
Origins of Extreme Resistance
• Functional divergence
• Evolution of novel genes/processes
• Acquire genes from other species
• Gene duplication and functional divergence
• Enhanced catalytic efficiency and/or
coordination

141. TIGRTIGR

142. TIGRTIGR
SNF2 Family of Proteins (1995)
• SNF2 family defined by presence of conserved DNA-
dependent ATPase domain
• 100s of proteins
• Diversity of functions:
– transcriptional activation (SNF2)
– transcriptional repression (MOT1)
– Recombination (RAD54)
– transcription-coupled repair (CSB)
– post-replication repair (RAD5)
– chromosome segregation (lodestar)
– Many with unknown functions
• Some species have 15+ representatives

143. TIGRTIGR
How to Sort Out Diversity in SNF2 Family
• Presence of additional motifs
– RING fingers
– Bromodomains
– Chromodomains
• Interactions with other proteins
• Evolutionary relationships
– Orthology and paralogy
– Subfamilies
– Relationships among subfamilies

144. TIGRTIGR
SNF2 Alignment
BRM
hBRM
hBRG1
mBRG1
STH1
SNF2
YB95
F37A4
ISWI
SNF2L
CHD1
SYGP
ETL1
FUN30
MOT1
ERCC6
RAD26
YB53
DNRPPX
hNUCP
mNUCP
RAD5
spRAD8
HIP116
RAD16
LODE
NPH42
HepA
B.cereus ORF
I Ia Ib II III V VI
C
C
R
R
R
R
Br
CHD1
SNF2
SNF2L
ETL1
RAD16
ERCC6
RAD54
RAD54
Br
Br
Br
Br
Br
Protein
Sub-
Family
SCALE (aa)
0 500
Helicase Motif s --
MOT1
IV

145. TIGRTIGR
SNF2 Subfamilies
Subfamily Conserved Function
SNF2 Transcription activation (Swi/Snf complex)
SNF2L Transcription activation (NURF complex)
CHD1 Chromatin remodelling
ETL1 Unknown
MOT1 Transcription repression
CSB Transcription-coupled repair
Rad54 Recombinational repair
Rad16 Chromatin access for DNA repair
HepA Bacterial RNA polymerase subunit
HepA2 Unknown

146. TIGRTIGR
What Evolutionary Analysis
Reveals About the SNF2 Family
• Ancient duplication into two lineages may distinguish
genes by type of activity
• Multiple subfamilies with distinct sequences and
functions.
• Presence of particular orthologs can be predicted in
species for which they have not been cloned.
• Predict functions of uncharacterized members by
orthology.
• Addition of motifs to SNF2 domain occurred early in
eukaryotic evolution.
• Many duplications within eukaryotes.
• Classificaiton into subfamilies helps search for
functional motifs

147. TIGRTIGR
ETL1_M.m
YA19_S.c
CHD1_M.m
SYGP4_S.c
MOT1_S.c
ERCC6_H.s
RAD26_S.c
NUCP_H.s
NUCP_M.m
YB53_S.c
RAD54_S.c
DNRPPX_S.p
RAD5_S.c
RAD8_S.p
HIP116A_H.s
RAD16_S.c
LODE._D.m
NPHCG_42
HEPA._E.c
YB95_S.c
F37A4_C.e
ISWI_D.m
SNF2L_H.s
BRM_D.m
BRM_H.s
BRG1_H.s
BRG1_M.m
STH1_S.c
SNF2_S.c
SNF2
SNF2L
CHD1
ETL1
CSB
R A D54
R A D16
LO DE
Evolution of the SNF2 Family of Proteins

148. TIGRTIGR