Abstract
In the present paper we show a survey of the Asterias rubens sea star genome for genes associated with NF-kappa-B proteins implied in the immune response. The NF-kappa B gene, into 2 subunits, was found in this invertebrate.
In recent papers, we have found that the A. rubens sea star genome has shown Ig kappa genes (Leclerc, et al., 2011, Vincent, N, et al., 2014) We discovered also the interleukin 17 receptor B (Leclerc et al., 2013) which mediated the activation of NF-kappa B in mammals (Hayden et al., 2006). So It seemed really interesting to study the NF-kappa B genes in such an invertebrate.
1. Material and methods
Immunized and non-immunized sea stars to HRP (Horse-radish peroxydase) were used (Leclerc et al., 2011). The sea star axial organs were removed. RNA was extracted using Trizol (Invitrogen) according to manufacturer's instructions. CDNA was normalized using double strand specific nuclease essentially as described by Zhulidov et al. (2004). cDNA was fragmented using DNA fragmentase (New England Biolabs), according to the manufacturer's instructions. After ligation of adapters for Illumina's GSII sequencing system, the cDNA was sequenced on the Illumina GSII platform sequencing 1.100 bp from one side of the approximately 200 bp fragments. Sequences were assembled using Velvet: Zerbino et al. (2007). Assembled nodes were used for further assembled including Beta vulgaris EST-Data from NCBI in MIRA.
2. Results
Results are summarized as following:
-
A)
Controls
The first contig shows the presence, in non-immunized A. rubens of the gene: NF-kappa-B one contig (Contig12117 | m.10680) could be annotated via BLASTX to mouse “Nuclear factor NF-kappa-B p100 subunit” from the SWISSPROT database, with an e-value of 7,00E-071. On an aligned region of 242 amino acids, 170 positive and 138 identical amino acids were found.
5′: AGAAGATTGAAAACCGGAAGAATTGTGACACTGCAACTTGTCACACTTGAAGCACCAATT
TAAAAGATGTCAGACGAGGAGAGGTCTGATTCCCTGCAGTCAGTTGAGATCCCAAATGAC
ATGTTACAGCAAGCATTCTTAAACAATAACGGAGCATCAAACCAATCCTCGGAAATGAAT
TTATCTGTTCTGCCAACTGACCTTCATCAACTTGAGGTCATGACAGGAATGAGTCAACAG
ACGCTAAATGACCTCTCTGCTCCCAAACTCCGCATCATAGAGCAACCCAAACAGCGTGGC
TTTCGGTTCCGTTACGACTGCGAAGGGCCATCACATGGTGGTCTCCCGGGTCAACATAGC
CAGAGAGGAAAGAAGACCTTCCCATCAGTTGAGATACTTAACTATACTGGACCAGCAAGA
ATTGTTGTTTCCTTGGTAACCAGTGAAGATCTACCTAAACCCCATGCACATAGCATCGTT
GGCAAACACTGCACTGATGGATCATGCACTGTGCAGGTCGGACCTACAGACATGACTGCA
AGCTTTCCAAACCTTGGCATCCTTCATGTAACTCGCAAGAATGTTGAGAGGGTCTTGAAA
GATCGTCTTCGTGAACAGCAGAAGCTTTATAGGACTTTTCTTGGTGATAAGGGATTCGTA
CCTGGAGCACAGCATCTGCTTGAGACTGAGCCAGCCCTTGCCAAGAAAGCCAAGGAAATG
GCCAAGGAGATGGATCTGTCTAAGGTGTGCTTGTGTTTTCAAGCCTACCTTCCAGACAGT
CAGGGACATTACACGCGTCCTCTGGCTCCTGTCACATCACTACCCATCTTTGACAGCAAG
GCCCCTGGTGCAACAACTTTAAAAATCTGCCGTATGGACAAGAGCTCAGGATGTGTGACA
GGGAATGACGAGGTATATCTGCTTTGTGATAAAGTTCAAAAGGACGACATTTATGTGAGG
TTTTTTGAGATGAACCCAAGCGGAAGTGGGCC 3′.
The second contig with its sequence reveals the presence of another subunit of NF Kappa-B (p105):
The contig (Contig675 | m.5295) could be annotated via BLASTX to mouse “Nuclear factor NF-kappa-B p105 subunit” from the SWISSPROT database, with an e-value of 4,00E-071. On an aligned region of 518 amino acids, 263 positive and 192 identical amino acids were found.
5′: CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAACAGCAAAAT
CACCAGGGTGCGGGCTTCCAGAGCCGTGGTATAAGCTTTGAAAAGGATCAAGACTTGAAG
ATTACCCGCGAGACTGAAGTGGTGCAGGACACACAACCAGCGGTTCTACCTACAGTCACT
CTTATTGGGGAGAGAGAGATTGATAGCGATCTTGAAGTAGATTCTCAAATACTTGAGGAT
GCAACAAAAGGCCCTGTCAGGCGATTTGAATCCTGTGTGCAGAGTGGAGTTGGAGTTAGC
ACTTTTGAACCAGCAAAAAGTGCCAGAGATATTTCTGTGCAGACTGATGATATTCCATTG
GATTATGAATCCCTCGCTTGGCGTCTTGCTGAGCGTACATCTCGTGCATTGCGTGACTTT
GCAATCACTGCTGATATGAAGATGCTGTTAGCGGTCCAGAGGCACCTGACTGCTGTGGAG
GATGAGAATGGTGACACACCTCTTCATCTGGCAATTATCCACAAGAAATACGACGTTGCT
TTGGGACTCTTAAATGTCATTATCAGCATTCCTCATCAGAAGATCATTAATCATCACAAC
AAACTTCATCAGACGCCCCTGCATGTTGCTGTAATCACAGAACAGCCTGACATTGTAGAG
TTACTTCTCCGTTGTGGAGCTGATCCAAACATCATGGATCATAACGGGAGTATGCCTATC
CACCTAGCTGCTCATAATCGTCTTAGTGATATAACAGAGGTCTTAGTCCAAGGGGTGGAA
CCACTTCCTGGAAAGTCCAACGCTTCTATTGATCCAATCCCTACTGAAATCAACCTCAAG
AATCTTGATGGCTTCACTGCAGCACACATTTCAGTTCAGATCTCTGATTTGAAGAACCTC.
AAAGTTTTGGTGAAAAACGGAGCTGATATTAATGTTCAGGATGGTAAAAGTGGTCGCACC
ACCCTCCATTACGCCGTGGAGCAGGCAAACTTCTCACTGCTTGGATACCTGATTGCTGAT
GCAGATGCAGATATCCACATTCAGACGTTTGCTGGTGATACCCCACTTCATCTTGCTTGC
AGCCTTGATTACGTTGCTGTTGCTGCAGTTCTGGTTAGTGCTGGAGCAGAACCAAGCATT
GAGAACTTTGACACCTCTTCCAACGAGATGGCTGGGGAGACTGGTGTAATATCTGATGAA
GGACATGTGGAGATATTTGAAGACGAGTACAATAATGAGATACTTAGTGGCAAGACATCA
GTGGATCTGGCATGCTCTGAGAGGATGTTGAAAATTTTGAATGGTGAGGACTACATATCC
AGTTCAGTGTCAGAATCCACACCTAGATTTGAAGAGAGTTTGGTATCAGCTGTTTCTGGT
TCCTACGCCTCTAAGACCAATATGTCTGATTCCTACACCTCTACTGCAAATAAATCAAGC
ACTAGCCTCCGCTTCACTCCCACAGGAGATTTGAATCGCCTGGATTCCTTAACTCGTCAG
AAATTATCGTCGCTCCTGGATGAGATAAAACCCCACAGGGATTGGTTGTCTCTTGCAGAC
CACTTGGGCCTTGGCAACATGATTAACCAATTAAAGCAGTTTGATAGCCCCACAAGTATA
CTGCTTGACCAGTATGAGGCCATGGATGGAACTATCACGGAGCTCACCTCAGTTCTCAAA
GGTCTAAATCGTGAAGACGCTATTAACGTCATTGCAGAAATAAGGAAGCCAATGAAAAAA
GCTGCTCATCCCTTACCCAAAGATTTGGATTGTACAACTCAAAACGATTCCGGTCTGGCA
TCCTCCCTTCAATCCATGCAGATTACAGGAGATTCTGGTTTGTCCCGTGGATGTATGACG
AATCAGTTCAACCAGTCGGATGCCAGGAATATTGAAGTTTAGCTATTTTGGTGTTAAGAA
ATAAATTTGATGCAAAAATAGGTGGTGTTTTGTTTCCCCAAAAGACTGTATAGTTGTACA
TAGTGCTTCGTAAATGCCTTGTGAAGTTTATTAAAGTGAGATATTCTACTTTGTATTATC
GGTTTAAATCTTAAGGTATTATGTAGTTGAAGAACTTGAAACGTCTTAATTTCTAACTAG
GTGTTTAAGATAACTGTTTTTGTTATCAGAAAATAACTGTGGGAAACATAGCAAAACGCT
GGAAGATTAGAATGATTTTTGGGAATATTTTCCTTGTGTAATAGGTTTTTTTTCTTTTTC
TGAAGATACATTTTTTACAGAATTTTGTCTGCAACATATATCATACAAATTTTCGGCTTA
ACTGTCGTCAGGACATTGTCTGTTATGTTTGTAAATGTAAATGTTTGTCGCTTTATTGTC
ATTGCATCAGTGAAACAAAAGGGAGCACTACTTTGTTAGTGCAGGGATCTTTTTATTGTA
CCAGCTTTGTAAAGCCTTGTTAGCCATAAAGAAATGTTTCTTAAATACTTGTTTTTCAAT
CTATTTGATAGTCGTCAAGAAGGATTTAAACAGAACATATTTTTAGGAACTGCTTGTTTT
AATTTTGTGGACAGTTCACTTTAAAGCAGAAACATGGTGATAGTTAGCTCATTGTTAAGT
AAAGGAGATGGCTGTGATGGGAATTGCATTGGTGAAAGCATAATAGGCCCAATGCTGTTT
ATCAACCTGTTTGTTTTATGAACAATGTGTAGCCTCCAAACACACTCTAGTTTAAATCAT
ATTAGTTTGGATTTGTGTACATCACTACCTATGAAAGAGTCTTGGACAAGTTGTGCAAAA
TGCTGCATCTTTTGTTTAAGAACATCATTATTGCTTTGTATCACATTACATAGGAGGTGA
TTGGTCTCTTGTGTCTCTAGATTGCCAATGTAATGGTGAGATTGACAATGTAATGTCGCA
TGAGTTCCAATTTAGCATCCATTGTGGTTCGAATAGATTTTTTTACATTTAAAGGCAGTG
GACACTATTGGTAATTGTCAAAGACTAGCATTCACAGTTGGTGTATCTCAACATTTGCAT
AAAATAACAACTCAGTAATAATTTCAAATTAAAGATTTATTAAATTTGTATTCAAGTTTG
CAAAATGTTACAGACTATTCTTTTACAATAGAAGATTTAAAAGCAATGTTGTGTAATACT
ACTGCATTTACCTTCAAAATAAAGAATTTATAATTTAAATTTGCAACCCTCCTACTGTTG
GTACAATTATCTAAAAAGTTGATGCACACTATCGTGGCTTCAAATACTAACCAGCTCCCC
CCCCCGCCCAGTTTTATAGAGCTCCTTTAGCAGTAAATATTGATATATATTTGGTTTGCA
GTAACACCATGTGTGTATCTACTTGCCAGGTAGAGTTTGTTCCTAGAGAACTGTCTTGCT
TTATTCAAATACCGCGGAGAAGATGTTCGGGGGATCGGGAGACTTCTCAGTTCAGAAAAG
AATTGTCCTGCTTTTTCAACTATTACCTGGGCGGATTGTATAGAACATATGCTGGGACTA
CTACTTAAGCTTATAGGATCGTAATTAACTG
CAAAGTATAGTACACTACAAATCCAAGATGCACAATTTAGTGACAACGGCAATTACGTCT
GCCAAGTAGGCAATAACAGAGCCTATGGTGATCTTACAGTAACGTATGATGGAATCGTCG
TAATTAAAGGCAATCGAGTCATTGAAATGGAGCAAGATATGAACTTCACATGCTTTATTG
ATGGCGACTCATCTGTTGAGGTCACATGGAAAAAGGAAAATACAATGCTACCATCTCCTG
AGCTGCCTAACATTGAAGTAAAGGGAAGCTCTCTGCTTATAAAGAAGGCATCGACTGCCA
ATGGTGGTAAATACACCTGCAGTGGATTGTACAACAAAACAATTCCAGTTGAACAAACTA
TTTATGTAGGAGGGGAAATGCGAATCAAGTCCCCCAATTCAGTGAAGTTTACAGAAGGAG
AGCGTGCTCGCTTTGAGTGTGTCATTCTCCAGCCAGAAGGCGACCCCAAACCTACTTTCC
AATGGATAGTGAGCACTGCAAACTACACCTTTGAGCTGACTGAGACGATTGAGGGAAGTC
GCTACTCCATTGAGCCGTCCAAGCACAAGACTGGATCCATCTTGCACATGCTGGATGTAG
TCAAGGAAGACAGAGGAAACTATACTTGCATTGCAACCAGTAACGT 3′
-
B)
HRP.
2 contigs (a and b) show the presence also of two subunits of nuclear factor in immunized A. rubens.
-
a)
One contig (Contig11285 | m.9708) could be annotated via BLASTX to mouse “Nuclear factor NF-kappa-B p100 subunit” from the SWISSPROT database, with an e-value of 1,00E-031. On an aligned region of 203 amino acids, 107 positive and 78 identical amino acids were found.
-
b)
Another contig (Contig2602 | m.5791) could be annotated via BLASTX to mouse “Nuclear factor NF-kappa-B p105 subunit” from the SWISSPROT database, with an e-value of 2,00E-058. On an aligned region of 398 amino acids, 209 positive and 153 identical amino acids were found.
3. Discussion and conclusion
Echinoderms occupy a critical and largely unexplored phylogenetic situation from the point of view of the evolution. According to the present study, it appears that NF kappa-B factor is present in sea star immune genome, under 2 subunits p100(daltons) and p105(daltons). It seems fundamental when compared to another invertebrate the arthropod in its specificity. Undoubtedly the NF-kappa B plays an important role in the immune function of A. rubens. Nevertheless this role remains enigmatic, when compared to mammal 's one (Scheinman et al., 1993). In conclusion, the sea star immune system, in its discovery appears more and more sophisticated.
References
- Hayden M.S. Oncogene. 2006;25:6758–6780. doi: 10.1038/sj.onc.1209943. [DOI] [PubMed] [Google Scholar]
- Leclerc M. Immunol. Lett. 2011;138(19):7–198. [Google Scholar]
- Leclerc M. Am. J. Immunol. 2013;9:65. [Google Scholar]
- Scheinman R.I. Mol. Cell. Biol. 1993;10:6089–6101. doi: 10.1128/mcb.13.10.6089. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vincent N. Metagene. 2014;2:320–322. [Google Scholar]
- Zerbino D.R. Genome Res. 2007;18:821–829. doi: 10.1101/gr.074492.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhulidov P.A. Nucleic Acids Res. 2004;3:32–37. doi: 10.1093/nar/gnh031. [DOI] [PMC free article] [PubMed] [Google Scholar]