Identification of a chromosomal region required for biosynthesis of the phytotoxin coronatine by Pseudomonas syringae pv. tomato

1989 ◽  
Vol 35 (10) ◽  
pp. 910-917 ◽  
Author(s):  
R. A. Moore ◽  
A. N. Starratt ◽  
S.-W. Ma ◽  
V. L. Morris ◽  
D. A. Cuppels
Keyword(s):  
Pseudomonas Syringae ◽  
Genomic Dna ◽  
Chromosomal Region ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Wild Type ◽  
Tomato Dc3000 ◽  
Indigenous Plasmid ◽  
Insertion Sites ◽  
Resistant Mutants

Pseudomonas syringae pv. tomato produces the chlorosis-inducing phytotoxin coronatine. Five of 3700 (0.13%) kanamycin-resistant mutants generated by random Tn5 mutagenesis were unable to synthesize this toxin. Clone pEC18, isolated from a cosmid pLAFR1 library of wild-type P. syringae pv. tomato DC3000 genomic DNA, complemented four of the mutants. Restriction enzyme analysis of pEC18 and corresponding clones from the four mutants indicated that the Tn5 insertion sites of these four mutants spanned a 19-kb region of DC3000 genomic DNA. Complementation tests with subclones of pEC18 confirmed the relative locations of the Tn5 insertions. Because pEC18 did not complement all five mutants or confer the ability to produce toxin on nonproducing P. syringae pathovars, sequences outside this cloned region must also be involved in toxin synthesis. As demonstrated by Southern blot analysis, this cloned region was not on the 68-kb indigenous plasmid of DC3000. The only P. syringae pathovars with DNA homologous to sequences within the coronatine gene cluster were the coronatine producers P. syringae pv. tomato, P. syringae pv. atropurpurea, and P. syringae pv. glycinea. Since the hybridization patterns of these toxin producers were identical, this locus is highly conserved and appears crucial to the synthesis of coronatine.Key words: coronatine, phytotoxin mutants, Pseudomonas syringae pv. tomato.

Restriction enzyme and DNA hybridization analysis of cellulolytic Streptomyces isolates of different origin

1997 ◽  
Vol 43 (4) ◽  
pp. 395-399 ◽  
Author(s):  
Laura Marri ◽  
Emanuela Barboni ◽  
Tiziana Irdani ◽  
Brunella Perito ◽  
Giorgio Mastromei
Keyword(s):  
Restriction Enzyme ◽  
Dna Hybridization ◽  
Genomic Dna ◽  
Restriction Pattern ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Restriction Maps ◽  
E Coli ◽  
The One ◽  
Different Sources

Streptomyces rochei A2 endoglucanase (eglS) and β-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities. The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S. rochei A2. The DNA from all strains, except one, hybridized with the bgsl probe and one strain showed the same restriction pattern as seen in S. rochei A2. The sequence localized by the eglS probe in S. thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E. coli in plasmids pTAE and pCSF203, respectively. The restriction maps showed that the cloned genes were identical to eglS and bgs1. The restriction enzyme analysis of genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments.Key words: Streptomyces, cellulase genes, hybridization, restriction enzyme analysis.

Restriction Enzyme Analysis of Campylobacter Genomic DNA

1988 ◽  
Vol 529 (1 Fourth Colloq) ◽  
pp. 279-282 ◽  
Author(s):  
J. H. BRYNER ◽  
I. V. WESLEY ◽  
L. A. POLLET
Keyword(s):  
Restriction Enzyme ◽  
Genomic Dna ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis

A novel mutation in the transmembrane region of glycoprotein IX associated with Bernard-Soulier syndrome

2004 ◽  
Vol 92 (09) ◽  
pp. 606-613 ◽  
Author(s):  
Xiaojuan Zhao ◽  
Weiming Duan ◽  
Jianxin Fu ◽  
Mingen Lu ◽  
Giamin Wang ◽  
...  
Keyword(s):  
Cho Cells ◽  
Novel Mutation ◽  
Flow Cytometric Analysis ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Wild Type ◽  
Transmembrane Region ◽  
Bernard Soulier Syndrome

SummaryWe describe here a novel mutation in glycoprotein (GP) IX transmembrane region in a patient with Bernard-Soulier syndrome (BSS). Flow cytometric analysis of the patient’s platelets showed that GP Iba and GP IX were expressed at decreased levels. Sequence analysis of the gene coding for GP IX revealed a homozygous (G to A) transition at nucleotide 2113, resulting in a Ala 140 (GCC) to Thr (ACC) replacement in the mature peptide, whereas no defects were found in the coding region of the GP Iba and GP Ib? gene. Allele-specific restriction enzyme analysis using HPYCH4 III revealed that the patient was homozygous and her mother and brother were heterozygous for the defect, and excluded the possibility that the mutation was a polymorphism of GP IX.To clarify the effect of this mutation on the surface expression of the GP Ib/IX complex, we introduced this mutation into the cDNA of GP IX by site-directed mutagenesis and performed in vitro transfection studies with plasmids harboring GP Iba, GP Ib? and wild-type GP IX or mutant GP IX. Mutant GP IX decreased the surface expression of GP Iba and GP IX, whereas both immunostaining and immunoblotting of the transfected Chinese hamster ovary (CHO) cells showed abundant GP Iba and GP IX in the cytoplasm of the CHO cells transfected with plasmids harboring GP Iba, GP Ib? and wild-type GP IX or mutant GP IX These findings indicate that the Ala140→Thr mutation in the transmembrane region of GP IX does not induce intracellular GP Ib/IX complex degradation, but prevents its insertion in the cytoplasmic membrane of platelets and CHO cells.

scholarly journals Presence of a Single Genotype of the Newly Described Species Mycobacterium immunogenum in Industrial Metalworking Fluids Associated with Hypersensitivity Pneumonitis

2002 ◽  
Vol 68 (11) ◽  
pp. 5580-5584 ◽  
Author(s):  
Richard J. Wallace, ◽  
Yansheng Zhang ◽  
Rebecca W. Wilson ◽  
Linda Mann ◽  
Harold Rossmoore
Keyword(s):  
Genomic Dna ◽  
Hypersensitivity Pneumonitis ◽  
The United States ◽  
Clinical Isolates ◽  
Metalworking Fluids ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Clinical Patient ◽  
Industrial Sites ◽  
Single Genotype

ABSTRACT Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species Mycobacterium immunogenum. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as M. immunogenum and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes DraI and XbaI, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of M. immunogenum, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.

scholarly journals Whole-Genome Expression Profiling Defines the HrpL Regulon of Pseudomonas syringae pv. tomato DC3000, Allows de novo Reconstruction of the Hrp cis Element, and Identifies Novel Coregulated Genes

2006 ◽  
Vol 19 (11) ◽  
pp. 1167-1179 ◽  
Author(s):  
Adriana O. Ferreira ◽  
Christopher R. Myers ◽  
Jeffrey S. Gordon ◽  
Gregory B. Martin ◽  
Monica Vencato ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
De Novo ◽  
Effector Proteins ◽  
Iterative Refinement ◽  
Host Cells ◽  
Whole Genome ◽  
Wild Type ◽  
Tomato Dc3000 ◽  
Type Iii ◽  
In The Wild

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and ΔhrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the ΔhrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated op-erons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phyto-hormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.

scholarly journals Glanzmann's thrombasthenia caused by homozygosity for a splice defect that leads to deletion of the first coding exon of the glycoprotein IIIa mRNA

Blood ◽  
1993 ◽  
Vol 81 (8) ◽  
pp. 2044-2049 ◽  
Author(s):  
S Simsek ◽  
H Heyboer ◽  
LG de Bruijne-Admiraal ◽  
R Goldschmeding ◽  
HT Cuijpers ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Donor Site ◽  
Molecular Genetic ◽  
Platelet Membrane ◽  
Splice Donor Site ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Inherited Disease ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

Abstract Glanzmann's thrombasthenia (GT) is the result of the absence or of an altered and dysfunctional expression on the platelet membrane of the fibrinogen receptor (glycoprotein [GP] IIb/IIIa complex). Various molecular genetic mechanisms have been found to be responsible for this inherited disease. In a patient with a severe type of GT, we have found a splice variant in the GP IIIa gene that leads to premature chain termination. Immunoprecipitation experiments, using monoclonal antibodies specific for GP IIb/IIIa, showed that GP IIb/IIIa was not detectable on the platelet membrane. Amplification of reversely transcribed platelet GP IIIa mRNA by the polymerase chain reaction and subsequent sequence analysis showed a 86-bp deletion, which corresponds to exon i of the GP IIIa gene. This deletion results in a shift of the reading frame leading to eight altered amino acids followed by a premature termination codon. Analysis of the corresponding genomic DNA fragments showed three mutations in the exon i-intron i boundary region of the GP IIIa gene. One of these mutations is a G-->T transition that eliminates the GT splice donor site in the wild type. This base pair change creates a restriction site for the enzyme Mse I. Allele-specific restriction enzyme analysis (ASRA) with Mse I of amplified genomic DNA of the parents and the proposita showed that both parents (who are first cousins) are heterozygous, whereas the proposita is homozygous for the G-->T substitution.

scholarly journals PCR-based Single-strand Conformation Polymorphism (SSCP) Analysis to Clone Nine Aquaporin Genes in Cucumber

2002 ◽  
Vol 127 (6) ◽  
pp. 925-930 ◽  
Author(s):  
Jiahua Xie ◽  
Todd C. Wehner ◽  
Mark A. Conkling
Keyword(s):  
Dna Sequence ◽  
Restriction Enzyme ◽  
Genomic Dna ◽  
Sequence Variation ◽  
Single Strand ◽  
Sscp Analysis ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Pcr Products ◽  
Dna Pcr

Combining the use of PCR and single-strand conformation polymorphisms (SSCP), nine sequences from the cucumber genome were successfully identified and cloned that encoded two well-conserved asparagine-proline-alanine (NPA) domain homologues to aquaporin genes. The sensitivity and detection efficiency of SSCP and restriction enzyme analysis for detecting DNA sequence variation were evaluated using similar-sized DNA fragments. The SSCP analysis was more sensitive and efficient for discriminating different clones than restriction enzyme analysis, although some sequence variation inside similar-sized DNA fragments could be identified by restriction analysis. Consideration of the results of SSCP analysis with DNA sequence information indicated that one or two base pair changes in the amplified regions could be detected. Moreover, the SSCP analysis results of genomic DNA PCR products that were amplified by degenerate primers can provide rough information about the number of member genes. If the SSCP bands of a cloned fragment (such as CRB7) did not have the corresponding bands from genomic DNA PCR products, that fragment might be a misamplified product. The PCR-based SSCP method with degenerate oligonucleotide primers should facilitate the cloning of member genes.

scholarly journals Molecular cloning and genetic mapping of the t complex responder candidate gene family.

Genetics ◽  
1990 ◽  
Vol 124 (4) ◽  
pp. 957-966
Author(s):  
D C Bullard ◽  
J C Schimenti
Keyword(s):  
Genetic Mapping ◽  
Gene Family ◽  
Molecular Cloning ◽  
Molecular Data ◽  
Transmission Ratio Distortion ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Wild Type ◽  
T Complex ◽  
Duplication Events

Abstract Male transmission ratio distortion (TRD) is a property of mouse t haplotypes requiring the t complex responder locus (Tcr). Tcr maps to the central region of t haplotypes, and is embedded within a series of large duplicated tracts of DNA known as "T66 elements." In previous work, a family of genes (the "T66" genes) was identified within this region that encodes male germ cell-specific transcripts. Genetic and molecular data indicate that one of these genes represents Tcr. Here, we describe the molecular cloning of the four members of the T66 gene family, the genetic mapping of these genes to three adjacent t haplotype loci, and comparative restriction enzyme analysis of the genes. The results indicate that these genes are highly similar to one another, and were created by recent, complex duplication events. This suggests that a minor alteration(s) could have been responsible for conferring "mutant" responder activity upon Tcr, while the other homologs retained "wild-type" biochemical function. In addition, we have identified and mapped three T66 genes in wild-type t complexes. They reside in two separate loci at the opposite ends of the proximal t complex inversion, and are separated by at least 3 cM.

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