The objective is to understand the molecular mechanisms involved in transposition of mobile genetic elements. Specifically, we are studying transposition of bacteriophage Mu DNA. The mechanisms used in Mu transposition are, in essence, highly analogous to those used by other mobile elements ranging from prokaryotic insertion sequences and transposons, which mediate movement of antibiotic resistance determinants, to the proviral forms of eukaryotic retroviruses. The unique properties of Mu as a lysogenic virus and a transposon allow the use of biochemical studies of transposition in the entirety of a population, studies which are difficult or impossible in systems with very low frequencies of transposition. We have developed improved techniques for in vivo studies, including the synchronization of a single round of Mu replicative transposition, and we are initiating in vitro studies using purified proteins. We will focus on two broad, major avenues of research: I. Studies on the molecular mechanism of transposition, including studies on the isolation and characterization of transposition intermediates, the products of a single round of transposition, selection of target sites for transposition, the influence of transposon length, and the isolation of Mu and host mutants defective in transposition. II. Studies on the role of Mu encoded """"""""accessory functions"""""""" which stimulate Mu DNA replication and transposition, including mapping and sequencing of the accessory genes kil and arm from Mu and the closely related heteroimmune phage D108, purification of thge kil and arm proteins, and studies on the mechanism of action of the stimulatory activities of kil and arm in vivo and in vitro.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024270-12
Application #
3272170
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-01-01
Project End
1990-12-31
Budget Start
1989-01-01
Budget End
1989-12-31
Support Year
12
Fiscal Year
1989
Total Cost
Indirect Cost
Name
National Jewish Health
Department
Type
DUNS #
City
Denver
State
CO
Country
United States
Zip Code
80206
Shortridge, V D; Pato, M L; Vasil, A I et al. (1991) Physical mapping of virulence-associated genes in Pseudomonas aeruginosa by transverse alternating-field electrophoresis. Infect Immun 59:3596-603
Pato, M L; Banerjee, M; Wagonner, B T (1990) Sequence of gene E15 of bacteriophage D108 and comparison with phage Mu. Nucleic Acids Res 18:6458
Gama, M J; Toussaint, A; Pato, M L (1990) Instability of bacteriophage Mu transposase and the role of host Hfl protein. Mol Microbiol 4:1891-7
Pato, M L; Howe, M M; Higgins, N P (1990) A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition. Proc Natl Acad Sci U S A 87:8716-20
Waggoner, B T; Sultana, K; Symonds, N et al. (1989) Identification of the bacteriophage Mu kil gene. Virology 173:378-89
Waggoner, B T; Wade, T; Pato, M L (1988) Identification of the bacteriophage D108 kil gene and of the second region of sequence nonhomology with bacteriophage Mu. Gene 62:111-9
Toussaint, A; Desmet, L; Faelen, M et al. (1987) In vivo mutagenesis of bacteriophage Mu transposase. J Bacteriol 169:5700-7
Pato, M; Banerjee, M; Desmet, L et al. (1987) Involvement of heat shock proteins in bacteriophage Mu development. J Bacteriol 169:5504-9