Poster Pubblicazione

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Conclusions 1. As in other teleost species, IgT isotype is present in both Antarctic and non-Antarctic species, T. bernacchii and B. diacanthus, with the complete or partial loss of the second constant domain in the Antarctic species. 2. Analysis on T. bernacchii cDNA showed the presence of three IgT variants, differing in the length of the region at the boundary between the first and second constant domain. 3. Genomic analysis revealed that within the intron between the first and the second costant exons a reminiscence of the ancestral second exon was present. This remnant falls within a duplicated region and shows different sizes. 4. The qPCR-based copy number analysis puzzled out the number of loci in T. bernacchii genome. These findings can be interpreted as another distinctive feature of the Antarctic fish genome evolution, providing also new insights into the structural variation of teleost immunoglobulin genes. Strange case of IgT from Antac1c fish Giacomelli S 1 , Albanese F 1 , Esposito De Lucia R 1 , Buonocore F 2 , Scapigliati G 2 , Oreste U 1 , Coscia MR 1 1 Institute of Protein Biochemistry, CNR, Napoli, Italy 2 Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia, Largo dell’Università snc, Viterbo, Italy Introduction Immunoglobulins (Ig) appeared when jawed vertebrates emerged about 450 millions years ago and represent one of the most studied molecules having a key role in the adaptive immune response. Different isotypes of Igs are known in teleosts: IgM and IgD, which are also present in other species, and the lastly discovered IgT/Z, which is a teleost specific isotype considered to be critical for mucosal immunity. IgT/Z heavy chain consists of a variable number of Constant (CH) domains according to the species: most species have four CH domains whereas G. aculeatus and T. rubripes have three and two CH domains, respectively. IgTs are expressed as secreted form, multimeric in mucus and monomeric in serum. Non-Antarctic Notothenioids Antarctic Notothenioids GGA AAA GGA ACT ACA GTC ACA GTA ACT ACAdAAC ACA GTT GCA TCA CCG ACT dG K G T T V T V T TddsN T V A S P Ts CTG TTC CCT TTG CTT CAG TGC AAT CCT GTG TCT GCA AGT ACA GTC ACG GTT L F P L L Q C N P V S A S T V T Vs GGT TGT CTT GCA AAC AAC TTC TAC CCA AAG AGT CTT ACT TTC CAG TGG ACC G C L A N N F Y P K S L T F Q W Ts GAT ACC AGT GGG ACC ACC CTG TCT TCT GTA CAA TAT CCT ACG GTT GAG AAA D T S G T T L S S V Q Y P T V E Ks AAC AAC AAT TAT ACG GGA GTC AGT TTG CTC CAA GTA TCA AAA TCT GAC TGG N N N Y T G V S L L Q V S K S D Ws GAT TCA AAG AAG TCT TTT ACT TGT TCT GTG ACT CAC GAT GGA AGC CAA AGA D S K K S F T C S V T H D G S Q Rs AGC GTG CAA TTG AGC AAAsGAT GTC TCA TTT CCT CCA AAG CTA ACC TTG GTA sS V Q L S KsssD V S F P P K L T L Vs GCA GTG CCA GGA GAA GAC GCT CAG GCC CTG GTG TGC ACC ATT GAG GAT TTT A V P G E D A Q A L V C T I E D Fs CTT CCA AAA AAT GTG TTG GTC AAA TGG AAA AAG AAT GGA AAC TCT GTG ACT L P K N V L V K W K K N G N S V Ts GGC TGC ATT GAT TGG GCC CCC AAA ATA ATT GGA AAC GTC TAT TCA GCT GTC G C I D W A P K I I G N V Y S A Vs AGT GTC CTG AAG GTT AAG AAC AAC GAC TGG GAC AGT AAT GCT GTT TAC ACA S V L K V K N N D W D S N A V Y Ts TGT GAG GTG GTG CAC CAA GGA ACA TCA TAT ACA ATG AGG GCC TCT AAAsGCT sC E V V H Q G T S Y T M R A S KsssAs CCT ATC ACA ATG ATA CTG AAC CAA CCA AGT CCC AAA GAA ATG TTC AAC AAC P I T M I L N Q P S P K E M F N Ns CAC CAG GCA AAG TTG GAG TGC ATC ATT AGT GGA CAG GAC CAG GCC ACT ATG H Q A K L E C I I S G Q D Q A T Ms AAT GAC ATT CAA ATC ACT TGG CAA ATG TAT GGA GAA AAT GTG ACC CAC AAC N D I Q I T W Q M Y G E N V T H Ns AAT ATT GAA ACA ACT TGG TCC GGA GGC GAC AAA GTC AGT GTG ATA ACA CTC N I E T T W S G G D K V S V I T Ls AGT GTC ACT GAG TGG CAG AAA GTC AAC AAA GTG CGC TGC TCT GCC ATG AGA S V T E W Q K V N K V R C S A M Rs GAC AAT ATG ACA CCA GTT ATT CAA GAT CTG ATT GTC CAT AAA GGAsGAT CAG sD N M T P V I Q D L I V H K GsssD Qs AGC AAG AGC AAG CCA AAA GCA ACA GTC CAC ATC CTC CCA GAG GAG GAC ATC S K S K P K A T V H I L P E E D Is GGC AAA GAT GCC TCA GCT GAT GTC ACT CTG GTG TGT CTG GTC TCC AGT GAT G K D A S A D V T L V C L V S S Ds GTG CAG CAG GAT TTC TAC ATC GCC TGG ACA GAA CAA ATC AGA GAA CTC GTC V Q Q D F Y I A W T E Q I R E L Vs AGC AAC TAC AGT GAT GGC ATT AAC TTC CCG TCT CAG AAG ACC CAA CAT GGC S N Y S D G I N F P S Q K T Q H Gs TAC TCA ATT ACA AGT CTT TAC ACC ACC ACC AAG GAA AAG TGG AAC AAG CTG Y S I T S L Y T T T K E K W N K Ls AAA ATG TTC TCC TGC CAC GCC TGG CCT GCT GGT AAC AAA GAG GCT ATG GTA K M F S C H A W P A G N K E A M Vs CAA AGA CAA GTG TCT AAG GCC CAT AGT AACsTCG ACT GAA TGT GAG CAT GTG sQ R Q V S K A H S NsssS T E C E H Vs TAGattcaactttaaatgtggcattttgtgctaatgtgttgttctgtctctgtctctgtgtactctctg TER ccatgagaacctgtatttgtgattgtaacaataacttttgtttgtcatcatattttgtttcaatctgtc tttgtcacattgtccaactgcttgtgaagtgtgctaactgtttcaaataaatgttgcattgagagacaa aaaaaaaaaaaaa ATG TTC TCT GTA GCT CTG CTG CTG CTG CTG GCT GCT GGA TCC TGT GTG AAG sM F S V A L L L L L A A G S C V Ks TGTdGAG CAG TTG ATA CAG CCA GCC TCT GTG ACT GTG CAG CCA GGT CAA CGT sCdddE Q L I Q P A S V T V Q P G Q Rs CTG ACC ATC ACC TGT CAG GTC TCT TAT TCT GTT AGC AGC TAC TTC ACA GCC L T I T C Q V S Y S V S S Y F T As TGG ATC AGA CAG CCT GCA GGG AAA GGA CTG GAG TGG ATA GGA ATG GCA GCT W I R Q P A G K G L E W I G M A As ACT GGA AAC CCC ACA CAC TTT AAA GAT TCA CTG AAG AGC AAG TTT AGT ATC T G N P T H F K D S L K S K F S Is GAC TTA GAC TCT TCC AGC AAC TCA GTG ACT CTG AAC GGA CAG AGC GTG CAG D L D S S S N S V T L N G Q S V Qs CCT GGA GAC TCT GCT GTG TAT TAC TGT GCC AGA GTC CAT GGA CGG GTG GGG P G D S A V Y Y C A R V H G R V Gs GGC CAT GAC TAC TGG GGA CAA GGA GCT AGA GTC ACA GTTsTCT AAT GAA ATC sG H D Y W G Q G A R V T VsssS N E Is GAT GCA TCT CCG ACT CTG TTC CCT CTG GTC CAG TGC AAC CCT GGC TCA GCA D A S P T L F P L V Q C N P G S As GAG ACA ATC ACG GTT GGT TGT CTT GCA AAC GAC TTC TCC CCA AAT AGT GTG E T I T V G C L A N D F S P N S Vs ACT TTT CAG TGG ACT GAC GCC AGT GGG ACA AAC CTG CCC GCT GTA CAG TAT T F Q W T D A S G T N L P A V Q Ys CCT CCA ACA GTG AAA AAC AAC AAG TAT ACA GGA ATC AGT GTT GTC AAC GTA P P T V K N N K Y T G I S V V N Vs CCA AAA TCT GAC TGG GAT TTA AGA AAG TCT TAC AAG TGT GCT GTG ACT CAC P K S D W D L R K S Y K C A V T Hs CCT GGA GGA CAA AAA AGT TTG AGC ATT AAA AAGsGTT AAA GCA CCT TCA TGT sP G G Q K S L S I K Kss V K A P S Cs TCA GAG ACT AAC GCT ACC ATC ACG ATG AAA CTG AAC AAA TCA AGT CCC AAA S E T N A T I T M K L N K S S P Ks GAA ATG TTC AAC AAC AAC CAG GCA AGG TTC GAT TGT GTC ATT AGT GGA GAT E M F N N N Q A R F D C V I S G Ds GAG GAG ACC ACT GTC GAT CAA TTT CAA ATC ACT TGG CAA GTC AAT GGA GCA E E T T V D Q F Q I T W Q V N G As ATT CGG ACC ACC AGC GAG CAA GGA AGA TCG GGA GGA ATA AAA ACC AGC ACA I R T T S E Q G R S G G I K T S Ts ATG AGG CTC AGT CTC CAT GAG AGC CTC AGT AAT GTG CGC TGT TCT GCC TCT M R L S L H E S L S N V R C S A Ss AAA GAT GAT GTG ACG GTT TTT CAA GAT CTG CCT CCC CCC AAA ATAsGAT GGA sK D D V T V F Q D L P P P K IsssD Gs AAG GAG CCA AAA GTA ACA GTC CAC ATC GTC CCA GAG GAG GAC ATC GAT GCA K E P K V T V H I V P E E D I D As GAC CCA TCC GAC GAG GTC ACT TTG GTG TGT CTG GTC TTC AGT AGT GAG AAG D P S D E V T L V C L V F S S E Ks GAT TAC TAC ATT GCA TGG GCT GAA CAT ATT GGA CAA AAC ACC CCA ATC TAC D Y Y I A W A E H I G Q N T P I Ys CAA GAT GGC ATC GAC TTA CCC CCT CAG AAG GCT AAC AAT GGC TAC TTT GTT Q D G I D L P P Q K A N N G Y F Vs ACA AGT ATT TAC ACC ACC AAC AAG ACA AAG TGG CAA AAT CAC ATG TTC TCC T S I Y T T N K T K W Q N H M F Ss TGC AAC GTC TGG CCT GCT GGC GCC AAA AAG GCA ATG GCA CCA AGA ACC GTG C N V W P A G A K K A M A P R T Vs TCT AAC TCA ATG AAT AACsTCC ATT GAA TGT AAG AAA TAG tagattcaacttttag sS N S M N NsssS I E C K Ks TER tgtgctattttgtgctattgtgttgttctgtctctatgtactctccgtcacgacaagctgtctttgtga ttgtaacaataacagttaacatgtcgattcacttgtgtttgtcataatactttgttaaatgttgtctct gtcaaattgtccgactgctttaaaagtgtgctgactgtttcaaataaatgttgcatggagaaaaaaaaaa aaaaaaa Results In order to investigate the mucosal immunity in Antarctic teleosts, IgT heavy chain gene of the Antarctic Notothenioid Trematomus bernacchii, was sequenced and characterized. cDNA analysis unraveled the presence of three IgT transcript variants, named L, S and Sts, differing in length of a remnant CH2 exon. According to the genomic sequences investigated, these differently sized L and S variants were found to be encoded by indel alleles. On the other hand Sts variant originated by the alternative splicing that led to the remnant CH2 exon skipping. Moreover, through the comparison between genomic and cDNA sequences we could hypothesize the presence of three different copies of the IgT heavy chain gene, one of which being considered the functional gene since the corresponding transcripts were identified. IgT heavy chain gene was also isolated in the non- Antarctic Notothenioid species, Bovichtus diacanthus, chosen for comparison since it represents the phyletically basal lineage of Notothenioid species. Bovichtus diacanthus vs Trematomus bernacchii Genomic sequence analysis g30.8 TCACGGTTGGTTGTCTTGCAAACGACTTCTACCCAAATAATGTGACTTTTCAGTGGACTG 60 g30.6 TCACGGTTGGTTGTCTTGCAAACGACTTCTCCCCAAATAGTGTGACTTTTCAGTGGACTG gBd TCACGGTTGGTTGTCTTGCAAACAACTTCTACCCAAAGAGTCTTACTTTCCAGTGGACCG *********************** ****** ****** * * * ***** ******** * g30.8 ACGCCAGTGGGACAAACCTGCCCGCTGTACAGTATCCTCCAACAGTGAAAAACAACAAGT 120 g30.6 ACGCCAGTGGGACAAACCTGCCCGCTGTACAGTATCCTCCAACAGTGAAAAACAACAAGT gBd ATACCAGTGGGACCACCCTGTCTTCTGTACAATATCCTACAGTTGAGAAAAACAACAATT * ********** * **** * ******* ****** ** * ************ * g30.8 ATACAGGAGTCAGTGTTGTCAACGTACCAAAATCTGACTGGGATTTTAAAAAGTCTTACA 180 g30.6 ATACAGGAGTCAGTGTTGTCAACGTACCAAAATCTGAGTGGGATTTAAGAAAGTCTTACA gBd ATACAGGAGTCAGTTTGCTCCAAGTATCAAAATCTGACTGGGATTCAAAGAAGTCTTTTA ************** * ** * *** ********** ******* * ******* * g30.8 ACTGTGCTGTGACTCACCCTGGAGGACAAAAAAGT------TTGAGCATTAAAAGTACGT 240 g30.6 AGTGTGCTGTGACTCACCCTGGAGGACAAAAAAGT------TTGAGCATTAAAAGTACGT gBd CTTGTTCTGTGACTCACGATGGAAGCCAAAGAAGCGTGCAATTGAGC---AAAGGTACGT *** *********** **** * **** *** ****** *** ****** g30.8 GATTTTATTTACTTGCTATCATGATCTTCTTTACTCTTTGAGTCCAAATGAAGCACATTC 300 g30.6 GATTTTATTTACTTGCTATCATGATCTTCTTTACTCTTTGAGTCCAAATGAAGCACATTC gBd GATTTT--TAACTTACTATCATGGCTCTCTTATGTCTATGAGTCCACATGCATCACCTTG ****** * **** ******** **** *** ******** *** * *** ** g30.8 TCAGTTTTATCCTGAA-----AATGTTCATTAACACTCTCACTTTCTCATGTTTATCTTC 360 g30.6 TCAGTTTTATCCTGAA-----AATGTTCATTAACACTCTCACTTTCTCATGTTTATCTTC gBd TTAATCTCGTTTGGAATGTTAAATGTTCAGTACTATTTTCTGTTTCTCATGTTTATCGTC * * * * * *** ******** ** * * ** *************** ** g30.8 ATCCATCATGTGTCTTAC---------TTAAACAAATCTAAAATGATGCAT---TTCAGA 420 g30.6 ATCCATCATGTGTCTTAC---------TTAAACAAATCTAAAATGATGCAT---TTCAGA gBd ATCTCTCTGGTATTTTACAGTCAAAACTTTAACATTTTTAAAATAATGAATAACTACAGA *** ** ** * **** ** **** * ****** *** ** * **** g30.8 CTGATATCAATAAAAGATATCTATTTGTACTCTTTCTTCCC-CATGCTTAGAGGCTAAAG 480 g30.6 CTGATATCAATAAAAGATATCTATTTGTACTCTTTCTTCCC-CATGCTTAGAGGTTAAAG gBd CTGATGTCAAT--------------------------------------------TAAA- ***** ***** **** g30.8 CACCTTTCTCATGTTTATCTTCATCCATCATGTGTCTTACTTAAACAAATCTAAAAAATA 540 g30.6 CACCTTTCTCATGTTTATCTTCATCCATCATGTGTCTTACTTAAACAAATCTAAAAAATA gBd ------------------------------------------AAACGGGTATCT------ **** * * g30.8 TCTATTTGTACTCTTTCTTCCCCATGCTTAGAGGTTAA---------------------- 600 g30.6 TCTATTTGTACTCTTTCTTCCCCATGCTTAGAGGTTATTCAACTTCTA------------ gBd CTTTTTCTGCTCTTCTTCTCCACATGCTTAGATGTCTCATTTCCTCCAAAGCTAACTTTG ** * * *** ********** ** g30.8 ------------------------------------------------------------ 660 g30.6 ------------------------------------------------------------ gBd GTAGCAGTGCCAGGAGAAGACGCTCAGGCCCTGTGTGCACCATTGAGGATTTTCTTCCAA g30.8 ------------------------------------------------------------ 720 g30.6 ------------------------------------------------------------ gBd AAAATGTGTTGGTCAAATGGAAAAAGAATGGAAACTCTGTGACTGGCTGCATTGATTGGG g30.8 ------------------------------------------------------------ 780 g30.6 ------------------------------------------------------------ gBd CCCCCAAAATAATTGGAAACGTCTATTCAGCTGTCAGTGTCCTGAAGGTTAAGAACAACG g30.8 -------------------------------------------------AGCACCTTCAT 840 g30.6 -----------------------------------------GGTGCTTTAGCACCTTCAT gBd ACTGGGACAGTAATGCTGTTTACACATGTGAGGTGGTGCACCAAGGAACATCATATACAA * ** * ** g30.8 GTCCCGAGACTAACGGTAAAGAGCTACTACTGTCATTACATAATACTGTTTTACTGTAAC 900 g30.6 GTCCAGAGACTAAAGGTAAAGAGCTACTACTGTCATTACATAATACTGTTTTACTGTAAC gBd TGAGGGCCTCTAAAGGTAAAGGGCTACTACAGTCATGACATAATACTGTTGTTCTGTAAC * **** **************** ***** ************* * ******* g30.8 T----------------------AATAGTATACATTATACTCAAATATAAG-AGATCTAC 960 g30.6 T----------------------AATAGTATACATTATACTCAAATATAAG-AGATCTAC gBd TCAGAGTAAGCTGTTACTGACATAATAGTATACAGTATGCTCAAATACAAGCATTTTTAC * ************************ *** * * *** g30.8 TGTTTGTATTTATTGATTTATTTCTGTTGATCTTGTTTCTGTAGCTACCATCACGATGAA 1020 g30.6 TGTTTGTATTTATTGATTTATTTCTGTTGATCTTGTTTCTGTAGCTACCATCACGATGAA gBd TGTT-GAACTTATTGGTTT-TCTCTGTTGCCCTCGTTTATGCAGCTCCTATCACAATGAT **** * * ****** ** ******** *** * *** ** **** * ***** **** g30.8 ACTGAACAAATCAAGTCCCAAAGAAATGTTCAACAACAACCAGGCAAGG 1069 g30.6 ACTGAACAAATCAAGTCCCAAAGAAATGTTCAACAACAACCAGGCAAGG gBd ACTGAACCAACCAAGTCCCAAAGAAATGTTCAACAACCACCAGGCAAAG ******* ** ************************** *********** g30.3 g30.1 g30.5 g30.7 Tb30.3 g30.8 Tb30.8 Tb30.7 Tb30.9 g30.6 0.002 qPCR-based Copy Number Analysis Copy number analysis was performed on genomic DNA from Tb30 individual using the qPCR method. IgT gene was normalized by the single-copy gene Sreb2. The results suggested the presence of three IgT heavy chain gene copies for haploid genetic makeup, in line with the phylogenetic analysis shown on the left. 0 0,5 1 1,5 2 2,5 3 3,5 IgT Sreb2 Deduced amino acid sequence alignment The deduced amino acid sequences of T. bernacchii and of another Antarctic Notothenioid species, Notothenia coriiceps, identified by screening its transcriptome, were aligned with B. diacanthus and other temperate teleost species IgT heavy chains. The results obtained strongly suggest that the loss in the Antarctic fish of almost the entire CH2 domain is balanced by the conservation of some amino acids typical of hinge regions, such as proline, glycine and cysteine, in the remaining domain. E. coioides (GU182366), O. mykiss (AY870263), S. salar (GQ907003), G. aculeatus, S. caurinus (GE798008), S. chuatsi (DQ016660), T. orientalis (KF713336), C. idella (DQ478943), D. rerio (AY643750), M. amblycephala (KC894946.1), N. coriiceps (KP876589), B. diacanthus (KP876590) and the three variants of T. bernacchii (S, KP876587; L, KP876588; Sts, KP876615). A: Phylogenetic tree built considering the Notothenioid species together with ten other species included in the alignment shown above, and excluding the CH2 domain. For T. bernacchii only one sequence was considered (S variant). The species are distributed in three main clades, depending on the order they belong to. B: Phylogenetic tree of the single IgT heavy chain CH domains from 13 teleost species. All the CH1 domains are grouped in the same clade; all the CH2 domains are gathered together in the same clade. T. bernacchii, N. coriiceps, and G. aculeatus, represent the exception since their second constant domain has been found to be evolutionarily related to the CH3 domain of the teleost species possessing four domains. Likewise, their third constant domain grouped with the CH4 domain of the other fishes. B A

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Conclusions

1. As in other teleost species, IgT isotype is present in both Antarctic and non-Antarctic species, T. bernacchii and B. diacanthus, with the complete or partial loss of the second constant domain in the Antarctic species.

2. Analysis on T. bernacchii cDNA showed the presence of three IgT variants, differing in the length of the region at the boundary between the first and second constant domain. 3. Genomic analysis revealed that within the intron between the first and the second costant exons a reminiscence of the ancestral second exon was present. This remnant falls within a duplicated region

and shows different sizes. 4. The qPCR-based copy number analysis puzzled out the number of loci in T. bernacchii genome. These findings can be interpreted as another distinctive feature of the Antarctic fish genome evolution, providing also new insights into the structural variation of teleost immunoglobulin genes.

Strange  case  of  IgT  from  Antac1c  fish  

Giacomelli S1, Albanese F1, Esposito De Lucia R1, Buonocore F2, Scapigliati G2, Oreste U1, Coscia MR1 1Institute of Protein Biochemistry, CNR, Napoli, Italy

2Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia, Largo dell’Università snc, Viterbo, Italy  

Introduction

Immunoglobulins (Ig) appeared when jawed vertebrates emerged about 450 millions years ago and represent one of the most studied molecules having a key role in the adaptive immune response. Different isotypes of Igs are known in teleosts: IgM and IgD, which are also present in other species, and the lastly discovered IgT/Z, which is a teleost specific isotype considered to be critical for mucosal immunity. IgT/Z heavy chain consists of a variable number of Constant (CH) domains according to the species: most species have four CH domains whereas G. aculeatus and T. rubripes have three and two CH domains, respectively. IgTs are expressed as secreted form, multimeric in mucus and monomeric in serum.  

Non-Antarctic Notothenioids

Antarctic Notothenioids

 

GGA AAA GGA ACT ACA GTC ACA GTA ACT ACAdAAC ACA GTT GCA TCA CCG ACT dG K G T T V T V T TddsN T V A S P Ts CTG TTC CCT TTG CTT CAG TGC AAT CCT GTG TCT GCA AGT ACA GTC ACG GTT L F P L L Q C N P V S A S T V T Vs GGT TGT CTT GCA AAC AAC TTC TAC CCA AAG AGT CTT ACT TTC CAG TGG ACC G C L A N N F Y P K S L T F Q W Ts GAT ACC AGT GGG ACC ACC CTG TCT TCT GTA CAA TAT CCT ACG GTT GAG AAA D T S G T T L S S V Q Y P T V E Ks AAC AAC AAT TAT ACG GGA GTC AGT TTG CTC CAA GTA TCA AAA TCT GAC TGG N N N Y T G V S L L Q V S K S D Ws GAT TCA AAG AAG TCT TTT ACT TGT TCT GTG ACT CAC GAT GGA AGC CAA AGA D S K K S F T C S V T H D G S Q Rs AGC GTG CAA TTG AGC AAAsGAT GTC TCA TTT CCT CCA AAG CTA ACC TTG GTA sS V Q L S KsssD V S F P P K L T L Vs GCA GTG CCA GGA GAA GAC GCT CAG GCC CTG GTG TGC ACC ATT GAG GAT TTT A V P G E D A Q A L V C T I E D Fs CTT CCA AAA AAT GTG TTG GTC AAA TGG AAA AAG AAT GGA AAC TCT GTG ACT L P K N V L V K W K K N G N S V Ts GGC TGC ATT GAT TGG GCC CCC AAA ATA ATT GGA AAC GTC TAT TCA GCT GTC G C I D W A P K I I G N V Y S A Vs AGT GTC CTG AAG GTT AAG AAC AAC GAC TGG GAC AGT AAT GCT GTT TAC ACA S V L K V K N N D W D S N A V Y Ts TGT GAG GTG GTG CAC CAA GGA ACA TCA TAT ACA ATG AGG GCC TCT AAAsGCT sC E V V H Q G T S Y T M R A S KsssAs CCT ATC ACA ATG ATA CTG AAC CAA CCA AGT CCC AAA GAA ATG TTC AAC AAC P I T M I L N Q P S P K E M F N Ns CAC CAG GCA AAG TTG GAG TGC ATC ATT AGT GGA CAG GAC CAG GCC ACT ATG H Q A K L E C I I S G Q D Q A T Ms AAT GAC ATT CAA ATC ACT TGG CAA ATG TAT GGA GAA AAT GTG ACC CAC AAC N D I Q I T W Q M Y G E N V T H Ns AAT ATT GAA ACA ACT TGG TCC GGA GGC GAC AAA GTC AGT GTG ATA ACA CTC N I E T T W S G G D K V S V I T Ls AGT GTC ACT GAG TGG CAG AAA GTC AAC AAA GTG CGC TGC TCT GCC ATG AGA S V T E W Q K V N K V R C S A M Rs GAC AAT ATG ACA CCA GTT ATT CAA GAT CTG ATT GTC CAT AAA GGAsGAT CAG sD N M T P V I Q D L I V H K GsssD Qs AGC AAG AGC AAG CCA AAA GCA ACA GTC CAC ATC CTC CCA GAG GAG GAC ATC S K S K P K A T V H I L P E E D Is GGC AAA GAT GCC TCA GCT GAT GTC ACT CTG GTG TGT CTG GTC TCC AGT GAT G K D A S A D V T L V C L V S S Ds GTG CAG CAG GAT TTC TAC ATC GCC TGG ACA GAA CAA ATC AGA GAA CTC GTC V Q Q D F Y I A W T E Q I R E L Vs AGC AAC TAC AGT GAT GGC ATT AAC TTC CCG TCT CAG AAG ACC CAA CAT GGC S N Y S D G I N F P S Q K T Q H Gs TAC TCA ATT ACA AGT CTT TAC ACC ACC ACC AAG GAA AAG TGG AAC AAG CTG Y S I T S L Y T T T K E K W N K Ls AAA ATG TTC TCC TGC CAC GCC TGG CCT GCT GGT AAC AAA GAG GCT ATG GTA K M F S C H A W P A G N K E A M Vs CAA AGA CAA GTG TCT AAG GCC CAT AGT AACsTCG ACT GAA TGT GAG CAT GTG sQ R Q V S K A H S NsssS T E C E H Vs TAGattcaactttaaatgtggcattttgtgctaatgtgttgttctgtctctgtctctgtgtactctctg TER ccatgagaacctgtatttgtgattgtaacaataacttttgtttgtcatcatattttgtttcaatctgtctttgtcacattgtccaactgcttgtgaagtgtgctaactgtttcaaataaatgttgcattgagagacaaaaaaaaaaaaaaa

!

ATG TTC TCT GTA GCT CTG CTG CTG CTG CTG GCT GCT GGA TCC TGT GTG AAG sM F S V A L L L L L A A G S C V Ks TGTdGAG CAG TTG ATA CAG CCA GCC TCT GTG ACT GTG CAG CCA GGT CAA CGT sCdddE Q L I Q P A S V T V Q P G Q Rs CTG ACC ATC ACC TGT CAG GTC TCT TAT TCT GTT AGC AGC TAC TTC ACA GCC L T I T C Q V S Y S V S S Y F T As TGG ATC AGA CAG CCT GCA GGG AAA GGA CTG GAG TGG ATA GGA ATG GCA GCT W I R Q P A G K G L E W I G M A As ACT GGA AAC CCC ACA CAC TTT AAA GAT TCA CTG AAG AGC AAG TTT AGT ATC T G N P T H F K D S L K S K F S Is GAC TTA GAC TCT TCC AGC AAC TCA GTG ACT CTG AAC GGA CAG AGC GTG CAG D L D S S S N S V T L N G Q S V Qs CCT GGA GAC TCT GCT GTG TAT TAC TGT GCC AGA GTC CAT GGA CGG GTG GGG P G D S A V Y Y C A R V H G R V Gs GGC CAT GAC TAC TGG GGA CAA GGA GCT AGA GTC ACA GTTsTCT AAT GAA ATC sG H D Y W G Q G A R V T VsssS N E Is GAT GCA TCT CCG ACT CTG TTC CCT CTG GTC CAG TGC AAC CCT GGC TCA GCA D A S P T L F P L V Q C N P G S As GAG ACA ATC ACG GTT GGT TGT CTT GCA AAC GAC TTC TCC CCA AAT AGT GTG E T I T V G C L A N D F S P N S Vs ACT TTT CAG TGG ACT GAC GCC AGT GGG ACA AAC CTG CCC GCT GTA CAG TAT T F Q W T D A S G T N L P A V Q Ys CCT CCA ACA GTG AAA AAC AAC AAG TAT ACA GGA ATC AGT GTT GTC AAC GTA P P T V K N N K Y T G I S V V N Vs CCA AAA TCT GAC TGG GAT TTA AGA AAG TCT TAC AAG TGT GCT GTG ACT CAC P K S D W D L R K S Y K C A V T Hs CCT GGA GGA CAA AAA AGT TTG AGC ATT AAA AAGsGTT AAA GCA CCT TCA TGT sP G G Q K S L S I K Kss V K A P S Cs TCA GAG ACT AAC GCT ACC ATC ACG ATG AAA CTG AAC AAA TCA AGT CCC AAA S E T N A T I T M K L N K S S P Ks GAA ATG TTC AAC AAC AAC CAG GCA AGG TTC GAT TGT GTC ATT AGT GGA GAT E M F N N N Q A R F D C V I S G Ds GAG GAG ACC ACT GTC GAT CAA TTT CAA ATC ACT TGG CAA GTC AAT GGA GCA E E T T V D Q F Q I T W Q V N G As ATT CGG ACC ACC AGC GAG CAA GGA AGA TCG GGA GGA ATA AAA ACC AGC ACA I R T T S E Q G R S G G I K T S Ts ATG AGG CTC AGT CTC CAT GAG AGC CTC AGT AAT GTG CGC TGT TCT GCC TCT M R L S L H E S L S N V R C S A Ss AAA GAT GAT GTG ACG GTT TTT CAA GAT CTG CCT CCC CCC AAA ATAsGAT GGA sK D D V T V F Q D L P P P K IsssD Gs AAG GAG CCA AAA GTA ACA GTC CAC ATC GTC CCA GAG GAG GAC ATC GAT GCA K E P K V T V H I V P E E D I D As GAC CCA TCC GAC GAG GTC ACT TTG GTG TGT CTG GTC TTC AGT AGT GAG AAG D P S D E V T L V C L V F S S E Ks GAT TAC TAC ATT GCA TGG GCT GAA CAT ATT GGA CAA AAC ACC CCA ATC TAC D Y Y I A W A E H I G Q N T P I Ys CAA GAT GGC ATC GAC TTA CCC CCT CAG AAG GCT AAC AAT GGC TAC TTT GTT Q D G I D L P P Q K A N N G Y F Vs ACA AGT ATT TAC ACC ACC AAC AAG ACA AAG TGG CAA AAT CAC ATG TTC TCC T S I Y T T N K T K W Q N H M F Ss TGC AAC GTC TGG CCT GCT GGC GCC AAA AAG GCA ATG GCA CCA AGA ACC GTG C N V W P A G A K K A M A P R T Vs TCT AAC TCA ATG AAT AACsTCC ATT GAA TGT AAG AAA TAG tagattcaacttttag sS N S M N NsssS I E C K Ks TER tgtgctattttgtgctattgtgttgttctgtctctatgtactctccgtcacgacaagctgtctttgtgattgtaacaataacagttaacatgtcgattcacttgtgtttgtcataatactttgttaaatgttgtctctgtcaaattgtccgactgctttaaaagtgtgctgactgtttcaaataaatgttgcatggagaaaaaaaaaaaaaaaaa !

 

Results

In order to investigate the mucosal immunity in Antarctic teleosts, IgT heavy chain gene of the Antarctic Notothenioid Trematomus bernacchii, was sequenced and characterized. cDNA analysis unraveled the presence of three IgT transcript variants, named L, S and Sts, differing in length of a remnant CH2 exon. According to the genomic sequences investigated, these differently sized L and S variants were found to be encoded by indel alleles. On the other hand Sts variant originated by the alternative splicing that led to the remnant CH2 exon skipping. Moreover, through the comparison between genomic and cDNA sequences we could hypothesize the presence of three different copies of the IgT heavy chain gene, one of which being considered the functional gene since the corresponding transcripts were identified. IgT heavy chain gene was also isolated in the non-Antarctic Notothenioid species, Bovichtus diacanthus, chosen for comparison since it represents the phyletically basal lineage of Notothenioid species.

Bovichtus diacanthus vs Trematomus bernacchii

 

Genomic  sequence  analysis  

g30.8 TCACGGTTGGTTGTCTTGCAAACGACTTCTACCCAAATAATGTGACTTTTCAGTGGACTG 60 g30.6 TCACGGTTGGTTGTCTTGCAAACGACTTCTCCCCAAATAGTGTGACTTTTCAGTGGACTG gBd TCACGGTTGGTTGTCTTGCAAACAACTTCTACCCAAAGAGTCTTACTTTCCAGTGGACCG *********************** ****** ****** * * * ***** ******** * g30.8 ACGCCAGTGGGACAAACCTGCCCGCTGTACAGTATCCTCCAACAGTGAAAAACAACAAGT 120 g30.6 ACGCCAGTGGGACAAACCTGCCCGCTGTACAGTATCCTCCAACAGTGAAAAACAACAAGT gBd ATACCAGTGGGACCACCCTGTCTTCTGTACAATATCCTACAGTTGAGAAAAACAACAATT * ********** * **** * ******* ****** ** * ************ * g30.8 ATACAGGAGTCAGTGTTGTCAACGTACCAAAATCTGACTGGGATTTTAAAAAGTCTTACA 180 g30.6 ATACAGGAGTCAGTGTTGTCAACGTACCAAAATCTGAGTGGGATTTAAGAAAGTCTTACA gBd ATACAGGAGTCAGTTTGCTCCAAGTATCAAAATCTGACTGGGATTCAAAGAAGTCTTTTA ************** * ** * *** ********** ******* * ******* * g30.8 ACTGTGCTGTGACTCACCCTGGAGGACAAAAAAGT------TTGAGCATTAAAAGTACGT 240 g30.6 AGTGTGCTGTGACTCACCCTGGAGGACAAAAAAGT------TTGAGCATTAAAAGTACGT gBd CTTGTTCTGTGACTCACGATGGAAGCCAAAGAAGCGTGCAATTGAGC---AAAGGTACGT *** *********** **** * **** *** ****** *** ****** g30.8 GATTTTATTTACTTGCTATCATGATCTTCTTTACTCTTTGAGTCCAAATGAAGCACATTC 300 g30.6 GATTTTATTTACTTGCTATCATGATCTTCTTTACTCTTTGAGTCCAAATGAAGCACATTC gBd GATTTT--TAACTTACTATCATGGCTCTCTTATGTCTATGAGTCCACATGCATCACCTTG ****** * **** ******** **** *** ******** *** * *** ** g30.8 TCAGTTTTATCCTGAA-----AATGTTCATTAACACTCTCACTTTCTCATGTTTATCTTC 360 g30.6 TCAGTTTTATCCTGAA-----AATGTTCATTAACACTCTCACTTTCTCATGTTTATCTTC gBd TTAATCTCGTTTGGAATGTTAAATGTTCAGTACTATTTTCTGTTTCTCATGTTTATCGTC * * * * * *** ******** ** * * ** *************** ** g30.8 ATCCATCATGTGTCTTAC---------TTAAACAAATCTAAAATGATGCAT---TTCAGA 420 g30.6 ATCCATCATGTGTCTTAC---------TTAAACAAATCTAAAATGATGCAT---TTCAGA gBd ATCTCTCTGGTATTTTACAGTCAAAACTTTAACATTTTTAAAATAATGAATAACTACAGA *** ** ** * **** ** **** * ****** *** ** * **** g30.8 CTGATATCAATAAAAGATATCTATTTGTACTCTTTCTTCCC-CATGCTTAGAGGCTAAAG 480 g30.6 CTGATATCAATAAAAGATATCTATTTGTACTCTTTCTTCCC-CATGCTTAGAGGTTAAAG gBd CTGATGTCAAT--------------------------------------------TAAA- ***** ***** **** g30.8 CACCTTTCTCATGTTTATCTTCATCCATCATGTGTCTTACTTAAACAAATCTAAAAAATA 540 g30.6 CACCTTTCTCATGTTTATCTTCATCCATCATGTGTCTTACTTAAACAAATCTAAAAAATA gBd ------------------------------------------AAACGGGTATCT------ **** * * g30.8 TCTATTTGTACTCTTTCTTCCCCATGCTTAGAGGTTAA---------------------- 600 g30.6 TCTATTTGTACTCTTTCTTCCCCATGCTTAGAGGTTATTCAACTTCTA------------ gBd CTTTTTCTGCTCTTCTTCTCCACATGCTTAGATGTCTCATTTCCTCCAAAGCTAACTTTG ** * * *** ********** ** g30.8 ------------------------------------------------------------ 660 g30.6 ------------------------------------------------------------ gBd GTAGCAGTGCCAGGAGAAGACGCTCAGGCCCTGTGTGCACCATTGAGGATTTTCTTCCAA g30.8 ------------------------------------------------------------ 720 g30.6 ------------------------------------------------------------ gBd AAAATGTGTTGGTCAAATGGAAAAAGAATGGAAACTCTGTGACTGGCTGCATTGATTGGG g30.8 ------------------------------------------------------------ 780 g30.6 ------------------------------------------------------------ gBd CCCCCAAAATAATTGGAAACGTCTATTCAGCTGTCAGTGTCCTGAAGGTTAAGAACAACG

g30.8 -------------------------------------------------AGCACCTTCAT 840 g30.6 -----------------------------------------GGTGCTTTAGCACCTTCAT gBd ACTGGGACAGTAATGCTGTTTACACATGTGAGGTGGTGCACCAAGGAACATCATATACAA * ** * ** g30.8 GTCCCGAGACTAACGGTAAAGAGCTACTACTGTCATTACATAATACTGTTTTACTGTAAC 900 g30.6 GTCCAGAGACTAAAGGTAAAGAGCTACTACTGTCATTACATAATACTGTTTTACTGTAAC gBd TGAGGGCCTCTAAAGGTAAAGGGCTACTACAGTCATGACATAATACTGTTGTTCTGTAAC * **** **************** ***** ************* * ******* g30.8 T----------------------AATAGTATACATTATACTCAAATATAAG-AGATCTAC 960 g30.6 T----------------------AATAGTATACATTATACTCAAATATAAG-AGATCTAC gBd TCAGAGTAAGCTGTTACTGACATAATAGTATACAGTATGCTCAAATACAAGCATTTTTAC * ************************ *** * * *** g30.8 TGTTTGTATTTATTGATTTATTTCTGTTGATCTTGTTTCTGTAGCTACCATCACGATGAA 1020 g30.6 TGTTTGTATTTATTGATTTATTTCTGTTGATCTTGTTTCTGTAGCTACCATCACGATGAA gBd TGTT-GAACTTATTGGTTT-TCTCTGTTGCCCTCGTTTATGCAGCTCCTATCACAATGAT **** * * ****** ** ******** *** * *** ** **** * ***** **** g30.8 ACTGAACAAATCAAGTCCCAAAGAAATGTTCAACAACAACCAGGCAAGG 1069 g30.6 ACTGAACAAATCAAGTCCCAAAGAAATGTTCAACAACAACCAGGCAAGG gBd ACTGAACCAACCAAGTCCCAAAGAAATGTTCAACAACCACCAGGCAAAG ******* ** ************************** ***********

 

g30.3

g30.1

g30.5

g30.7

Tb30.3

g30.8

Tb30.8

Tb30.7

Tb30.9

g30.60.002

qPCR-based Copy Number Analysis

Copy number analysis was performed on genomic DNA from Tb30 individual using the qPCR method. IgT gene was normalized by the single-copy gene Sreb2. The results suggested the presence of three IgT heavy chain gene copies for haploid genetic makeup, in line with the phylogenetic analysis shown on the left.

 

0

0,5

1

1,5

2

2,5

3

3,5

IgT Sreb2

 

Deduced amino acid sequence alignment

The deduced amino acid sequences of T. bernacchii and of another Antarctic Notothenioid species, Notothenia coriiceps, identified by screening its transcriptome, were aligned with B. diacanthus and other temperate teleost species IgT heavy chains. The results obtained strongly suggest that the loss in the Antarctic fish of almost the entire CH2 domain is balanced by the conservation of some amino acids typical of hinge regions, such as proline, glycine and cysteine, in the remaining domain. E. coioides (GU182366), O. mykiss (AY870263), S. salar (GQ907003), G. aculeatus, S. caurinus (GE798008), S. chuatsi (DQ016660), T. orientalis (KF713336), C. idella (DQ478943), D. rerio (AY643750), M. amblycephala (KC894946.1), N. coriiceps (KP876589), B. diacanthus (KP876590) and the three variants of T. bernacchii (S, KP876587; L, KP876588; Sts, KP876615).

 

 

A: Phylogenetic tree built considering the Notothenioid species together with ten other species included in the alignment shown above, and excluding the CH2 domain. For T. bernacchii only one sequence was considered (S variant). The species are distributed in three main clades, depending on the order they belong to. B: Phylogenetic tree of the single IgT heavy chain CH domains from 13 teleost species. All the CH1 domains are grouped in the same clade; all the CH2 domains are gathered together in the same clade. T. bernacchii, N. coriiceps, and G. aculeatus, represent the exception since their second constant domain has been found to be evolutionarily related to the CH3 domain of the teleost species possessing four domains. Likewise, their third constant domain grouped with the CH4 domain of the other fishes.

B

A