A key problem in the generation of representational, full length cDNA libraries is the poor processivity of the copying enzyme, reverse transcriptase (RT). We propose to utilize mutants of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMuLV) RT that display improved processivity and/or better polymerase fidelity in the construction of representational, full-length cDNA libraries. We will compare selected HIV-1 RT mutants to identify the RT that displays the greatest increase in processivity and introduce point mutations that are known to increase the fidelity of DNA synthesis. Using procedures that we will develop to evaluate the representativeness of the DNA products, we will test a variety of conditions that will augment the representativeness of the cDNAs synthesized. These will include the use of two different RTs to saturate binding to mRNAs, addition of viral nucleocapsid protein to further increase the processivity as well as the use of a 5'-m7G cap-affinity procedure to enrich full-length mRNAs. Following the identification of the best RT enzyme and the optimization of conditions for processive synthesis, we will attempt to construct full-length representational cDNA libraries from transformed cell lines.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA076922-02
Application #
2796372
Study Section
Special Emphasis Panel (ZCA1-RLB-Y (O3))
Program Officer
Couch, Jennifer A
Project Start
1997-09-30
Project End
2000-09-29
Budget Start
1998-09-30
Budget End
1999-09-29
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461