Xenopus rhodopsin promoter - Identification of immediate upstream sequences necessary for high level, rod-specific transcription

To understand the mechanisms that control the cell-specific visual pigment gene transcription, the Xenopus rhodopsin 5' regulatory region has been cha racterized in vivo using transient transfection of Xenopus embryos and tran sgenesis. The principal control sequences were located within -233/+41, a r egion with significant conservation with mammalian rhodopsin genes. DNase f ootprinting indicated seven distinct regions that contain potential cis-act ing elements. Sequences near the initiation site (-45/+41, basal region) we re essential, but not sufficient, for rod-specific transcription. Two negat ive regulatory regions were found, one between -233 to -202, with no appare nt similarity to known elements, and a second Ret-l-like CAAT (-136/-122) m otif. Deletion of either sequence led to a 2-3-fold increase in expression levels, without a change in rod specificity. Sequences between -170 to -146 , which contain an E-box motif, were necessary for high level expression in transgenic tadpoles but not in transient transfections. Sequences between -84 and -58, which contained an NRE-like consensus were found to be necessa ry for high level expression in both assays. Although expression levels wer e modulated by various proximal sequences in the rhodopsin promoter, none o f the tested sequences were found to be necessary for rod specificity. Prom oter constructs with a consensus BAT-1 sequence in conjunction with an NRE- like element upstream of the basal promoter directed low level green fluore scent protein expression in the central nervous system in transgenic tadpol es. These results suggest that rod cell-specific expression of rhodopsin is controlled by redundant elements in the proximal promoter.