Abstract
Abstract: :
Purpose: We have studied gene expression differences between wildtype and rd mouse retinae to identify genes that may be associated with the retinal circadian clock. The perturbation of the circadian clock in the rd retina may be associated with decreased expression of specific genes. Methods: Wildtype and rd radiolabelled cDNA probes were reverse transcribed from mRNA pooled from circadian time (ZT) 0, 4, 8, 12, 16, 20 and used to screen pairs of human and mouse macroarrays (GDA, Incyte). Differential positives were confirmed with slot blot and northern hybridisation. The identity and human chromosomal localisation of one candidate positive was determined by BLAST search. Genomic DNA flanking the gene was analysed for conserved promoter motifs. Potential promoter fragments were amplified and inserted into luciferase reporter constructs, cotransfected into COS cells with combinations of known circadian transcription factors, and detected with a dual-luciferase assay kit. Results: BLAST search identified the candidate as the mouse embryo development protein kinase (EDPK) or human Krct, novel serine/threonine orthologs with unknown function. Krct localised to human chromosome 2 immediately flanking serine threonine kinase 16 (STK16). The upstream DNA contains two highly similar sequences that show conservation with an opsin promoter, sections of the mouse Period gene promoter and the Drosophila circadian regulatory sequence (CRS). The sequences also contain conserved E-box, Crx, and E-opsin/MASH motifs. Each promoter sequence showed orientation-specific enhancement of luciferase activity. One sequence is unaffected by the circadian transcription factors CLOCK and BMAL1 but enhanced by PER1 and PER2 while the other appears to be enhanced by CLOCK. Conclusion: Mouse EDPK / human Krct is a novel kinase with unknown function in the retina. It maps adjacent to another serine/threonine kinase on human chromosome 2. Flanking regions show conservation with known retinal/circadian promoter elements and two sections of the promoter region exhibit differential responses to known circadian transcription factors. Current experiments are localising the in-situ expression of this gene in the retina to help further characterise its function.
Keywords: 417 gene/expression • 556 retina: neurochemistry • 525 protein modifications-post translational