A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.
AUTOR(ES)
Elliott, T
RESUMO
This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=205702Documentos Relacionados
- Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.
- Single-Copy Green Fluorescent Protein Gene Fusions Allow Accurate Measurement of Salmonella Gene Expression In Vitro and during Infection of Mammalian Cells
- Conditionally Amplifiable BACs: Switching From Single-Copy to High-Copy Vectors and Genomic Clones
- Single-copy transgenic mice with chosen-site integration.
- Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization.