The aims of these experiments are to define those aspects of gene structure that determine the various steps in gene expression, and to define at the DNA level the types of genetic events that lead to mutation in mammalian cells. We will continue to generate Chinese hamster ovary cell mutants deficient in dihydrofolate reductase (DHFR) activity. These mutants will be selected starting with a recently isolated hemizygous clone that carries only one copy of the dhfr gene. Structural changes at the dhfr locus will be documented by Southern blotting analysis using cloned probes covering a 0 kb region that includes the 27 kb wild-type gene. An initial emphasis will be to accummulate spontaneous mutants in order to determine how often a mammalian gene is interrupted by the insertion of an endogenous mobile genetic element. Induced mutations will be studied using mutagens that are likely to induce small deletions detectable by Southern blot analysis. These include ionizing radiation, ultraviolet irradiation and diepoxyoctane, as well as carcinogens that form DNA adducts. The phenotypes of these DHFR-deficient mutants will be analyzed at the molecular level. Northern blot analysis will be used to detect mutants that fail to produce mature DHFR mRNA or that produce mRNA's abnormal in size or amount. Attempts will be made to pinpoint the defective step in such mutants, i.e., transcription, splicing, polyadenylation, termination, or nuclear transport. A major effort will be made to establish homologous recombination systems for the purpose of mapping point mutations in the dhfr gene. One approach will be to rescue endogenous mutant genes using cloned gene fragments introduced into the cell as episomes. A second approach will be to clone the mutant genes and allow homologous recombination to take place during co-transfer with cloned wild type gene fragments. For this last project, a cloning vector specialized for the isolation of single-copy dhfr genes is being constructed.
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