Clones of mouse fibroblasts have been isolated that contain hybrid DNA molecules composed of sequences from polyoma virus DNA and the prokaryotic recombinant plasmid pYeleulO. These cells were able to survive in leucine-free medium because the recombinant DNA contributed genetic information for an enzyme involved in leucine biosynthesis. This functional gene provides the basis for the biological selection of cells containing recombinant DNA. The studies proposed here will define the potential of this polyoma-pYeleulO hybrid DNA as a eukaryotic cloning vehicle. The number of copies per cell, and whether the recombinant DNA has integrated into the host cell DNA or replicates autonomously, will be established by renaturation kinetics, cesium chloride-ethidium bromide buoyant density gradient analysis, restriction endonuclease digestion and agarose gel electrophoresis. A physical map of restriction enzyme sites on one isolate will be constructed and used to compare several isolates, as well as to identify sites at which unnecessary DNA may be deleted and/or additional sequences inserted in future experiments. Sequences coding for at least two known genes are present and the expression of their products will be analyzed. The number of cells producing polyoma's early (T) antigen will be determined by indirect immunofluorescence and the amount of T antigen per culture measured by complement fixation. The cold labile yeast enzyme (beta-isopropylmalate (beta-IPM) dehydrogenase) can be identified and quantitated in cell extracts by photometrically assaying the production of alpha-ketoisocaproic acid from beta-IPM. Experiments will be conducted to determine optimum growth conditions for recombinant DNA-containing cells, transfection methods for mouse fibroblasts and other eukaryotic cells, and the response, if any, of the enzyme activity to various concentrations of substrate (beta-IPM) or end product (leucine).