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Results for “Multilocus sequence typing”

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PubMed1993

Geographic and temporal distribution and molecular characterization of two highly pathogenic clones of Streptococcus pyogenes expressing allelic variants of pyrogenic exotoxin A (Scarlet fever toxin).

The molecular population genetics and pathogenic potential of North American and European invasive strains of Streptococcus pyogenes were assessed. Isolates from recent invasive infections and from infections in the 1920s and 1930s were characterized for multilocus enzyme genotype and allelic variation in the gene (speA) that encodes streptococcal pyrogenic exotoxin (SPE) A (scarlet fever toxin). A subset of strains was studied for allelic variation in genes that encode SPE B and streptokinase. All contemporary strains assigned to electrophoretic types (ETs) 1 and 2 that synthesize SPE A have the speA2 and speA3 allelic variants, respectively, and their relative virulence in two mouse models is similar to that of strains of the same ET and M protein types recovered earlier. In contrast, ET 1 and 2 isolates from disease episodes in the 1920s and 1930s contain the speA1 allele. The data suggest there may be temporal and geographic variation in the occurrence of clone--virulence factor allele combinations, an observation that may in part explain fluctuations in disease frequency, severity, and character.

Alleles↗

PubMed1993

Genetic characterization of six parasitic protozoa: parity between random-primer DNA typing and multilocus enzyme electrophoresis.

We have assayed genetic polymorphisms in several species of parasitic protozoa by means of random amplified polymorphic DNA (RAPD). One goal was to ascertain the suitability of RAPD markers for investigating genetic and evolutionary problems, particularly in organisms, such as the parasitic protozoa, unsuitable for traditional methods of genetic analysis. Another goal was to test certain hypotheses concerning Trypanosoma cruzi, and other protozoa, that have been established by multilocus enzyme electrophoresis. The RAPD results corroborate the hypothesis that the population structure of T. cruzi is clonal and yield a phylogeny of the clonal lineages in agreement with the one obtained by enzyme electrophoresis. This parity between the two sets of results confirms that RAPD markers are reliable genetic markers. The RAPD markers are also suitable for reconstructing species phylogenies and as diagnostic characters of species and subspecific lineages. The number of DNA polymorphisms that can be detected by the RAPD method seems virtually unlimited, since the number of primers can be increased effectively at will. The RAPD method is well suited for investigating genetic and evolutionary questions in certain organisms, because it is cost effective and demands no previous genetic knowledge about the organism.

Animals↗

PubMed2005

Multilocus consensus genetic maps (MCGM): formulation, algorithms, and results.

In process of creating genetic maps different labs/research groups obtain overlapping parts of the map. Merging these parts into one integrative map is based on looking for maximum shared marker orders among the maps. Really, not all shared markers of such maps have consensus order that obstructs building of the integrative maps. In this paper we propose a new approach to build verified multilocus consensus genetic maps in which shared markers always are integrated in stable consensus order. The approach is based on combined analysis of initial mapping data rather than manipulating with previously constructed maps. We show that more effective and reliable solutions may be obtained based on "synchronized ordering" facilitated by cycles of "re-sampling-->ordering-->removing unstable markers". The proposed formulation of consensus genetic mapping can be considered as a version of traveling salesperson problem (TSP) that we refer to as synchronized-TSP. From the viewpoint of optimization, synchronized-TSP belongs to discrete constrained optimization problems. Earlier we developed new powerful and fast guided evolution strategy algorithms for some types of discrete constrained optimization. These algorithms were used here as a basis for solving more challenging problems of consensual marker ordering.

Algorithms↗

PubMedDate not supplied

Rapid molecular genetic subtyping of serotype M1 group A Streptococcus strains.

Serotype M1 group A Streptococcus, the most common cause of invasive disease in many case series, generally have resisted extensive molecular subtyping by standard techniques (e.g., multilocus enzyme electrophoresis, pulsed-field gel electrophoresis). We used automated sequencing of the sic gene encoding streptococcal inhibitor of complement and of a region of the chromosome with direct repeat sequences to unambiguously differentiate 30 M1 isolates recovered from 28 patients in Texas with invasive disease episodes temporally clustered and thought to represent an outbreak. Sequencing of the emm gene was less useful for M1 strain differentiation, and restriction fragment length polymorphism analysis with IS1548 or IS1562 as Southern hybridization probes did not provide epidemiologically useful subtyping information. Sequence polymorphism in the direct repeat region of the chromosome and IS1548 profiling data support the hypothesis that M1 organisms have two main evolutionary lineages marked by the presence or absence of the speA2 allele encoding streptococcal pyrogenic exotoxin A2.

Amino Acid Sequence↗

PubMed2004

Renin-angiotensin system gene polymorphisms and atrial fibrillation.

BACKGROUND: The activated local atrial renin-angiotensin system (RAS) has been reported to play an important role in the pathogenesis of atrial fibrillation (AF). We hypothesized that RAS genes might be among the susceptibility genes of nonfamilial structural AF and conducted a genetic case-control study to demonstrate this. METHODS AND RESULTS: A total of 250 patients with documented nonfamilial structural AF and 250 controls were selected. The controls were matched to cases on a 1-to-1 basis with regard to age, gender, presence of left ventricular dysfunction, and presence of significant valvular heart disease. The ACE gene insertion/deletion polymorphism, the T174M, M235T, G-6A, A-20C, G-152A, and G-217A polymorphisms of the angiotensinogen gene, and the A1166C polymorphism of the angiotensin II type I receptor gene were genotyped. In multilocus haplotype analysis, the angiotensinogen gene haplotype profile was significantly different between cases and controls (chi2=62.5, P=0.0002). In single-locus analysis, M235T, G-6A, and G-217A were significantly associated with AF. Frequencies of the M235, G-6, and G-217 alleles were significantly higher in cases than in controls (P=0.000, 0.005, and 0.002, respectively). The odds ratios for AF were 2.5 (95% CI 1.7 to 3.3) with M235/M235 plus M235/T235 genotype, 3.3 (95% CI 1.3 to 10.0) with G-6/G-6 genotype, and 2.0 (95% CI 1.3 to 2.5) with G-217/G-217 genotype. Furthermore, significant gene-gene interactions were detected by the multifactor-dimensionality reduction method and multilocus linkage disequilibrium tests. CONCLUSIONS: This study demonstrates the association of RAS gene polymorphisms with nonfamilial structural AF and may provide the rationale for clinical trials to investigate the use of ACE inhibitor or angiotensin II antagonist in the treatment of structural AF.

Aged↗

PubMed1996

Characterization of multilocus lesions in human cells exposed to X radiation and radon.

Human TK6 lymphoblasts were exposed to X radiation or radon, and thymidine kinase negative (TK-/-) mutants were selected, isolated and harvested for analysis of structural changes in the TK gene. A large majority (82%) of the radon-induced mutants, 74% of the X-radiation-induced mutants and 45% of the spontaneous mutants lost the entire active TK allele. To analyze these mutants further we measured the loss of heterozygosity at several loci neighboring the TK locus on chromosome 17q. A greater proportion (61%) of the radon-induced mutants than X-radiation-induced or spontaneous mutants harbored the smaller lesions involving the TK allele alone or extending from the TK locus to one or both of the closest neighboring sequences tested. Further, 21% of the X-radiation-induced mutants but only 5% of the radon-induced mutants lost heterozygosity at the col1A1 locus, 31 Mb from the TK gene. These results are in agreement with a recent analysis of radon- and X-radiation-induced lesions inactivating the HPRT gene of TK6 cells, in which we reported that a lower percentage of radon- than X-radiation-induced mutants showed lesions extending to markers 800 kb or more from the HPRT gene on the X chromosome (Bao et al., Mutat. Res. 326, 1-13, 1995). In the present study, we observed that the percentage of slowly growing and very slowly growing TK-/- mutants was greater after treatment with radon than after treatment with X radiation, regardless of the type of lesion present. It is possible, therefore, that the radon-induced lesions are complex and/or less easily repaired, leading to slow growth in a large proportion of the surviving mutant cells.

B-Lymphocytes↗

PubMed1996

Analysis of genetic relationships among strains of groups A, C and G streptococci by random amplified polymorphic DNA analysis.

Random amplified polymorphic DNA (RAPD) analysis was evaluated for its capacity to distinguish species and strains within species of groups A, C and G streptococci. The 99 strains tested, previously typed by multilocus enzyme electrophoresis (MLEE), included 41 group A streptococci (Streptococcus pyogenes), 25 group G Streptococcus spp. (GGS), seven S. dysgalactiae, 11 S. equisimilis, four S. canis, three S. equi and eight S. zooepidemicus. The combined data obtained with three single primers distinguished 82 types. RAPD analysis provided taxonomic results that were in general agreement with previous species classification based on DNA-DNA homology and MLEE. The intraspecies typing efficiency of the technique was significantly improved by the parallel use of several primers. RAPD analysis had greater discriminatory power than MLEE for GAS and GGS. There was not total agreement between the two techniques as RAPD distinguished strains with identical electrophoretic types, whereas MLEE differentiated strains with identical PCR types. RAPD analysis did not distinguish all GAS strains with different biotypes and its already high discriminatory power was further enhanced by concomitant biotyping.

Animals↗

PubMed1995

Genetic diversity and population structure of Trypanosoma brucei: clonality versus sexuality.

Genomic fingerprinting by arbitrarily primed PCR was used to analyze the genetic variability among 59 Trypanosoma brucei stocks representing the three T. brucei subspecies isolated from various hosts and different countries in Africa. 14 oligonucleotide primers revealed 355 polymorphic binary characters which were used for phenetic and phylogenetic analysis and to perform recombination tests exploring the linkage disequilibrium in the sample. There was good concordance between arbitrarily primed PCR polymorphisms and isoenzyme data previously collected for many of the same strains [1]. However, the arbitrarily primed PCR typing was more discerning than multilocus enzyme electrophoresis typing. Phenetic and phylogenetic analysis using arbitrarily primed PCR markers did not confirm T. brucei brucei and T. brucei rhodesiense as separate subspecies, but T. brucei gambiense group I was monophyletic, confirming this group as suitable for the subspecies status. With this exception, there were no clear lineages among the sample, other than clustering of East African stocks and clustering of West African stocks. Some features of the phylogenetic analysis suggested that the population structure was not strictly clonal though recombination tests showed linkage disequilibrium, even in the absence of repeated genotypes. While genotypes appear stable enough for tracking in applied studies, sexuality will impact at the evolutionary time scale, and may be more frequent under some ecological conditions. The arbitrarily primed PCR approach should be an effective and simple approach to follow epidemics and to quantify the role of sexuality in T. brucei populations.

Africa↗

PubMed2002

Frequent and multiple mutations at minisatellite loci in sporadic human colorectal and gastric cancers--possible mechanistic differences from microsatellite instability in cancer cells.

Minisatellites (MNs), composed of 5 to 100 nucleotide repeat units, range from 0.5 to 30 kb in length, and have been reported to be mutated in various human malignancies. In this study, frequencies of MN mutations in sporadic human colorectal (34 cases) and gastric cancers (24 cases) at various clinicopathological stages were assessed by multilocus DNA fingerprint analysis with three MN probes, Pc-1, 33.6 and 33.15. MN mutations were observed in both colorectal and gastric cancers, but at a significantly higher frequency in the former (56%) than in the latter (25%). Multiplicities of MN mutations were 1.50 +/- 1.81 and 0.46 +/- 1.10 in colorectal and gastric cancers, respectively, and the difference was also significant. Neither the presence nor multiplicity of MN mutations in either colorectal or gastric cancer cases had any correlation with the pathological stage, histological grading or the presence of microsatellite instability (MSI). Although the biological relevance of MN mutations still remains to be clarified, a subset of colorectal and gastric cancers could feature a new type of genomic instability, distinct from MSI.

Adult↗

PubMed1994

Further evidence for the non-clonal population structure of Neisseria gonorrhoeae: extensive genetic diversity within isolates of the same electrophoretic type.

Analysis of data from multilocus enzyme electrophoresis has revealed that Neisseria gonorrhoeae populations are non-clonal. Fifteen percent of 227 isolates of N. gonorrhoeae had an identical multilocus genotype (ET1) and were recovered world-wide over a 26 year period. The recovery of isolates of identical multilocus genotype from geographically and temporally unassociated hosts is a common criterion of a clonal population structure. However, in a recombining (non-clonal) population, isolates with the same multilocus genotype can arise by the random association of the most common alleles in the population. Analysis of the variation in two further enzymes, in the restriction patterns obtained from the glutamine synthetase gene, and in the DNA fragments obtained using an arbitrarily primed polymerase chain reaction, was used to show that members of ET1 were almost as variable as randomly selected N. gonorrhoeae isolates. Unlike the situation in a strongly clonal species, the 26 ET1 isolates examined were increasingly sub-divided to give 19 distinguishable groups as variation at further loci was examined, and 24 distinguishable groups when auxotypes were also considered. We conclude that, as expected of a non-clonal population, the most commonly encountered N. gonorrhoeae multilocus genotype does not define a clone.

Bacterial Proteins↗

PubMed2006

Polyphasic analysis of Thermus isolates from geothermal areas in Iceland.

Genetic relationships and diversity of 101 Thermus isolates from different geothermal regions in Iceland were investigated by using multilocus enzyme electrophoresis (MLEE) and small subunit ribosomal rRNA (SSU rRNA) sequence analysis. Ten polymorphic enzymes were used and seven distinct and genetically highly divergent lineages of Thermus were observed. Six of seven lineages could be assigned to species whose names have been validated. The most diverse lineage was Thermus scotoductus. In contrast to the other lineages, this lineage was divided into very distinct genetic sublineages that may represent subspecies with different habitat preferences. The least diverse lineage was Thermus brockianus. Phenotypic and physiological analysis was carried out on a subset of the isolates. No relationship was found between growth on specific single carbon source to the grouping obtained by the isoenzyme analysis. The response to various salts was distinguishing in a few cases. No relationship was found between temperature at the isolation site and the different lineages, but pH indicated a relation to specific lineages.

Adaptation, Physiological↗

PubMed2006

Multilocus microsatellite typing as a new tool for discrimination of Leishmania infantum MON-1 strains.

The Leishmania donovani complex, which consists of L. donovani, L. infantum-L. chagasi, and L. archibaldi, is responsible for visceral manifestations of leishmaniasis. Multilocus enzyme electrophoresis is the standard method for the characterization and identification of strains of Leishmania. For L. infantum, the predominance of zymodeme MON-1 significantly reduces the discriminative power of this approach. In the present study, we developed 17 independent polymorphic microsatellite markers for the typing of strains of L. infantum, with the main emphasis on zymodeme MON-1. The discriminative powers of 11 markers selected from among these markers were tested by using a panel of 63 isolates of the L. donovani complex. Unique multilocus genotypes were observed for the strains analyzed, with only three exceptions. Model-based and distance-based analyses of the data set showed comparable results. It was possible to discriminate between L. donovani sensu stricto, a non-MON-1 group of L. infantum isolates, and a MON-1 group of L. infantum isolates. Within MON-1, three clusters with geographical correlations became apparent. The frequency of heterozygosity in the alleles analyzed varied extremely between the different groups of isolates. The main clusters described are not consistent with species definitions based on isoenzyme analysis but confirm the results of former PCR-based investigations.

Animals↗

PubMed2006

Multiple gene genealogical analyses suggest divergence and recent clonal dispersal in the opportunistic human pathogen Candida guilliermondii.

Candida guilliermondii is a haploid opportunistic pathogen accounting for about 2 % of human blood yeast infections. Recent analyses using multilocus enzyme electrophoresis and karyotyping suggest that strains from human sources traditionally designated C. guilliermondii in fact include at least two species, C. guilliermondii and Candida fermentati. However, the patterns of molecular variation within and between these two species remain largely unknown. In this study, DNA fragments were sequenced from five genes for each of 37 strains collected from Canada, China, the Philippines and Tanzania. The analyses identified significant sequence differences between C. guilliermondii and C. fermentati. The five gene genealogies showed no apparent incongruence, suggesting a predominantly clonal reproductive structure for both species in nature. Indeed, two large clones of C. guilliermondii were identified, with one from Ontario, Canada, and the other from China. Interestingly, the results indicate that strains currently designated C. guilliermondii may contain additional divergent lineages. On the practical side, the results revealed several diagnostic molecular markers that can be used in clinical microbiology laboratories to distinguish C. guilliermondii and C. fermentati. The multiple gene genealogical analyses conducted here revealed significant divergence and clonal dispersal in this important pathogenic yeast complex.

Actins↗

PubMed1995

Identification of multiple clones of extended-spectrum cephalosporin-resistant Streptococcus pneumoniae isolates in the United States.

We characterized 12 isolates of Streptococcus pneumoniae with various levels of susceptibility of penicillin and extended-spectrum cephalosporins by antimicrobial susceptibility patterns, serotypes, ribotypes, chromosomal DNA restriction patterns by pulsed-field gel electrophoresis, multilocus enzyme electrophoresis patterns, penicillin-binding protein (PBP) profiles, and DNA restriction endonuclease cleavage profiles of pbp1a, pbp2x, and pbp2b. Seven cefotaxime-resistant (MIC, > or = 2 micrograms/ml) serotype 23F isolates were related on the basis of ribotyping, pulsed-field gel electrophoresis, and multilocus enzyme electrophoresis, but they had two slightly different PBP patterns: one unique to strains for which the MIC of penicillin is high (4.0 micrograms/ml) and one unique to strains for which the MIC of penicillin is low (0.12 to 1.0 micrograms/ml). The pbp1a and pbp2x fingerprints were identical for the seven isolates; however, the pbp2b fingerprints were different. An eighth serotype 23F isolate with high-level resistance to cephalosporins was not related to the other seven isolates by typing data but was a variant of the widespread, multiresistant serotype 23F Spanish clone. The PBP profiles and fingerprints of pbp1a, pbp2x, and pbp2b were identical to those of the Spanish clone isolate. An additional serotype 6B isolate with high-level resistance to cephalosporins had unique typing profiles and was unrelated to the serotype 23F cephalosporin-resistant isolates but was related on the basis of genetic typing methods to a second serotype 6B isolate that was cephalosporin susceptible. The serotype 6B isolates had different PBP profiles and fingerprints for pbp1a, but the fingerprints for pbp2x and pbp2b were the same.

Aminoacyltransferases↗

PubMedDate not supplied

Is Leishmania (Viannia) peruviana a distinct species? A MLEE/RAPD evolutionary genetics answer.

A set of 38 Leishmania stocks from the Andean valleys of Peru was characterized by both Multilocus Enzyme Electrophoresis (MLEE) and Random Amplified Polymorphic DNA (RAPD). Data were analyzed in terms of taxonomy and evolutionary genetics. Synapomorphic MLEE and RAPD characters, clear-cut clustering, and strong agreement between the phylogenies inferred from either MLEE or RAPD supported the view that Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis correspond to two closely related, but distinct monophyletic lines (clades) and can therefore be considered as "discrete typing units" (DTUs). The question whether the L. (V.) peruxviana DTU deserves species status is dependent upon the desirability of it, in terms of epidemiological and medical relevance. A previous Orthogonal Field Alternating Gel Electrophoresis (OFAGE) analysis of the same L. (V.) peruviana isolates was published by Dujardin et al. (1995b). The data from the different markers (i.e. MLEE, RAPD and OFAGE) were compared by population genetics analysis. RAPD and OFAGE provided divergent results, since RAPD showed a strong linkage disequilibrium whereas OFAGE revealed no apparent departure from panmictic expectation. MLEE showed no linkage disequilibrium. Nevertheless, contrary to OFAGE, this is most probably explainable by the limited variability revealed by this marker in L. (V.) peruviana (statistical type II error). RAPD data were consistent with the hypothesis that the present L. (V.) peruviana sample displays a basically clonal population structure with limited or no genetic exchange. Disagreement between RAPD and OFAGE can be explained either by accumulation of chromosomal rearrangements due to amplification/deletion of repeated sequences, or by pseudo-recombinational events.

Animals↗

PubMed2005

Ten years of AFLP in ecology and evolution: why so few animals?

Researchers in the field of molecular ecology and evolution require versatile and low-cost genetic typing methods. The AFLP (amplified fragment length polymorphism) method was introduced 10 years ago and shows many features that fulfil these requirements. With good quality genomic DNA at hand, it is relatively easy to generate anonymous multilocus DNA profiles in most species and the start-up time before data can be generated is often less than a week. Built-in dynamic, yet simple modifications make it possible to find a protocol suitable to the genome size of the species and to screen thousands of loci in hundreds of individuals for a relatively low cost. Until now, the method has primarily been applied in studies of plants, bacteria and fungi, with a strong bias towards economically important cultivated species and their pests. In this review we identify a number of research areas in the study of wild species of animals where the AFLP method, presently very much underused, should be a very valuable tool. These aspects include classical problems such as studies of population genetic structure and phylogenetic reconstructions, and also new challenges such as finding markers for genes governing adaptations in wild populations and modifications of the protocol that makes it possible to measure expression variation of multiple genes (cDNA-AFLP) and the distribution of DNA methylation. We hope this review will help molecular ecologists to identify when AFLP is likely to be superior to other more established methods, such as microsatellites, SNP (single nucleotide polymorphism) analyses and multigene DNA sequencing.

Animals↗

PubMed1999

Cleavase fragment length polymorphism analysis of Neisseria meningitidis basic metabolic genes.

Cleavase fragment length polymorphism (CFLP) is a subtyping system based on the property of the enzyme cleavase to recognize junctions between single- and double-stranded regions of DNA formed after denaturation and cooling. To assess the capacity of CFLP for discriminating Neisseria meningitidis serogroup B strains belonging to the electrophoretic type (ET) 5 (ET-5) complex from other serogroup B strains, 30 serogroup B N. meningitidis isolates were subtyped by CFLP with internal fragments of five housekeeping genes, adk, aspC, carA, dhp, and glnA. Two genes (glnA and carA) which demonstrated a high degree of diversity for the serogroup B isolates were then used to further evaluate the suitability of CFLP for screening 50 serogroup C N. meningitidis outbreak-associated and sporadic-case isolates with a single metabolic gene. The results were compared to those from multilocus enzyme electrophoresis (MEE), the current standard subtyping method. CFLP was able to distinguish the ET-5 complex isolates from other serogroup B isolates as efficiently as MEE. Furthermore, CFLP analysis of a single gene was sufficient to identify and cluster the serogroup C isolates belonging to the ET-37 complex from other, unrelated serogroup C isolates but was not capable of differentiating between the isolates of the major individual ETs of this complex (ET-17 and ET-24) causing most serogroup C meningococcal disease outbreaks in the United States. CFLP based on a single gene with a high degree of diversity but not under selective pressure can be applied to the rapid screening of a large number of isolates related to the recognized epidemic complex ET-5 or ET-37. Additionally, CFLP can be used as an initial screening tool to survey the amount of diversity in genes that might be used to develop a DNA sequence-based subtyping system.

Genes, Bacterial↗

PubMed2000

Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli.

Repetitive-element PCR (rep-PCR) fingerprinting is a promising molecular typing tool for Escherichia coli, including for discriminating between pathogenic and nonpathogenic clones, but is plagued by irreproducibility. Using the ERIC2 and BOXA1R primers and 15 E. coli strains from the ECOR reference collection (three from each phylogenetic group, as defined by multilocus enzyme electrophoresis [MLEE], including virulence-associated group B2), we rigorously assessed the effect of extremely elevated annealing temperatures on rep-PCR's reproducibility, discriminating power, and ability to reveal MLEE-defined phylogenetic relationships. Modified cycling conditions significantly improved assay reproducibility and discriminating power, allowing fingerprints from different cyclers to be analyzed together with minimal loss of resolution. The correspondence of rep-PCR with MLEE with respect to tree structure and regression analysis of distances was substantially better with modified than with standard cycling conditions. Nonetheless, rep-PCR was only a fair surrogate for MLEE, and when fingerprints from different days were compared, it failed to distinguish between different clones within all-important phylogenetic group B2. These findings indicate that although the performance and phylogenetic fidelity of rep-PCR fingerprinting can be improved substantially with modified assay conditions, even when so improved rep-PCR cannot fully substitute for MLEE as a phylogenetic typing method for pathogenic E. coli.

DNA Fingerprinting↗