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PubMed2004

Genetic characterization of Erwinia amylovora strains by amplified fragment length polymorphism.

AIMS: Erwinia amylovora is one of the most important pathogens of pear and apple and is subject to strict quarantine regulations worldwide, although its patterns of dispersal are largely unknown. Previous attempts to fingerprint E. amylovora strains by molecular techniques have detected very little polymorphism because of the high genetic homogeneity of this bacterium. Our aim was to establish and test a typing method to quantify genetic diversity among strains of this plant pathogen. METHODS AND RESULTS: Twenty-two strains from different hosts and geographical locations were examined by PCR fingerprinting with four primers and by amplified fragment length polymorphism (AFLP) with four selected combinations of primers with a single base extension. PCR fingerprinting revealed little polymorphism producing the same amplification patterns for 17 strains, while the combined AFLP patterns yielded 78 polymorphic bands (34% of total bands) and allowed the differentiation of all but two strains. Clustering of strains in the resulting dendrogram was not correlated with host, year or country of isolation, and questions previous genealogies based on PFGE patterns. CONCLUSIONS: The AFLP technique allowed the detection of an unprecedented number of genetic markers in E. amylovora and proved to be the most useful tool so far for discriminating among strains of this pathogen. The results obtained in this study strongly suggest the occurrence of multiple introductions of the pathogen in Spain and other European countries. SIGNIFICANCE AND IMPACT OF THE STUDY: A major limitation in understanding the ecology of fire blight is the lack of typing techniques with a high power of discrimination. This study demonstrates the high resolution and the usefulness of the AFLP technique to differentiate among E. amylovora strains.

Biodiversity↗
PubMed2002

Genetic characterization of the oral Actinomyces.

Actinomyces are difficult to identify using serological and biochemical methods but genotyping is an efficient and reliable means of bacterial characterization and can be used to determine clonal identity. The purpose here was to genotype 13 American type culture collection (ATCC) reference strains representing six different oral Actinomyces spp. by using chromosomal DNA fingerprinting (CDF), arbitrarily primed-polymerase chain reaction (AP-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In CDF analysis, BamHI, BstEII and SmaI yielded digestion patterns revealing characteristic differences among the known Actinomyces spp., with SmaI demonstrating optimal resolution. Amplicons generated by AP-PCR with primer OPB-07 displayed banding patterns that permitted discrimination of all Actinomyces strains tested. PCR-RFLP with MnlI digests generated fragment patterns that also characterized the reference strains. Collectively, genotypic profiles generated by CDF, AP-PCR and PCR-RFLP permitted differentiation of all 13 ATCC Actinomyces strains. SmaI CDF analysis of 18 clinical isolates of catalase-positive A. naeslundii genospecies 2 revealed extensive genetic diversity among these strains. These molecular approaches may be useful in determining genetic diversity within oral Actinomyces populations and fidelity of Actinomyces transmission between mother and child.

Actinomyces↗
PubMed1990

Molecular and genetic characterization of an ornithine decarboxylase-deficient Chinese hamster cell line.

The ornithine decarboxylase (ODC)-deficient Chinese hamster ovary (CHO) cell line C55.7 has normal amounts of ODC mRNA with very low amounts of immunologically detectable ODC protein, suggesting a structural mutation; however, 5-azacytidine treatment leads to phenotypical reversion (Steglich, C., and Scheffler, I. E. (1985) Somat. Cell Mol. Genet. 11, 11-23). We have demonstrated by chemical cleavage a single base mismatch in DNA heteroduplexes composed of wild-type and mutant cDNA strands. DNA sequencing showed that the mutant phenotype results from an aspartate-glycine substitution at amino acid 381 of the protein. When 5-azacytidine-revertant cell lines were selected for resistance to alpha-difluoromethylornithine, the resulting amplified ODC gene was structurally indistinguishable from the wild type gene. These results suggested the existence of a single active ODC locus in CHO cells. Using the methylation-sensitive restriction endonucleases AvaI and HpaII, we found evidence for two differentially methylated alleles in wild type, ODC-deficient and alpha-difluoromethylornithine-resistant cells. One of the alleles appeared completely inactivated by hypermethylation but could be reactivated by demethylation in spontaneous or 5-azacytidine-induced revertants.

Animals↗
PubMed2004

Genetic characterization of the dibenzofuran-degrading Actinobacteria carrying the dbfA1A2 gene homologues isolated from activated sludge.

Thirteen dibenzofuran (DF)-utilizing bacteria carrying the DF terminal dioxygenase genes homologous to those of Terrabacter sp. strain DBF63 (dbfA1A2) were newly isolated from activated sludge samples. The amplified ribosomal DNA restriction analysis and the hybridization analyses showed that these strains were grouped into five genetically different types of bacteria. The sequence analyses of the 16S rRNA genes and the dbfA1A2 homologues from these five selected isolates revealed that the isolates belonged to the genus Rhodococcus, Terrabacter or Janibacter and that they shared 99-100% conserved dbfA1A2 homologues. We investigated the genetic organizations flanking the dbfA1A2 homologues and showed that the minimal conserved DNA region present in all five selected isolates consisted of an approximately 9.0-kb region and that their outer regions became abruptly non-homologous. Among them, Rhodococcus sp. strain DFA3 possessed not only the 9.0-kb region but also the 6.2-kb region containing dbfA1A2 homologues. Sequencing of their border regions suggested that some genetic rearrangement might have occurred with insertion sequence-like elements. Also, within their conserved regions, some insertions or deletions were observed.

Actinobacteria↗
PubMed2003

Detection of a Salmonella enterica serovar California strain spreading in spanish feed mills and genetic characterization with DNA microarrays.

We performed an epidemiological study on Salmonella isolated from raw plant-based feed in Spanish mills. Overall, 32 different Salmonella serovars were detected. Despite its rare occurrence in humans and animals, Salmonella enterica serovar California was found to be the predominant serovar in Spanish feed mills. Different typing techniques showed that isolates of this serovar were genetically closely related, and comparative genomic hybridization using microarray technology revealed 23 S. enterica serovar Typhimurium LT2 gene clusters that are absent from serovar California.

Animal Feed↗
PubMed1990

Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2.

One of the regions responsible for the stable inheritance of the broad-host-range plasmid RK2 is contained within the PstI C fragment, located from coordinates 30.8 to 37.0 kb (P.N. Saurugger, O. Hrabak, H. Schwab, and R.M. Lafferty, J. Biotechnol. 4:333-343, 1986). Genetic analysis of this 6.2-kb region demonstrated that no function was present that stabilized by selectively killing plasmid-free segregants. The sequence from 36.0 to 37.0 kb mediated a twofold increase in plasmid copy number, but this region was not required for stabilization activity. The PstI C fragment was shown to encode a multimer resolution system from 33.1 to 35.3 kb. The resolution cis-acting site was mapped to 140 bp, sequenced, and observed to contain two directly repeated sequences of 6 and 7 bases and two perfect inverted repeats of 6 and 8 bases. The trans-acting factor(s) was mapped and functionally determined to encode a resolvase capable of catalyzing recombination at high frequency between cis-acting sites in either direct or inverted orientation. Multimer resolution alone did not account for complete plasmid stabilization by the PstI C fragment, since removal of regions adjacent to the 35.3-kb border of the minimal mrs locus dramatically reduced stabilization. The minimal region required for complete stabilization, from 32.8 to 35.9 kb, was capable of fully stabilizing plasmids independently of the replicon or the recA proficiency of the host. Stabilization activity was also fully expressed in several diverse gram-negative bacteria, whereas the F plasmid par locus functioned only in Escherichia coli. On the basis of these observations, we conclude that under the growth conditions used, the minimal stabilization locus encodes both an mrs activity and a stabilization activity that has the properties of a par locus.

Azotobacter↗
PubMed1998

Biochemical and genetic characterization of a Prevotella intermedia/nigrescens-like organism.

Thirty-three previously non-typable faintly pigmented Gram-negative anaerobic bacterial isolates, biochemically most closely related to Prevotella intermedia and Prevotella nigrescens, were analysed for enzymic reactions, cellular fatty acid (CFA) composition, electrophoretic mobility of malate and glutamate dehydrogenases, hybridization with P. intermedia and P. nigrescens species-specific oligonucleotide probes and, for genetic heterogeneity, by arbitrarily primed PCR (AP-PCR). P. intermedia ATCC 25611T and P. nigrescens ATCC 33563T were run in parallel for comparison. Twenty-nine isolates originated from the normal oral flora of 18 subjects (including five mother-child pairs), and four isolates from various infections. Except for a negative lipase reaction, enzymic profiles of the test isolates were similar to those of P. intermedia and P. nigrescens. Clustering of CFAs, electrophoretic mobility patterns, hybridization with DNA probes for P. intermedia and P. nigrescens, and AP-PCR band patterns of the test isolates differed from those of the type strains of P. intermedia and P. nigrescens, suggesting the existence, in humans, of a new anaerobic species of pigmented, moderately saccharolytic, indole-positive Gram-negative rods.

Bacterial Proteins↗
PubMed2004

Genetic characterization and specific detection of beer-spoilage Lactobacillus sp. LA2 and related strains.

AIMS: Lactobacillus sp. LA2 (DSM15502) and related strains (LA2 group) possess strong beer-spoilage ability. The 16S rDNA sequence of LA2 strain is virtually indistinguishable from that of L. collinoides, generally considered to be nonbeer-spoilage bacteria. The aim of this study was to identify the genetic marker to distinguish between Lactobacillus sp. LA2 group and L. collinoides and to provide a rapid means of identifying beer-spoilage strains belonging to Lactobacillus sp. LA2 group. METHODS AND RESULTS: The 16-23S rDNA intergenic spacer (ITS) regions of Lactobacillus sp. LA2 and L. collinoides JCM1123T were sequenced to identify a genetic marker to distinguish between the two groups. As a result, 300 and 500 bp ITS regions of Lactobacillus sp. LA2 were found to be almost identical with those of L. collinoides JCM1123T. Sequence comparison analysis between Lactobacillus sp. LA2 and L. collinoides JCM1123T revealed that the two contiguously located nucleotides are absent in both ITS regions of Lactobacillus sp. LA2. Based on the sequence difference, we have designed specific PCR primers with a minor modification to the primer sequence that can differentiate between beer-spoilage Lactobacillus sp. LA2 group and nonbeer-spoilage L. collinoides. CONCLUSIONS: The PCR-based method has been developed to identify Lactobacillus sp. LA2 group, providing a rapid and sensitive means of determining the beer-spoilage ability of detected bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The substitution of one nucleotide, located at the third position to the 3'-end in the primer sequence, enhanced the specificity of the PCR method while retaining sufficient sensitivity. The nucleotide gap identified in this study appeared to serve as a useful genetic marker that can differentiate 12 beer-spoilage Lactobacillus sp. LA2 group strains from its close relatives that exhibit no beer-spoilage ability.

Bacterial Typing Techniques↗
PubMed1978

Genetic characterization of Mu-like bacteriophage D108.

Infection of Escherichia coli by bacteriophage D108 was shown to result in the generation of apparently random chromosomal mutations. Approximately 1% of the cells lysogenized by D108, as with Mu, acquired new auxotrophic mutations. D108-induced mutations were nonreverting and were most probably the result of insertion of the D108 genome into regions of genetic function. D108 and Mu shared many similar properties but were heteroimmune and had different host ranges. Lytic infections of Mu lysogens with D108 and D108 lysogens with Mu resulted in 100-fold increases in release of phage with prophage markers over those due to spontaneous induction. Phenotypic mixing was common, with most phage carrying the prophage immunity being packaged in particles with the host range of the superinfecting phage. A fraction of the superinfecting phage genomes were, however, packaged in particles with the prophage-specified host range. Although 10% of the prophage progeny were D108-Mu genetic hybrids, superinfecting phage-induced release of the prophage with reciprocal phenotypic mixing occurred in recA hosts, in which the frequency of D108-Mu genetic hybrids was reduced 100-fold.

Coliphages↗
PubMed2002

Molecular genetic characterization of the EWS/ATF1 fusion gene in clear cell sarcoma of tendons and aponeuroses.

Clear cell sarcoma (CCS) is a rare malignant soft tissue tumor particularly associated with tendons and aponeuroses. The cytogenetic hallmark is the translocation t(12;22)(q13;q12) resulting in a chimeric EWS/ATF1 gene in which the 3'-terminal part of EWS at 22q is replaced by the 3'-terminal part of ATF1 at 12q. To date, only 13 cases of CCS have been analyzed for fusion genes at the transcription level, and there is no information about the breakpoints at the genomic level. In the present study, we describe the molecular genetic characteristics of CCS from 10 patients. Karyotypes were obtained from 10 cases, 7 of which showed the characteristic t(12;22). As an initial step in the characterization of the EWS/ATF1 and ATF1/EWS chimeras, we constructed an exon/intron map of the ATF1 gene. The entire ATF1 gene spanned >40 kb and was composed of 7 exons. Intron 3, in which most of the genomic breakpoints occurred, was to a large extent (83%) composed of repetitive elements. RT-PCR amplified EWS/ATF1 cDNA fragments in all patients and ATF1/EWS cDNA fragments in 6 of 10 patients. Four types of EWS/ATF1 chimeric transcript, designated types 1-4, were identified. The most frequent chimeric transcript (type 1) was an in-frame fusion of exon 8 of EWS with exon 4 of ATF1. This was the only chimeric transcript in 5 patients but found together with other variants in 3 tumors. The type 2 transcript of EWS/ATF1, an in-frame fusion of exon 7 of EWS with exon 5 of ATF1, was detected in 4 patients, as the only transcript in 1 case and together with other variants in 3 cases. An in-frame fusion of exon 10 of EWS with exon 5 of ATF1 (type 3) was found in 1 patient as the only transcript, and an out-of-frame fusion of EWS exon 7 with ATF1 exon 7 (type 4) was detected in 1 patient together with type 1 and type 2 transcripts. Sequencing of the amplified ATF1/EWS cDNA fragments showed in 5 patients that ATF1 exon 3 was fused with EWS exon 10, resulting in an out-of-frame chimeric transcript. In 1 case, nt 428 of ATF1 (exon 4) was fused with EWS exon 8; at the junction, there was an insertion of 4 nucleotides, also resulting in an out-of-frame transcript. Genomic extra long PCR and sequence analysis mapped the genomic breakpoints to introns 7, 8 and 9 of EWS and intron 3 and exon 4 of ATF1. While a simple end-to-end fusion was observed in 2 cases, additional nucleotides were found at the junctions in 2 other cases. In addition, topoisomerase I consensus sequences were found close to the junctions, suggesting that this enzyme may participate in the genesis of the EWS/ATF1 fusion.

Adolescent↗
PubMed2002

Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strains from cases presenting various clinical conditions.

Porcine circovirus type 2 (PCV2) has been associated with a newly identified and described disease in swine called the postweaning multisystemic wasting syndrome (PMWS). An association of PCV2 with various other clinical conditions in pigs are also increasingly being reported. To date molecular studies to determine the extent of genetic variations of this virus have been limited. To more fully understand the extent of genetic diversity we report the sequencing of 34 PCV2 strains from Eastern Canada originating from a variety of clinical conditions spanning years 1990-2001 along with the phylogenetic analysis of these strains and that of 36 PCV2 sequences published in GenBank. Sequence analysis of the complete genome indicated that the Canadian PCV2 strains analyzed in the present study are closely related to each other but also to other PCV2 strains originating from Western Canada, US, Europe and Asia. Sequence analysis of ORF1 and ORF2 genes of the 34 strains revealed that the extent of nucleotide variation was greater for the ORF2 than for the ORF1. The amino acid sequences alignment of the PCV2 capsid protein identified three major regions of amino acid heterogeneity, two of which correspond with dominant immunoreactive areas. No repeatable amino acid motifs for these two regions could be associated with PCV2 strains identified from cases of PMWS or other clinical conditions. Phylogenetic analysis of all 70 strains revealed one large cluster composed of strains from Europe, Taiwan, China and Canada. This large cluster could be divided in several sub clusters, in two of which Canadian strains were found closely related to strains from Germany. The remaining strains from Canada and the US were spread in small groupings along the phylogenetic tree and an association with geographic origin could not be established. The genomic characterizations performed in this study indicate that PCV2 strains associated with PMWS are scattered throughout the phylogenetic tree often in groupings including PCV2 strains identified from cases other that PMWS such as, porcine reproductive and respiratory syndrome (PRRS), generalized tremors, porcine dermatitis and nephropathy syndrome (PDNS), arthritis, nervous signs, erysipelas and even healthy pigs.

Amino Acid Sequence↗
PubMed1982

Identification of the structural genes for glutamate synthase and genetic characterization of this region of the Salmonella typhimurium chromosome.

Salmonella typhimurium cells require glutamate synthase activity for growth in media containing a growth rate-limiting nitrogen source. Although this enzyme plays a critical role in ammonia assimilation, little is known about the organization and regulation of the structural genes for its two subunits. To identify the location of the structural genes, mutants having heat-labile glutamate synthase activities were isolated and characterized. Mutations that altered glutamate synthase activity were mapped at 69 U on the S. typhimurium chromosome. Four strains with independent Tn10 insertions in this region were constructed and used for mutant selection and for positioning mutations affecting glutamate synthase activity relative to other genetic markers. In contrast to results obtained with Escherichia coli mutants, there was no linkage between mutations affecting glutamate synthase activity and the argG gene. The results of a combination of transduction experiments demonstrated the gene order argG-glnF-gltB-cod-argR-envB-aroE for S-typhimurium.

Chromosome Mapping↗
PubMed2006

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety.

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.

Alleles↗
PubMed2000

Genetic characterization of a new variant within the ET-37 complex of Neisseria meningitidis associated with outbreaks in various parts of the world.

A new variant within the electrophoretic type (ET)-37 complex of Neisseria meningitidis, ET-15, first detected in Canada in 1986, has been associated with severe clinical infections and high mortality rates in several European countries, Israel and Australia. To ascertain the genetic and epidemiological relationships of ET-15 strains from different geographical areas, 72 ET-15 isolates from 10 countries were compared to 13 isolates representing other clones of the ET-37 complex. The 85 strains were analysed by pulsed-field gel electrophoresis (PFGE) using 2 restriction endonucleases and Southern hybridization with 10 genetic markers. Four ET-15 strains and 4 other strains of the ET-37 complex were further examined using an additional restriction enzyme and a total of 18 genetic markers. PFGE fingerprints of the ET-15 strains were closely related. Strains within each country were even more closely related, suggesting single introductions of the clone. Physical mapping of genes in ET-15 and other strains of the ET-37 complex demonstrated that large genetic rearrangements of the genome have occurred in association with the appearance of the ET-15 variant.

DNA Primers↗
PubMed1998

A clinical, echocardiographic and genetic characterization of a Danish kindred with familial amyloid transthyretin methionine 111 linked cardiomyopathy.

AIMS: To identify carriers and non-carriers of the mutant transthyretin methionine 111 linked familial amyloid disease, to detect early signs of the restrictive cardiomyopathy and other clinical manifestations characteristic of this inheritable disease. METHODS AND RESULTS: Out of 125 living family members 99 were available for clinical, echocardiographic and genetic examination. Twenty-five family members were heterozygous carriers of the mutant transthyretin methionine 111 genotype, while 74 were non-carriers. Among the 25 carriers, none had overt clinical signs of heart disease. Eight carriers, all above the age of 35, showed echocardiographic abnormalities suggestive of developing or manifest restrictive cardiomyopathy. Three had biopsy-verified transthyretin-related amyloid cardiomyopathy. None of the 15 carriers in the younger age group exhibited aberrant echocardiographic patterns. Nine carriers had carpal tunnel syndrome as opposed to none of the non-carriers. CONCLUSION: For early detection of familial amyloid cardiomyopathy, echocardiography is the investigation of choice. The first sign is diastolic dysfunction detected as an abnormal relaxation pattern. The appearance of echocardiographic aberrations solely in the older age group suggests that the cardiomyopathy is a late onset disease. Carpal tunnel syndrome appears to be the earliest presenting clinical symptom. A curative treatment seems to be an early liver transplantation.

Adolescent↗
PubMed2006

Further clinical and genetic characterization of SPG11: hereditary spastic paraplegia with thin corpus callosum.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders leading to progressive spasticity of the lower limbs. Clinically, HSPs are divided into "pure" and "complicated" forms. In pure HSP, the spasticity of the lower limbs is the sole symptom, whereas in complicated forms additional neurological and non-neurological features are observed. Genetically, HSPs are divided into autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) forms. Up to date, 30 different HSPs are linked to different chromosomal loci and 11 genes could be defined for AR-HSP, AD-HSP and XL-HSP. SPG11, an AR-HSP (synonym: HSP11), is a complicated HSP associated with a slowly progressive spastic paraparesis, mental impairment and the development of a thin corpus callosum (TCC) during the course of the disease. SPG11 has been previously linked to chromosomal region 15q13 - 15. First, we applied rigid diagnostic criteria to systematically examine 20 Turkish families with autosomal recessive HSP for characteristic features of SPG11. We detected four large Turkish families with AR-HSP and TCC consistent with SPG11. Subsequent genetic linkage analysis of those 4 families refines the SPG11 locus further down to a small region of 2.93 cM with a maximum lod score of 11.84 at marker D15S659 and will guide further candidate gene analysis.

Adolescent↗