MMTV GENE PRODUCTS AND TRANSFORMATION

We will continue investigations into the role of the mouse mammary tumor virus (MMTV) long terminal repeat region (LTR) and LTR open reading frame (ORF) products in tumorigenesis and in the life cycle of MMTV. The phenomenon of tissue tropism by MMTV will be addressed by studying the interaction of specific nuclear DNA binding proteins with MMTV LTR DNA. This interaction will be studied with the full length as well as the truncated forms of the LTR found in amplified proviruses of certain leukemia cells. DNA- protein binding will be tested with nuclear extracts from various cell types, before and after treatment of the cells with glucocorticoids and phorbol esters. Cloned MMTV LTR cDNAs will be ligated into an inducible mammalian expression vector to study the post-translational processing of the LTR proteins as well as the effects of LTR ORF gene expression in various cell types. The interaction of the LTR protein with the MMTV genome will be studied using ORF translation product purified from a bacteriophage lambda gt11 expression vector. The altered LTR of the amplified provirus found in DBA/2 leukemia cells will be molecularly cloned and sequenced in order to define the alteration in the LTR region. The cloned LTR DNA will then be used for studying DNA binding protein interactions. Enhancer activity of the altered LTR will be measured by fusion into a plasmid containing the chloramphenicol acetyl transferase gene (CAT), followed by transfection of the construct into various cell types and measurement of CAT activity. The CAT constructs will also be co-transfected with the LTR cDNA expression vectors in order to test for possible effects of the ORF translation product on transcription. MMTV antigen expressing tumor cell Lines other than mammary tumor cells will be analyzed for the presence of amplified MMTV proviruses. If amplified proviruses are detected then restriction endonuclease analysis will be performed in order to determine the presence of alterations in the LTRs of the proviruses. Such alterations will then be studied in more detail with the aim of understanding at a molecular level, factors which determine tissue tropism.