The mechanism of generalized genetic recombination carried out by the bacteriophage P22 essential recombination function (erf gene) will be studied by biochemical and genetic means. The interaction of purified Erf protein with DNA will be examined by gel electrophoretic techniques and enzymatic probes of the state of the DNA. Fragments of the Erf protein, generated by proteolysis in vitro and by chain terminating mutations in vivo, will be purified. Their structural and DNA binding activities will be examined in parallel with those of intact Erf. The abc gene, a newly discovered P22 recombination-related gene, will be sequenced, and expressed at high levels by genetic engineering techniques. The Abc protein will be purified and its interaction with DNA, Erf protein, and possibly, host cell recombination functions will be examined. Similar studies of Y, another new recombination-related gene of P22, will be carried out. erf-dependent supercoiling of linear, terminally repetitious P22 chromosomes will be examined in order to determine which phage and host cell components are necessary for this process. Attempts will be made to assay this process in vitro, in order to permit reconstruction of the erf recombination system from purified components. Such a reconstruction would greatly facilitate studies of mechanism. The long-term goal of this research is an understanding of the mechanisms of genetic recombination. Recombination is a process of fundamental importance in the generation of genetic variation and, in higher organisms, of diversity in the immune response.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI018234-05
Application #
3127776
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-07-01
Project End
1989-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
5
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
Murphy, K C (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063-71
Murphy, K C (1994) Biochemical characterization of P22 phage-modified Escherichia coli RecBCD enzyme. J Biol Chem 269:22507-16
Ranade, K; Poteete, A R (1993) A switch in translation mediated by an antisense RNA. Genes Dev 7:1498-507
Ranade, K; Poteete, A R (1993) Superinfection exclusion (sieB) genes of bacteriophages P22 and lambda. J Bacteriol 175:4712-8
Poteete, A R; Fenton, A C (1993) Efficient double-strand break-stimulated recombination promoted by the general recombination systems of phages lambda and P22. Genetics 134:1013-21
Murphy, K C; Lewis, L J (1993) Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity. J Bacteriol 175:1756-66
Poteete, A R; Fenton, A C; Semerjian, A V (1991) Bacteriophage P22 accessory recombination function. Virology 182:316-23
Murphy, K C (1991) Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme. J Bacteriol 173:5808-21
Hardy, L W; Poteete, A R (1991) Reexamination of the role of Asp20 in catalysis by bacteriophage T4 lysozyme. Biochemistry 30:9457-63
Semerjian, A V; Malloy, D C; Poteete, A R (1989) Genetic structure of the bacteriophage P22 PL operon. J Mol Biol 207:1-13

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