Conditional dihydrofolate reductase deficiency due to transposon Tn5tac1 insertion downstream from the folA gene in Escherichia coli

doi:10.1016/0378-1119(93)90747-Q

Gene

Volume 125, Issue 1, 15 March 1993, Pages 69-73

https://doi.org/10.1016/0378-1119(93)90747-Q Get rights and content

Abstract

Transposon Tn5 tac 1 can generate conditional mutations by virtue of an outward-facing tac promoter, which is regulated by the lac repressor and isopropyl-β-D-thiogalactopyranoside (IPTG). We report here on a Tn5 tac 1 insertion in Escherichia coli that results in a conditional (IPTG-elicited) folA mutant phenotype: During aerobic growth, IPTG caused decreased synthesis of dihydrofolate reductase (DHFR; encoded by the folA gene) and hypersensitivity to trimethoprim (a DHFR inhibitor); during anaerobic growth, IPTG elicited auxotrophy that was satisfied by thymine or glycine or threonine. The Tn5 tacl insertion was downstream from folA, with the tac promoter pointing into the gene (antisense direction). Complementation tests indicated that the conditional folA deficiency was a cis effect of transcription from the tac promoter, perhaps due to head-to-head collision between converging RNA polymerases.

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4 Isolation and Development of Transposons
1999, Methods in Microbiology
Mutant isolation is central to the genetic analysis of any organism and, in the last twenty years, transposable elements have become indispensable tools for this purpose, particularly in the context of where there is little or no information about the nature of the genes under investigation. Transposition is a DNA recombination reaction resulting in translocation of a discrete DNA segment termed the transposable element, insertion sequence or transposon from a donor site to one of many non-homologous target sites. By virtue of this insertion into coding sequences, these elements provide a means of potential random mutagenesis, with the inherent advantage to the investigator that the mutated gene is “tagged” by the element. Transposon-based genetic tools have been developed and exploited to the greatest extent in bacteria, and are now an essential part of the armory to genetically dissect these organisms. As it diversifies and investigates a broader spectrum of microbial species, so it will become necessary to continue these developments. Likewise, new applications can be expected to follow from technical advances and the wealth of information generated by genome-sequencing projects. These will all impinge on our understanding of the biology of the prokaryotes, how bacteria can be exploited in biotechnology, and the approaches we adopt to combat bacterial diseases.
Insertion of transposon Tn5tac1 in the Sinorhizobium meliloti malate dehydrogenase (mdh) gene results in conditional polar effects on downstream TCA cycle genes
2004, Molecular Plant-Microbe Interactions
The 3' untranslated regions of chloroplast genes in Chlamydomonas reinhardtii do not serve as efficient transcriptional terminators
1996, Molecular and General Genetics

^∗: Present addresses: (A.F.N.) National Center for Biotechnology Information, National Library of Medicine, Building 38A, National Institutes of Health, 8600 Rockville Pike, Bethesda, MD 20894, USA. Tel. (301) 496-2475; (B.R.K.) Department of Medicine, Washington University Medical School, Box 8122, 660 S. Euclid Ave., St. Louis, MO 63110, USA. Tel. (314) 362-9038; (P.M.A.) Amersham Corp., 2636 S. Clearbrook Dr., Arlington Heights, IL 60005, USA. Tel. (708) 593-6300, ext. 467.

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Abstract

References (30)

Patterns of convergent and overlapping transcription in the b2 region of coliphage λ

Virology

Sensitive mutants of bacteriophage λ

Virology

Antisense RNA: its functions and applications in gene regulation — a review

Gene

The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library

Cell

Serine transhydroxymethylase: identification as the threonine and allothreonine aldolases

J. Biol. Chem.

Naturally occurring antisense RNA control — a brief review

Gene

Acetylornithinase of Escherichia coli: partial purification and some properties

J. Biol. Chem.

Convergent transcription in bacterio-phage lambda: interference with gene expression

J. Mol. Biol.

Construction of a fol mutant strain of Escherichia coli for use in dihydrofolate reductase mutagenesis experiments

J. Bacteriol.

Effects of point mutations in a hinge region on the stability, folding, and enzymatic actvity of Escherichia coli dihydrofolate reductase

Biochemistry

Linkage map of Escherichia coli K-12

Microbiol. Rev.

Transposable elements and the genetic engineering of bacteria

The biochemistry of folic acid and related pteridines

The gene for Escherichia coli diadenosine tetraphosphatase is located immediately clockwise tofolA and forms an operon with ksgA

Mol. Gen. Genet.

Tn5 tac l, a derivative of transposon Tn5 that generates conditional mutations

Cited by (3)

4 Isolation and Development of Transposons

Insertion of transposon Tn5tac1 in the Sinorhizobium meliloti malate dehydrogenase (mdh) gene results in conditional polar effects on downstream TCA cycle genes

The 3' untranslated regions of chloroplast genes in Chlamydomonas reinhardtii do not serve as efficient transcriptional terminators

Short communicationConditional dihydrofolate reductase deficiency due to transposon Tn5tac1 insertion downstream from the folA gene in Escherichia coli

Abstract

Virology

Virology

Gene

Cell

J. Biol. Chem.

Gene

J. Biol. Chem.

J. Mol. Biol.

Construction of a fol mutant strain of Escherichia coli for use in dihydrofolate reductase mutagenesis experiments

J. Bacteriol.

Effects of point mutations in a hinge region on the stability, folding, and enzymatic actvity of Escherichia coli dihydrofolate reductase

Biochemistry

Linkage map of Escherichia coli K-12

Microbiol. Rev.

Transposable elements and the genetic engineering of bacteria

The biochemistry of folic acid and related pteridines

The gene for Escherichia coli diadenosine tetraphosphatase is located immediately clockwise tofolA and forms an operon with ksgA

Mol. Gen. Genet.

Tn5 tac l, a derivative of transposon Tn5 that generates conditional mutations

Short communication
Conditional dihydrofolate reductase deficiency due to transposon Tn5tac1 insertion downstream from the folA gene in Escherichia coli