Examples of Amino Acid Changes in Notothenioids Methods To

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 <ul style="display: flex;"><li style="flex:1">Substitutions in other fish </li><li style="flex:1">Notes </li></ul>Calmodulin No Notothenioid‐specific substitutions. 4 Notothenioid species‐specific substitutions Superoxide dismutase 6 Notothenioid species‐specific substitutions <ul style="display: flex;"><li style="flex:1">198 </li><li style="flex:1">Leu </li><li style="flex:1">Ile </li><li style="flex:1">No significant change </li></ul>Neuroglobin 3 Notothenioid species‐specific substitutions. Last 13 aa at 3’ end very different. 8 forms across 18 species. <ul style="display: flex;"><li style="flex:1">85 </li><li style="flex:1">Pro </li><li style="flex:1">Thr </li><li style="flex:1">No significant change </li></ul>Dihydrofolate reductase 4 Notothenioid species‐specific substitutions. Notothenioid 3’ end slightly longer by 2‐6 aa <ul style="display: flex;"><li style="flex:1">4</li><li style="flex:1">Met </li></ul>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 <ul style="display: flex;"><li style="flex:1">Ile </li><li style="flex:1">Leu Lys </li></ul><ul style="display: flex;"><li style="flex:1">Thr </li><li style="flex:1">Ala Val </li></ul>128 129 141 169 Gln Phe Met His Glu Gly Ser Arg Ala Gly Leu Variable Variable No significant change <ul style="display: flex;"><li style="flex:1">Variable </li><li style="flex:1">Gln Glu Leu Val </li></ul>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 <ul style="display: flex;"><li style="flex:1">Asp </li><li style="flex:1">No significant change </li></ul>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 <ul style="display: flex;"><li style="flex:1">Val Ala </li><li style="flex:1">No significant change </li></ul>Variable Variable Variable Non‐polar aliphatic to polar Positive to negative/neutral Positive to neutral No significant change <ul style="display: flex;"><li style="flex:1">99 </li><li style="flex:1">Glu Gln Gly Lys His </li></ul>Ala Ser Val Gln Leu Glu Thr Lys Glu Asp Asn Gln Asn Gln 109 110 134 145 181 <ul style="display: flex;"><li style="flex:1">183 </li><li style="flex:1">Ile </li></ul>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 <ul style="display: flex;"><li style="flex:1">Variable </li><li style="flex:1">Lys Ile </li></ul>Lys Ile Pro Ser <ul style="display: flex;"><li style="flex:1">Ser </li><li style="flex:1">Variable </li></ul>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 <ul style="display: flex;"><li style="flex:1">Ile </li><li style="flex:1">Met Val </li></ul>Ala Leu Ile Ala Leu Leu Ile Ser Thr Leu Pro Arg Met Phe Gln Pro Thr Val Glu Phe Val <ul style="display: flex;"><li style="flex:1">Ala Val </li><li style="flex:1">Thr </li></ul>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 <ul style="display: flex;"><li style="flex:1">1 . </li><li style="flex:1">.. ......... .. ....... .......**........ **.*...*...* </li></ul>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 ...... ..... .

Examples of Amino Acid Changes in Notothenioids Methods To

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