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 Development ofa Physical and Genetic Map of the Virulent WolbachiaStrainwMelPop

2003 ◽  
Vol 185 (24) ◽  
pp. 7077-7084 ◽  
Author(s):  
Ling V. Sun ◽  
Markus Riegler ◽  
Scott L. O'Neill
Keyword(s):  
Genetic Map ◽  
Gel Electrophoresis ◽  
Southern Hybridization ◽  
Gene Organization ◽  
Pulsed Field Gel Electrophoresis ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Restriction Fragments ◽  
Chromosome Fragments ◽  
Physical And Genetic Map

ABSTRACT We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.

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.

scholarly journals Use of pulsed field gel electrophoresis and transposon mutagenesis to estimate the minimal number of genes required for motility in Caulobacter crescentus.

Genetics ◽  
1989 ◽  
Vol 123 (4) ◽  
pp. 649-654 ◽  
Author(s):  
B Ely ◽  
T W Ely
Keyword(s):  
Gel Electrophoresis ◽  
Caulobacter Crescentus ◽  
Pulsed Field Gel Electrophoresis ◽  
Restriction Fragments ◽  
Pulsed Field ◽  
Transposon Insertion ◽  
Number Of Genes ◽  
Physical And Genetic Map ◽  
Insertion Mutations ◽  
Flagellar Genes

Abstract To facilitate the mapping of transposon insertion mutations in Caulobacter crescentus, we have used pulsed field gel electrophoresis to construct a detailed physical and genetic map of the C. crescentus genome. Restriction fragments were generated by DraI, AseI, or SpeI which cleave the C. crescentus 40, 13, and 26 times, respectively, and Tn5 insertions were used to align the restriction fragments generated by each of the enzymes. The utility of the resulting map was demonstrated by determining the chromosomal locations of a collection of flagellar mutations. As a result of this study, we were able to identify ten new flagellar genes at various locations on the chromosome. Thus, at least 48 genes are required for the assembly of a functional flagellum in C. crescentus.

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 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.

Two-dimensional motion of DNA bands during 120° pulsed-field gel electrophoresis. I. Effect of molecular weight

Biopolymers ◽  
1995 ◽  
Vol 35 (3) ◽  
pp. 297-306 ◽  
Author(s):  
M. Shane Hutson ◽  
George Holzwarth ◽  
Thomas Duke ◽  
Jean-Louis Viovy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Two Dimensional ◽  
Pulsed Field

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.

A physical and genetic map of theMycoplasma hyopneumoniaestrain J genome

2000 ◽  
Vol 46 (9) ◽  
pp. 832-840 ◽  
Author(s):  
Walter A Blank ◽  
Gerald W Stemke
Keyword(s):  
Genetic Map ◽  
Dna Hybridization ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Physical Map ◽  
Mycoplasma Hyopneumoniae ◽  
Pulsed Field Gel Electrophoresis ◽  
Cosmid Library ◽  
Physical And Genetic Map ◽  
Genomic Map

A macrorestriction map of the genome of Mycoplasma hyopneumoniae strain J, the type strain of the causative agent of enzootic pneumonia in pigs, was constructed using pulsed-field gel electrophoresis (PFGE) and DNA hybridization. The size of the genome as determined by PFGE was approximately 1070 kb. Assembly of the M. hyopneumoniae genomic map was facilitated and complimented by the simultaneous construction of an ordered cosmid library. Five contigs of overlapping cosmids were assembled, which together represent coverage of approximately 728 kb. Forty-two genetic markers (including three types of repeated elements) were placed on the M. hyopneumoniae map. Closer examination of an ApaI restriction fragment contained entirely within a single cosmid insert suggests that the genome size may be overestimated by PFGE.Key words: Mycoplasma hyopneumoniae, mollicutes, physical map, genetic map.

scholarly journals Comparison of the beta-lactamase gene cluster in clonally distinct strains of Enterococcus faecalis.

1996 ◽  
Vol 40 (5) ◽  
pp. 1170-1174 ◽  
Author(s):  
J F Tomayko ◽  
K K Zscheck ◽  
K V Singh ◽  
B E Murray
Keyword(s):  
Gene Cluster ◽  
Enterococcus Faecalis ◽  
Gel Electrophoresis ◽  
Considerable Change ◽  
Pulsed Field Gel Electrophoresis ◽  
Enzyme Analysis ◽  
Beta Lactamase ◽  
Pulsed Field ◽  
Large Clone ◽  
Lactamase Gene

Ten beta-lactamase-producing Enterococcus faecalis isolates were examined for the presence of the staphylococcal beta-lactamase repressor and antirepressor genes. Four isolates, previously shown to be unrelated to each other by pulsed-field gel electrophoresis analysis, were positive for both genes by PCR, although beta-lactamase production was not induced with methicillin. Six isolates, previously shown to be clonally related, were negative for both genes by PCR. The blaZ sequences of eight beta-lactamase-producing E. faecalis isolates were determined. Seven isolates from five distinct clones had sequences identical to that previously reported for E. faecalis HH22, regardless of whether the repressor or antirepressor was demonstrated by PCR. However, blaZ from one isolate differed from those of the other enterococci by 11 nucleotides; this isolate is part of the large clone, as defined by pulsed-field gel electrophoresis and multilocus enzyme analysis, that includes HH22. These findings suggest either that enterococci have acquired the bla gene cluster from more than one source or that the gene cluster has undergone considerable change since acquisition by this clone.

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