In order to study many different types of genes and genetic elements, genetic procedures that can be used conveniently and rapidly are being developed. Intermeshed with this approach the specific subject of DNA transposition is being studied, both for a fundamental understanding of the process and as a basis for further genetic applications. Because of the ease of manipulations in E. coli, these studies are started in E. coli and are further engineered for applications in other organisms including yeast and higher eukaryotic cells. Many of the procedures used involve the construction of special vectors and transposons that can be used to fuse parts of well-characterized genes to the complementary parts of other genes. This gene fusion process can be used to identify gene segments or domains, and the resulting gene fusions can be used for further genetic and biochemical studies. To increase the versatility of gene fusions, new types of genes are being sought with properties different from the most commonly used gene, the E. coli B-galactosidase gene. These include genes for other hydrolytic enzymes which like B-galactosidase have a wide range of colorimetric substrates, as well as composite or hybrid genes between the B-galactosidase gene and other types of genes such as for aminoglycoside resistance or thymidine kinase. Studies and applications of transposable elements are focusing on the Tn3 and the bacteriophage Mu transposons. Of particular interest are the transposon terminal sequences where the transposase proteins bind, and the aberrant transposition structures that are formed when these terminal sequences are altered. New technical applications can be developed with transposon termini by incorporating between them various genetic entities such as promoters, structural genes, replicons, recombination sites, packaging sites, and transfer origins. The resulting transposable constructs have many possible applications, including the formation of various types of gene fusions, DNA cloning entirely in vivo, directed DNA cloning (walking), localized recombination, site directed DNA transfer, new types of genetic selections, and rapid DNA sequencing. Studies of aberrant transposition products will increase our understanding of the process of DNA transposition and may allow the development of new applications for transposable elements. Additional experiments proposed involve testing transposition of Tn3 and Mu elements in other species to extend their uses to higher species, as has been done with the B-galactosidase gene-fusion technology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029067-09
Application #
3276534
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-04-01
Project End
1991-03-31
Budget Start
1989-04-01
Budget End
1991-03-31
Support Year
9
Fiscal Year
1989
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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