Prolactin (PRL) has been shown to effect reproduction, lactation and immunological function of many species, including humans. This research proposal describes how we will elucidate PRL signaling in the pigeon cropsac in the proximal promoter of the cp35 gene. Whole cell extracts obtained from pigeon cropsacs receiving PRL or saline (controls) will be used in gel mobility shift analysis with oligonucleotide probes generated from the G-rich and N-box containing region of the cp35 promoter-proximal region. Mutations of the sequences will be generated to identify nucleotide sequences which are absolutely required as determined by competition experiments and gel mobility shift procedures. High affinity oligonucleotide probes will be used to screen a pigeon cropsac cDNA expression library and/or for the affinity purification of factors binding this region of the cp35 promoter. Purified proteins will be subjected to microsequencing and the resulting amino acid sequence used to generate oligonucleotide probes to screen a cDNA library. Biological activity of proteins binding the promoter-proximal region of the cp35 gene will be assessed by using the cell-free transcription system employed by-this laboratory. Mutations of the G-rich and N-box regions will be compared to wild type sequences in the transcription assay. This will allow us to directly compare protein binding to promoter activity. The identification of PRL responsive proteins which bind the proximal region of cp35 will then allow us to test interactions between proximal and distal regions in PRL induction of the cp35 gene.
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