Purchase this article with an account.
K. L. Feathers, J. A. David, J. D. Chrispell, D. A. Thompson; Promoter Analysis of the Genes Encoding Human and Mouse RDH11 and RDH12. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5431.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
RDH11 and RDH12 belong to the family of short-chain alcohol dehydrogenases/reductases expressed in photoreceptor cells. Mutations in the gene encoding RDH12 cause autosomal recessive retinal dystrophy in patients, but not in mice, while the closely-related gene encoding RDH11 is not associated with any known disease. The purpose of this study is to identify the promoters that regulate the expression of the genes encoding RDH11 and RDH12, in order to further understand their roles in photoreceptor function.
The 5’-untranslated regions of the transcripts encoding RDH11 and RDH12 were identified using 5'-RACE with total RNA from human and mouse retina, ligating into pCR4-TOPO and sequencing. Reporter constructs were generated by amplifying genomic DNA located 5’ of each putative transcription start-site and cloning into pGL3-Basic upstream of the luciferase gene. Promoter activity was evaluated in co-transfected Y-79 and CHO cells in assays of firefly luciferase normalized to Renilla luciferase from the control vector.
The 5’-untranslated regions identified by RACE ranged from 65-152 bp in length and corresponded to sequences located immediately upstream of the initiation codon of each gene, with no evidence of intervening sequences. For both human and mouse genes, transfections with reporter constructs containing ~1.5 - 2.0 kb of upstream sequence generated at least 10-fold greater luciferase activity than transfections with empty vector. For RDH12, analysis of nested deletions showed that promoter activity was retained in the proximal ~400 bp for both human and mouse genes. In contrast, for the human gene encoding RDH12, no promoter activity was detected in assays of a ~2.0 kb fragment located upstream of a proposed exon at -4845/-4760 relative to the initiation codon.
Our findings suggest that the genes encoding RDH11 and RDH12 each have a single transcription start-site in human and mouse retina. In all cases, promoter activity was present in the genomic region located directly upstream of the initiation codon. Future studies will focus on comparative analysis of the specific elements that regulate gene expression in human and mouse, with an interest in understanding the differences in outcomes resulting from loss-of-function in each species.
This PDF is available to Subscribers Only