The growth hormone set of genes includes those for growth hormone (GH), chorionic somatomammotropin (CS) and prolactin (Prl). These genes are crucial for several aspects of growth and development. In addition, two other developmental hormones, thyroid and glucocorticoid hormones regulate their expression. The overall objective of this project is to understand the transcription of the GH set of genes in a reconstituted cell-free system. Such a systemm would provide a quick assay for probing the necessary and sufficient parameters required for differential expression by either RNA polymerase II or III of this set of genes by simply monitoring the appearance of specific transcripts on RNA gels. These genes appear to have arisen from a common evolutionary precursor and have substantial nucleic acid sequence homology. However, the rat GH gene contains RNA polymerase III promoters imbedded within a repeated DNA structure located in the second intron. Preliminary results reveal preferential RNA polymerase III transcription from this rGH repetitive DNA, whereas the hGH template has no such activity, and one hCS variant is transcribed by both polymerases. Furthermore, these genes are expressed in different tissues and their expression is dynamically controlled by various factors, including hormones. In the proposed studies, I hope to further characterize the rGH repetetive DNA transcripts and attempt to define their function. I also plan to study how the two promoters might possibly interact, establish conditions that will allow differential polymerase II or III expression and attempt to correlate promoter strength with specific sequences. In addition, a complete analysis of the transcriptional activity of the human GH and CS genes and their variants will be made. Furthermore, evolutionary changes in promoter number and strength will be determined by a comparison of transcription efficiency and products synthesized by the same genes from two different species (rGH vs hGH, and rPrl vs hPrl). Finally, I plan to assay for tissue specific expression by using homologous extracts prepared from cells that express the gene being tested. The cell free transcription assay, therefore, provides a powerful assay to test these ideas by simply combining the various templates in different extracts with or without added factors.
