Our goal is to understand the mechanisms and regulation of gene amplification in mammalian cells. The primary mechanisms of amplification involve recombination rather than over-replication. Unequal distribution of the recombined chromosomes into two daughter cells leads to an increase in gene copy number in one (amplification) and a decrease in the other (genetic loss). Thus the same basic process is also likely to be responsible, at least in part, for the loss of tumor suppressor alleles in cancer. Mechanisms. The primary events of amplification seem to be region- specific recombinations between sister chromatids involving T-T, C-C and possibly C-T pairings. The first amplified structures formed at several different loci in several different species will be studied and the novel joints will be identified. Recombination reactions involving these sequences will be studied in mammalian cells. Permissivity. Normal cell strains do not give stable drug-resistant colonies containing amplified DNA whereas most immortalized cells give such colonies readily. Cooperating oncogenes confer permissivity on amplification-negative cells. What normal proteins interact with these oncogene products? The roles of temperature-sensitive or conditionally expressed oncogenes such as the SV40 large T antigen and tumor suppressor genes such as p53 will be explored. Stimulation. Cell lines that amplify genes at a significant rate can be stimulated to increase that rate transiently by many treatments that damage DNA or arrest DNA synthesis, or stably in spontaneous "amplificator" mutants. Genes whose products stimulate amplification will be cloned by expression. The clones will be used to explore how stimulated amplifications occur.