Work described in this application will involve the use of amnion cells to elucidate principal mechanisms of up and down regulation of the OTR. The investigator will: (1) isolate the rabbit OTR gene from rabbit genomic libraries, knowledge of the 5-prime flanking and coding sequences will allow the completion of aims 2 to 5; (2) determine the effects of regulatory agents on OTR gene expression. These studies involve measurement of OTR mRNA levels by RNAase protection and transcription initiation rates by nuclear runoff assays. The investigator will also study OTR mRNA stability; (3) describe the promoter region of the rabbit OTR gene and use transient tranfection of cultured cells with deletion and replacement mutants to characterize regulatory elements; (4) determine the effects of regulatory agents on foot printing sites in the OTR promoter and 5' flanking sequence. These studies will define which of the potential regulatory elements (deduced from consensus sequence analysis and transfection studies) are functional when the OTR gene is transcriptionally active and elucidate essential regulators of OTR gene expression; and (5) determine the effects of OTR regulatory agents on the synthesis of OTR protein by immunoabsorption after labelling amnion proteins with (35S)methionine. Antisera will be produced to three separate domains of the OTR.
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