Genispheres 3DNA dendrimer labeling technology, which overcomes some difficulties associated with direct or indirect labelling selleck chem Regorafenib methods, was used in this study. This technology allows the labelling of cDNA via a tar get capture sequence oligonucleotide rather than incor porating the fluorescent dye directly during cDNA preparation, thus avoiding inefficient synthesis and hybridization of the cDNA to the array that results from the incorporation of fluorescent dye nucleotide conju gates into the reverse transcript. Additionally, the signal generated from each message will be independent of base composition or length of the transcript. The sequencing of 556 clones from two libraries generated from P. pelagi cus, revealed that a significant portion of these cDNAs could not be annotated via the GenBank database.
However, these transcripts may nevertheless represent genes with a significant role in the process of moulting in crustaceans, as they were isolated within the scope of the moult cycle related Inhibitors,Modulators,Libraries differential gene expression analysis study. Cellular energy requirements across the moult cycle Transcripts representing mitochondrial proteins, such as ATP synthase, cytochrome oxidase, and NADH dehy drogenase form the second largest group of cDNAs isolated during this microarray study. Such proteins are required for cellular energy homeosta sis as they are a part of the mitochondrial respiratory chain. NADH dehydrogenase and cytochrome c oxidase are two of the three energy transducing enzymes in the mitochondrial electron transport chain.
Mitochondrial ribosomal and elon gation factor transcripts, were identified in Cluster A which display an expression profile of relatively low expression during ecdysis then a gradual increase across the rest of the moult cycle generally peaking in early pre moult then decreasing slightly in late pre moult. The expression profile of these tran scripts appears to reflect an increase Inhibitors,Modulators,Libraries in the energy requirements of the animal Inhibitors,Modulators,Libraries as the moult cycle pro gresses. Lower levels of mitochondrial gene expression at ecdysis suggests reduced energy requirements during moulting, with a recovery in metabolic activity appearing in the post moult stage and returning to nor mal during interphase, reflected in the increase Inhibitors,Modulators,Libraries in mito chondrial gene expression observed in Cluster A.
Interestingly, studies into the ecdysteroid responsive genes of Cherax quadricarinatus revealed that moult induction through endocrine manipulation resulted in differential expression of genes predominantly belonging to a group known to be involved Inhibitors,Modulators,Libraries selleck in metabolic functions such as digestive enzymes, carbohydrate metabolism and mitochondrial respiration. These transcripts how ever, were down regulated in pre moult when compared to control animals in intermoult, possibly indicating that moult induction creates metabolic stress which may impact on metabolic function.