Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11.
Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.
Johnson
J M
JM
Department of Microbiology, Southern Illinois University, Carbondale, IL 62901-6508, USA.
Ding
W
W
Henkhaus
J
J
Fix
D
D
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D002875:Chromosomes; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics*; D005038:Ethylnitrosourea; D007763:Lac Operon; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009153:Mutagens; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000378:metabolism
D029968:Escherichia coli Proteins; D009153:Mutagens; C440169:tas protein, E coli; D001616:beta-Galactosidase; D005038:Ethylnitrosourea