Biomedical subjects
E Eisenstadt
Publications and source records attributed to E Eisenstadt.
Salmonella typhimurium LT7 and LT2 strains carrying the imp operon on colIa.
The imp operon is carried on a transmissible plasmid, ColIa, in original isolates of Salmonella typhimurium LT7. LT2 strain recipients of F' factors from LT7 strains harboring ColIa can acquire ColIa and imp under nonselective conditions. Thus, S. typhimurium LT2 strains that have received plasmids by conjugal transfer from LT7 strains might be inadvertently harboring ColI factors.
Mutations in topA interfere with the inducible expression of DNA damage response loci in Salmonella typhimurium.
Strains of Salmonella typhimurium deficient in topoisomerase I activity (topA mutants) are UV sensitive and non-mutable (Overbye and Margolin: J Bacteriol 146:170-178, 1981). Using lac-operon fusions to DNA damage inducible (din) loci we investigated whether these observations could be explained by an inability of topA strains to efficiently induce DNA damage responses. Mitomycin C (MMC)-induced expression of lac-operon fusions to uvrB and to a second SOS locus, din-9, was largely eliminated in topA bacteria. The inducible expression of several other din-fusions was also diminished. This inducibility defect was mimicked by growth of din-9 topA+ bacteria in media of high osmolarity, a condition that leads to increased DNA supercoiling. Inhibitors of DNA gyrase efficiently induced din-9 in topA bacteria. Together, these results suggest that the topA effect on din expression may be mediated at the level of DNA supercoiling. The sensitivities of a number of din-fusions to topA paralleled the degree to which they were repressed by excess LexA, suggesting that mutations in topA might influence LexA-operator interactions and/or increase lexA expression.
UV mutagenesis in Salmonella typhimurium is umuDC dependent despite the presence of samAB.
We investigated the role of the umuDC and samAB operons in the UV mutability of Salmonella typhimurium. umuDC is located on the chromosome, whereas samAB resides on the virulence plasmid pSLT. Using allele replacement and plasmid curing techniques, we found that UV mutability was eliminated when any of three different umuDC alleles (umuD1, umuC1, or umuD1 umuC1) were on the chromosome even when samAB was present. We conclude that samAB normally does not complement umuDC function in S. typhimurium.
Induction and cleavage of Salmonella typhimurium UmuD protein.
SOS mutagenesis in prokaryotes is dependent upon the inducible activity of the chromosomally encoded UmuDC proteins, or homologous proteins such as MucAB or ImpCAB which are found on naturally occurring plasmids. Relative to Escherichia coli, however, Salmonella typhimurium is much less responsive to the mutagenic effects of DNA-damaging agents, despite the fact that it possesses both chromosomally and plasmid encoded umu-like operons. In E. coli, activation of the UmuD mutagenesis protein to UmuD' via RecA-mediated proteolysis is a critical step in the mutation fixation pathway. We have used a polyclonal antiserum raised against the E. coli UmuD and UmuD' proteins to show that S. typhimurium expresses cross-reacting material only after treatment with the DNA-damaging agent mitomycin C. The S. typhimurium umuDC operon, therefore, appears to be regulated by mechanisms similar to the E. coli umuDC operon. After induction, the S. typhimurium UmuD protein was processed to UmuD' in both S. typhimurium and E. coli. However, the S. typhimurium UmuD protein appears to be cleaved more efficiently than the E. coli UmuD protein under similar conditions. The data suggest that conversion of UmuD to the mutagenically active UmuD' is not the rate-limiting factor accounting for the weakly mutable phenotype of S. typhimurium.
DNA damage-inducible loci in Salmonella typhimurium.
lac operon fusions to DNA damage-inducible (din) loci were generated in Salmonella typhimurium LT2. Many of these din fusions were efficiently repressed by cloned Escherichia coli LexA, while others were not; all required RecA for induction. Several din fusions exhibited strong inducibility and will be useful in developing an SOS induction assay in S. typhimurium to detect genotoxins.
Sequence analysis and mapping of the Salmonella typhimurium LT2 umuDC operon.
In Escherichia coli, efficient mutagenesis by UV requires the umuDC operon. A deficiency in umuDC activity is believed to be responsible for the relatively weak UV mutability of Salmonella typhimurium LT2 compared with that of E. coli. To begin evaluating this hypothesis and the evolutionary relationships among umuDC-related sequences, we cloned and sequenced the S. typhimurium umuDC operon. S. typhimurium umuDC restored mutability to umuD and umuC mutants of E. coli. DNA sequence analysis of 2,497 base pairs (bp) identified two nonoverlapping open reading frames spanning 1,691 bp that were were 67 and 72% identical at the nucleotide sequence level to the umuD and umuC sequences, respectively, from E. coli. The sequences encoded proteins whose deduced primary structures were 73 and 84% identical to the E. coli umuD and umuC gene products, respectively. The two bacterial umuDC sequences were more similar to each other than to mucAB, a plasmid-borne umuDC homolog. The umuD product retained the Cys-24--Gly-25, Ser-60, and Lys-97 amino acid residues believed to be critical for RecA-mediated proteolytic activation of UmuD. The presence of a LexA box 17 bp upstream from the UmuD initiation codon suggests that this operon is a member of an SOS regulon. Mu d-P22 inserts were used to locate the S. typhimurium umuDC operon to a region between 35.9 and 40 min on the S. typhimurium chromosome. In E. coli, umuDC is located at 26 min. The umuDC locus in S. typhimurium thus appears to be near one end of a chromosomal inversion that distinguishes gene order in the 25- to 35-min regions of the E. coli and S. typhimurium chromosomes. It is likely, therefore, that the umuDC operon was present in a common ancestor before S. typhimurium and E. coli diverged approximately 150 million years ago. These results provide new information for investigating the structure, function, and evolutionary origins of umuDC and for exploring the genetic basis for the mutability differences between S. typhimurium and E. coli.
Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium.
Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.
Identification of a umuDC locus in Salmonella typhimurium LT2.
The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV light and many other DNA-damaging agents. The existence of a umuDC analog in Salmonella typhimurium has been questioned. With DNA probes to the E. coli umuD and umuC genes, we detected, by Southern blot hybridization, sequences similar to both of these genes in S. typhimurium LT2. We also confirmed that the presence of cloned E. coli umuD enhances the UV mutability and resistance of S. typhimurium. Our data strongly suggest that S. typhimurium contains a functional umuDC operon.
Induction of base substitution mutations by aflatoxin B1 is mucAB dependent in Escherichia coli.
Recovery of aflatoxin B1-induced base substitution mutations in Escherichia coli was almost completely dependent on the presence of the SOS-mutagenesis-enhancing operon mucAB+; the normal E. coli analog, umuDC+, was not sufficient. Yet aflatoxin B1 induced the SOS response, including the umuDC operon, as well as did UV light. Neither preinduction of the SOS response nor the presence of additional copies of umuDC+ allowed the recovery of aflatoxin B1-induced base substitutions. Thus, the premutagenic DNA lesions induced by aflatoxin B1 reveal a functional difference between UmuDC and MucAB. We estimate that in the presence of MucAB the probability that aflatoxin B1-induced DNA lesions will be converted into mutations is increased at least 10-fold.
Suppressible base substitution mutations induced by angelicin (isopsoralen) in the Escherichia coli lacI gene: implications for the mechanism of SOS mutagenesis.
Angelicin- plus near-UV-induced mutations were umuC dependent in Escherichia coli K-12. Angelicin, a monofunctional psoralen derivative, is believed to damage DNA almost exclusively at pyrimidine bases. To broaden our knowledge about the mutagenic specificity of SOS-dependent mutagens, we determined the mutational specificity of 233 suppressible lacI mutations induced by angelicin. More than 90% of the nonsense mutations arose via transversion substitutions. The three most frequently mutated sites were at A-T base pairs and accounted for more than one-third of all induced nonsense mutations. The two hottest sites were at the only occurrences of the 5'-TATA-3' tetranucleotide in lacI, a sequence expected to be a preferred binding site for a psoralen. Both A-T-to-T-A and A-T-to-C-G transversions were well induced by angelicin treatment, but the frequency of each transversion depended on the particular site. We also detected significant induction of transversion mutations at G-C sites. The induction of transversions by an SOS-dependent mutagen that generates lesions at pyrimidines supports the idea that DNA lesions influence the selection of bases that are incorporated via the process of SOS repair.
An inducible pathway is required for mutagenesis in Salmonella typhimurium LT2.
UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA+ gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate (K. Overbye and P. Margolin, J. Bacteriol. 146:170-178, 1981; K. Overbye, S. M. Basu, and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 47:785-791, 1983). Mitomycin C induction of the phi(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead us to deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability.
Enhanced sensitivity of Escherichia coli umuC to photodynamic inactivation by angelicin (isopsoralen).
Escherichia coli umuC cells were inactivated four times more rapidly than umuC+ cells by angelicin (a monofunctional psoralen) plus near-UV irradiation. With other DNA-damaging treatments, either no or much smaller differences in sensitivity were observed. These results show that functions associated with the UmuC+ phenotype contribute to the repair (or tolerance) of some categories of DNA damage more efficiently than others.
Induction of transversion mutations in Escherichia coli by N-methyl-N'-nitro-N-nitrosoguanidine is SOS dependent.
Escherichia coli alkA mutants, which are deficient for an inducible DNA glycosylase, 3-methyladenine-DNA glycosylase II, are sensitive to mutagenesis by low doses of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). As many as 90% of the alkA-dependent mutations induced by MNNG are also umuC+ dependent and thus are due to DNA lesions that are substrates for the mutagenic functions of the SOS response. A great number of these mutations are base substitutions at A . T sites, particularly A . T transversions. We discuss which DNA lesions may be responsible for these mutations. Our results show that the induction of 3-methyladenine-DNA glycosylase II, which occurs as part of the adaptive response to alkylating agents such as MNNG, significantly reduces the mutagenicity as well as the lethality of alkylation damage.
A simple modification of the Salmonella liquid-incubation assay. Increased sensitivity for detecting mutagens in human urine.
A simple modification of the Salmonella/microsome liquid-incubation procedure improves the sensitivity of the assay for detecting mutagens in human urine. Extracts from cigarette smokers' urine were used as a model complex mutagenic mixture for validation of the assay. The modification consists of adding increased numbers of bacterial cells (approximately 10(9] in a concentrated suspension to liver homogenate mix and urine extract, all in 0.2-ml volume. After 90 min incubation at 37 degrees C, the mixture is processed according to the standard Ames test protocol. This procedure is 20 times more sensitive than the standard plate-incorporation test and 13 times more sensitive than a previously reported liquid-incubation protocol. The number of spontaneous revertants did not increase under these conditions and, compared to the plate-incorporation test, 10-fold less liver homogenate and 5-fold less enzymatic cofactors were needed per plate. The procedure was approximately 14 times more sensitive in detecting the mutagenic activity of benzo[ a ]pyrene. We also used the modification to determine mutagenic activity in urine from a group of nonsmokers. The method may be generally useful for investigations of mutagenic activity in human urine samples.
Base substitution mutations induced by metabolically activated aflatoxin B1.
We have determined the base substitutions generated by metabolically activated aflatoxin B1 in the lacI gene of a uvrB- strain of Escherichia coli. By monitoring over 70 different nonsense mutation sites, we show that activated aflatoxin B1 specifically induced GxC leads to TxA transversions. One possible pathway leading to this base change involves depurination at guanine residues. We consider this mechanism of mutagenesis in the light of our other findings that the carcinogens benzo[a]pyrene diol epoxide and N-acetoxyacetylaminofluorene also specifically induce GxC leads to TxA transversions.
Distribution and specificity of mutations induced by neocarzinostatin in the lacI gene of Escherichia coli.
Although neocarzinostatin (NCS) attacks DNA almost exclusively at adenine and thymine residues in vitro, exposure of Escherichia coli to this antitumor drug resulted in a high frequency of mutations at guanine:cytosine base pairs in the lacI gene. Thus, NCS-induced base substitution mutations do not appear to result from the major DNA lesions that have been biochemically characterized. The overall distribution of nonsense mutations produced by NCS was distinctly nonrandom, consisting in part of a few "hotspots" and a large number of "coldspots." The existence of these coldspots implies that untargeted mutagenesis does not make a significant contribution to the mutations induced by this SOS-dependent mutagen.
Biological assays for mutagens in human samples.
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