Meeting Abstract
With increasing availability of crustacean transcriptome data and a few currently available sequenced genomes (Daphnia pulex and Daphnia magna), we can now study the crustacean circadian system in this group more fully with regard to the TIM-PER/CYC-CLK transcription-translation feedback loop. Our prior studies have shown that crustaceans possess predicted orthologous genes and transcripts for all of the core and ancillary clock component proteins known in Drosophila and other arthropods. Interestingly, Daphnia pulex – known for its high degree of general gene duplication – possessed numerous copies of a timeless (tim) gene homolog, versus a single copy in Drosophila. We selected for further study the 8 paralogs with statistics that were predictive of potential homology. Transcription: Daphnia pulex were collected at 3 hr intervals over a 24 hr LD period. Using qPCR with primers designed uniquely for each of the 8 tim paralogs, we detected transcription of all 8 genes, with variability over 24 hr. Bioinformatics: A manual annotation and curation workflow was used to further predict the likely functionality of the resulting amino acid sequences. This workflow included promoter prediction, refinement of intron/exon borders, functional domain and motif conservation, and 3-dimensional structural prediction using coordinates of well-characterized arthropod homologs. These results suggest that several of the predicted TIM products are functional proteins.
