The proposed experiments are directed towards a molecular understanding of the specialized recombination events attributeable to the prokaryotic kanamycin resistance transposon Tn5 in E. coli and, more generally, to the understanidng of the evolution of antibiotic resistance transposons and their component genes. We will employ genetic and molecular approaches to: (19 generate a transposase gene (tnp)-1ac operon fusion in Tn5 and use it to learn how tnp transcription is regulated, obtain tnp nonsense mutants, and produce large quantities of transposase protein in vivo: (2) determine conditions under which the Tn5 encoded transpospase limits the rate of transposition in vivo: (3) detect transposase activity in vitro; (4) test current models of transposon stimulated deletion formation; (5) examine the structural, physiological and genetic factors which control the rate of Tn5 excision; (6) learn whether components of Tn5 retain the capacity for transposition by themselves, in the absence of intact Tn5: and (7) detect and characterize new aminoglycoside resistance transposons in Gram negative clinical isolates.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Washington University
Schools of Medicine
Saint Louis
United States
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Krishnan, B R; Kersulyte, D; Brikun, I et al. (1993) Transposon-based and polymerase chain reaction-based sequencing of DNAs cloned in lambda phage. Methods Enzymol 218:258-79
Neuwald, A F; Krishnan, B R; Brikun, I et al. (1992) cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth. J Bacteriol 174:415-25
Kersulyte, D; Krishnan, B R; Berg, D E (1992) Nonrandom orientation of transposon Tn5supF insertions in phage lambda. Gene 114:91-6
Weston-Hafer, K; Berg, D E (1991) Deletions in plasmid pBR322: replication slippage involving leading and lagging strands. Genetics 127:649-55
Lodge, J K; Weston-Hafer, K; Berg, D E (1991) Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA. Mol Gen Genet 228:312-5
Kulakauskas, S; Wikstrom, P M; Berg, D E (1991) Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome. J Bacteriol 173:2633-8
Krishnan, B R; Blakesley, R W; Berg, D E (1991) Linear amplification DNA sequencing directly from single phage plaques and bacterial colonies. Nucleic Acids Res 19:1153
Phadnis, S H; Kulakauskas, S; Krishnan, B R et al. (1991) Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage. J Bacteriol 173:896-9
Weston-Hafer, K; Berg, D E (1991) Limits to the role of palindromy in deletion formation. J Bacteriol 173:315-8
Krishnan, B R; Kersulyte, D; Brikun, I et al. (1991) Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones. Nucleic Acids Res 19:6177-82

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