Three of most important genes in growth and development are those for growth hormone (GH), prolactin (Prl) and chorionic somatomammotropin (CS). This gene family provides a model system to investigate tissue-specific gene expression since these genes contain very similar nucleic acid sequences (including the non-coding, 5' flanking regions) and yet are transcribed in different tissues. The molecular mechanisms which regulate the differential activity of the same or closely related genes in different tissues are not well understood. Studies have shown that chromatin structure, DNA methylation, and DNA-binding proteins may all play a significant role in directing cell-specific gene activity. The importance of discrete genomic regions (5' flanking and intron DNA) which may bind tissue-specific factors has been shown directly by gene-transfer studies. However, none of these factors have been purified. My preliminary results indicate that subclones of the rat growth hormone (rGH) and rat prolactin (rPrl) genes are transcribed in whole cell extracts from cultured pituitary cells, but not in Hela cell extracts. I have also complemented the Hela cell extract with an unfractionated pituitary cell extract and obtained transcription of the rPrl gene. Thus, the complementation assay can be used to purify pituitary-specific factors. I plan to identify transcription factors mediating the pituitary-specific expression of the rGH and rPrl genes by fractioning pituitary cell extracts on various affinity, sizing and ion-exchange columns. Factor purification will be monitored by a transcription-complementation assay. I plan to identify the DNA region required for tissue-specific expression by transcribing rGH and rPrl 5' deletion subclones in both homologous (pituitary) and heterologous (fibroblast) whole cell extracts. Furthermore, each assay will be controlled for variability in DNA input and extract basal activity by including an internal control template which contains a """"""""neutral"""""""" promoter active in many cell types. The precise factor-binding sequence will be determined by filter-binding and DNA-footprinting studies. Finally, I hope to establish protein blotting conditions so that I can probe blots of the various fractions with defined 5' flanking DNA fragments of the rGH and rPrl genes. The transcription-complementation assay provides a powerful tool to purify, and biochemically characterize, pituitary-specific transcription factors, and sets the stage for further studies which can directly address their mechanism of action at the molecular level.
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