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Results for “mutational profiling”

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PubMed1997

Prenatal diagnosis of beta-thalassemia among Indians using denaturing gradient gel electrophoresis.

We have offered first trimester prenatal diagnosis to 55 couples at risk for beta-thalassemia, originating from various parts of India, using polymerase chain reaction and denaturing gradient gel electrophoresis. Apart from the six common mutations, codon 30 (CAG-->CAA), Cap site +1 (A-->C), as well as three uncharacterized mutations were seen among the parents. In the majority of cases, the diagnosis was possible by scanning only one fragment (B) where most of the Indian mutations are situated. In 18 out of 55 cases, framework analysis could also have been used to offer prenatal diagnosis without characterizing the beta-thalassemia mutations. In the two cases where the mutations were uncharacterized, prenatal diagnosis was done only on the basis of the anomalous denaturing gradient gel electrophoresis patterns seen in the parents and in previously affected children. This is the first attempt of prenatal analysis using denaturing gradient gel electrophoresis in the extremely diverse Indian population where the profile of mutations has not yet been fully elucidated.

Electrophoresis↗

PubMed1998

Polygenic mutation in Drosophila melanogaster: genotype x environment interaction for spontaneous mutations affecting bristle number.

A highly inbred line of Drosophila melanogaster was subdivided into replicate sublines that were subsequently maintained independently with 10 pairs of parents per generation. The parents were randomly sampled for 19 'unselected' sublines and artificially selected for high or low abdominal or sternopleural bristle number for 12 'selected' sublines (with 3 replicate selection lines/trait/direction of selection). Divergence in mean bristle number among the unselected sublines, and response of the selected sublines to selection, are attributable to the accumulation of new mutations affecting bristle number. The input of mutational variance per generation, VM, can be estimated from the magnitude of response or divergence, assuming neutrality of mutations affecting the bristle traits. We reared unselected lines at generations 222 and 224, and selected lines at generations 182-184 of mutation accumulation at each of three temperatures (18 degrees C, 25 degrees C, 28 degrees C), and estimated the mutational variance common to all environments and the mutational variance from genotype x environment interaction. For sternopleural bristle number, the mutational interaction variance was 26% of the mutational variance common to all temperatures, and the interaction variance was due to temperature x line interaction. For abdominal bristle number, the mutational interaction variance was 142% of the mutational variance common to all temperatures, and the interaction variance was due to interactions of temperature x line, sex x line, and temperature x sex x line. It is possible that segregating variation for bristle number is maintained partly by genotype x environment interaction, but information on the fitness profiles of mutations affecting bristle number in each environment will be necessary to evaluate this hypothesis quantitatively.

Abdomen↗

PubMed1982

Point mutations change the thermal denaturation profile of a short DNA fragment containing the lactose control elements. Comparison between experiment and theory.

To understand the denaturation process of short DNA segments we have chosen a 203-base pair (bp) restriction fragment containing the lactose control region. A steady decrease in GC content exists between its i proximal and z proximal ends. We confirm that this fragment melts at low salt in two subtransitions. A GC to AT mutation in the AT-rich region (mutation UV5) increases the number of denatured base pairs in the first subtransition and decreases the cooperativity of the melting process. A GC to AT mutation in the GC-rich region (mutation L8) decreases the number of denatured base pairs in the first subtransition and increases the cooperativity. These mutations induce the same shift in the temperature of half denaturation. The effects of both mutations are additive. A short deletion at the z end of the fragment affects only the first subtransition. When four GC pairs are added to both end, the fragment melts in one transition. Comparison with the results obtained with a larger 789-bp lac fragment reveals strong end effects on base pair stability and suggests that denaturation of the 203-bp fragment proceeds unidirectionally from the z end. Good agreement is shown with the predictions made with the "z ipper model" of Crothers et al. (1965).

Base Sequence↗

PubMed2005

The Ataxia-telangiectasia mutated gene and breast cancer: gene expression profiles and sequence variants.

The role of the Ataxia-telangiectaisa mutated (ATM) gene, as a risk factor for breast cancer has been a consistent theme in the literature since the first reports by Swift and colleagues who reported that ATM heterozygotes in AT families had increased risks of developing breast cancer. Loss of heterozygosity at the ATM locus has been reported in 30-40% of breast tumours and 50-70% show altered ATM protein levels. Germline ATM sequence variants have been reported in breast cancer cases, however, it is difficult to fully evaluate the increased risk associated with their presence. The potential role of such variants needs to be further assessed, together with functional studies to model their impact on ATM function.

Animals↗

PubMed2005

Altering protein specificity: techniques and applications.

Protein engineering constitutes a powerful tool for generating novel proteins that serve as catalysts to induce selective chemical and biological transformations that would not otherwise be possible. Protocols that are commonly employed for altering the substrate specificity and selectivity profiles by mutating known enzymes include rational and random methods as well as techniques that entail evolution, selection and screening. Proteins identified by these techniques play important roles in a variety of industrial and medicinal applications and in the study of protein structure-function relationships. Herein we present a critical overview of methods for creating new functional proteins having altered specificity profiles and some practical case studies in which these techniques have been applied to solving problems in synthetic and medicinal chemistry and to elucidating enzyme function and biological pathways.

Biotechnology↗

PubMed2000

Despite high concordance, distinct mutational and phenotypic drug resistance profiles in human immunodeficiency virus type 1 RNA are observed in gastrointestinal mucosal biopsy specimens and peripheral blood mononuclear cells compared with plasma.

The gastrointestinal mucosa is a major lymphoid tissue reservoir for human immunodeficiency virus (HIV) replication. Genotypic and phenotypic resistance patterns of HIV type 1 (HIV-1) RNA isolated from colonic mucosa were compared with those from the plasma and peripheral blood mononuclear cells (PBMC) of 7 patients. Genotyping was performed using full-sequence analysis, and phenotyping was performed using a recombinant virus assay. Mutations in the reverse-transcriptase (kappa=.84) and protease (kappa=.73) genes were highly concordant among compartments. Similarly, phenotypic resistance patterns were highly concordant among compartments (intraclass correlation coefficient,.91). In 5 instances among 3 patients, a different genotypic result was observed between plasma and the other tissue compartments. Mixtures of wild-type and mutated HIV-1 RNA were present in the mucosa and PBMC but not in the plasma. Despite significant concordance among compartments, mucosal- and PBMC-derived viral RNA showed instances of discordance with plasma-derived virus that may suggest compartmentalization of virus.

Adult↗

PubMed2001

The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone.

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).

4-Butyrolactone↗

PubMed1995

Burkitt's lymphomas express VH genes with a moderate number of antigen-selected somatic mutations.

The normal counterpart of the neoplastic B cells occurring in Burkitt's lymphomas (BL) is an issue of controversial debate. To clarify this matter, a semi-nested primer polymerase chain reaction was performed to amplify the VDJ rearrangements of the immunoglobulin heavy chain (VH) gene of DNA extracts from 10 (8 sporadic and 2 endemic) BL cases. The resulting amplificates were sequenced for comparison with known germ line VH segments. The control cases comprised six cases of B cell chronic lymphocytic leukemia and six cases of mantle cell lymphoma known to display naive nonmutated, ie, pre-germinal center VH configurations; and eight cases of follicular center lymphoma known to display mutated VH genes with signs of a still-ongoing mutation reaction, characteristic for germinal center cells and lymphomas that derive therefrom. The results of this approach revealed that both sporadic and endemic BL express mutated VH genes with a mutation frequency considerably lower (4.9% and 5.4%, respectively) than that observed in follicular center lymphoma (11.8%). In addition, after subcloning the amplificates, sequence analysis revealed no signs of ongoing mutations. These results led us to conclude that the derivation of neoplastic B cells in BL is definitely not from naive, nonmutated pre-germinal center B cells. Instead, our findings support the view that BL cells stem either from early centroblasts that are arrested after an initial hypermutation reaction, or from germinal center B cells that have differentiated in terms of surface immunoglobulin profile and mutation pattern but not in terms of morphology and proliferation toward SIgM+ IgD- memory B cells because of the deregulated c-myc gene expression.

Burkitt Lymphoma↗

PubMed2002

Inhibitor development in previously untreated patients with hemophilia A: a prospective long-term follow-up comparing plasma-derived and recombinant products.

In order to assess inhibitor development in previously untreated patients (PUPs) with severe (factor VIII [FVIII]<1%) and moderate (FVIII 1 to 5%) hemophilia A, a prospective study was initiated in 1976. During the 23-year study period, 72 hemophiliacs were frequently exposed prophylactically or on demand to plasma-derived (pd) (n = 51) or recombinant FVIII (rFVIII) (n = 21) concentrates (median 270 exposure days [ED]). Inhibitor testing was performed before the first exposure and at regular intervals thereafter. Of the 72 hemophilia A patients, 22 (32%) developed an inhibitor after 15 ED in median (range 4 to 195); 17 (77%) were high responders (>5 Bethesda Units [BU]), and the remaining 5 patients (23%) were low responders (>0.6 to 5 BU). The severely affected patients (n = 46) showed a significantly higher frequency of inhibitor formation (43%) than did the moderate ones (8%). Comparing the severely affected patients receiving pd products exclusively (n = 35) with those treated with recombinant concentrate (n = 11), 37% of the pd group developed a high-titer inhibitor (>5 BU, median 290 ED in noninhibitor patients) and 36% of the recombinant group (median 49 ED in the noninhibitor patients). However, the exposure status of the recombinant noninhibitor patients is rather low and therefore remains a high risk of developing further inhibitors in the future. The mutation type profile revealed no difference between the pd- and the recombinant-treated patients.

Autoantibodies↗

PubMed2005

Hypermutation is a key factor in development of multiple-antimicrobial resistance in Pseudomonas aeruginosa strains causing chronic lung infections.

Pseudomonas aeruginosa is the most relevant pathogen producing chronic lung infections in patients with chronic underlying diseases such as cystic fibrosis (CF), bronchiectasis, and chronic obstructive pulmonary disease (COPD). Hypermutable (or mutator) P. aeruginosa strains, characterized by increased (up to 1,000-fold) spontaneous mutation rates due to alterations of the DNA mismatch repair (MMR) system have been found at high frequencies in the lungs of CF patients, but their role in other chronic processes is still unknown. Sixty-two P. aeruginosa isolates from 30 patients with underlying non-CF chronic respiratory diseases (22 with bronchiectasis and 8 with COPD) and documented chronic infection were studied. Antibiotic susceptibility profiles and mutation frequencies were determined, and complementation assays using the cloned wild-type mutS gene and molecular epidemiology studies (pulsed-field electrophoresis, [PFGE]) were performed with these strains. Thirty-three (53%) of the isolates were hypermutable, and 17 (57%) of the 30 patients were colonized by hypermutable strains. Strains from 11 of the 17 patients were found to be defective in the MMR mutS gene by complementation assays. Interpatient transmission of strains was ruled out by PFGE. Multiple-antimicrobial resistance was documented in 42% of the hypermutable strains in contrast to 0% resistance in the nonhypermutable strains (P < 0.0001). Hypermutable P. aeruginosa strains are extremely prevalent in chronic infections in contrast to what has been described in acute processes, suggesting a role of hypermutation in bacterial adaptation for long-term persistence. Furthermore, hypermutation is found to be a key factor for the development of multiple-antimicrobial resistance, and therefore these findings are expected to have important consequences for the treatment of chronic infections.

Adenosine Triphosphatases↗

PubMed2001

Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns.

The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 microm i.d. monolithic poly(styrene-divinylbenzene) capillary columns was utilized for mutation screening in polymerase chain reaction amplified polymorphic loci. Recognition of mutations is based on the separation of homo- and heteroduplex species by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) under partially denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Six different single nucleotide substitutions and combinations thereof were confidently identified in 413 bp amplicons from six heterozygous individuals each of which yielded a different unique chromatographic profile. Alternatively, mutations were identified in short, 62 bp PCR products upon their complete on-line denaturation at 75 degrees C taking advantage of the ability of IP-RP-HPLC to resolve single-stranded nucleic acids of identical length that differ in a single nucleotide. Separations in monolithic capillary columns can be readily hyphenated to electrospray ionization mass spectrometry and promise increased sample throughput by operating in arrays similar to those already used in capillary electrophoresis.

Alleles↗

PubMed2004

Allelic genes of blood group antigens: a source of human mutations and cSNPs documented in the Blood Group Antigen Gene Mutation Database.

In this report, we analyze data assembled in the Blood Group Antigen Gene Mutation Database (www.bioc.aecom.yu.edu/bgmut/index.htm), which describes sequence information on human genes associated with expression of the various serologically-determined blood group phenotypes. The database documents 38 genetic loci and a total of 624 alleles that together encode a large repertoire of proteins and constitute 27 serologically-defined blood group systems. Analysis of sequence variation patterns across alleles of a number of genes is focused on their molecular profiles, including mutational sites and recurrence, patterns of gene rearrangements in duplicated gene families, correlation of predicted location of epitopes in extracellular loops with sites of alterations, and effects of mutations on protein expression. That information, and the relative ease of identifying individuals bearing variant alleles, has led to the proposal that genes encoding blood group antigens are an important and unique resource for studies of human DNA variation. Another focus is on mutations in regions that encode the antigenic epitopes and on their occurrence in world populations. These mutations may be viewed as coding single nucleotide polymorphisms (cSNPs). We propose that one group of these cSNPs, which are known to occur with significant frequency in all world populations, could serve as well-validated genetic markers. In addition, specific mutations in a number of "low incidence" and rare alleles could serve as cSNPs specific for a given population. The allelic frequencies of these mutations and knowledge of their world-wide occurrence add a valuable dataset to the existing cSNP pools documented in SNP databases.

Alleles↗

PubMed2001

[Gene chip technology and its advances in medical science].

Gene (DNA) chip or DNA microarray is a new technique developed during human genomic research programs. When DNA chip containing thousands of DNA or oligonucleotide are hybridized to labeled samples, gene expression, DNA sequencing as well as DNA mutation and polymorphism can be analyzed with high efficiency in large scale. We are able to perform this technique on dozens of specimens at once in tumor differentiation, tumor typing, tumor diagnosis, tumorigenesis, and new tumor-associated genes discovery.

Gene Expression Profiling↗

PubMed2004

Global expression profiling of strains harbouring null mutations reveals that the five rpf-like genes of Mycobacterium tuberculosis show functional redundancy.

SETTING: Aged, dormant cultures of Mycobacterium tuberculosis can be resuscitated by a secreted, proteinaceous growth factor from Micrococcus luteus, known as resuscitation-promoting factor (Rpf). M. tuberculosis contains five rpf-like genes, rpfA (Rv0867c), rpfB (Rv1009), rpfC (Rv1884c), rpfD (Rv2389c) and rpfE (Rv2450c), that bear significant similarity to Mi. luteus rpf, suggesting that these too may play a role in growth and/or reactivation from a quiescent state. OBJECTIVE AND DESIGN: Unmarked deletion mutants of each of the five rpf-like genes of M. tuberculosis H37Rv were constructed and their phenotypes and global gene expression profiles were assessed. RESULTS AND CONCLUSIONS: Deletion of any one of the rpf-like genes did not affect growth or survival of M. tuberculosis in liquid culture, but some alterations in colony-forming ability and colonial morphology were observed. Global gene expression profiling suggested that loss of rpfC affected the expression of the largest number of genes and there was a significant overlap in the differential gene expression profile of the rpfC mutant with those of the rpfB, rpfD and rpfE mutants. The expression profile of the rpfA mutant was notably less similar, but inverse associations with genes affected in the other mutants were observed. These results, together with those obtained by real-time, quantitative RT-PCR, suggest that the rpf-like genes serve wholly or partially overlapping functions in M. tuberculosis.

Bacterial Proteins↗

PubMed2005

The transcriptional consequences of mutation and natural selection in Caenorhabditis elegans.

The evolutionary importance of gene-expression divergence is unclear: some studies suggest that it is an important mechanism for evolution by natural selection, whereas others claim that most between-species regulatory changes are neutral or nearly neutral. We examined global transcriptional divergence patterns in a set of Caenorhabditis elegans mutation-accumulation lines and natural isolate lines to provide insights into the evolutionary importance of transcriptional variation and to discriminate between the forces of mutation and natural selection in shaping the evolution of gene expression. We detected the effects of selection on transcriptional divergence patterns and characterized them with respect to coexpressed gene sets, chromosomal clustering of expression changes and functional gene categories. We directly compared observed transcriptional variation patterns in the mutation-accumulation and natural isolate lines to a neutral model of transcriptome evolution to show that strong stabilizing selection dominates the evolution of transcriptional change for thousands of C. elegans expressed sequences.

Animals↗

PubMed2003

A common truncated variant of lipoprotein lipase in the Japanese population is characterized by pattern B phenotype.

To investigate the relationship between the lipoprotein lipase Ser447Ter (S447X) mutation, lipid profiles and risk of atherosclerotic disease, we studied two groups of Japanese subjects. These groups consisted of a dyslipidemic group (triglyceride (TG) > 1.69 mmol/l and high-density lipoprotein-cholesterol (HDL-C) < or = 0.91 mmol/l; n = 106) and a control group (TG < or = 1.69 mmol/l and HDL-C > or = 1.16 mmol/l; n = 106). All subjects in the control group were confirmed to the pattern A phenotype (normal low-density lipoprotein (LDL) pattern), and 21 individuals in this group were heterozygous for the S447X mutation, but not homozygous. In the patient group, pattern A was shown by 44 of 106 patients. The rest were of the pattern B phenotype, which is associated with an increased risk of coronary heart disease. Homozygosity was concentrated in the patient group (p < 0.05) and in those with the pattern B phenotype (p < 0.05). In contrast, heterozygosity between both groups was not statistically significant. In conclusion, heterozygous and homozygous status with respect to the LPL S447X mutation appears to have different meanings with respect to biochemical and clinical phenotypes of atherosclerosis.

Adolescent↗

PubMed2026

A guide to understanding tumour evolution through the lens of population genetics.

Every cancer carries the history of its own evolution, hidden in its genome. Modern DNA sequencing can catalogue millions of mutations and profile tumours across space and time, but sequencing alone struggles to answer the questions that matter most: when did key adaptations emerge, how strongly were they selected, why do some tumours relapse whereas others do not, and how will the cancer evolve next? The reason is fundamental: sequencing is a snapshot, whereas evolution is a dynamic process. Bridging this gap requires moving beyond descriptive cancer genomics towards quantitative evolutionary inference. In this Review, we argue that population genetics provides the mathematical framework needed to extract evolutionary dynamics from cancer genomes. We show how models of mutation, selection and drift transform allele frequencies from descriptive measurements into quantitative estimates of clonal fitness and evolutionary timings. We discuss how these principles extend to epigenetic inheritance, plasticity and ecological interactions within the tumour ecosystem, and examine the assumptions and limitations for their application to modern sequencing data. By reframing cancer genomes as quantitative records of evolutionary processes rather than catalogues of mutations, researchers have used population genetics to provide a foundation for understanding - and ultimately predicting - the trajectories of cancer evolution.

Journal Article↗

PubMed2004

The binding mode of epothilone A on alpha,beta-tubulin by electron crystallography.

The structure of epothilone A, bound to alpha,beta-tubulin in zinc-stabilized sheets, was determined by a combination of electron crystallography at 2.89 angstrom resolution and nuclear magnetic resonance-based conformational analysis. The complex explains both the broad-based epothilone structure-activity relationship and the known mutational resistance profile. Comparison with Taxol shows that the longstanding expectation of a common pharmacophore is not met, because each ligand exploits the tubulin-binding pocket in a unique and independent manner.

Binding Sites↗