Identification of Rat Ovarian Nuclear Factors That Interact with the cAMP-inducible Lactate Dehydrogenase A Subunit Promoter

Utilizing the gel electrophoresis/DNA binding assay and a new technique of direct binding of radioactive DNA to protein blots, we have investigated putative factors selective for the cAMP-responsive element (CRE) of the lactate dehydrogenase A subunit promoter in rat ovary nuclear extracts. Analysis of linker-scanning mutants of lactate dehydrogenase A subunit promoter fragments by DNA binding assay identified DNA binding activity selective for the 11-nucleotide sequence 5′ TCTGACGTCAG 3′ located between positions −51 and −41 relative to the transcription initiation site. This sequence contains the previously identified CRE 5′ TGACGTCA 3′. Probing of protein blots with radioactive promoter fragments containing the CRE demonstrated that ovarian nuclear extracts contain a protein of relative molecular mass 47,000 (Mr47,000) which exhibits selective binding affinity for the CRE. The 47-kDa CRE binding protein was found to be present in comparable levels in the ovaries of normal and hypophysectomized rats. Furthermore, our data suggest the presence of a 37,000-dalton (Mr37,000) protein which possesses selective binding affinity for part of the CRE sequence. The binding activity/level of the 37-kDa CRE binding protein appeared to be modulated by short-term hypophysectomy/follicle-stimulating hormone administration. These results provide evidence for the presence of CRE binding factors in rat ovarian nuclei, which may be involved in the molecular events responsible for transcriptional regulation of ovarian cAMP-inducible genes.