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Examples of Amino Acid Changes in Notothenioids Methods To

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Supplementary Information S1

Candidate genes: examples of amino acid changes in notothenioids Methods

To evaluate notothenioid amino acid changes in a cross‐section of genes, six candidates from a range of functional categories of interest were chosen for a more detailed examination. The selected genes comprised small, well‐characterised genes present in multiple fish species (mainly teleosts, but in some cases these analyses were extended to the Actinopterygii when sequences from the spotted gar (Lepisosteus oculatus) were available) and available in at least two notothenioids. The amino acid composition of a series of six candidate genes were further analysed in depth at the amino acid sequence level. These genes were superoxide dismutase 1 (SOD1), neuroglobin, dihydrofolate reductase (both paralogues), p53 and calmodulin. Clustal X alignments were constructed from orthologues identified in the four Notothenioid transcriptomes along with other fish orthologues extracted from either SwissProt (Bateman et al., 2017) or Ensembl release 91 (Aken et al., 2017). Alignment data were visualised and annotated in BoxShade version 3.21 (https://embnet.vital‐ it.ch/software/BOX_form.html). Notothenioid‐specific changes were noted and the type of substitution in terms of amino acid properties was noted.

Results

A range of amino acid changes were revealed, from virtually complete conservation between the notothenioids and other fish (calmodulin), to one amino acid substitution present in neuroglobin and SOD1, to more extensive changes in dihydrofolate reductase, dihydrofolate reductase‐like and p53. In the case of the latter three genes, notothenioid‐specific changes were at positions that were often highly variable in temperate fish species with up to six different amino acid substitutions and did not result in a definable pattern of directional substitution. In all cases, the notothenioid protein coding regions did not have consistent length differences compared to other fish.

Table showing amino acid substitutions in candidate proteins

Amino acid changes in Notothenioid candidate proteins compared with other fish. Colour code: blue: polar positive; red: polar negative; green: polar neutral; white: non‐polar aliphatic; purple: non‐polar aromatic; brown: small (proline and glycine); yellow: cysteine. Notes: ‘variable’ is recorded when substitutions in non‐Notothenioid fish have 2‐3 different amino acids with different properties in the same position; ‘no significant change’ is recorded where there is a change in amino acid in the notothenioids, which has the same physical properties as that of the non‐notothenioid fish species.

Amino acid position
Notothenioid substitution

  • Substitutions in other fish
  • Notes

Calmodulin

No Notothenioid‐specific substitutions. 4 Notothenioid species‐specific substitutions

Superoxide dismutase

6 Notothenioid species‐specific substitutions

  • 198
  • Leu
  • Ile
  • No significant change

Neuroglobin

3 Notothenioid species‐specific substitutions. Last 13 aa at 3’ end very different. 8 forms across 18 species.

  • 85
  • Pro
  • Thr
  • No significant change

Dihydrofolate reductase

4 Notothenioid species‐specific substitutions. Notothenioid 3’ end slightly longer by 2‐6 aa

  • 4
  • Met

Asp Gln Val
Val Pro Ile Asn His Lys Asn Asp
No significant change Negative to neutral/positive No significant change No significant change Polar neutral to non‐polar aliphatic
20 22 28 111

  • Ile
  • Leu Lys

  • Thr
  • Ala Val

128 129 141 169
Gln Phe Met His
Glu Gly Ser Arg Ala Gly Leu
Variable Variable No significant change

  • Variable
  • Gln Glu Leu Val

Dihydrofolate reductase‐like

12 Notothenioid species‐specific substitutions. First 10 aa highly variant 9 different forms in 11 species. Last 8 aa at 3’ end highly variant 9 forms in 11 species. 28 30 48 55 78 85 88 90
Glu Gly Thr Phe Thr Leu Asp Pro Ser Gly Ser Leu Lys Lys Val

  • Asp
  • No significant change

Variable Variable Non‐polar to polar Variable Non‐polar aliphatic to polar
Gln Arg Thr Ile Arg Gly Ala Ser Met Val Lys Ser Cys Val Ile Lys Arg Thr Glu Phe Ser Thr Ile Gly Variable

  • Val Ala
  • No significant change

Variable Variable Variable Non‐polar aliphatic to polar Positive to negative/neutral Positive to neutral No significant change

  • 99
  • Glu Gln Gly Lys His

Ala Ser Val Gln Leu Glu Thr Lys Glu Asp Asn Gln Asn Gln
109 110 134 145 181

  • 183
  • Ile

p53

11 Notothenioid species‐specific substitutions in core region. 5’ end highly variant with only 9 conserved residues in 93 aa. 3’ end highly variant with only 29 conserved residues in 81 aa. 107 109 113 126 145 148 153 181 216 223 254 257 264 268 299
Gln Gln Thr Gln Gly Ile
Asn Glu Lys Ala Arg His Glu Ser
No significant change Variable No significant change

  • Variable
  • Lys Ile

Lys Ile Pro
Ser

  • Ser
  • Variable

Non‐polar aliphatic to small No significant change Variable Variable No significant change Variable No significant change Aliphatic to aromatic No significant change Neutral to non‐polar aliphatic

  • Ile
  • Met Val

Ala Leu Ile Ala Leu Leu Ile
Ser Thr Leu Pro Arg Met Phe Gln Pro Thr Val Glu Phe Val

  • Ala Val
  • Thr

References

Aken BL, Achuthan P, Akanni W, Amode MR, Bernsdorff F, Bhai J, Billis K, Carvalho‐Silva D, Cummins C, Clapham P, et al. 2017. Ensembl 2017. Nucl Acids Res 45:D635‐D642.

Bateman A, Martin MJ, O'Donovan C, Magrane M, Alpi E, Antunes R, Bely B, Bingley M, Bonilla C, Britto R, et al. 2017. UniProt: the universal protein knowledgebase. Nucl Acids Res 45:D158‐D169.

Consensus line symbols: * = absolute conservation, . = up to 50% conservation across the alignment at that position. Dark squares are absolute conservation of amino acids. Light squares are conservative changes and white squares are divergent changes. Antarctic species names are in blue, whilst red name denotes a temperate Notothenioid.

Calmodulin

DANRE ELEEL CTEID ONCSP TORCA

NEOIO

POEFO ASTMX TAKRU ORYLA XIPMA

PARCH

consensus

1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG

1 ************************************************************
DANRE ELEEL CTEID ONCSP TORCA

NEOIO

POEFO ASTMX TAKRU ORYLA XIPMA

PARCH

61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMAKKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE 61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGFISAAELRHVMTNLGEKLTDE

consensus 61 **************.************************.******************** DANRE ELEEL CTEID ONCSP TORCA

NEOIO

POEFO ASTMX TAKRU ORYLA XIPMA

PARCH

121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVQMMTAK 121 EVDEMIREADIDGDGQVNYEEFVTMMMSK

consensus 121 ***********************.**..*

Superoxide dismutase 1 (SOD1)

XIPHE XIPMA POEFO TAKRU DANRE HYPMO SALSA ONCMY GADMO ASTMX

CHIHA TREBE NEOIO

GASAC LEPOC ORYLA ORENI consensus

1 MVLKAVCVLKGAGETTGTVHFEQEIESAPVKVTGEISGLTPGDHGFHVHAFGDNTNGCIS 1 MVLKAVCVLKGAGETTGTVHFEQENESAPVKVTGEISGLTPGDHGFHVHAFGDNTNGCIS 1 MVLKAVCVLKGAGETTGTVHFEQENESAPVKVTGEIGGLTPGEHGFHVHAFGDNTNGCIS 1 MAMKAVCVLKGAGDTSGTVYFEQENESAPVKLTGEIKGLTPGEHGFHVHAFGDNTNGCIS 1 MVNKAVCVLKGTGEVTGTVYFNQEGEKKPVKVTGEITGLTPGKHGFHVHAFGDNTNGCIS 1 MVNKAVCVLKGDGQVTGTVYFEQEAEKSPVKLSGEITGLTAGKHGFHVHAFGDNTNGCIS 1 MALKAVCVLKGTGEVTGTVFFEQEGDGAPVKLTGEIAGLTPGEHGFHVHAFGDNTNGCMS 1 MAMKAVCVLKGTGEVTGTVFFEQEGADGPVKLIGEISGLAPGEHGFHVHAYGDNTNGCMS 1 MVLKAVCVLKGTGDVTGTVFFEQEGDGAPVKLSGQIAGLAAGEHGFHVHVFGDNTNGCIS 1 MVHKAVCVLKGTGEVTGTVFFEQVGDGAPVKVSGEITGLTPGLHGFHVHAFGDNTNGCIS 1 ---KAVCVFKGAGEASGTVFFEQETDSCPVKLTGEIKGLTPGEHGFHVHAFGDNTNGCIS 1 ---KAVCVFKGTGEASGTVFFEQENDSAPVKLTGEIKGLTPGEHGFHVHAFGDNTNGCIS 1 MVIKAVCVLKGAGEASGTVFFEQENDSSPVKLTGEIKGLTPGEHGFHVHAFGDNTNGCIS 1 MVVKAVCVLKGAGETTGTIYFEQESDKAAVKLTGEIKGLTPGEHGFHVHAFGDNTNGCIS 1 MVLKAVCVLKGSGEVSGTVHFEQQNGDAPVKVTGKISGLTPGDHGFHVHAFGDNTNGCVS 1 MVLKAVCVLKGTGETNGVVNFEQESDSAPVKVTGEIKGLTPGKHGFHIHVYGDNTNGCVS 1 MVLKAVCVLKGTGDTSGTVYFEQENESAPVKLTGEIKGLTPGEHGFHVHAFGDNTNGCIS

1 ...*****.**.*. .*...*.*. ....**..*.* **..*.****.*..*******.*
XIPHE XIPMA POEFO TAKRU DANRE HYPMO SALSA ONCMY GADMO ASTMX

CHIHA TREBE NEOIO

GASAC LEPOC ORYLA ORENI

61 AGPHYNPFTKNHGGPTDVERHVGDLGNVTAGADNIAKIDIKDTFIKLSGPNSIIGRTMVI 61 AGPHYNPFTKNHGGPTDVERHVGDLGNVTAGADNIAKIDIKDTFIKLSGPNSIIGRTMVI 61 AGPHYNPFSKKHGGPTDEERHVGDLGNVTAGADNIAKIDIKDSFIKLSGPNSIIGRTMVI 61 AGPHYNPHNKTHAGPTDADRHLGDLGNVTAGADNIAKIDIKDSMLTLTGPYSIIGRTMVI 61 AGPHFNPHDKTHGGPTDSVRHVGDLGNVTADASGVAKIEIEDAMLTLSGQHSIIGRTMVI 61 AGPHFNPYSKNHGGPTDSERHVGDLGNVTAGENGVAKIDIVDKMLTLSGPDSIIGRTMVI 61 AGPHFNPHNHTHGGPTDTVRHVGDLGNVTAAADSVAKINIQDEILSLAGPHSIIGRTMVI 61 AGPHFNPHNQTHGGPTDAVRHVGDLGNVTAGADNVAKINIQDKMLTLTGPDSIIGRTMVI 61 AGPHFNPHKKDHAGPNDVDRHVGDLGNVTAGADKVAKIDITDKMLSLNGPFSIVGRTMVI 61 AGPHYNPHNKTHGGPTDEIRHVGDLGNVTAGADGVAKINIEDKMLSLSGPHSIVGRTMVI 58 AGPHFNPHNKTHAGPTDENRHVGDLGNVTAAADNVAKLDITDKMITLAGQYSIIGRTMVI 58 AGPHFNPHNKTHAGPTDEDRHVGDLGNVTAAADNVAKLNITDKMITLAGQYSIIGRTMVI 61 AGPHFNPHNKTHAGPTDENRHVGDLGNVTAAADNVAKLDITDKMITLAGQYSIIGRTMVI 61 AGPHFNPHSKTHAGPNDEIRHVGDLGNVTAGGDNIAKIDITDKVITLTGQHSIIGRTMVI 61 AGPHYNPHNKTHGGPRDENRHVGDLGNVTAGEDKVANINIEDKHITLTGQFSIIGRTMVI 61 AGPHFNPYNKNHGGPEDAERHVGDLGNVTAGDNNVAKIDITDKLIRLSGPDSIVGRTVVV 61 AGPHFNPYNKNHGGPKDAERHVGDLGNVTAADN-VAKIEITDKVITLTGPDSIIGRTMVI

consensus 61 ****.**....*.**.* .**.********.....*...* *....*.*. **.***.*. XIPHE XIPMA POEFO TAKRU DANRE HYPMO SALSA ONCMY GADMO ASTMX

CHIHA TREBE NEOIO

GASAC LEPOC ORYLA ORENI

121 HEKADDLGKGGNEESLKTGNAGGRLACGVIGITQ------ 121 HEKADDLGKGGNEESLKTGNAGGRLACGVIGIAQ------ 121 HEKADDLGKGGNEESLKTGNAGGRLACGVIGITQ------ 121 HEKADDLGKGGNEESLKTGNAGGRLACGVIGITQ------ 121 HEKEDDLGKGGNEESLKTGNAGGRLACGVIGITQ------ 121 HEKEDDLGKGNNEESLKTGNAGGRLACGVIGIAQ------ 121 HEKADDLGKGDNEESRKTGNAGSRLACGVIGIAQ------ 121 HEKADDLGKGGNEESLKTGNAGGRQACGVIGIAQ------ 121 HEKADDLGKGGNEESLKTGNAGSRLACGVIGI-------- 121 HEKEDDLGKGGDEESLKTGNAGARLACGVIGIAQ------ 118 HEKADDLGKGGNDESLKTGNAGGRLACGVIGIAQMDRRPT 118 HEKADDLGKGGNDESLKTGNAGGRLACGVIGIAQ------ 121 HEKADDLGKGGNDESLKTGNAGGRLACGVIGIAQ------ 121 HEKADDLGKGGNEESLKTGNAGSRLACGVIGIAQ------ 121 HEKADDLGKGGDDESLKTGNAGGRLACGVIGIAQ------ 121 HEKVDDLGKGGNDESLKTGNAGARLACGVIGIAQ------ 120 HEKVDDLGKGGNEESLKTGNAGGRLACGVIGITQ------

consensus 121 ***.******...**.******.*.*******..

Neuroglobin

DANRE ASTMX POEFO XIPMA ORYLA TAKRU TETNG

PARCH PSEGE CHAAC DISMA CHIMY NEOIO GYMAC

BOVVA

ONCMY1 ONCMY2 LEPOC consensus

1 MEKLSEKDKGLIRDSWESLGKNKVPHGIVLFTRLFELDPALLTLFSYSTNCGDAPECLSS 1 MEKLTGKDKELIRDSWESLGKNKVPHGIVMFTRLFELDPSLLTLFNYKTNCGVVPECLSS 1 MGELSVKDKELIRGSWESLGKNKVPHGVIMFSRLFELDPALLNLFHYSTNCDSKQDCLSS 1 MGELSVKDKELIRGSWESLGKNKVPHGVIMFSRLFELDPALLNLFHYSTNCDSKQDCLSS 1 MEKLSGKDKELIRGSWESLGKNKVPHGVIMFSRLFELDPALLSLFNYNTNCGSTQDCLSS 1 MEKLSSKDKELIRGSWDSLGKNKVPHGVIMFSRLFELDPELLSLFHYTTNCGSTQDCLSS 1 MEKLSSKDKELIRGSWDSLGKNKVPHGVILFSRLFELDPELLNLFHYTTNCGSTQDCLSS

1 ------------------------------------------------------------ 1 ---------------------------VFVCGRLFELDPELLTLFHYTTNCGSTQDCLSS

1 MEKLSEKDKELIRGSWESLGKNKVPHGVVMFSRLFELDPELLTLFHYTTNCGSTQDCLSS 1 MEKLSEKDKELIRGSWESLGKNKVPHGVVMFSRLFELDPELLTLFHYTTNCGSTQDCLSS 1 MEKLSGKDKELIRGSWESLGKNKVPHGVVMFSRLFELDPELLTLFHYTTNCGSTQDCLSS 1 MEKLSGKDKELIRGSWESLGKNKVPHGVVMFSRLFELDPELLTLFHYTTNCGSTQDCLSS 1 MEKLSGKDKELIRGSWESLGKNKVPHGVVMFSRLFELDPELLTLFHYTTNCGSTQDCLSS 1 MEKLSGKDKELIRGSWESLGKNKVPHGVVMFSRLFELDPELLTLFHYTTNCGSTQDCLSS 1 MEKLTEKEKELIRVSWESLGKDKVPHGVIMFSRLFELEPALLNLFHYNTNCGTIQDCLSS 1 MEKLTEKDKELIRGSWESLGKNKVPHGVVMFSRLFELEPALLNLFHYNTNCSPTQDCLSS 1 MEKLTAKDKEMIRDSWERLGKNKLTHGTIMFTRLFELDPELLGLFHYNTPYSSPQECLSS

1 ..... ......................................................
DANRE ASTMX POEFO XIPMA ORYLA TAKRU TETNG

PARCH PSEGE CHAAC DISMA CHIMY NEOIO GYMAC

BOVVA

ONCMY1 ONCMY2 LEPOC

61 PEFLEHVTKVMLVIDAAVSHLDDLHTLEDFLLNLGRKHQAVGVNTQSFALVGESLLYMLQ 61 PEFLEHVTKVMLVVDAAVSHLDDLHTLEDFLLNLGKKHQAVGVNTQSFALVGESLLYMLQ 61 PEFLDHVTKVMLVIDAAVSHLDDLHSLEDFLLNLGRKHQAVGVSTQSFTEVGESLLYMLQ 61 PEFLDHVTKVMLVIDAAVSHLDDLHSLEDFLLNLGRKHQAVGVSTQSFTVVGESLLYMLQ 61 PEFLDHVTKVMLVIDAAVNHLDDLHSLEDFLLNLGRKHQAVGVSTQSFAVVGESLLYMLQ 61 PEFLEHVTKVMLVIDAAVSHLDDLHSLEDFLLNLGRKHQAVGVNPQSFATVGESLLYMLQ 61 PEFLEHVTKVMLVIDAAVSHLDDLHSLEDFLLNLGRKHQAVGVKPQSFAMVGESLLYMLQ
1 ---------VMLVIDAAVSHLDDLPSLEDFLLNLGRKHQAVGVNTQSFAEVGESLLYMLQ
34 PEFLEHVTKVMLVIDAAVSHLDDLPSLEDFLLNLGRKHQAVGVNTQSFAEVGESLLYMLQ 61 PEFLEHVTKVMLVIDAAVSHLDDLPSLEDFLLNLGRKHQAVGVNTQSFAEVGESLLYMLQ 61 PEFLEHVTKVMLVIDAAVSNLDDLPSLEDFLLNLGRKHQAVGVNTQSFAEVGESLLHMLQ 61 PEFLEHVTKVMLVIDAAVSHLDDLPSLEDFLLNLGRKHQAVGVNTQSFAEVGESLLYMLQ 61 PEFLEHVTKVMLVIDAAVSHLDDLPSLEDFLLNLGRKHQAVGVNTQSFAEVGESLLYMLQ 61 PEFLEHVTKVMLVIDAAVSHLDDLPSLEDFLLNLGRKHQAVGVNTQSFAEVGESLLYMLQ 61 PEFLEHVTKVMLVIDAAVSNLDDLPSLEDFLLNLGGKHQAVGVNTQSFAEVGESLLYMLQ 61 PEFLDHVTKVMLVIDAAVSHLDNLHTLEDFLLNLGKKHQAVGVNTQSFAVVGESLLYMLQ 61 PEFLDHVTKVMLVIDAAVSHLDDLHTLEDFLLNLGKKHQAVGVNTQSFAVVGESLLYMLQ 61 PEFVDHINKVMLVVDAAVTHLDNLHSLEEYLVNLGRKHQAVGVQTQSFAAVGESLLYMLE

consensus 61 .........****.****..**.*..**..*.***.*******..***..******.**. DANRE ASTMX POEFO XIPMA ORYLA TAKRU TETNG

PARCH PSEGE CHAAC DISMA CHIMY NEOIO GYMAC

BOVVA

ONCMY1 ONCMY2 LEPOC

121 SSLGPAYTTSLRQAWLTMYSIVVSAMTRGWAKNGEHKSN 121 CSLGTAYTTALRQAWLNMYTIVVSAMTRGWAKNGEHKSN 121 CSLGQAYTAPLRQAWLNMYSIVVAVMSRGWAKNGEDKAD 121 CSLGQAYTAPLRQAWLNMYTIVVAVMSRGWSKNGEDKAD 121 CSLGQAYTAALSQAWLNMYSIVVAAMSRGWAKNGEDKAD 121 CSLGQAYTASLRQAWLNMYSVVVAAMSRGWAKNGEDKAD 121 CSLGQAYTASLRQAWLNMYSVVVASMSRGWAKNGEDKAD
52 CSLGQAYTAPLRQAWLNLYSIVVAAMSQGWAKNGEDKAD 94 CSLGQAYTAPLRQAWLNLYSIVVAAMSQGWAKNGEDKAD
121 CSLGQAYTAPLRQAWLNLYSIVVAAMSQGWAKNGEDKAD 121 CSLGQAYTAPLRQAWLNLYSIVVAAMSQGWAKNGEDKAD 121 CSLGQAYTAPLRQAWLNLYSIVVAAMMPRLG-------- 121 CSLGQAYTAPLRQAWLNLYSIVVAAMMPRLG-------- 121 CSLGQAYTAPLRQAWLNLYSIVVAAMMPRLG-------- 121 CSLGQAYTAPLRQAWLNLYSIVVAAMSRGWA-------- 121 CSLGQGYTAPLRQAWLNMYTIVVAAMSRGWAKNGEHKTD 121 CSLGHGYTGPLRQAWLNMYTIVVAAMSRGWAKNGEHKTD 121 RSLGPAYTSTLRHAWLTLYSVVVEAMSSGWTKNGKSQTD

consensus 121 .***..**..*..***..*..**..*.............

Dihydrofolate reductase

TAKRU TETNI

HARAN NEOIO

GASAC ORENI LEPOC DANRE consensus

1 MSRVVNAIVAVCPDLGIGMNGDLPWHPIRLNKEFVHFRKMTSTPSVNGKQNVVIMGRKTW 1 MARVLNAIVAVCPDLGIGRNGDLPWHPIRLDNEFKHFRKMTSTPSVNGKQNVVIMGRKTW 1 MSRMLNAIVAVCPDLGIGMDGQLPWHPVRLNNEFKHFRKITATPSVEGKQNVVIMGRKTW 1 MSRMLNAIVAVCPDLGIGMDGQLPWHPVRLNNEFKHFRKMTATPSVEGKQNVVIMGRKTW 1 MSRVLNGIVAVCPDLGIGCHGNLPWHPLRLSNEFKHFRRMTATASVKDKQNVVIMGRKTW 1 MPRVLNAIVAVCPDRGIGNKGNLPWHPIRLSKEFAHFRKMTATPSVKGKQNVVIMGKKTW 1 MPRPINCIVAVCPNMGIGHNGNLPWHPKRLSNEFKYFQKMTMTPTLEGQQNAVIMGRKTW 1 MSRILNCIVAVCPDMGIGKNGNLPWHPIRLSNELKHFQKMTMTPSDEGKKNVVIMGRKTW

1 *.*..*.******..*** .*.*****.**..*...*...*.*.......*.****.***
TAKRU TETNI

HARAN NEOIO

GASAC ORENI LEPOC DANRE

61 FSIPERNRPLANRINIVLSRRCREPPAGAHHLARDLPSALRLVDS-ELAEQADQVWVIGG 61 FSIPEKHRPLANRINIVLSRRSREPPAGAHHLAHDLSSALQLVET-QLADR-DQVWVIGG 61 FSIPEKNRPLNNRINIVLSRECRAPPAGAHHLAPDFNSALRLVET-ELAERTDQVWVIGG 61 FSIPEKNRPLNNRINIVLSRECRAPPAGAHHLAPDFSSALRLVET-ELAERTAQVWVIGG 61 FSIPEKNRPLNNRINIVLSRELKAPPAGAHHLAPDFSSALRLVDT-ELAEQADQVWVIGG 61 YSIPEKNRPLSNRINIVLSRECKVPPAGAHYLASDFSSALRLIDT-ELADQADQVWVIGG 61 FSIPERNRPLKNRINIVLSRELKNPPEGAHYLASDFSSALRLLDSAPQTDQVDQVWVIGG 61 FSIPAAHRPLKNRINIVLSRELKTAPEGAHYLASDFSSALHLLDSGELEKLVDQVWIIGG

consensus 61 .***...*** *********... .*.***.** *..***.*... ..... .***.*** TAKRU TETNI

HARAN NEOIO

GASAC ORENI LEPOC DANRE

120 SSLYQELMEGTGTTRLFVTRILKQFECDTFLPEINPARYRRLPQFPGVAQELQEEEGIQY 119 SSLYQEMMERPGTSRLFVTHVLKQFDCDTFLPEIRPQRYRLLPQFPGVPQELQEEKGIQY 120 SSLYKELMQFPGTRRLFITRIMKQFECDTFLPEISLDKYRLLPQFPGVPHELQEENGIQY 120 SSLYKELMQFPGTRRLFVTRIMKQFECDTFLPEISLDRYRLLPQFPGVPHELQEENGIQY 120 SSLYKELLESPGTRRLFVTRILKQFECDTFLPEICPDRYRLLPEFPGVPEELQEENGVQY 120 SSLYKEMMGSTGMRRLFVTQILKQFECDTFLPEISLDKYRLLPEFPDVPQELQEENGIQY 121 SSVYKEAMEAPGWCRLFVTQILQDFECDTFLPEINLNKYRLLPEFPGVPLEVQEENGIRY 121 SSLYKEVMERSGHRRLFVTRILKQFDCDTFIPNFDMDKYKLLPEFPGVPVGLQEDNGVQY

consensus 121 **.*.*... .*..***.*.....*.****.*.. ...*..** **.*. ..**..*..* TAKRU TETNI

HARAN NEOIO

GASAC ORENI LEPOC DANRE
180 RFQVYESIEH------ 179 RYQVYESLE------- 180 RFEVYESIDELC---- 180 RFEVYESIDELCNLNK 180 RFEVYESIDNK----- 180 RFKVYESIQE------ 181 KFEVYESIQN------ 181 VFEVYESIEH------ consensus 181 ...****..

Dihydrofolate reductase‐like

HARAN PARCH NEOIO

GASAC TAKRU TETNG POEFO XIPMA ORYLA DANRE LEPOC consensus

1 MENTQNKVQRKPVRLIAAACNDMGIGKEGGMPWSLPSEFQWFLNKVTTVSRPGKFNMMVW 1 MENTQNKVQRKPVRIIAAACNDMGIGKEGGMPWSLPSEFQWFLNKVTTVSRPGKFNMMVW

1 ------------------------------------SEFQSFLNRVTTVSRPGKFNMMVW

1 --MSGDNVQKKPVRLIAAACSGGGIGKDGQMPWDLPSEFRYFRNHVQGVSRPGKMNMMVW 1 --MEAAKGNKKPVRVIAAVCNNRGIGKDNQMPWSIPAEFQYFLNTVTRVSRPGNMNMMVW 1 --MEPAKVIKKPVRLIAAKCNGGGIGKDNRMPWSLPSEFRFFLSTITRVSRPGKMNMIIW 1 MEKDREKVQKKPVRLIAATCNNMGMGKDGTLPWSLPSEFQYFLNTITRVSRPGNMNLLIW 1 MEQDRETVRKKPVQLIAAICNNMGMGKDGTLPWSLPSEFQYFLNTITRVSRPGNMNLLIW 1 MEN-RLEERRKPVRLIAAVCRNNGIGKDGTLPWSLPSEFQYFLNTITRVSRPGNVNLMIW 1 MMNIEGEEMRKPIRLIAAACRDMGIGKDGQIPWCLPKEFQFLLDTITAVSAPGKKNLIVW 1 MSDCEGKVHRKSIRLIAAACNGMGIGKDGILPWSLPKEFKFFLDTITSVSSPDKKNLLIW

  • 1 .
  • .. ......... .. ....... .......**........ **.*...*...*

HARAN PARCH NEOIO

GASAC TAKRU TETNG POEFO XIPMA ORYLA DANRE LEPOC

61 GKKSWFNHPESTFPLPNTLHAVLSLTLDSPPDHAHFVGSDLEAAVRLAGSPPLADLIETI 61 GKKCWFSHPESTFPLPNTLHAVLSLTLDSPPDHAHFVCSDLEAAVRLAGSPPLADLIETI 25 GKTCWFSHPESTFPLPNTFHAVLSLTLDSPPDHAHFVCSDLEAAVCLAGSPPLADLIETI 59 GKLCWYSVPKPDFPLPNVLHVVLSKTLESVPDHAHFLCEDLDAAARLAVQPPLADLIETI 59 GKQCWVSHPDSTFPLPNILHAVLSKTLFTVPDHAHFLCESLDAAVRLASEPPLADLIEII 59 GKQCWISHPESTFPLPNVLHTVLSTTLFAVPDHAHFVCETLDAAVRLASEPPLADLIEIV 61 GRLCWNSHPENIFPLANSLHVVLSKTLSSVPDHAHFLCQDFESAVRLAAQPPLSGIIETV 61 GRLCWNSHSENMFPLANSLHVVLSKTLSSAPDHAHFLCQDFESAVRLAAQPPLSDIIETV 60 GRLCWFSHPDDLFPLPNSLHVVLSKTLTSVPNHAQFLCGDFESAVRLAALPPLADIIETI 61 GRICWFSCPETVFPLANCINLVLSRKMISVPPHAHYLCKDFDSIIRLVSEPPLCHTVEVI 61 GKRCWISFPESLHPLANCIHVVLSRTMGCVPDHAHYLCHDLPSVIQLGSTHPLSDKIETI

consensus 61 *. .*...... .**.*....***... ..*.**.... .......*.. .**....*..

HARAN PARCH NEOIO

GASAC TAKRU TETNG POEFO XIPMA ORYLA DANRE LEPOC

121 WIVGGMQVYKEALLHQWCDLVYLTKVMADFDCDVFFPEFDRELFKLQEGFPGVPSEIQEE 121 WIVGGVQVYKEALLHRWCDLVYLTKVMADFDCDVFFPKFDRELFKLQEG-----------
85 WIVGGVQVYKEALLHRWCDLVYLTKVMADFDCDVFFPEFDRELFKLQEGFPGVPTEIQEE
119 WVVGGTQVYEVALKHPWCDLVYLTDVMADFDCNVFFPEFDRGLFKVQEGFPGVPSEIQEE 119 WIVGGVQVYKEAMEHPRCDLIYLTDIMAEFECDVFFPEFDKKLFEVQDSFPDVPNGIQED 119 WIVGGVQVYKEAMEHPWCDLIYLTDIMAEFECDVFFPEFDRQLFQVQDGFPDVPDGIQEE 121 WILGGTQVYEDALKHPWCDLLYLTDVMADFDCDVFFPDFDRELFKLQETFPDVPIEIQEE 121 WILGGTQVYEDALKHPWCDLLYLTDVMADFDCDVFFPEFDRELFKLQEKFPDVPSEIQEE 120 WVLGGTKVYEEALKHPCCDLLYFTDVMADFDCDVFFPDFDRELFRVQEEFPDVPSEVQEE 121 WILGGTEVYKESLEHPWCDLIYLTNIMANFECDVFFPEFDPNIFRKQKSFPGVPDEIIEE 121 WILGGAELYKESLKHPWCDYIYLTQVMADFDCDTFFPEFDREIYKLQDEFPGVPSEIQED

consensus 121 *..** ..*.... *..**..*.*..**.*.*..***.**......*. .. ........

HARAN PARCH NEOIO

GASAC TAKRU TETNG POEFO XIPMA ORYLA DANRE LEPOC
181 KGVKYQFQVFKRETGDNV-
-------------------
145 KGVKYQFQVFKRETGNKV- 179 NGIKYKFQVFKKETGDAV- 179 NGIKYKCQVYKRKTAVCLL 179 NGIKYKCQVYKRETAAEDQ 181 NGIRFKCQVFKKKTDDTM- 181 NGIRFKCQVFKKKTDDAF- 180 NGIKFKCQVFKRVTMEDHL 181 NGIKFQFQVFKKIKN---- 181 QGIKLKFQVFKKDMH---- consensus 181 ...... ..... .

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