We shall be studying the mechanism whereby cultured animal cells become resistant to methotrexate as a result of selective amplification of DNA sequences coding for dihydrofolate reductase. The research is directed at the fundamental mechanism(s) whereby this occurs and results in amplified DNA sequences that are either present on chromosomes, or are present at extrachomosomal elements called """"""""double minute chromosomes."""""""" Gene amplification occurs on evolutionary time scale, as well as one mechanism for developmental regulatino of protein synthesis. We wish to use the experimental system of cultured mammalian cells to study the mechanism of amplification. The studies will employ varoius cells lines of cultured hamseter and mouse cells. Mechanisms to be studied include uptake-replicaton-integration of DNA from cells, as well as a saltatory replication model. Techniques to be employed include use of fluorescence-activated cell sorter to study DNA synthesis, as well as isolate cells with elevated dihydrofolate reductase levels. Dihydrofolate reductase DNA sequences will be detected and isolate employing both messenger-derived, as well as genomic recombinant DNA clones presently available in the laboratory. Ultimately, we are interested in studing how genes are amplified, and what intrinsic and extrinsic factors may affect the amplification process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM014931-20
Application #
3268702
Study Section
Biochemistry Study Section (BIO)
Project Start
1976-09-01
Project End
1986-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
20
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Stanford University
Department
Type
Schools of Arts and Sciences
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305