The majority of the sequencing how ever, was carried out subsequent to microarray analysis to identify genes that demonstrated differential expres no sion profiles across the moult cycle. 396 clones were randomly selected from a list that displayed differential expression patterns between moult stages. This approach enabled the identification of genes likely to be involved in, and important for, crustacean moult ing. The 556 cDNAs were assembled in Sequencher based on sequence similarity, this resulted in 175 sin gletons and 62 contigs, Inhibitors,Modulators,Libraries representing 237 unique puta tive genes. Sequence annotation was via BLASTn, BLASTx and Pfam domain analysis.
The expressed gene sequences were grouped according to the follow ing biological functions, cuticular proteins associated Inhibitors,Modulators,Libraries with arthropod exoskeletons, FaMeT, proteins belong ing to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators, ribosomal proteins, and other sequences that did not fall into these groups. Unanno tated transcripts, were so termed, because they dis played no significant sequence similarity with sequences deposited in the NCBI database and were therefore not able to be annotated by BLAST analysis. The percentage distribution of the 556 sequenced cDNAs is depicted in Figure 1. The largest group of transcripts depicted here represents cDNAs that could not be annotated via the GenBank database. Transcripts encoding mitochondrial proteins such ATP synthase, cytochrome oxidases and NADH dehydrogenase make up 24% of the total cDNAs isolated in this study.
Cuticular protein transcripts con stitute 14%, Inhibitors,Modulators,Libraries while transcripts of the hemocyanin gene family and those related to muscle function Inhibitors,Modulators,Libraries and devel opment comprise 6% each, of the total cDNA popula tion. Inhibitors,Modulators,Libraries Phenoloxidase activators such as serine proteases, antimicrobial and clotting protein transcripts contribute to 5% of all sequenced cDNAs. Other tran scripts encoding diverse proteins not classified into the other groups include ovary development related protein, opsin, ferritin, heat shock protein, tubulin, notch pro tein, arginine and pyruvate dehydrogenase kinase, and transcripts that contained CT, GT or AC repeats, repre sent 5% of the total population. Lectins, such as the C type lectin receptor and mannose binding protein, as well as ribosomal proteins, each contributed to 3% of all sequenced cDNAs.
Fatty acid binding protein and diaze pam binding inhibitor transcripts, that are associated with lipid metabolism, constitute 2% of selleck chemicals the overall tran script population, while FaMeT transcripts represent the smallest group that form 1% of all cDNAs sequenced within the scope of this microarray study. Gene expression profiles across the moult cycle of P.