Y relationship of the alternative exons from D. melanogaster, A. gambiae
Y relationship of the alternative exons from D. melanogaster, A. gambiae, B. mori, T. castaneum and A. mellifera nAChR alpha6 genes.Page 2 of(page number not for citation purposes)BMC Evolutionary Biology 2007, 7:http://www.biomedcentral.com/1471-2148/7/For these analyses we used D. melanogaster as the representative Drosophila species. Phylogenetic analyses indicated that A. mellifera did not contain an ortholog to the second alternative exon 3 while B. mori and A. gambiae lacked an ortholog to the first alternative exon 8[17,24] PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28380356 (Figure 1). The orthologs to the second alternative exon 8 were very highly conserved, with all amino acid sequences identical (Figure 1), although they are not highly conserved at the nucleotide level. The hierarchy of amino acid conservation of the alternative exons 8 of nAChR subunit alpha6 genes was exon 8b > exon 8a > exon 8c. Phylogenetic analysis of the protein products of equivalent duplicated exons showed that members of a duplicated pair were more similar to each other than to the exons from other genes (Figure 1). This evidence suggests that exonduplication predated at least the radiation of insect orders spanning 300 million years of evolution. We next analyzed the evolutionary relationship of the alternative exons within the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27486068 Drosophila species. All three exon 8 variants had orthologs in each species. All three exon 8 orthologs were very highly conserved in these species, and were identical at the amino acid level. Surprisingly, the orthologs to the alternative exon 8b were identical even at the nucleotide level. We determined a similar hierarchy of nucleotide conservation of the alternative exons 8 of nAChR subunit alpha6 genes within the Drosophila species, namely exon 8b > exon 8a > exon 8c.Conservation and divergence of alternative splicing We were interested in understanding the alternative splicing of the nAChR alpha6 transcripts and in particular whether this is regulated. We first analyzed how the alternative exons 3 were regulated. The vast majority of tandemly duplicated exons (99.4 ) are likely to be involved in mutually exclusive alternative splicing events [14]. The Reverse Transcription Polymerase Chain Reaction (RTPCR) showed a very clear band in D. melanogaster and T. castaneum adult cDNA, as in A. mellifera with constitutive exon 3 (Figure 2A). Direct sequencing of these amplification products confirmed that these duplicated exons were alternatively spliced (Figure 2B). Sequence analysis of 30 cDNAs also showed that no duplicated exons were spliced together. These results indicated that the vast majority of exons were likely to be involved in mutually exclusive alternative splicing, which was consistent with EST and cDNA data analysis, although a duplicate of exon 3 was even found in cDNA [16].Figure alignments and phylogenetic analysis of duplicated exon nucleotide sequences Multiple1 Multiple alignments and phylogenetic analysis of duplicated exon nucleotide sequences. (A, B) Multiple alignments of amino acid sequences of duplicated exon 3 (A) and exon 8 (B) sequences and their counterparts from orthologs in other species, respectively. The alternative exons are labeled ‘a’ and ‘b’ or ‘c’. (C, D) Cladogram of insect duplicated exon DNA sequences and vertebrate orthologous constitutive exons corresponding to the alignments shown in (A, B). For each cluster, the amino acid sequences of each alternative exon from each VelpatasvirMedChemExpress GS-5816 species were aligned using the Clustal W program and phylogeneti.
