INVERSE PCR-MEDIATED CLONING OF THE PROMOTER FOR THE RAT VASOPRESSIN V-2 RECEPTOR GENE

The adenylyl cyclase-coupled vasopressin V-2 receptor has been cloned recently and shown, in rats, to be produced from the predominant form of two alternate spliced variants. To begin to unravel the transcripti onal regulation of this receptor, we have isolated the 5' flanking reg ion of the rat vasopressin V-2 receptor gene and characterized its pro moter sequence. The method of inverse polymerase chain reaction (PCR), which allows the amplification of DNA fragments adjacent to a segment of known sequence, was used as an alternative approach to genomic DNA library screening. Using a probe encompassing part of the coding regi on, first we identified by Southern blot analysis, a single BstX I hyb ridizing fragment of 2.3 kilobases (kb). This size predicted a BstX I restriction site 1.5 kb upstream to the gene coding region. Cloning of this fragment was accomplished through circularization of BstX I rest riction digests and inverse PCR-mediated amplification. Sequence analy sis of the gene 5' flanking domain enabled the design of oligonucleoti de primers with the usual forward/reverse orientation, and additional clones were generated from native genomic DNA using a high fidelity th ermoresistant DNA polymerase. Reverse transcription-PCR (RT-PCR) and p rimer extension analysis mapped the major transcription start site 422 nucleotides upstream to the translation initiation codon. The promote r region lacks a TATA box but contains a CAAT box and a consensus bind ing site for transcription factor Sp1. Multiple potential binding site s for the transcription factor PEA3 are clustered in two DNA portions located 0.6 kb and 1 kb upstream to the coding region. In addition, se quences homologous to glucocorticoid response elements are present and might be responsible for the regulation by adrenal steroids of vasopr essin-dependent adenylyl cyclase activity in the kidney.