Gene fusions can be used for basic studies of gene expression, structure, and function. Our purpose is to develop and improve methods for fusing genes in the bacterium E. coli, and to use these and other general methods to study in particular the genetic system of DNA transposition. These methods involve the fusion of transcription and/or translation gene control signals from genes under investigation to the structural part of a well-characterized gene such as the lactose operon lacZ gene for B-galactosidase. With these fusions the biochemical and genetic procedures available for a gene such as lacZ can be used to study other genes. Fusion methods involve both in vitro cloning vectors and in vivo bacteriophage Mu transposable elements containing various segments of the B-galactosidase gene. The cloning vectors are designed with technical improvements including many restriction sites, more types of gene segments, smaller sizes, and various replicons for different applications. The Mu elements will be designed for use in the chromosome, in plasmids, and with replicons inside the Mu element for autogenous replication. Mu fusion elements will also be designed for promoting transcription, with and without translation, for expression of adjacent structural genes. Studies of transposition involve the Tn3 ampicillin resistance DNA transposon and the bacteriophage Mu transposon. Tn3 studies are divided into studies fo the trpA transposase and its site of action for transposition, the trpR repressor-resolvase and its tri-partite binding site, the phenomena of transposition immunity, and an investigation of a fourth gene on Tn3. Mu studies involved identification of the genes and sites essential for high frequency replication and transposition, particularly as they pertain to Mu gene fusion elements.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029067-05
Application #
3276531
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-04-01
Project End
1986-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
5
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Roncero, C; Casadaban, M J (1992) Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus. J Bacteriol 174:3250-60
Groisman, E A; Pagratis, N; Casadaban, M J (1991) Genome mapping and protein coding region identification using bacteriophage Mu. Gene 99:1-7
Roncero, C; Sanderson, K E; Casadaban, M J (1991) Analysis of the host ranges of transposon bacteriophages Mu, MuhP1, and D108 by use of lipopolysaccharide mutants of Salmonella typhimurium LT2. J Bacteriol 173:5230-3
Castilho, B A; Casadaban, M J (1991) Specificity of mini-Mu bacteriophage insertions in a small plasmid. J Bacteriol 173:1339-43
Tu, H; Casadaban, M J (1990) The upstream activating sequence for L-leucine gene regulation in Saccharomyces cerevisiae. Nucleic Acids Res 18:3923-31
Roncero, C; Darzins, A; Casadaban, M J (1990) Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 require pili and surface growth for adsorption. J Bacteriol 172:1899-904
Kans, J A; Casadaban, M J (1989) Nucleotide sequences required for Tn3 transposition immunity. J Bacteriol 171:1904-14
Darzins, A; Casadaban, M J (1989) In vivo cloning of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage. J Bacteriol 171:3917-25
Darzins, A; Casadaban, M J (1989) Mini-D3112 bacteriophage transposable elements for genetic analysis of Pseudomonas aeruginosa. J Bacteriol 171:3909-16
Darzins, A; Kent, N E; Buckwalter, M S et al. (1988) Bacteriophage Mu sites required for transposition immunity. Proc Natl Acad Sci U S A 85:6826-30

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