In-vitro studies of the control of renin gene-expression in the mouse.
thesisposted on 19.11.2015, 09:08 by Angus Thomas. De Souza
Certain inbred strains of mice can be divided into two groups on the basis of their submandibular gland (SMG) renin activity. High renin producing strains have two genes Ren-1d and Ren-2d, whereas low producers have a single gene Ren-1c. To search for the presence of regulatory elements involved in renin control, approximately 1Kb of both Ren-ld and Ren-2d 5' flanking sequence containing the major transcription start-site P3 were fused to a "G-free" reporter cassette, and the resulting templates transcribed in various nuclear-protein extracts. In rat liver, mouse kidney and mouse testis extracts the Ren-2d promoter was 2-fold more active than the Ren-1d promoter whereas in mouse liver, both promoters were transcribed equally well. Deletion-mapping data are consistent with the presence of a functional negative control element(s) within Ren-1d but not Ren-2d. Polymerase catalysed chain reaction (PCR) generated deletions have shown that a fragment of 62bp, containing only the P3 promoter, was sufficient for accurate in-vitro transcription. Disruption of the P3 TATAAAA resulted in total loss of activity. Deletion of an AP-2 consensus sequence from the Ren-ld promoter had little effect on in-vitro transcription. Furthermore, sequences 3' to the Ren-1d P3 start-site when positioned either upstream or downstream to the Ren-1d promoter/G-free fusion, appeared to have a minimal effect on promoter activity suggesting that these regions are not involved in in-vitro regulation of the Ren-1d gene. Finally, patterns of specific protein-DNA interactions observed with the mobility-shift assays, are also consistent with an overall negative control hypothesis.