In this proposal Dr. Brent intends to use a novel set of artificially produced inhibitors called aptamers to investigate the structural parameters which modulate protein-protein interactions and to investigate cell cycle regulation. Aptamers are generated by inserting random sequences encoding 20 amino acids into a stable loop which normally extends out away from surface of thioredoxin. To screen this random library for fusion proteins, which interact with specific target proteins of interest, the thioredoxin proteins or """"""""prey"""""""" also contain the B112 transcription domains and the target proteins or bait lex A. Screening for interaction between prey and bait is then achieved by a modified version of the two-hybrid screen. The technology has been used to identify 14-aptamers out of 6 x 106 which interact with high affinity (Kd = 10 -7 -10 -8) to cdk2. The investigator has shown that these cdk2 aptamers block cdk2-cyclin E kinase activity when H1 is a substrate. Thus, the methodology can be used effectively to identify and isolate sequences which bind to specific proteins and inhibit their activity. In this grant the Principal Investigator proposes to use this new set of reagents to better characterize and define the function of 3 cell cycle regulators Cdc2, Cdk2, and Cdi1.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053636-03
Application #
2725890
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Program Officer
Forry-Schaudies, Suzanne L
Project Start
1997-01-01
Project End
2000-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Molecular Sciences Institute
Department
Type
DUNS #
941716045
City
Berkeley
State
CA
Country
United States
Zip Code
94704