Genetic instability associated with the aroC321 allele in Salmonella typhimurium involves genetic duplication

Strains of Salmonella typhimurium that contain the aroC321 allele require phenylalanine, tyrosine, and tryptophan for growth but revert to tryptophan-prototrophy at high frequencies (about 10(-4) per cell plated). The Trp+ derivatives remain auxotrophic for phenylalanine and tyrosine and are genetically unstable, in that they readily give rise to cells that require all three aromatic amino acids. On the basis of growth characteristics and genetic instability, it has been proposed that reversion to tryptophan-prototrophy in aroC321 strains occurs by genetic duplication. This paper provides genetic evidence in support of that hypothesis. The data indicate, moreover, that the tryptophan prototrophs contain a duplication that extends at least from glpT to xyl, a region of greater than 30% of the Salmonella chromosome. The aroC locus is found within the duplicated region, and aroC321/aroC321 merodiploids apparently grow as tryptophan prototrophs because of a gene-dosage effect.     

Lowered induction of genetic tandem duplications in Salmonella by the pKM101 plasmid

Summary Selection for 3-amino-1,2,4-triazole (AT) resistance in certain strains of Salmonella typhimurium has been previously shown to select for genetic tandem duplications of the histidine operon. We show here that agents which induce tandem duplications are less effective in such induction in the presence of the pKM101 plasmid. The presence of the plasmid also produces an increase in AT-resistance due to mechanisms other than duplication, presumably because pKM101 produces high levels of error-prone repair. We suggest that high levels of error-prone repair may cause decreases in tandem duplication induction and propose that error-prone repair and tandem duplication may be alternative cellular responses to certain DNA lesions.     

Induction of genetic duplications and frameshift mutations in Salmonella typhimurium by acridines and acridine mustards: dependence on covalent binding of the mutagen to DNA

Summary The aroC321 allele permits positive selection for the detection of a large genetic duplication that arises in the Salmonella typhimurium chromosome by homologous recombination. Strains that contain both aroC321 and the hisC3076 allele were constructed so that the induction of genetic duplications and frameshift mutations in a run of GC base pairs could be studied simultaneously by selecting for tryptophan and histidine prototrophy, respectively. Using these strains, we examined the ability of 9-aminoacridine, quinacrine, four acridine mustards (ICR-170, ICR-191, ICR-372, and quinacrine mustard) and the nitroacridine Entozon to induce genetic duplications and frameshift mutations. Although all these compounds induce reversion of hisC3076, only the four mustards and Entozon are effective as inducers of genetic duplications under identical treatment conditions. The induction of genetic duplications by acridine mustards, like the toxic and mutagenic effects of these compounds, is enhanced by a deficiency for excision repair caused by a deletion through the uvrB gene. The ineffectiveness of 9-aminoacridine and quinacrine in the test for genetic duplications indicates that simple intercalation is sufficient for the mutagenic effect measured with the hisC3076 allele but that the induction of duplications by the acridine mustards and Entozon requires covalent binding of the chemical to DNA.     

Duplication Mutation as an Sos Response in Escherichia Coli: Enhanced Duplication Formation by a Constitutively Activated Reca

The SOS response in Escherichia coli involves the induction of a multioperon regulatory system, which copes with the presence of DNA lesions that interfere with DNA replication. Induction depends on activation of the RecA protein to cleave the LexA repressor of SOS operons. In addition to inducible DNA repair, the SOS system produces a large increase in the frequency of point mutations. To examine the possibility that other types of mutations are induced as part of the SOS response, we have studied the production of tandem duplications. To avoid the complications of indirect effects of the DNA lesions, we have activated the SOS response by a constitutive mutation in the recA gene, recA730. The introduction of the recA730 mutation results in an increase in duplications in the range of tenfold or greater, as judged by two different criteria. Based on its genetic requirements, the pathway for induced duplication formation is distinct from the point mutation pathway and also differs from the major normal recombination pathway. The induction of pathways for both duplications and point mutations shows that the SOS system produces a broad mutagenic response. We have suggested previously that many types of mutations might be induced by severe environmental stress, thereby enhancing genetic variation in an endangered population.     

Genetic duplications in bacteria and their relevance for genetic toxicology

Tandem genetic duplications of various lengths occur at high frequency and at many chromosomal locations in bacteria. Most duplications are formed and lost by recombinational mechanisms. Since they readily give rise to haploid segregants, duplications are characteristically unstable. Various selection procedures permit measurements of duplication frequencies, and several mutagens have been shown to induce the formation of duplications in haploid and the loss of duplications from merodiploid bacteria. Although the data base is not extensive, it includes agents that interact with DNA by a variety of molecular mechanisms. Grounds on which the induction of genetic duplications in bacteria can be relevant for genetic toxicology are discussed.     

Effects of chemical and physical mutagens on the frequency of a large genetic duplication in Salmonella typhimurium. I. Induction of duplications.

In Salmonella typhimurium a simple selection has been described to detect bacteria that are merodiploid for almost one-third of the chromosome. The selective procedure is based upon improved utilization of L-malate as the sole carbon source in merodiploid strains. The spontaneous frequency of the duplication in haploid strains is approximately 10(-4) per cell plated. Following the exposure of a haploid strain to mutagenic agents, there is a dose-dependent increase in the duplication frequency above the spontaneous level. In this paper we describe the induction of genetic duplications in Salmonella typhimurium by X-rays, ultraviolet light (UV), ethyl methanesulfonate (EMS), nitrous acid, and the azaacridine half mustard, ICR-372.     

Modification of chromosome instability in Aspergillus nidulans

Strains of Aspergillus nidulans with a chromosome segment in duplicate show instability at mitosis; their colonies produce faster-growing sectors which arise from nuclei with spontaneous deletions in either duplicate segment. In an attempt to probe the deletion process, the effects of mutations causing sensitivity to UV treatment, and those of manganous ions, have been studied in strains carrying either Dp(I,II) or Dp(III,VIII). For comparison, the effects of Mn2+ on balanced and unbalanced diploids have also been examined. The uvsE allele, which decreases intragenic mitotic crossing over in diploids, increased deletion frequency in strains with either duplication. The uvsB allele, which increases intragenic mitotic crossing over in diploids, increased deletion frequency only in Dp(I,II) strains; in addition, by causing early mitotic crossing over between the homologous segments, it produced some novel deletion products. Mn2+ substantially decreased the deletion frequency in Dp(I,II) strains and decreased mitotic crossing over in diploids; it had no effect on Dp(III,VIII) strains. The results suggest that in haploid duplication strains there are two classes of spontaneous DNA lesions, recombinogenic and non-recombinogenic, both of which, failing repair, lead to deletion.     

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli III. Enhancement of excision repair in UV-treated bacteria

Summary The question of whether induction of the SOS response in Escherichia coli increases the efficiency of excision repair was addressed by measuring repair of UV-damaged nonreplicating lambda phage DNA in previously irradiated bacteria. Prior UV irradiation of lex ⁺ bacteria enhanced both the rate of regeneration of infective phage DNA (about 10-fold) and the rate of cyclobutane dimer removal early in repressed infections. Indirect induction of SOS-regulated repair activities by the nonreplicating irradiated phage DNA itself seemed negligible. Prior bacterial irradiation reduced the frequency of recombination (loss of a tandem chromosomal duplication) of nonreplicating UV-irradiated DNA. In this respect UV-stimulated recombination of nonreplicating DNA differs from RecF-dependent recombination processes that are stimulated by increased SOS expression.Surprisingly, prior UV irradiation of lexA3 bacteria caused a small but reproducible increase in the regeneration of infective phage DNA.     

The characteristics of amino acid catabolism in bacteria of the genus Citrobacter

The comparison of the occurrence of enzymes effecting the deamination of dicarbon, aromatic and oxyamino acids, as well as transamination enzymes, in Citrobacter bacteria and the activity levels of these enzymes was made. The constant sign of such bacteria was the presence of serine and threonine dehydratase activity. 92% of the strains showed the presence of phenylalanine deaminase. No tryptophan activity was established. 96-98% of Citrobacter strains possessed phenylalanine, tyrosine and tryptophan aminotransferases with alpha-ketoglutaric acid functioning as the acceptor of the amino group.     

Recombinations occurring in the process of DNA replication in Escherichia coli

In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.     

Mutagenesis by normal metabolites in Escherichia coli: phenylalanine mutagenesis is dependent on error-prone DNA repair

In search of a model for the production of 'spontaneous' mutations induced by DNA damage produced during normal metabolism, 19 amino acids were tested for mutagenicity in Escherichia coli K-12 uvrB. Cystine, and, to a lesser extent, arginine and threonine were found to be antimutagenic; only phenylalanine was found to be mutagenic. At 2 mM, phenylalanine induced mutants at 1.5-2-fold above background [lacZ53(amber)----Lac+, rifampicin resistance (missense), and bacteriophage T6 resistance]. Tyrosine and, to a lesser extent, tryptophan (each at 2 mM) inhibited the mutagenicity of phenylalanine. Phenylalanine mutagenesis was detected in the uvrB strain, but not in the wild-type, uvrB umuC or uvrB lexA strains. Thus, phenylalanine seems to cause the production of excisable lesions ('UV-like'?) in DNA, which, if not excised, can induce mutations via error-prone DNA repair.     

Effect of nucleotide excision repair in human cells on intrachromosomal homologous recombination induced by UV and 1-nitrosopyrene.

To study the role of nucleotide excision repair in the induction of intrachromosomal homologous recombination in mammalian cells, we introduced a plasmid containing a substrate for recombination into three human cell lines that differ in their repair capacity and compared the frequency of recombination induced by UV radiation and by 1-nitrosopyrene. One strain had a normal capacity for nucleotide excision repair, the second exhibited an intermediate rate of repair, and the third, derived from a patient with xeroderma pigmentosum, had no ability to repair UV- or 1-nitrosopyrene-induced DNA damage. The endogenous thymidine kinase genes in these cell strains had been inactivated, and the cells contained an integrated copy of a plasmid carrying duplicated copies of the herpes simplex virus type 1 thymidine kinase (Htk) gene, each inactivated by an 8-base-pair XhoI site inserted at a unique site. A functional tk gene can only be generated by a productive recombination event between the two Htk genes. In all three stains, UV and 1-nitrosopyrene induced dose-dependent increases in the frequency of recombinants. However, the doses required to cause a specific increase in recombination in the repair-deficient strains were 10 to 30 times lower than the dose required for the cell strain with a normal capacity for repair. These results strongly suggest that unexcised DNA lesions, rather than excision repair per se, stimulate intrachromosomal homologous recombination. Southern blot analysis of DNA from representative recombinants indicated that in all cases one of the two Htk genes had become wild type (XhoI resistant). The majority (90%) retained the Htk duplication, consistent with nonreciprocal transfer of genetic information (gene conversion).     

The function of recombinations occurring in the process of DNA replication in Escherichia coli

In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.     

Ultraviolet B-Sensitive Rice Cultivar Deficient in Cyclobutyl Pyrimidine Dimer Repair

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair.     

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.     

Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I.

The induction of mutations to valine resistance and to rifampin resistance occurs after UV irradiation in bacteria carrying a deletion through the polA gene (delta polA), showing that DNA polymerase I (PolI) is not an essential enzyme for this process. The PolI deletion strain showed a 7- to 10-fold-higher spontaneous mutation frequency than the wild type. The presence in the deletion strain of the 5'----3' exonuclease fragment on an F' episome caused an additional 10-fold increase in spontaneous mutation frequency, resulting in mutation frequencies on the order of 50- to 100-fold greater than wild type. The mutator effect associated with the 5'----3' exonuclease gene fragment together with much of the effect attributable to the polA deletion was blocked in bacteria carrying a umuC mutation. The mutator activity therefore appears to reflect constitutive SOS induction. Excision-proficient polA deletion strains exhibited increased sensitivity to the lethal effect of UV light which was only partially ameliorated by the presence of polA+ on an F' episome. The UV-induced mutation rate to rifampin resistance was marginally lower in delta polA bacteria than in bacteria carrying the polA+ allele. This effect is unlikely to be caused by the existence of a PolI-dependent mutagenic pathway and is probably an indirect effect caused by an alteration in the pattern of excision repair, since it did not occur in excision-deficient (uvrA) bacteria. An excision-deficient polA deletion strain possessed UV sensitivity similar to that of an isogenic strain carrying polA+ on an F' episome, showing that none of the functions of PolI are needed for postreplication repair in the absence of excision repair. Our data provide no evidence for a pathway of UV mutagenesis dependent on PolI, although it remains an open question whether PolI is able to participate when it is present.     

Conserved motifs II to VI of DNA helicase II from Escherichia coli are all required for biological activity.

There are seven conserved motifs (IA, IB, and II to VI) in DNA helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed.     

Length Variation in Mitochondrial DNA of the Minnow Cyprinella Spiloptera

Length differences in animal mitochondrial DNA (mtDNA) are common, frequently due to variation in copy number of direct tandem duplications. While such duplications appear to form without great difficulty in some taxonomic groups, they appear to be relatively short-lived, as typical duplication products are geographically restricted within species and infrequently shared among species. To better understand such length variation, we have studied a tandem and direct duplication of approximately 260 bp in the control region of the cyprinid fish, Cyprinella spiloptera. Restriction site analysis of 38 individuals was used to characterize population structure and the distribution of variation in repeat copy number. This revealed two length variants, including individuals with two or three copies of the repeat, and little geographic structure among populations. No standard length (single copy) genomes were found and heteroplasmy, a common feature of length variation in other taxa, was absent. Nucleotide sequence of tandem duplications and flanking regions localized duplication junctions in the phenylalanine tRNA and near the origin of replication. The locations of these junctions and the stability of folded repeat copies support the hypothesized importance of secondary structures in models of duplication formation.