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PubMed2002

The influence of sequence divergence between alleles of the human MS205 minisatellite incorporated into the yeast genome on length-mutation rates and lethal recombination events during meiosis.

Certain minisatellites exhibit hypervariability with respect to the number of repeat units and, thus, allele length. Such polymorphism is generated by germline-specific recombinational events that occur at high frequencies and lead to the gain or loss of repeat units. In order to elucidate the molecular details of mutagenesis in minisatellites, we have integrated human minisatellites into the yeast genome in the vicinity of a hotspot for meiotic double-strand breaks (DSBs). Here, we describe the results of tetrad analyses of mutations in the human MS205 minisatellite in yeast strains heterozygous for alleles composed of 51 and 31 repeat units, as well as in a strain homozygous for the same 51 repeat unit allele. The length-mutation rate was twice as high in the heterozygous strain as in the homozygous strain, suggesting that sequence divergence between alleles enhances the generation of length mutations. In the case of heterozygotes, the frequency of length mutants resulting from inter-allelic exchange was significantly higher in tetrads with three viable spores than in tetrads with four viable spores, indicating that there is a higher probability for spore mortality in tetrads originating from meioses during which inter-allelic exchange of repeat units occurs. In an attempt to explain these findings, we propose a model for minisatellite mutation involving recombination, in which sequence divergence between alleles results in a heteroduplex containing numerous mismatches. We suggest that convergent mismatch-repair tracts in this heteroduplex give rise to a DSB that may be repaired by an additional round of recombination resulting in mutation of a third allele, or be lethal if such recombination fails. It appears probable that the formation of such additional mutants is the major explanation for the difference in meiotic length-mutation rates between the heterozygous and homozygous yeast strains, and that this phenomenon contributes to high germline length-mutation frequencies at minisatellites in humans.

Alleles↗

PubMed2005

Genomic divergence of Escherichia coli strains: evidence for horizontal transfer and variation in mutation rates.

This report describes the sequencing in the Escherichia coli B genome of 36 randomly chosen regions that are present in most or all of the fully sequenced E. coli genomes. The phylogenetic relationships among E. coli strains were examined, and evidence for the horizontal gene transfer and variation in mutation rates was determined. The overall phylogenetic tree indicated that E. coli B and K-12 are the most closely related strains, with E. coli O157:H7 being more distantly related, Shigella flexneri 2a even more, and E. coli CFT073 the most distant strain. Within the B, K-12, and O157:H7 clusters, several regions supported alternative topologies. While horizontal transfer may explain these phylogenetic incongruities, faster evolution at synonymous sites along the O157:H7 lineage was also identified. Further interpretation of these results is confounded by an association among genes showing more rapid evolution and results supporting horizontal transfer. Using genes supporting the B and K-12 clusters, an estimate of the genomic mutation rate from a long-term experiment with E. coli B, and an estimate of 200 generations per year, it was estimated that B and K-12 diverged several hundred thousand years ago, while O157:H7 split off from their common ancestor about 1.5-2 million years ago.

Codon↗

PubMed2004

Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the virus mutation rate and for replication in macrophages.

Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.

Blotting, Western↗

PubMed1992

Spontaneous chromosome rearrangements in the protozoan Giardia lamblia: estimation of mutation rates.

Subcloned lines of the WB strain of Giardia lamblia contain polymorphic ribosomal RNA (rRNA) encoding chromosomes (Le Blancq et al., Nucl. Acids Res. 1991, 19, 4405-4412). We show that in a continuously propagated culture of G.lamblia trophozoites the proportion of trophozoites with rearranged rRNA encoding chromosomes gradually increases, consistent with the high mutation rate of about 1% per cell per division cycle. This conclusion is based on the finding in one experiment that after about 8 division cycles 20% of the population consisted of independent mutants, while after approximately 100 division cycles 87.5% of the population were independent mutants. In a second experiment, approximately 38% and 71.5% of the trophozoites were independent mutants after approximately 9 and approximately 100 division cycles, respectively. The data show that the genome of the WB strain of G.lamblia has a highly recombinogenic phenotype. Extensive karyotype heterogeneity has also been observed among recently isolated G.lamblia strains obtained from a defined geographic area (Korman et al., J. Clin. Invest. 1992, 89, 1725-1733) suggesting that a high mutation rate might also occur in vivo.

Animals↗

PubMed1999

A low mutation rate for chloroplast microsatellites.

We used chloroplast simple sequence repeats (cpSSRs) to examine whether there is any variation present in the chloroplast genome of Pinus torreyana (Parry ex Carrière) that may previously not have been detected using RFLPs. Analysis of 17 cpSSR loci showed no variation, which is consistent with previous cpRFLP work and confirms that the species is descended from an original, highly monomorphic population following a bottleneck. This lack of biological variation in the chloroplast genome of P. torreyana allowed us to estimate the mutation rates at cpSSR loci as between 3. 2 x 10(-5) and 7.9 x 10(-5). This estimate is lower than published mutation rates at nuclear SSR loci but higher than substitution rates elsewhere in the chloroplast genome.

Chloroplasts↗

PubMed2002

Mutation rate and specificity analysis of tetranucleotide microsatellite DNA alleles in somatic human cells.

We have systematically varied microsatellite sequence composition to determine the effects of repeat unit size, G+C content, and DNA secondary structure on microsatellite stability in human cells. The microsatellites were inserted in frame within the 5' region of the herpes simplex virus thymidine kinase (HSV-tk) gene. The polypyrimidine/polypurine microsatellites displayed enhanced S1 nuclease sensitivity in vitro, consistent with the formation of non-B-form DNA structures. Microsatellite mutagenesis studies were performed with a shuttle vector system in which inactivating HSV-tk mutations are measured after replication in a nontumorigenic cell line. A significant increase in the HSV-tk mutation frequency per cell generation was observed after insertion of [TTCC/AAGG]9, [TTTC/AAAG]9, or [TCTA/AGAT]9 sequences (P <or= 0.0002), relative to the HSV-tk gene control. We observed that the G + C content of the microsatellite may affect mutagenesis, as the mean microsatellite mutation rates of the [TTTC/AAAG]9 and [TCTA/AGAT]9 alleles were sevenfold and 11-fold higher, respectively, than the [TTCC/AAGG]9 allele. A bias toward expansion mutations was noted for the majority of clones bearing the [TTCC/AAGG]9 allele as well as a [TC/AG]17 microsatellite of similar allele length. The mean microsatellite mutation rate of the [TTCC/AAGG]9 allele did not differ significantly from that for a [TC/AG]11 allele, demonstrating that these tetranucleotide and dinucleotide alleles are of equivalent stability. It is known that microsatellite mutagenesis is affected by the number of repeat units within an allele. Our data suggest that additional biochemical factors may regulate both the rate and specificity of somatic cell microsatellite mutagenesis.

Alleles↗

PubMed2003

Mutation rate and the efficacy of antimicrobial drug treatment.

Despite rapid progress in drug development, microbial infections in general are becoming increasingly difficult to treat as a result of the emergence of drug-resistant strains. In some cases, such as HIV-1, the early goal of eradicating infections with antimicrobial drugs is, for now, being replaced with the more pragmatic goal of controlling infections over long periods of time through a succession of transiently effective treatments. Because treatment efficacy is often incomplete, studying the degree of treatment efficacy has great relevance to clinical disease management. We derived a model describing the association between the mutation rate of the pathogen and the degree of treatment efficacy. We found that drug treatment is most effective when the mutation rate of the pathogen is either very low or, perhaps counterintuitively, very high. We discuss this finding in the light of a promising new treatment strategy for RNA viruses that combines antiviral compounds with a mutagen.

Antiviral Agents↗

PubMed2004

The B chromosome polymorphism of the grasshopper Eyprepocnemis plorans in North Africa: III. mutation rate of B chromosomes.

B chromosome variation in nine Moroccan populations of the grasshopper Eyprepocnemis plorans was analysed for 3 consecutive years. In addition to B1, which was the predominant B chromosome in all nine populations, we found 15 other B variants, albeit at very low frequency. Eight variants were found in adults caught in the wild, four appeared in adults reared in the laboratory and seven were found in embryo progeny of controlled crosses between a 0B male and a B-carrying female. Some variants were found in more than one kind of material. At least the seven B variants that appeared in embryo progeny of females carrying a different B type arose de novo through mutation of the maternal B chromosome. The mutation rate of B chromosomes was 0.73%, on average, which explains the high variety of morphs and banding patterns found. The most frequent de novo mutations observed in these chromosomes were centromere misdivision with or without chromatid nondisjunction, which generates iso-B-chromosomes or telocentric Bs, respectively, as well as translocations with A and B chromosomes and deletions. But the whole variation observed, including that found in adult individuals, suggests that other mutations such as duplications, inversions and centric fusions do usually affect B chromosomes. Finally, B chromosome mutation rate was remarkably similar in both Moroccan and Spanish populations, which suggests that it might be dependent on B chromosome intrinsic factors.

Africa, Northern↗

PubMed2005

[In vitro activity of furazolidone and nitrofurantoin in Helicobacter pylori clinical isolates and study of mutation rate].

Clarithromycin, amoxicillin, tetracycline and metronidazole are the most frequently used antimicrobials for Helicobacter pylori infection treatment. While tetracycline and amoxicillin resistance are rare, clarithromycin and metronidazole resistance vary in different populations and are considered factors for treatment failure. The aim of this study was to determine the in vitro activity of furazolidone and nitrofurantoin in 164 H. pylori clinical isolates by agar dilution and to determine the spontaneous mutation rate. Metronidazole and clarithromycin resistance were 23.77% (CI95%: 18.96-29.14) and 16.78% (CI95%: 12.64-21.62), respectively; moreover, 1.4% (CI95%: 0.38-3.54) were intermediate to clarithromycin. All the isolates were susceptible to amoxicillin and tetracycline. Furazolidone and nitrofurantoin resistance rates were 1.82% (CI95%: 0.37-5.25) and 0.6% (CI95%: 0-3.35), respectively. The three furazolidone-resistant strains were nitrofurantoine-susceptible (MIC 4 mg/l for furazolidone and 2 mg/l for nitrofurantoin) and the nitrofurantoin-resistant strains were furazolidone-susceptible (MIC 4 mg/l for nitrofurantoin and 1 mg/l for furazolidone). These four strains were metronidazole-resistant (MIC 16 mg/l). Furazolidone or nitrofurantoin spontaneous mutants were not detected in the eight H. pylori strains tested. However, mutants with resistance to metronidazole were found with all the strains with a mutation rate of 7.4 x 10(-10) to 9.4 x 10(-10). Furazolidone and nitrofurantoin showed an excellent in vitro activity against the H. pylori clinical isolates included herein, supporting the usefulness of furazolidone as second-line antimicrobial after treatment failure or as first-line therapy in populations with low economical resources.

Anti-Infective Agents↗

PubMed1991

On the dependence of spontaneous mutation rates on the functional state of genes.

Spontaneous mutation of some genes was studied in haploid adenine and leucine auxotrophic yeast Saccharomyces. It was shown that a decrease in the amount of adenine (from 500 to 0 mg l-1) or leucine (from 300 to 0.3 mg l-1) in the medium, simultaneously with the transition from repression to derepression of the biosynthesis of these metabolites, resulted in a 15- to 150-fold increase in the reversion rate of genes ade 2 and leu2, respectively, for different strains. At the same time the mutation rate of suppressor genes varied relatively little (up to five-fold), and that of gene lys did not change at all. It was also demonstrated (on gene leu2) that the mutation rate is determined by the composition of the nutrient medium at the time of the S-phase of the cell cycle and it does not depend on the cultivation conditions during the presynthetic period. We discuss the hypothesis that derepressed genes mutate with a significantly higher rate than genes in the repressed state.

Adenine↗

PubMed2005

Mutation rates, spectra and hotspots in mismatch repair-deficient Caenorhabditis elegans.

Although it is clear that postreplicative DNA mismatch repair (MMR) plays a critical role in maintaining genomic stability in nearly all forms of life surveyed, much remains to be understood about the genome-wide impact of MMR on spontaneous mutation processes and the extent to which MMR-deficient mutation patterns vary among species. We analyzed spontaneous mutation processes across multiple genomic regions using two sets of mismatch repair-deficient (msh-2 and msh-6) Caenorhabditis elegans mutation-accumulation (MA) lines and compared our observations to mutation spectra in a set of wild-type (WT), repair-proficient C. elegans MA lines. Across most sequences surveyed in the MMR-deficient MA lines, mutation rates were approximately 100-fold higher than rates in the WT MA lines, although homopolymeric nucleotide-run (HP) loci composed of A:T base pairs mutated at an approximately 500-fold greater rate. In contrast to yeast and humans where mutation spectra vary substantially with respect to different specific MMR-deficient genotypes, mutation rates and patterns were overall highly similar between the msh-2 and msh-6 C. elegans MA lines. This, along with the apparent absence of a Saccharomyces cerevisiae MSH3 ortholog in the C. elegans genome, suggests that C. elegans MMR surveillance is carried out by a single Msh-2/Msh-6 heterodimer.

Animals↗

PubMed2002

Low-level hypermutation in T cell-independent germinal centers compared with high mutation rates associated with T cell-dependent germinal centers.

Exceptionally germinal center formation can be induced without T cell help by polysaccharide-based antigens, but these germinal centers involute by massive B cell apoptosis at the time centrocyte selection starts. This study investigates whether B cells in germinal centers induced by the T cell-independent antigen (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to Ficoll undergo hypermutation in their immunoglobulin V region genes. Positive controls are provided by comparing germinal centers at the same stage of development in carrier-primed mice immunized with a T cell-dependent antigen: NP protein conjugate. False positive results from background germinal centers and false negatives from non-B cells in germinal centers were avoided by transferring B cells with a transgenic B cell receptor into congenic controls not carrying the transgene. By 4 d after immunization, hypermutation was well advanced in the T cell-dependent germinal centers. By contrast, the mutation rate for T cell-independent germinal centers was low, but significantly higher than in NP-specific B cells from nonimmunized transgenic mice. Interestingly, a similar rate of mutation was seen in extrafollicular plasma cells at this stage. It is concluded that efficient activation of hypermutation depends on interaction with T cells, but some hypermutation may be induced without such signals, even outside germinal centers.

Adoptive Transfer↗

PubMed1991

Nucleotide sequence and mutation rate of the H strain of hepatitis C virus.

Patient H is an American patient who was infected with hepatitis C virus (HCV) in 1977. The patient became chronically infected and has remained so for the past 13 years. In this study, we compared the nucleotide and predicted amino acid sequences of the HCV genome obtained from plasma collected in 1977 with that collected in 1990. We find that the two HCV isolates differ at 123 of the 4923 (2.50%) nucleotides sequenced. We estimate that the mutation rate of the H strain of HCV is approximately 1.92 x 10(-3) base substitutions per genome site per year. The nucleotide changes were exclusively base substitutions and were unevenly distributed throughout the genome. A relatively high rate of change was observed in the region of the HCV genome that corresponds to the non-structural protein 1 gene region of flaviviruses, where 44 of 960 (4.6%) nucleotides were different. Within this region there was a 39-nucleotide domain in which 28.2% of the nucleotides differed between the two isolates. In contrast, relatively few nucleotide substitutions were observed in the 5' noncoding region, where only 2 of 276 (0.7%) nucleotides were different. Our results suggest that the mutation rate of the HCV genome is similar to that of other RNA viruses and that genes appear to be evolving at different rates within the virus genome.

Amino Acid Sequence↗

PubMed1992

Genomic characterization and mutation rate of hepatitis C virus isolated from a patient who contracted hepatitis during an epidemic of non-A, non-B hepatitis in Japan.

To investigate the genomic characterization of hepatitis C virus (HCV) isolated from patient who contracted hepatitis during an epidemic of non-A, non-B (NANB) hepatitis in Shimizu city, Japan, we have cloned the nucleotide sequence of the viral genome (HCV-KF) spanning the structural domain. When compared to other previously reported HCV isolates, HCV-KF showed an overall identity at the amino acid level of 90.0 to 92.1% with Japanese isolates and 80.9 to 82.1% with American-like isolates. The HCV-KF genome displays an insertion of three nucleotides in-frame (corresponding to one amino acid) found at the junction between the E1 and E2/NS1 region. The mutation rate of the HCV-KF genome was assessed by comparing the nucleotide and deduced amino acid sequences of the viral RNA obtained from the serum of the original patient with viral sequences derived from the serum of a chimpanzee inoculated with the same serum 9 years previously. The substitution rate of the viral genome was estimated at 0.9 x 10(-3) nucleotides per site per year for the HCV structural region. The highest mutation rate was found in the hypervariable region within the E2/NS1 domain. It is suggested that the outbreak in Shimizu city was caused by a strain of HCV closely related to the Japanese-like subgroup of isolates.

Americas↗

PubMed1978

Spontaneous mutation rate to thioguanine resistance is decreased in polyploid hamster cells.

The mutation rate to thioguanine resistance was 3.11 X 10(-6) in a near diploid V79 hamster cell line and 7.58 X 10(-8) in a near tetraploid derivative produced with colchicine. The specific activities of glucose-6-phosphate dehydrogenase and phosphoglycerate kinase of the tetraploid line were greater than that of the diploid which suggests that twice the number of active X chromosomes were present in the tetraploid. These results are compatible with the hypothesis that spontaneous variants resistant to thioguanine arise through mutation and chromosomal segregation, as has been suggested for induced mutations in tetraploid hamster cells.

Cell Line↗

PubMed1983

The frequency and mutation rate of balanced autosomal rearrangements in man estimated from prenatal genetic studies for advanced maternal age.

The frequencies of balanced chromosome rearrangements were estimated from three series of advanced maternal-age prenatal genetic studies, and were compared to the frequencies that had been estimated from consecutive newborn surveys. In the maternal-age prenatal studies, the frequencies were: Robertsonian translocations, 0.11%; reciprocal translocations, 0.17%; and inversions, 0.12%. The total frequency of balanced rearrangements in the prenatal genetic studies performed with banding (0.40%, or 1 in 250) was twice that in the consecutive newborn surveys performed without banding (0.19%, or 1 in 526). The difference was limited to inversions and reciprocal translocations; the frequency of Robertsonian translocations was similar in the prenatal series and the newborn surveys. Both familial and de novo rearrangements were more common than anticipated. The de novo cases provided a mutation rate estimate of 4.3 per 10,000 gametes per generation (compared with 1.78 to 2.2 per 10,000 gametes in other surveys). These higher estimates may more reliably approximate the true mutation rate and frequencies of balanced rearrangements in the newborn population than do the newborn surveys.

Chromosome Aberrations↗

PubMed1994

Mutation rates and mechanisms of resistance to etoposide determined from fluctuation analysis.

BACKGROUND: The major known mechanisms of resistance to etoposide include altered expression of its target enzyme, topoisomerase II (Topo II), and the multidrug-resistant phenotypes encoded by the mdr1 and MRP (multidrug resistance-associated protein) genes. There is little information regarding the distribution, frequency, and origin of these mechanisms in cancer cells. PURPOSE: We performed fluctuation analysis experiments with the human sarcoma cell line, MES-SA, to assess 1) if selection or induction mechanisms are involved in resistance to etoposide, 2) mutation rates for cellular resistance to etoposide, and 3) the nature of the single-step selected surviving clones. METHODS: Three groups of 10 flasks were seeded with more than 2000 cells each and allowed to grow to near confluence (approximately 3 x 10(6) cells per flask). After reseeding, each group received etoposide for 1 week at a final concentration of 0.5 microM (group A), 1.0 microM (group B), and 5.0 microM (group C). Surviving colonies in each of the 30 populations were scored and individually harvested. RESULTS: Mutation rates were estimated at 2.9 x 10(-6) (group A), 5.7 x 10(-7) (group B), and 1.7 x 10(-7) (group C) per cell generation. Of 61 propagated colonies, four of 26 from group A, five of 19 from group B, and none of 16 from group C were stably resistant. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction, conferring etoposide resistance in groups A and B. Five of the stably resistant clones were cross-resistant to doxorubicin. Analysis by polymerase chain reaction failed to detect the expression of the multidrug-resistant gene mdr1 messenger RNA (mRNA) in any of the clones. No increase in expression of the MRP gene was observed. However, a significant decrease in both Topo II alpha and II beta mRNA (30%-70%) was found in six of seven stably resistant and six of six unstably resistant mutants. CONCLUSIONS: Our study demonstrates that resistance to etoposide arises spontaneously, with most clones surviving either stochastically or through very labile mechanisms of resistance. The experimental design has derived a set of resistant mutants from a single-step selection. In those clones, decreased expression of Topo II is the predominant mechanism selected. IMPLICATIONS: These findings suggest that stable resistance to etoposide chemotherapy may be acquired by selection of spontaneously arising mutants rather than induction by drug exposure. The stably resistant clones may represent descendants from a single mutational event in each population.

DNA, Complementary↗

PubMed1993

An improved estimator of spontaneous mutation rates in Luria-Delbrück fluctuation experiments.

In estimating the spontaneous mutation rate, mu, in cultured mammalian cells, the number of mutant cells in several parallel cultures, each a clone from a single cell, is determined. Luria and Delbrück (1943), and subsequently Lea and Coulson (1949), proposed several estimators of mu using data from such experiments. These methods of analysis were originally proposed to apply to mutants arising in microbial cultures, and some of the limitations in applying them to mammalian cell cultures have been outlined by Featherstone et al. (1987), and by Kendal and Frost (1988). For a given value of mu, the number of mutants, r, found in a culture is exceedingly variable, so that r contains very little information concerning mu. It is important, therefore, that an estimator of mu be efficient; that it not waste the limited information available in the experimental data. To this end, a new estimator is proposed which compounds the information previously utilized by the so-called P0 and median estimators. Using a spreadsheet program such as that available with MINITAB, the algorithm can be programmed in as few as 26 instructions. Tested against simulated data across a range of values of mu, the proposed compound estimator is more efficient than the currently used estimators.

Algorithms↗