Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “mutant identification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 505 records · Page 28Linked to original sources
PubMed1996

Selection, identification, and genetic analysis of random mutants in the cloned primase/helicase gene of bacteriophage T7.

T7 gene 4 specifies two overlapping proteins 4A, a 566-amino acid primase/helicase, and 4B, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. The 4A' gene, which has a leucine codon replacing the 4B initiation codon, specifies a single 566-amino acid protein that can provide the primase and helicase functions required for normal T7 growth. We selected N-methyl-N'-nitro-N-nitrosoguanidine mutants in the cloned 4A' gene that no longer support the growth of a phage that completely lacks gene 4. Genetic mapping of the 76 mutations found them to be distributed throughout the protein, including both the N-terminal and C-terminal halves of the molecule thought to represent primase and helicase domains, respectively. Complementation tests with partially and completely defective phage showed that all but five of the mutants lacked helicase function but retained primase function. The other five, which lacked both functions, all made short proteins, including one missing only 60 amino acids. No mutations lacked only primase function, and none mapped within the first 105 amino acids, which includes the 63-amino acid region unique to 4A that contains elements required to recognize primase sites. Forty-six mutations were sequenced and included 27 missense mutations affecting 25 amino acids. Many mutations in the N-terminal half of the protein affected its solubility in cell extracts. Mutations in the C-terminal half clustered in or near five helicase consensus sequences. Biochemical analysis of nine of the mutant proteins is described in the accompanying paper (Washington, M. T., Rosenberg, A. H., Griffin, K., Studier, F. W., and Patel, S. S. (1996) J. Biol. Chem. 271, 26825-26834).

Amino Acid Sequence↗
PubMed1997

Differences in the incidence of the CYP2C19 polymorphism affecting the S-mephenytoin phenotype in Chinese Han and Bai populations and identification of a new rare CYP2C19 mutant allele.

The incidence of the S-mephenytoin polymorphism was compared in two Chinese ethnic groups, Han (n = 101) and Bai (n = 202) by phenotype and genotype analysis. The frequency of poor metabolizers (PMs) in Han vs. Bai subjects was 19.8% vs. 13.4%. Han subjects had a higher frequency of the mutant CYP2C19m1 allele (0.366 vs. 0.257, P < .01) and a lower frequency of the wild-type allele (0.559 vs. 0.688, P < .01) than Bai subjects, which is consistent with the difference in the frequencies of PMs between the two ethnic groups. This results in a lower percentage of homozygous wild-type extensive metabolizers of mephenytoin (EMs) in Han subjects than in Bai subjects (40% vs. 59%, P = .005). Therefore, Han subjects may be more susceptible than Bai subjects to the drugs metabolized by the CYP2C19 enzyme. Ratios of urinary S/R-mephenytoin in homozygous EMs were lower than those of heterozygous EMs for both Han and Bai subjects, which shows a gene-dosage effect. Genotype analysis identified all but one PM as homozygous or heterozygous for the two known mutant CYP2C19m1 and/or CYP2C19m2 alleles. A single Bai PM outlier was shown to be heterozygous for CYP2C19m1 and a new mutant CYP2C19 allele containing a single amino acid change of Arg433 --> Trp433. A genotyping test demonstrated that only this one individual carried this rare allele (frequency of 0.0025 in Bai subjects).

Alleles↗
PubMed1998

Identification of SV40 T-antigen mutants that alter T-antigen-induced chromosome damage in human fibroblasts.

The SV40 T antigen causes numerical (aneuploidy) and structural (aberrations) chromosome damage when expressed in human diploid fibroblasts. This chromosome damage precedes the acquisition of neoplastic traits such as anchorage independence, colony formation in reduced serum growth factors, immortalization, or tumorigenicity. Therefore, chromosome damage may be important in acquiring these traits because it could provide a mutational mechanism. To determine how the T antigen causes chromosome damage, point mutations were constructed that altered previously defined biochemical functions of the T protein. Mutant T antigen constructs were introduced into human diploid fibroblasts and selected by using G418. Clones of G418r cells that expressed mutant T antigens were expanded and scored for chromosome damage. Most of these mutant T antigens caused [corrected] levels of chromosome damage similar to those caused by [corrected] the wild-type T antigen. However, some T-antigen mutants induced fewer chromosome changes. A subset of these clones that induced less chromosome damage than wild-type T were examined further. Mutant T-antigen protein levels from this subset were quantified with flow cytometry and compared with wild-type protein expression levels. Mutations of T antigen shown previously to form less stable complexes with p53 caused less chromosome damage. A mutation in the zinc finger domain of T antigen also caused less chromosome damage. Interestingly, a mutant that caused loss of the ATPase activity of T antigen caused an increase in endoreduplicated cells. Also, a correlation was noted between cells expressing very low levels of T antigen (below detection limits when using flow cytometry) and an undamaged karyotype. This correlation indicates that there is a threshold level of T-antigen expression that induces chromosome damage and that expression levels on a per-cell basis rather than on a population basis should be considered in subsequent studies.

Amino Acid Substitution↗
PubMed2005

Mutations in HlyD, part of the type 1 translocator for hemolysin secretion, affect the folding of the secreted toxin.

HlyD, a member of the membrane fusion protein family, is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli. Random point mutations affecting HlyA secretion were obtained, distributed in most periplasmic regions of the HlyD molecule. Analysis of the secretion phenotypes of different mutants allowed the identification of regions in HlyD involved in different steps of HlyA translocation. Four mutants, V349-I, T85-I, V334-I and L165-Q, were conditionally defective, a phenotype shown to be linked to the presence of inhibitory concentrations of Ca2+ in extracellular medium. Hly mutant T85-I was defective at an early stage in secretion, while mutants V334-I and L165-Q appeared to accumulate HlyA in the cell envelope, indicating a block at an intermediate step. Mutants V349-I, V334-I, and L165-Q were only partially defective in secretion, allowing significant levels of HlyA to be transported, but in the case of V349-I and L165-Q the HlyA molecules secreted showed greatly reduced hemolytic activity. Hemolysin molecules secreted from V349-I and V334-I are defective in normal folding and can be reactivated in vitro to the same levels as HlyA secreted from the wild-type translocator. Both V349-I and V334-I mutations mapped to the C-terminal lipoyl repeat motif, involved in the switching from the helical hairpin to the extended form of HlyD during assembly of the functional transport channel. These results suggest that HlyD is an integral component of the transport pathway, whose integrity is essential for the final folding of secreted HlyA into its active form.

Amino Acid Motifs↗
PubMed1999

[Huntington's disease: clinical and molecular genetics].

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms. An unstable CAG trinucleotide repeat expansion within the first exon of the responsible gene "IT15", encoding huntingtin, was identified. The clinical phenotype strongly correlates with the number of CAG repeat. The mutant huntingtin is expressed ubiquitously as same as the wild-type huntingtin, suggesting a toxic gain of function of the mutant huntingtin. The identification of huntingtin-interacting proteins and intranuclear aggregates containing a fragment of mutant huntingtin provide new insights into the pathophysiological mechanisms underlying HD. Moreover, the progress in transgenic animal models for HD will be critical for understanding the development of HD and for the testing of new therapeutics.

Animals↗
PubMed1991

Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells.

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes.

Animals↗
PubMed1980

Genetic analysis of Escherichia coli K-12 chromosomal mutants defective in expression of F-plasmid functions: identification of genes cpxA and cpxB.

Two temperature-sensitive, chromosomal mutants of Escherichia coli were selected for their inability to express deoxyribonucleic acid donor activity and other activities associated with the conjugative plasmid F. These mutants were also auxotrophic for isoleucine and valine at 41 degrees C. Each mutant strain contained two altered genes: cpxA, located at 88 min on the E. coli K-12 genetic map, and cpxB, located at 41 min. Mutations in both genes were required for maximal expression of mutant phenotypes. The parent strain of mutants KN401 and KN312 already contained the cpxB mutation that is present in both mutants (cpxB1). This mutation by itself was cryptic. The cpxA mutations represent different mutant alleles since they are of independent origin. A cpxA mutation by itself significantly affected the expression of plasmid functions and growth at 41 degrees C in the absence of isoleucine and valine, but strains containing both a cpxA and cpxB mutation were more severely affected. Along with the observation that both cpxA mutations were revertable, the temperature sensitivity of cpxA cpxB+ cells suggests that both cpxA alleles contain point mutations that do not completely destroy the activity of the cpxA gene product.

Chromosome Mapping↗
PubMed1993

William L. McGuire Memorial Symposium. Estrogen receptors: ligand discrimination and antiestrogen action.

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labelling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. Cys530A1a mutant), C381 becomes the site of estrogen-compatible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed and region-specific chemical mutagenesis have identified a region around C530 important in discrimination between estrogens and antiestrogens, and other mutants have allowed identification of residues important in hormone-dependent transcriptional activation. Some transcriptionally inactive ER mutants also function as potent dominant negative ERs, suppressing the activity of wild-type ERs at low concentrations. These studies are beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer.

Amino Acid Sequence↗
PubMed1993

Novel method for identifying bacterial mutants with reduced epiphytic fitness.

In order to identify novel traits involved in epiphytic colonization, a technique for the rapid identification of bacterial mutants with quantitatively different population sizes in a natural habitat based on measurements of ice nucleation activity was developed. The threshold freezing temperatures of leaves harboring different numbers of cells of ice nucleation-active Pseudomonas syringae B728a differed substantially. While few leaves containing less than about 10 cells per g (fresh weight) froze at assay temperatures of -2.75 degrees C or higher, nearly all leaves froze at these temperatures when population sizes of this strain increased to about 10 cells per g (fresh weight). Presumptive epiphytic fitness mutants could readily be identified as strains which initiated freezing in fewer leaves than did other strains within a given experiment. Most Tn5-induced mutants of strain B728a which conferred a low frequency of ice nucleation on inoculated bean leaves generally had a smaller population size than the parental strain at the time of the leaf freezing assay. The leaf freezing assay was capable of differentiating samples which varied by approximately three- to fivefold in mean bacterial population size.

Journal Article↗
PubMed1980

Approach to recognition of regulatory mutants of cyanobacteria.

Antimetabolite analogs of essential amino acids are useful as selective agents for isolation of regulatory mutants of cyanobacteria, although we observed striking microbiological differences from other widely used eubacterial systems. Regulatory mutants shown to overproduce and excrete tryptophan, phenylalanine, tyrosine, methionine, or arginine were isolated from four cyanobacteria: Anabaena sp. 29151, Synechococcus sp. 602, Synechococcus sp. AN Tx20, and Synechocystis sp. 29108. Surprisingly, regulatory-mutant colonies did not support a halo of cross-fed wild-type growth on selective medium. Since regulatory mutants were shown to excrete substantial levels of amino acids, it was deduced that poor cross-feeding must reflect a generally low nutritional responsiveness of the cyanobacterial background. This conclusion was confirmed by results which showed that regulatory-mutant cells of cyanobacteria dispersed among wild-type populations of Bacillus subtilis did produce halo colonies on solid analog-containing medium. Cross-feeding between one cyanobacterial pair (a phenylalanine excretor and a phenylalanine auxotroph) was successfully demonstrated in the absence of the analog under conditions in which relatively large masses of each cell population type were spread near one another on agar plates. These results suggest that amino acid excreted by regulatory mutants of cyanobacteria on analog-containing selective medium is transported into nearby wild-type cells too inefficiently to overcome the antimetabolite effects of the analog, thereby failing to generate halos of physiologically resistant background cells. Consistent with this interpretation was the finding that the pheA1 auxotroph from Synechococcus sp. 602 exhibited a linearly proportional dependence of growth rate upon exogenous concentration of l-phenylalanine (below 20 muM). Wild-type B. subtilis serves as a convenient and sensitive test lawn for screening obvious regulatory mutants from among collections of analog-resistant cyanobacterial mutants. Appropriate B. subtilis auxotrophs can be used as convenient indicator strains for the identification of regulatory mutants in cyanobacteria through the observation of syntrophic growth responses.

Amino Acids↗
PubMed2006

Identification of chromosomal fusion sites in Arabidopsis mutants using sequential bicolour BAC-FISH.

Double stranded chromosomal breaks are repaired by homologous recombination or nonhomologous end joining (NHEJ). When broken chromosome ends are fused together by NHEJ, the resulting dicentric chromosomes can be detected as anaphase bridges during the subsequent mitosis. Telomeres in the absence of functional telomerase shorten, became unprotected, and are eventually recognized by the cell repair system as double stranded breaks. As result, chromosomes of Arabidopsis thaliana plants that are deficient in the gene for telomerase reverse transcriptase (TERT) are prone to chromosome fusions. We use Arabidopsis tert-/- mutants as a model system for analyzing terminal chromosome fusions. Here we report a novel and sensitive cytogenetic assay for the identification and characterization of chromosome-terminal fusion events by employing fluorescence in situ hybridization (FISH) with multiple probes and a repeated hybridization approach. A mixture of chromosome-specific subtelomeric probes is applied successively in 3 FISH reactions to the slides containing mitotic anaphase figures with anaphase bridges. Each figure is registered by a CCD camera after each in situ hybridization procedure. By comparing the signals presented on the bridge in successive images the assessment of the particular chromosome arms involved in fusion is possible. This experimental setup enables unambiguous identification of individual chromosome ends employed in fusion events.

Arabidopsis↗
PubMedDate not supplied

Surveillance activity for canine parvovirus in Italy.

Recent identification of unusual canine parvovirus (CPV) mutants in cats and dogs suggests that CPV type 2 (CPV-2), which emerged suddenly in the late 1970s, is undergoing continual genetic and antigenic variations. A peculiarity of parvoviruses is that single-nucleotide substitutions may determine drastic phenotypic changes. The effects of either natural or artificial mutations on CPV phenotypic properties have been largely investigated, and this sets up CPV as an interesting model to study virus evolution. By monitoring the evolution of CPV-2 in Italy, we observed the onset and quick spread of a Glu-426 mutant, antigenically different from the pre-existing variants that were partially displaced within a few years of the initial identification of the new mutant. The identification of CPV-2 variants raises several questions concerning their impact on the efficacy of the current CPV-2 vaccines, based on the original CPV-2 strain that no longer exists in the field.

Animals↗
PubMed1998

The mutant plasmacytoma cell line S107 allows the identification of distinct pathways leading to NF-kappaB activation.

Studies on the mechanisms of inducible and constitutive activity of NF-kappaB transcription factors have been hampered by the lack of appropriate mutant cell lines. We have analyzed the defect in the murine S107 plasmacytoma cell line, which was previously found to lack both constitutive and inducible NF-kappaB activity. Our analysis shows that these cells bear a specific defect that interferes with NF-kappaB induction by many diverse stimuli, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, UV light, x-rays, and H2O2. This does not however represent a general signal transduction defect, because AP-1 transcription factors are readily induced by the same stimuli. Phosphatase inhibitors such as okadaic acid as well as calyculin A can efficiently induce NF-kappaB in S107 cells via a pathway apparently insensitive to the radical scavenger pyrrolidine dithiocarbamate. Furthermore, MEKK1 a protein kinase supposedly induced by some of the above stimuli, is also capable of activating NF-kappaB. Interestingly, both the potent physiological inducer of NF-kappaB TNFalpha as well as endoplasmic reticulum overload can induce NF-kappaB via a PDTC sensitive pathway. In all cases, DNA-binding NF-kappaB complexes are comprised predominantly of p50-RelA heterodimers, and NF-kappaB activation results in the induction of transiently transfected or resident reporter genes. In summary, these results suggest that the pathways for many NF-kappaB-inducing stimuli converge at a specific junction, and this pivotal step is mutated in the S107 cell line. Yet there are alternative routes bypassing this critical step that also lead to NF-kappaB induction. These routes utilized by tumor necrosis factor alpha and endoplasmic reticulum overload are still intact in this cell line.

Animals↗
PubMed1992

Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly.

We describe the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas). These mutants of P. pastoris can be identified solely by their inability to grow on methanol and oleic acid, the utilization of which requires peroxisomal enzymes, and are defined by the absence of normal peroxisomes as judged by electron microscopy and biochemical fractionation experiments. These mutants are the result of genetic defects at single loci and represent at least eight different complementation groups. The isolation of pas mutants of P. pastoris by a simple screen for mutants unable to use methanol and oleic acid represents a significantly more efficient method for identification of pas mutants than is possible in other organisms. To exploit this advantage fully we also developed new reagents for the genetic and molecular manipulation of P. pastoris. These include a set of auxotrophic strains with an essentially wild-type genetic background, plasmids that act as Escherichia coli-P. pastoris shuttle vectors, and genomic DNA libraries for isolation of P. pastoris genes by functional complementation of mutants or by nucleic acid hybridization. The availability of numerous pas mutants and the reagents necessary for their molecular analysis should lead to the isolation and characterization of genes involved in peroxisome assembly.

Biological Transport↗
PubMed1992

dnaR function of the prs gene of Escherichia coli in initiation of chromosome replication.

A new Escherichia coli mutant named dnaR, which was temperature sensitive in initiation of DNA replication, has been characterized through identification of the mutant gene. A 1.65 x 10(3) base-pair chromosomal DNA fragment isolated from wild-type cells, but not the corresponding fragment from the dnaR mutant, exhibited an activity that reversed temperature-sensitive growth of the mutant. The DNA fragment was found to include the entire prs-coding sequence and specify a 34,000 M(r) protein with phosphoribosylpyrophosphate synthetase activity. The dnaR mutation resided within the prs-coding segment and made the synthetase thermolabile. The coding segment for the dnaR product was determined, by introduction of various mutations into the cloned fragment, to be the same as that for the synthetase. The dnaR function of the prs gene product in DNA replication is discussed on the basis of an observation that thermal treatment of the dnaR mutant caused a delay in initiation of chromosome replication after the downshift, despite the presence of the synthetase activity at the preheat level.

Base Sequence↗
PubMed2005

A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum.

Rapid progress in fungal genome sequencing presents many new opportunities for functional genomic analysis of fungal biology through the systematic mutagenesis of the genes identified through sequencing. However, the lack of efficient tools for targeted gene replacement is a limiting factor for fungal functional genomics, as it often necessitates the screening of a large number of transformants to identify the desired mutant. We developed an efficient method of gene replacement and evaluated factors affecting the efficiency of this method using two plant pathogenic fungi, Magnaporthe grisea and Fusarium oxysporum. This method is based on Agrobacterium tumefaciens-mediated transformation with a mutant allele of the target gene flanked by the herpes simplex virus thymidine kinase (HSVtk) gene as a conditional negative selection marker against ectopic transformants. The HSVtk gene product converts 5-fluoro-2'-deoxyuridine to a compound toxic to diverse fungi. Because ectopic transformants express HSVtk, while gene replacement mutants lack HSVtk, growing transformants on a medium amended with 5-fluoro-2'-deoxyuridine facilitates the identification of targeted mutants by counter-selecting against ectopic transformants. In addition to M. grisea and F. oxysporum, the method and associated vectors are likely to be applicable to manipulating genes in a broad spectrum of fungi, thus potentially serving as an efficient, universal functional genomic tool for harnessing the growing body of fungal genome sequence data to study fungal biology.

Agrobacterium tumefaciens↗
PubMed2001

p53 mutants can often transactivate promoters containing a p21 but not Bax or PIG3 responsive elements.

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fisher's exact test), more rarely mutated (P<0.006, Fisher's exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fisher's exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fisher's exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.

Amino Acid Sequence↗
PubMed2003

Detection of a point mutation associated with high-level isoniazid resistance in Mycobacterium tuberculosis by using real-time PCR technology with 3'-minor groove binder-DNA probes.

Tuberculosis remains one of the leading infectious causes of death worldwide. The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health threat. Resistance to isoniazid (INH) is the most prevalent form of resistance in M. tuberculosis and is mainly caused by mutations in the catalase peroxidase gene (katG). Among high-level INH-resistant isolates (MIC > or = 2), 89% are associated with a mutation at codon 315 of katG. There is a need to develop rapid diagnostic tests to permit appropriate antibiotic treatment and to improve clinical management. Therefore, a single-tube real-time PCR, using a novel kind of probe (3'-minor groove binder-DNA probe), was developed to detect either the wild-type or the mutant codon directly in Ziehl-Neelsen-positive sputum samples. The detection limit of the assay for purified DNA was 5 fg per well (one mycobacterial genome), and with spiked sputum samples, it was 20 copies per well, corresponding to 10(3) mycobacteria per ml of sputum. Sputum samples from 20 patients living in Kazakhstan or Moldova and infected with monodrug- or multidrug-resistant M. tuberculosis and 20 sputum samples from patients infected with INH-susceptible M. tuberculosis were tested. The sensitivities and specificities of the probes were 70 and 94% for the wild-type probe and 82 and 100% for the mutant probe. Binding to either probe was nonambiguous. This real-time PCR allows the rapid identification of a mutant katG allele and can easily be implemented in a clinical microbiology laboratory.

3' Untranslated Regions↗