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PubMed1998

Ultraviolet-light induced p53 mutational spectrum in yeast is indistinguishable from p53 mutations in human skin cancer.

Ultraviolet (UV) light has been associated with the development of human non-melanoma skin cancers (NMSC). Such cancers often exhibit mutations in the p53 tumour suppressor gene. In order to determine the UV-induced p53 mutation spectrum, a yeast expression vector that harbours a human wild-type p53 cDNA was UV-irradiated in vitro and transfected into a yeast strain that contained the ADE2 gene regulated by a p53-responsive promoter. Forty-five mutant clones contained 51 mutations. Seven mutations were tandem base pair substitutions, four of which being CC-->TT, hallmark mutations of UV mutagenesis. Eighty percent (41/51) of the mutations were single or non-tandem base pair substitutions, the majority of which (27/41) were C-->T transitions. Ninety-five percent of such mutations occurred at dipyrimidine sites. Through a rigorous statistical test, the UV-induced p53 mutation spectrum appears to differ significantly (P < 0.008) from the one induced by the antineoplastic drug chloroethyl-cyclohexyl-nitrosourea, and to be indistinguishable from the one observed in NMSC (P = 0.4). These results demonstrate that the assay allows the determination of carcinogen-specific p53 mutation fingerprints and represents a new tool for molecular epidemiology.

DNA, Complementary↗
PubMed2000

Candidate mutator genes in mismatch repair-deficient thymic lymphomas: no evidence of mutations in the DNA polymerase delta gene.

DNA mismatch repair (MMR) proteins recognize nucleotides that are incorrectly paired. Deficiencies in MMR lead to increased genomic instability reflected in an increased mutation frequency and predisposition to tumorigenesis. Mice lacking the MMR gene, Msh2, develop thymic lymphomas that exhibit much higher mutational frequencies than other Msh2(-/-) tumours and Msh2(-/-) normal thymic tissue, suggesting that an additional mutator may have been acquired in a tissue-specific manner. Clustered mutations observed exclusively in the thymic lymphomas suggests that a gene(s) associated with the replication machinery might have become altered during tumorigenesis. Based on mutation studies in Saccharomyces cerevisiae lacking Msh2 and DNA polymerase delta (DNA pol delta), we hypothesized that the acquisition of mutations in DNA pol delta could contribute to the hypermutator phenotype and tumorigenesis in Msh2(-/-) thymic tissue. Furthermore, previous reports have suggested that genes containing mononucleotide repeats are non-random mutational targets in the absence of MMR. Therefore, we sequenced all 26 exons of the DNA pol delta catalytic subunit, including the six exons containing mononucleotide repeats of >5 bp, from nine Msh2(-/-) thymic lymphomas and two wild-type controls. No DNA pol delta pathogenic mutations were found in the thymic lymphomas, although several DNA base differences compared with published DNA pol delta sequences were observed. We conclude, therefore, that inactivating mutations in DNA pol delta are not a contributing factor in the development of the hypermutator phenotype in MMR-deficient murine thymic lymphomas.

Animals↗
PubMedDate not supplied

A mutator factor in a strain of Drosophila melanogaster: identified by use of mutation, reversion rates and male recombination.

A set of 1,000 "mutation accumulation" lines Drosophila melanogaster, which originated from two different wild-type, lethal-bearing second chromosomes (Yamaguchi and Mukai 1974; Mukai and Cockerham 1977), was examined for evidence of a mutator factor by using the occurrence of recessive visible mutations and male recombination to identify its presence. The 1,000 lines were screened at approximately generation 240 for the presence of recessive visible mutation at twelve loci, by outcrossing to a balanced multiply marked second chromosome stock (Muller's "12ple" Bowling Green). Twenty-three lines were found to carry a visible mutation at one of the vg locus. mutations found in three lines, two at the dp locus and one at the vg locus, demonstrated instability as revertants to the wild type and were recovered and verified in these three cases. The three revertant lines, and three lines showing no reversion, were tested for their ability to induce male recombination. Male recombination was observed in the three lines in which revertants were recovered. Male and female sterility assays indicated conclusively that these "hybrid dysgenic" characteristics could not be used to identify lines potentially carrying mutator factors, whereas the consistent ability of the lines to induce high rates of reversion and male recombination was successful in determining that the "mutation accumulation lines" do possess mutator factors.

Animals↗
PubMed1982

A cis-dominant mutation in aspergillus nidulans affecting the expression of the amdS gene in the presence of mutations in the unlinked gene, amdA.

A mutant producing very high levels of the acetamidase enzyme encoded by the amdS gene has been isolated in a strain containing the amdA7 mutation, which itself causes high levels of this enzyme. Genetic analysis has shown that this mutation, designated amdI66, is adjacent to the amdS gene and is cis-dominant in its effect. The amdI66 mutation has little effect on amdS expression when present in strains not containing the amdA7 mutation. Two other amdA mutations investigated also interact with the amdI66 mutation to result in high acetamidase levels. No interaction between amdI66 and any of the other putative regulatory genes affecting amdS expression has been observed. The amdI66 mutation has been located by fine structure mapping at the extreme end of the controlling region, which has previously been defined by genetic mapping (HYNES 1979). Analysis of this region has been extended by mapping new mutations resulting in loss of amdS expression. One of these defines the most extreme site capable of mutation to loss of gene function found so far.

Amidohydrolases↗
PubMed1995

An alpha tubulin mutation suppresses nuclear migration mutations in Aspergillus nidulans.

Microtubules and cytoplasmic dynein, a microtubule-dependent motor, are required for nuclei to move along the hyphae of filamentous fungi. Nuclear migration in Aspergillus nidulans is blocked by heat-sensitive (hs-) mutations in the nudA gene, which encodes dynein heavy chain, and the nudF gene, which encodes a G protein beta-subunit-like protein. Hs- mutations in the nudC and nudG genes also prevent nuclear migration. We have isolated extragenic suppressor mutations that reverse the hs- phenotypes caused by these mutations. Here we show that one nudF suppressor also suppresses hs- mutations in nudA, nudC, and nudG and deletions in nudA and nudF. This suppressor mutation is in the tubA alpha tubulin gene, and its characteristics suggest that it destabilizes microtubules. The mutation alters microtubule staining and confers sensitivity to cold and benomyl, two treatments that destabilize microtubules. Treatment with low concentrations of benomyl also suppresses the hs- nudA, nudC, nudF, and nudG mutations and the nudA and nudF deletions. Suppression of the hs- nudA mutation and the nudA deletion is especially interesting because these strains lack active dynein heavy chain. Together, these results suggest that microtubule destabilization allows nuclei to migrate even in the absence of cytoplasmic dynein motor function.

Aspergillus nidulans↗
PubMed1998

Inference of genome-wide mutation rates and distributions of mutation effects for fitness traits: a simulation study.

The properties and limitations of maximum likelihood (ML) inference of genome-wide mutation rates (U) and parameters of distributions of mutation effects are investigated. Mutation parameters are estimated from simulated experiments in which mutations randomly accumulate in inbred lines. ML produces more accurate estimates than the procedure of Bateman and Mukai and is more robust if the data do not conform to the model assumed. Unbiased ML estimates of the mutation effects distribution parameters can be obtained if a value for U can be assumed, but if U is estimated simultaneously with the distribution parameters, likelihood may increase monotonically as a function of U. If the distribution of mutation effects is leptokurtic, the number of mutation events per line is large, or if genotypic values are poorly estimated, only a lower limit for U, an upper limit for the mean mutation effect, and a lower limit for the kurtosis of the distribution can be given. It is argued that such lower (upper) limits are appropriate minima (maxima). Estimates of the mean mutational effect are unbiased but may convey little about the properties of the distribution if it is leptokurtic.

Animals↗
PubMed2001

The rate of mutation and the homozygous and heterozygous mutational effects for competitive viability: a long-term experiment with Drosophila melanogaster.

The effect of 250 generations of mutation accumulation (MA) on the second chromosome competitive viability of Drosophila melanogaster was analyzed both in homozygous and heterozygous conditions. We used full-sib MA lines, where selection hampers the accumulation of severely deleterious mutations but is ineffective against mildly deleterious ones. A large control population was simultaneously evaluated. Competitive viability scores, unaffected by the expression of mutations in heterozygosis, were obtained relative to a Cy/L(2) genotype. The rate of decline in mean DeltaM approximately 0.1% was small. However, that of increase in variance DeltaV approximately 0.08 x 10(-3) was similar to the values obtained in previous experiments when severely deleterious mutations were excluded. The corresponding estimates of the mutation rate lambda > or = 0.01 and the average effect of mutations E(s) < or = 0.08 are in good agreement with Bateman-Mukai and minimum distance estimates for noncompetitive viability obtained from the same MA lines after 105 generations. Thus, competitive and noncompetitive viability show similar mutational properties. The regression estimate of the degree of dominance for mild-to-moderate deleterious mutations was approximately 0.3, suggesting that the pertinent value for new unselected mutations should be somewhat smaller.

Animals↗
PubMed1991

A genotypic mutation system measuring mutations in restriction recognition sequences.

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.

Base Sequence↗
PubMed1999

Venous thromboembolism at a young age in a brother and sister with coinheritance of homozygous 20210A/A prothrombin mutation and heterozygous 1691G/A factor V Leiden mutation.

We report on members of a Turkish thrombophilic family with coinheritance of the prothrombin mutation PT20210A and the factor V Leiden mutation. The 23-year-old propositus and his elder sister both had episodes of venous theomboembolism at a young age (23 years and 26 years, respectively) and are homozygous for the PT20210A mutation and heterozygous for the factor V Leiden mutation. The 51-year-old father is suffering from coronary heart disease and is heterozygous for both thrombophilic mutations. The asymptomatic 43-year-old mother is heterozygous for the PT20210A mutation, but without activated protein C resistance. Two other children, a 20-year-old girl who is homozygous for the PT20210A mutation and a 13-year-old boy who is heterozygous for the PT20210A mutation, are both free from activated protein C resistance and thrombosis. This report provides further evidence for an early onset of thromboembolic disorders in individuals with an homozygous state of the prothrombin variant 20210A/A and coinheritance of another thrombophilic mutation. Consensus guidelines are required for the treatment and prophylaxis of patients and subjects who remain asymptomatic with homozygous or more than one heterozygous genetic defect associated with thrombophilia.

Adult↗
PubMed2004

CARD15 mutation analysis in an Italian population: Leu1007fsinsC but neither Arg702Trp nor Gly908Arg mutations are associated with Crohn's disease.

BACKGROUND: CARD15 gene mutations have been demonstrated to confer a high risk of Crohn's disease (CD). Despite this, recent studies reported variable associations between CD and CARD15 mutations in distinct ethnic groups, thus raising the hypothesis that genetic and/or allelic heterogeneity may influence the relationship between CARD15 and CD. The purpose of this study was to evaluate the frequency of the main mutations of the CARD15 gene (Leu 1007fsinsC, Arg702Trp, and Gly908Arg) in Italian CD patients and to establish possible genotype-phenotype correlations. METHODS: One hundred sixty-five CD patients and 125 healthy subjects were consecutively enrolled from January to November 2001. The Leu1007fsinsC mutation was assessed by denaturing high-performance liquid chromatography and Arg702Trp and Gly908Arg mutations by Pyrosequencing technology. RESULTS: Among the CARD15 gene mutations tested, only the Leu1007fsinsC was associated with CD (30/165 CD patients, 18%, versus 3/125 healthy subjects, 2.4%; p < 0.001). In particular, 23 CD patients were heterozygotes and 7 were homozygotes. No healthy subject exhibited the mutant homozygous genotype. Odds ratios for CD were 6.9 for heterozygotes and 41.0 for homozygotes. The genotype-phenotype analysis revealed that a fibrostenosing CD of the distal ileum was more frequent in patients carrying the Leu1007fsinsC mutation. CONCLUSIONS: This study confirms the association between CARD15 gene mutations and CD and shows that only the Leu1007fsinsC mutation is a risk factor of CD in an Italian population.

Adolescent↗
PubMed1999

The approach to mutation-selection balance in an infinite asexual population, and the evolution of mutation rates.

A method is described for calculating the dynamics of the distribution of fitness in an infinite asexual population which is subject to unconditionally deleterious mutations with independent effects. This method is applied to the problem of calculating the frequency of a mutator subpopulation, at equilibrium between mutation and indirect selection due to association with deleterious mutations. Many mutator alleles are produced by loss-of-function mutations in polymerase or mismatch repair genes. Previous calculations have ignored the fact that this creates a flux of higher fitness individuals into the mutator subpopulation. This flux raises the mean fitness of the mutator subpopulation, and when this factor is taken into account, the frequency of the mutator may be more than an order of magnitude greater than recent theoretical work has suggested.

Alleles↗
PubMed2005

Mutation analysis of POUF-1, PROP-1 and HESX-1 show low frequency of mutations in children with sporadic forms of combined pituitary hormone deficiency and septo-optic dysplasia.

OBJECTIVES: Mutations in the genes encoding the transcription factors PROP1 and POUF-1 (Pit-1) have been reported as common causes of combined pituitary hormone deficiency (CPHD), and HESX1 mutations have been identified in children with septo-optic dysplasia (SOD). There are few data on UK children. We have performed mutation analysis in a large cohort of affected children within the West Midlands region to assess the feasibility of a screening strategy for molecular diagnosis in CPHD and SOD. DESIGN AND PATIENTS: The three coding exons of PROP1, and six exons of POUF-1 in 27 children from 26 families with CPHD, and three exons of HESX1 in 23 children from 22 families with SOD were directly sequenced from a well-characterized regional cohort. RESULTS: We identified a C to T transition in exon 6 of POUF-1, resulting in a known missense mutation (R271W) in a mother and daughter from one family with CPHD. We also found a novel homozygous T to C transition in exon 6 of POUF-1, resulting in a missense mutation (F233L) in a twin with CPHD. This mutation was excluded in 100 ethnically matched control alleles. We did not identify any mutations in the PROP1 gene or HESX1. The median maternal age at delivery for the CPHD children was 27 years, compared to 21 years for the mothers of SOD children (P = 0.04). CONCLUSIONS: Mutations in POUF-1, PROP1 and HESX1 are rare causes of CPHD and SOD, respectively, in children from the West Midlands. In particular, we did not confirm the reported 'hotspot' in PROP1. A screening strategy that targets familial cases is highly likely to increase the mutation yield. The young maternal age at conception of children with SOD and potential teratogen exposure indicate the predominance of environmental factors in this condition compared with CPHD.

Child↗
PubMed2006

Multiple thrombophilic gene mutations rather than specific gene mutations are risk factors for recurrent miscarriage.

PROBLEM: Recurrent miscarriage is a heterogeneous condition. While the role of acquired thrombophilia has been accepted as an etiology of recurrent miscarriage, the contribution of specific inherited thrombophilic genes to this disorder has remained controversial. We compared the prevalence of 10 thrombophilic gene mutations among women with a history of recurrent miscarriages and fertile control women. METHOD OF STUDY: A total of 150 women with a history of two or more recurrent pregnancy losses and 20 fertile control women with no history of pregnancy losses had buccal swabs taken for DNA analyses of 10 gene mutations [factor V G1691A, factor V H1299R (R2), factor V Y1702C, factor II prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, HPA1 a/b (L33P), MTHFR C677T, MTHFR A1298C]. The prevalence of these mutations was compared between women experiencing recurrent miscarriage and controls. RESULTS: No differences in the frequency of specific gene mutations were detected when women with recurrent miscarriage were compared with control women. However, the prevalence of homozygous mutations and total gene mutations among patients with recurrent miscarriage was significantly higher than among controls. Homozygous mutations were found in 59% of women with a history of recurrent pregnancy loss contrasted to 10% of control women. More than three gene mutations among the 10 genes studied were observed in 68% of women with recurrent miscarriage and 21% of controls. CONCLUSION: Inherited thrombophilias are associated with recurrent miscarriage. This association is manifest by total number of mutations rather than specific genes involved.

Abortion, Habitual↗
PubMed1995

Detection of gyrA and gyrB mutations in quinolone-resistant clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations.

Twelve quinolone-resistant clinical isolates of Escherichia coli (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.25 to 8 micrograms/ml) were transformed with plasmid pJSW101 carrying the gyrA+ gene and with plasmid pJB11 carrying the gyrB+ gene to examine the proportion of gyrA and gyrB mutations. Transformation with pJSW101 resulted in complementation (nalidixic acid MICs, 4 to 32 micrograms/ml; norfloxacin MICs, 0.06 to 0.25 micrograms/ml). In contrast, no change in MICs were observed after transformation with pJB11. A 418-bp fragment of gyrA from the 12 strains was amplified by PCR. Direct DNA sequencing of that fragment identified the causes of quinolone resistance in eight strains as a single point mutation leading to a substitution of the serine at position 83 (Ser-83) to Leu and in four strains as a single point mutation leading to a substitution of Asp-87 to Gly. Exchange of the fragment from one of these strains with that of gyrA+ and transformation of resistance with the hybrid gyrA plasmid indicated the contribution of Gly-87 to resistance and the stabilities of mutants containing GyrA (Gly-87). Thus, gyrA gene mutations are probably encountered more often than gyrB gene mutations in clinical isolates of E. coli. In addition, the substitution of Asp-87 to Gly can be encountered in such strains. On the basis of the level of resistance found in the fragment exchange experiment, the quinolone resistance attributable to Gly-87 appears to be comparable to that attributable to Leu-83. The levels of resistance found in the clinical isolates shown to have a Gly-87 mutation (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.5 to 4 micrograms/ml) suggest that the Gly-87 mutation causes resistance at the level of the nalidixic acid MIC (64 micrograms/ml) or the norfloxacin MIC (0.5 micrograms/ml or less) and that the additional increments in resistance seen in the other strains with higher levels of resistance may be attributable to additional mutations. The single-strand conformational polymorphism analysis with PCR products readily detected te Leu-83 and Gly-87 mutations.

Amino Acid Sequence↗
PubMed2004

Single-nucleotide polymorphism mutation spectra and resistance to quinolones in Salmonella enterica serovar Enteritidis with a mutator phenotype.

Resistance to quinolone antibiotics has been associated with single-nucleotide polymorphisms (SNPs) in the quinolone resistance-determining region (QRDR) of gyrA. Mutations in the gyrA gene were compared by using mutant populations derived from wild-type Salmonella enterica serovar Enteritidis and its isogenic mutS::Tn10 mutator counterpart. Spontaneous mutants arising during nonselective growth were isolated by selection with either nalidixic acid, enrofloxacin, or ciprofloxacin. QRDR SNPs were identified in approximately 70% (512 of 695) of the isolates via colony hybridization with radiolabeled oligonucleotide probes. Notably, transition base substitution SNPs in the QRDR were dramatically increased in mutants derived from the mutS strain. Some, but not all, antibiotic-resistant mutants lacking QRDR SNPs were resistant to tetracycline and chloramphenicol, consistent with alterations in nonspecific efflux pumps or other membrane transport mechanisms. Changing the selection conditions shifted the mutation spectrum. Selection with ciprofloxacin was least likely to yield a mutant harboring either a QRDR SNP or chloramphenicol resistance. Selection with enrofloxacin was more likely to yield mutants containing Ser83-->Phe mutations, whereas selection with ciprofloxacin or nalidixic acid favored recovery of Asp87-->Gly mutants. Fluoroquinolone-resistant Salmonella strains isolated from veterinary or clinical settings frequently display a mutational spectrum with a preponderance of transition SNPs in the QRDR, the pattern found in vitro among mutS mutator mutants reported here. Both the preponderance of transition mutations and the varied mutation spectra reported for veterinary and clinical isolates suggest that bacterial mutators defective in methyl-directed mismatch repair may play a role in the emergence of quinolone and fluoroquinolone resistance in feral settings.

Anti-Bacterial Agents↗
PubMed1986

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3'----5' exonuclease (proofreading) activity.

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).

Base Sequence↗
PubMed2000

Positive selection for mutations affecting bioconversion of aromatic compounds in Agrobacterium tumefaciens: analysis of spontaneous mutations in the protocatechuate 3,4-dioxygenase gene.

A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain of Agrobacterium tumefaciens A348. The nucleotide sequence of the A348 pcaHGB genes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and beta-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into the pcaB gene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described for Acinetobacter sp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker in pcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained with Acinetobacter sp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting the pcaHG genes of A. tumefaciens for spontaneous mutation. Sixteen strains carrying putative second-site mutations in pcaH or -G were subjected to sequence analysis. All single-site events, their mutations revealed no particular bias toward multibase deletions or unusual patterns: five (-1) frameshifts, one (+1) frameshift, one tandem duplication of 88 bp, one deletion of 92 bp, one nonsense mutation, and seven missense mutations. PcaHG is considered to be the prototypical ferric intradiol dioxygenase. The missense mutations served to corroborate the significance of active site amino acid residues deduced from crystal structures of PcaHG from Pseudomonas putida and Acinetobacter sp. as well as of residues in other parts of the enzyme.

Agrobacterium tumefaciens↗
PubMed2001

Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype Typhimurium DT104 isolates.

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC-->AAC) mutation, an Asp-87-to-Gly (GAC-->GGC) mutation, and a Ser-83-to-Phe (TCC-->TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC-->TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC-->TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

Animals↗