The genomes of all mouse strains contain multiple copies of DNA sequences related to murine leukemia viruses. We propose to continue our studies of the endogenous MuLV proviruses of Balb/C mice. We propsoe to use unique """"""""integration site"""""""" probes derived from cellular sequences adjacent to cloned endogenous proviruses to screen the DNA of two panels of recombinant inbred mice. From these studies we will determine the chromosomal map location of individual endogenous MuLV proviruses. We will test the promoter-enhancer functions of the LTR sequences associated with endogenous MuLV's by inserting these sequences into a test vector, pSV104gpt, in which expression of bacterial galactokinase is dependent upon promoter functions supplied by the inserted DNA. We will select gal K mutants of rat, mouse, mink, and human cells in order to test the species specificity of promoter-enhancer functions associated with exogenous and endogenous MuLV LTR sequences. Additional recombinant DNA clones of endogenous MuLV proviruses will be selected from a mouse cosmid library in order to isolate intact integrated proviral genomes, both for the previously isolated partial provirus clones as well as additional MuLV proviruses. Clones of intact endogenous MuLV's will be tested for expression as viral RNA, virus particles, and infectious virus following reintroduction into mouse or mink cells. Certain endogenous proviruses will be tested for specific functions suggested by their chromosomal location. We will test whether BE 7, located on chromosome 1, will give rise to infectious xenotropic virus, while cosmid clones of BE 23 will be tested for their ability to confer Fv-1 B resistance on Fv-1 negative SC-1 cells. In order to identify endogenous proviruses which are expressed, including those which are expressed only as RNA, or noninfectious particles, we will construct a cDNA library from the poly A+ RNA of Balb/C spleen cells stimulated in vitro with LPS and BudR. C DNA clones complementary to MuLV's will be characterized by restriction enzyme digestion and limited DNA sequencing in order to match up these expressed endogenous MuLV's with genomic clones of intact integrated MuLV's. These experiments are directed towards the goal of defining the potential for expression of individual endogenous MuLV's, determining which MuLV proviruses actually are expressed, and defining host cell genetic functions which may be encoded by endogenous proviruses.
