We have developed techniques of efficient DNA-mediated transfer of genes into primate cells and used these techniques to search for DNA sequences which regulate mammalian cell DNA replication. Expression vectors based on the Tn9 chloramphenicol acetyltransferase (cat) gene served to optimize conditions for transient expression of exogenous DNA as well as to provide evidence that the promoter in the long terminal repeat or Rous sarcoma virus (RSV) is exceptionally active in primate cells. Based on cat plasmid data we constructed the dominant selectable marker plasmids pRSVgpt and pRSVneo. pRSVgpt was found to mediate stable "transformation" of monkey CV-1 cells with an efficiency of 5-8x10 to the -1; pRSVneo proved to be a superior selectable marker for stable transformation of WI38 human embryo fibroblasts and HeLa cells. pRSVneo transfection in combination with genomic DNA from E. coli, HeLa and WI38 cells has been used to obtain preliminary evidence for human DNA sequences that inhibit HeLa cell replication. Retrovirus vectors based on these markers will be constructed to facilitate gene transfer into hematopoietic and primary cell types that are inefficiently transformed by conventional vectors. In addition, the potential of novel LambdaSV2/cosmid vectors to improve gene shuttling techniques will be further studied.