|Budget Amount *help
¥6,800,000 (Direct Cost : ¥6,800,000)
Fiscal Year 1990 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1989 : ¥4,800,000 (Direct Cost : ¥4,800,000)
To clarify fully the gene expression in the mammary gland which synthesizes many kinds of protein by multi-hormonal interactions, it is essential first to establish assay systems to detect translation products as well as a serum-free culture system of mammary epithelial cells, second to clone probes to detect transcription products of genes encoding milk protein, and third to characterize prolactin receptors for the first step to clarify the signal transduction system of prolactin which triggers lactogenesis. Our results of research on this line are as follows.
1. Mouse mammary epithelial cells were cultured on floating collagen gels and caseins synthesized and secreted were detected by western blotting in the medium. We have further selected two hybridoma cell lines recognizing each one of alpha and beta casein. Using this system, we confirmed that insulin + glucocorticoid + prolactin can induce lactogenesis.
2. Genomic DNA and cDNA encoding beta casein are already cloned. We tried to clone cDNA for alpha casein. We have obtained several clones for milk protein, and analyzing nucleotide sequences. We also have cloned a genomic DNA clone for a milk protein with a molecular weight of 40,000 to 50,000.
3. Two hybridoma which produce monoclonal antibodies recognizing rabbit mammary gland prolactin receptors were selected. Using these antibodies, we characterized mammary prolactin receptors. Two distinct receptors with molecular weight of 40,000 and 80,000 were detected by western blotting. After partial digestion of these recptors with several proteases, almost all digestion products of the receptor with a molecular weight of 40,000 were found in the digests of the receptor with a molecular weight of 80,000. Therefore, the one with a molecular weight of 40,000 is a part of the one with a molecular weight of 80,000.