Construction of a genomic map ofMoraxella (Branhamella) catarrhalisATCC 25238 and physical mapping of virulence-associated genes

1999 ◽  
Vol 45 (4) ◽  
pp. 299-303 ◽  
Author(s):  
K T Nguyen ◽  
E J Hansen ◽  
M A Farinha
Keyword(s):  
Gel Electrophoresis ◽  
Moraxella Catarrhalis ◽  
Southern Hybridization ◽  
Physical Map ◽  
Outer Membrane Proteins ◽  
Restriction Enzymes ◽  
Pulsed Field Gel Electrophoresis ◽  
Respiratory Pathogens ◽  
Circular Chromosome ◽  
Pulsed Field

A physical genome map of the Moraxella catarrhalis type strain (ATCC 25238) has been constructed using pulsed field gel electrophoresis. Macrorestriction analyses of the genome of M. catarrhalis were performed by digestion with the restriction enzymes SmaI, NotI, and RsrII, which cleave the single circular chromosome into 9, 10, and 6 fragments, respectively. The chromosomal fragments generated by pulsed field gel electrophoresis were converted to a linkage map utilizing a combination of partial digestions, and cross-hybridizations. Moraxella catarrhalis, like a number of other respiratory pathogens, has a relatively small genome estimated at 1750 kilobase pairs or about 40% of the size of the Escherichia coli genome. The locations of the four ribosomal RNA operons (rrnLS) were determined by Southern hybridization and by digestion with I-CeuI endonuclease. A number of genes involved in virulence have been placed onto the physical map by Southern hybridization including those encoding the predominant outer-membrane proteins and the chromosomal gene encoding beta-lactamase.Key words: Moraxella catarrhalis, physical map, genome analysis, pulsed-field gel electrophoresis, virulence.

Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome

2006 ◽  
Vol 52 (9) ◽  
pp. 857-867 ◽  
Author(s):  
Ellen L Dally ◽  
Thereza S.L Barros ◽  
Yan Zhao ◽  
ShaoPing Lin ◽  
Bruce A Roe ◽  
...  
Keyword(s):  
Genetic Map ◽  
Gel Electrophoresis ◽  
Southern Hybridization ◽  
Pulsed Field Gel Electrophoresis ◽  
Enzyme Analysis ◽  
Two Dimensional ◽  
Pulsed Field ◽  
Spiroplasma Kunkelii ◽  
Corn Stunt ◽  
Physical And Genetic Map

Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.Key words: Spiroplasma kunkelii CR2-3x, corn stunt spiroplasma, mollicutes, genome mapping, two-dimensional pulsed-field gel electrophoresis.

scholarly journals Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

2013 ◽  
Vol 79 (12) ◽  
pp. 3856-3859 ◽  
Author(s):  
Zhen Zhang ◽  
Hannamari Hintsa ◽  
Ying Chen ◽  
Hannu Korkeala ◽  
Miia Lindström
Keyword(s):  
Gene Cluster ◽  
Gel Electrophoresis ◽  
Clostridium Botulinum ◽  
Southern Hybridization ◽  
Gene Clusters ◽  
Pulsed Field Gel Electrophoresis ◽  
Content Type ◽  
Pulsed Field ◽  
Large Plasmids

ABSTRACTA collection of 36Clostridium botulinumtype E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted tobotEandorfX1in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.

scholarly journals Usefulness of Pulsed-Field Gel Electrophoresis in Tracking Two Outbreaks of Invasive Meningococcal Disease Serogroup C in British Columbia

2007 ◽  
Vol 18 (6) ◽  
pp. 363-367 ◽  
Author(s):  
Grahame Quan ◽  
Mark Gilbert ◽  
Samara T David ◽  
Tazim Rahim ◽  
Kathy Adie ◽  
...  
Keyword(s):  
British Columbia ◽  
Meningococcal Disease ◽  
Gel Electrophoresis ◽  
Outer Membrane Proteins ◽  
Case Fatality ◽  
Pulsed Field Gel Electrophoresis ◽  
Invasive Meningococcal Disease ◽  
Sequencing Data ◽  
Outbreak Strain ◽  
Pulsed Field

Two major outbreaks of invasive meningococcal disease serogroup C (IMD-C) were identified in British Columbia between 2000 and 2004. Pulsed-field gel electrophoresis (PFGE) andporAgene sequencing of all retained IMD-C isolates were used to assess correlations between genotypes and epidemiological patterns. PFGE patterns of IMD-C genotypes correlated with epidemiological patterns between 2000 and 2004 in British Columbia, and demonstrated that PFGE can identify outbreak-related cases. Both IMD-C outbreaks correlated with a respective PFGE pattern. PFGE analysis demonstrated that the 2004 British Columbia outbreak strain in men who have sex with men was closely related to the 2001 Abbotsford outbreak strain.PorAsequencing data indicated low diversity of class 1 outer membrane proteins in British Columbia, and did not correlate with epidemiological trends. There was a trend for outbreak-associated PFGE types to demonstrate higher case fatality rates.

scholarly journals Metallo-β-Lactamases in ClinicalPseudomonas Isolates in Taiwan and Identification of VIM-3, a Novel Variant of the VIM-2 Enzyme

2001 ◽  
Vol 45 (8) ◽  
pp. 2224-2228 ◽  
Author(s):  
Jing-Jou Yan ◽  
Po-Ren Hsueh ◽  
Wen-Chien Ko ◽  
Kwen-Tay Luh ◽  
Shu-Huei Tsai ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Gel Electrophoresis ◽  
Southern Hybridization ◽  
Pulsed Field Gel Electrophoresis ◽  
Pulsed Field ◽  
Novel Variant ◽  
Pcr Assays ◽  
Positive Results

ABSTRACT A total of 209 clinical isolates of Pseudomonas (193Pseudomonas aeruginosa, 10 P. putida, 4P. stutzeri, and 2 P. fluorescensisolates) with reduced susceptibilities to imipenem and/or ceftazidime were subjected to PCR assays with primers specific forbla IMP-1, bla IMP-2,bla VIM-1, and bla VIM-2and sequence analysis to identify the metallo-β-lactamases (MBLs) prevalent among these organisms in Taiwan; and 21 isolates gave positive results. Five isolates including two P. putida and three P. stutzeri isolates were found to carrybla IMP-1, and six isolates including fiveP. putida and one P. stutzeri isolates harboredbla VIM-2. The remaining 10 isolates wereP. aeruginosa, and all were found to carry a novel variant of bla VIM-2, designatedbla VIM-3. There are only two nucleotide differences between bla VIM-2 andbla VIM-3, leading to two amino acid alterations. Our findings indicate that VIM-2 and its variant have become the most prevalent metalloenzymes in Pseudomonas in Taiwan. Southern hybridization with thebla VIM-2-, bla VIM-3-, and bla IMP-1 -specific probes revealed that only two VIM-2-producing P. putida isolates appeared to carry the MBL gene on plasmids. Pulsed-field gel electrophoresis showed that six VIM-3-producing P. aeruginosa isolates and two IMP-1-producing P. stutzeri isolates were genetically related, suggesting that the spread of these MBL genes in Taiwan could be due to clonal dissemination as well as genetic exchange between different clones.

scholarly journals A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP.

1988 ◽  
Vol 167 (2) ◽  
pp. 664-669 ◽  
Author(s):  
J Rey-Campos ◽  
P Rubinstein ◽  
S Rodriguez de Cordoba
Keyword(s):  
Gene Cluster ◽  
Complement Activation ◽  
Gel Electrophoresis ◽  
Physical Map ◽  
Pulsed Field Gel Electrophoresis ◽  
Complement Components ◽  
Pulsed Field ◽  
Human Genes ◽  
Tight Linkage ◽  
Genes Encoding

We report the organization of the human genes encoding the complement components C4-binding protein (C4BP), C3b/C4b receptor (CR1), decay accelerating factor (DAF), and C3dg receptor (CR2) within the regulator of complement activation (RCA) gene cluster. Using pulsed field gel electrophoresis analysis these genes have been physically linked and aligned as CR1-CR2-DAF-C4BP in an 800-kb DNA segment. The very tight linkage between the CR1 and the C4BP loci, contrasted with the relative long DNA distance between these genes, suggests the existence of mechanisms interfering with recombination within the RCA gene cluster.

scholarly journals Use of Pulsed-Field Gel Electrophoresis for Molecular Epidemiologic and Population Genetic Studies ofMycobacterium tuberculosis †

1999 ◽  
Vol 37 (6) ◽  
pp. 1927-1931 ◽  
Author(s):  
Samir P. Singh ◽  
Hugh Salamon ◽  
Carol J. Lahti ◽  
Mehran Farid-Moyer ◽  
Peter M. Small
Keyword(s):  
Genetic Diversity ◽  
Molecular Epidemiology ◽  
Gel Electrophoresis ◽  
Population Biology ◽  
Rflp Analysis ◽  
Analytical Techniques ◽  
Restriction Enzymes ◽  
Pulsed Field Gel Electrophoresis ◽  
Molecular Biology Technique ◽  
Pulsed Field

Pulsed-field gel electrophoresis (PFGE) is a powerful molecular biology technique which has provided important insights into the epidemiology and population biology of many pathogens. However, few studies have used PFGE for the molecular epidemiology ofMycobacterium tuberculosis. A laboratory protocol was developed to determine the typeability, stability, and reproducibility of PFGE typing of M. tuberculosis. Formal data-analytical techniques were used to assess the genetic diversity elucidated by PFGE analyses using four separate restriction enzymes and by IS6110 RFLP analyses, as well as to assess the concordance among these typing methods. One hundred epidemiologically characterized clinical isolates of M. tuberculosis were genotyped with four different PFGE enzymes (AseI, DraI,SpeI, and XbaI), as well as by RFLP analysis with IS6110. Identical patterns were found among 34 isolates known to be genetically related, suggesting that the PFGE protocol is robust and reproducible. Among 66 isolates representing population-sampled cases, heterozygosity and information content dependency estimates indicate that all five genotyping systems capture quantitatively similar levels of genetic diversity. Nevertheless, comparisons between PFGE analyses and IS6110 typing reveals that PFGE provided more discrimination among isolates with fewer than five copies of IS6110 and less clustering in isolates with five or more copies. The comparisons confirm the hypothesis that the resolution of IS6110 RFLP genotyping is dependent upon the number of IS6110 elements in the genome of isolates. The general concordance among the results obtained with four independent enzymes suggests that M. tuberculosis is a clonal organism. The availability of a robust genotyping technique largely independent of repetitive elements has implications for the molecular epidemiology of M. tuberculosis.

scholarly journals Genomic Complexity among Strains of the Facultative Photoheterotrophic Bacterium Rhodobacter sphaeroides

1999 ◽  
Vol 181 (5) ◽  
pp. 1684-1688 ◽  
Author(s):  
Kirsten Siedenburg Nereng ◽  
Samuel Kaplan
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Gel Electrophoresis ◽  
Southern Hybridization ◽  
Pulsed Field Gel Electrophoresis ◽  
Restriction Endonucleases ◽  
Pulsed Field ◽  
Genomic Complexity ◽  
Genome Complexity

ABSTRACT Pulsed-field gel electrophoresis following the use of rare cutting restriction endonucleases together with Southern hybridization, using markers distributed on chromosomes I and II of Rhodobacter sphaeroides 2.4.1, has been used to examine approximately 25 strains of R. sphaeroides in an effort to assess the occurrence of genome complexity in these strains. The results suggest that genome complexity is widespread and is accompanied by substantial genomic heterogeneity.

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