International Mammalian Genome Society

logo18th International Mouse Genome Conference

17-22 October 2004, Seattle, USA


Silverstein RS 1, Tempel BL 2

1 Graduate Program in Neurobiology & Behavior, University of Washington, Seattle, United States, 2 Department of Otolaryngology, University of Washington, Seattle, United States

Deafwaddler (dfw) mice are deaf and exhibit wobbly gait. The dfw mutation was localized to Atp2b2, encoding a plasma membrane Ca2+-ATPase, PMCA2. Heterozygous mutants of Atp2b2, while not deaf, have significant high-frequency hearing loss, implying that tight regulation of its expression is required. PMCA2 is highly expressed in stereocilia of cochlear outer hair cells, cerebellar Purkinje cells, and epithelial cells of lactating mammary gland, suggesting that it is important for hair cell Ca2+ homeostasis. To understand the pathways that control PMCA2 expression, we have examined transcriptional regulation of the Atp2b2 gene. Using antisense primers in the open reading frame, 5’-RACE was performed on RNA from multiple tissues in CBA mice. Sequencing of clones identified three primary alternative exons of transcriptional initiation, implying regulation through distinct promoters. Two of these (1c and 1f) were observed in cochlea and cerebellum. A third (1m) was restricted to lactating mammary gland. Quantitative real-time RT-PCR (qPCR) confirmed the findings from 5’-RACE. To determine the promoter used in hair cells, cochleae were microdissected by separating organ of corti (OC) from modiolus (MOD). qPCR was performed on the OC and MOD samples, using marker genes to confirm quality of dissection. This analysis revealed that exon 1f dominates in the OC (hair cells), while exon 1c is most abundant in MOD (spiral ganglion neurons). In silico analysis of putative regulatory sequences revealed high levels of conservation between mouse, rat, and human. Further analysis by in situ hybridization will confirm cell-specificity of promoter use in the different tissues.

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