scholarly journals Characterization of a Gene Identified in Pathotype 5 of the Clubroot Pathogen Plasmodiophora brassicae

2015 ◽  
Vol 105 (6) ◽  
pp. 764-770 ◽  
Author(s):  
H. Zhang ◽  
J. Feng ◽  
V. P. Manolii ◽  
S. E. Strelkov ◽  
S.-F. Hwang
Keyword(s):  
Plasmodiophora Brassicae ◽  
Blot Hybridization ◽  
Field Isolate ◽  
Pcr Analysis ◽  
In Planta ◽  
Dot Blot ◽  
Reverse Transcription Quantitative Pcr ◽  
Polymerase Chain ◽  
Differential Hosts

Clubroot caused by Plasmodiophora brassicae is an important disease of crucifers worldwide. Isolates of the pathogen can be classified into pathotypes according to their pathogenicity on differential hosts. In this study, the presence or absence of all database-available nonhousekeeping P. brassicae genes (118 in total) were assessed by polymerase chain reaction (PCR) analysis in isolates belonging to five P. brassicae pathotypes (2, 3, 5, 6, and 8 according to Williams’ differential set). One gene, designated Cr811, was present exclusively in the isolate of pathotype 5. This was further confirmed by dot blot hybridization and by PCR using alternative DNA preparations and primers. Reverse transcription quantitative PCR analysis indicated that in planta expression of Cr811 was up-regulated during canola infection, especially in the stage of secondary plasmodia. Primers specific to Cr811 could distinguish a field isolate of P. brassicae belonging to pathotype 5 from two other field isolates representing pathotypes 3 and 8. These findings suggest that Cr811 is a gene that is potentially involved in clubroot pathogenesis and that it also might serve as a molecular marker for differentiation of pathotype 5 from other pathotypes.

scholarly journals Characterization of Five Molecular Markers for Pathotype Identification of the Clubroot Pathogen Plasmodiophora brassicae

2018 ◽  
Vol 108 (12) ◽  
pp. 1486-1492 ◽  
Author(s):  
Jing Zheng ◽  
Xuliang Wang ◽  
Qian Li ◽  
Shu Yuan ◽  
Shiqing Wei ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Plasmodiophora Brassicae ◽  
Expression Patterns ◽  
Chain Reaction ◽  
Clubroot Disease ◽  
Polymerase Chain ◽  
Differential Hosts ◽  
Reaction Cycles ◽  
Important Disease

Clubroot disease is an important disease on cruciferous crops caused by Plasmodiophora brassicae infections. The pathotypes have been classified based on the reactions of differential hosts. However, molecular markers of particular pathotypes for P. brassicae are limited. In this study, we found five genetic markers in association with different pathotypes. Different gene expression patterns among different pathotypes (P4, P7, P9, and P11) were assayed according to the transcriptome data. The assay indicated that molecular markers PBRA_007750 and PBRA_009348 could be used to distinguish P11 from P4, P7, and P9; PBRA_009348 and Novel342 could distinguish P9 from P4, P7, and P11; and PBRA_008439 and Novel342 could represent a kind of P4. Polymerase chain reaction cycles ranging from 25 to 30 were able to identify the predominant pathotype in general. Therefore, these molecular markers would be a valuable tool to identify and discriminate pathotypes in P. brassicae population.

scholarly journals Characterization of the RND-Type Multidrug Efflux Pump MexAB-OprM of the Plant Pathogen Pseudomonas syringae

2008 ◽  
Vol 74 (11) ◽  
pp. 3387-3393 ◽  
Author(s):  
Savina O. Stoitsova ◽  
Yvonne Braun ◽  
Matthias S. Ullrich ◽  
Helge Weingart
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Pathogens ◽  
Efflux Pump ◽  
Blot Hybridization ◽  
In Planta ◽  
Multidrug Efflux ◽  
Dot Blot ◽  
Multidrug Efflux Pump

ABSTRACT In gram-negative bacteria, transporters belonging to the RND family are the transporters most relevant for resistance to antimicrobial compounds. In Pseudomonas aeruginosa, a clinically important pathogen, the RND-type pump MexAB-OprM has been recognized as one of the major multidrug efflux systems. Here, homologues of MexAB-OprM in the plant pathogens Pseudomonas syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. syringae pv. tomato DC3000 were identified, and mexAB-oprM-deficient mutants were generated. Determination of MICs revealed that mutation of MexAB-OprM dramatically reduced the tolerance to a broad range of antimicrobials. Moreover, the ability of the mexAB-oprM-deficient mutants to multiply in planta was reduced. RNA dot blot hybridization revealed growth-dependent regulation of the mexAB-oprM operon in P. syringae; the expression of this operon was maximal in early exponential phase and decreased gradually during further growth.

scholarly journals Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids

2000 ◽  
Vol 66 (11) ◽  
pp. 4854-4862 ◽  
Author(s):  
Kornelia Smalla ◽  
Holger Heuer ◽  
Antje Götz ◽  
Dagmar Niemeyer ◽  
Ellen Krögerrecklenfort ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Ralstonia Eutropha ◽  
Blot Hybridization ◽  
Restriction Pattern ◽  
Pcr Analysis ◽  
Dot Blot ◽  
High Prevalence ◽  
Resistance Plasmids ◽  
Incq Plasmids ◽  
K 12

ABSTRACT Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors inEscherichia coli CV601 and Pseudomonas putidaUWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in severalP. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriTprobes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobactersp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.

Characterization of alpha-adrenoceptor gene expression in arterial and venous smooth muscle

1993 ◽  
Vol 265 (5) ◽  
pp. H1501-H1509 ◽  
Author(s):  
P. Ping ◽  
J. E. Faber
Keyword(s):  
Smooth Muscle ◽  
Messenger Rna ◽  
Vena Cava ◽  
Pcr Analysis ◽  
Relative Distribution ◽  
Alpha Adrenoceptor ◽  
Aortic Media ◽  
Unique Restriction ◽  
Polymerase Chain

Six genes coding for three unique alpha 1- (1A, 1B, 1C) and three unique alpha 2- (2A, 2B, 2C) adrenergic receptor (AR) subtypes have been cloned. Ligand binding and contractile studies have demonstrated that both alpha 1- and alpha 2-ARs can exist on vascular smooth muscle (VSM) cells, although less is known about the relative distribution and specific subtypes in different vascular segments. In the present study polymerase chain reaction (PCR) analysis was used to characterize the species of alpha-AR messenger RNA (mRNA) present in freshly isolated rat thoracic aortic media and vena cava and in cultured VSM cells (passage 2) derived from both sources. To prevent possible contamination of VSM mRNA, aortic media was separated from adventitia, and vessels were denuded of endothelial cells. Oligonucleotide primers specific for each of the six adrenergic genes were synthesized and used to probe for the presence of alpha-AR mRNA species after reverse transcription of total cellular RNA to cDNA. PCR-amplified AR transcripts were distinguished by the size of amplified DNA fragments and unique restriction endonuclease cleavage. Expression of alpha 1C- or alpha 2C-mRNA was not detected in vascular tissues or cultured VSM cells, although the alpha 2C-primers detected the expected alpha 2C expression in cerebral cortex. Only alpha 1A-mRNA was detected in aortic adventitia. VSM from aorta expressed alpha 1A-, alpha 1B-, and alpha 2A-mRNA, and this pattern was preserved in cultured aortic VSM. Vena cava also expressed both alpha 1A and alpha 1B; however only alpha 2B-mRNA was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Laboratory Evidence of Norwalk Virus Contamination on the Hands of Infected Individuals

2013 ◽  
Vol 79 (24) ◽  
pp. 7875-7881 ◽  
Author(s):  
Pengbo Liu ◽  
Blanca Escudero ◽  
Lee-Ann Jaykus ◽  
Julia Montes ◽  
Rebecca M. Goulter ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Rna Extraction ◽  
High Pressure Processing ◽  
Norwalk Virus ◽  
Dot Blot ◽  
Definitive Evidence ◽  
Reverse Transcription Quantitative Pcr ◽  
Outbreak Investigations ◽  
Pcr Products ◽  
Polyethylene Glycol Precipitation

ABSTRACTHuman norovirus (NoV) outbreak investigations suggest that the hands of infected individuals play an important role in NoV transmission. However, there is no experimental evidence documenting the likelihood and degree of NoV contamination on hands. As part of a clinical trial designed to evaluate the efficacy of high-pressure processing for Norwalk virus (NV) inactivation in oysters, 159 hand rinse samples were collected from 6 infected and 6 uninfected subjects. NV was concentrated from the samples by polyethylene glycol precipitation, followed by RNA extraction using an automated guanidinium isothiocyanate-silica method. NV RNA was detected and quantified using multiple NV-specific reverse transcription-quantitative PCR (RT-qPCR) assays. A total of 25.4% (18/71) of the hand rinse samples collected from 6 infected volunteers were presumptively positive for NV, with an average of 3.86 log10genomic equivalent copies (GEC) per hand. Dot blot hybridization of PCR products obtained using a different primer set, and DNA sequencing of selected amplicons, provided further confirmation of the presence of NV in the hand rinses. NV contamination was also detected in two hand rinse samples obtained from one uninfected subject. These findings provide definitive evidence of NV contamination on the hands of infected subjects observed under controlled clinical research conditions. Such data support the need for better hand hygiene strategies to prevent NoV transmission.

scholarly journals A simple approach to prenatal diagnosis of beta-thalassemia in a geographic area where multiple mutations occur

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1357-1360 ◽  
Author(s):  
SP Cai ◽  
JZ Zhang ◽  
DH Huang ◽  
ZX Wang ◽  
YW Kan
Keyword(s):  
Southern China ◽  
Globin Gene ◽  
Blot Hybridization ◽  
Geographic Area ◽  
Simple Approach ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Dot Blot ◽  
Polymerase Chain

Abstract We describe a simple approach for detecting beta-thalassemia mutations in geographic areas such as southern China where multiple mutations are known to occur. Segments of the beta-globin gene were amplified in vitro by using the polymerase chain reaction. Dot blot hybridization of the amplified DNA with oligonucleotide probes corresponding to the six mutations found in southern China could directly identify the mutations causing beta-thalassemia in the affected families. The increased number of target sequences after amplification allows the use of 35S-labeled probes, which are reusable for up to 3 months. The mutations can be determined in two days.

Characterization of Marine Temperate Phage-Host Systems Isolated from Mamala Bay, Oahu, Hawaii

1998 ◽  
Vol 64 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Sunny C. Jiang ◽  
Christina A. Kellogg ◽  
John H. Paul
Keyword(s):  
Water Samples ◽  
Blot Hybridization ◽  
Temperate Phage ◽  
Dot Blot ◽  
Dot Blot Hybridization ◽  
Double Stranded Dna ◽  
The Family ◽  
Marine Viruses

ABSTRACT To understand the ecological and genetic role of viruses in the marine environment, it is critical to know the infectivity of viruses and the types of interactions that occur between marine viruses and their hosts. We isolated four marine phages from turbid plaques by using four indigenous bacterial hosts obtained from concentrated water samples from Mamala Bay, Oahu, Hawaii. Two of the rod-shaped bacterial hosts were identified as Sphingomonas paucimobilis andFlavobacterium sp. All of the phage isolates were tailed phages and contained double-stranded DNA. Two of the phage isolates had morphologies typical of the family Siphoviridae, while the other two belonged to the families Myoviridae andPodoviridae. The head diameters of these viruses ranged from 47 to 70.7 nm, and the tail lengths ranged from 12 to 146 nm. The burst sizes ranged from 7.8 to 240 phage/bacterial cell, and the genome sizes, as determined by restriction digestion, ranged from 36 to 112 kb. The members of the Siphoviridae, T-φHSIC, and T-φD0, and the member of the Myoviridae, T-φD1B, were found to form lysogenic associations with their bacterial hosts, which were isolated from the same water samples. Hybridization of phage T-φHSIC probe with lysogenic host genomic DNA was observed in dot blot hybridization experiments, indicating that prophage T-φHSIC was integrated within the host genome. These phage-host systems are available for use in studies of marine lysogeny and transduction.

scholarly journals Overexpression of chalcone isomerase A gene in Astragalus trigonus for stimulating apigenin

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nagwa I. Elarabi ◽  
Abdelhadi A. Abdelhadi ◽  
Ahmed G. M. Sief-Eldein ◽  
Ismail A. Ismail ◽  
Naglaa A. Abdallah
Keyword(s):  
Cell Suspension ◽  
Yeast Extract ◽  
Blot Hybridization ◽  
Fold Increase ◽  
Chalcone Isomerase ◽  
Pcr Analysis ◽  
Dot Blot ◽  
Antitumor Activities ◽  
Transgenic Cell ◽  
Important Enzyme

AbstractApigenin is one of the most studied flavonoids and is widely distributed in the plant kingdom. Apigenin exerts important antioxidant, antibacterial, antifungal, antitumor activities, and anti-inflammatory effects in neurological or cardiovascular disease. Chalcone isomerase A (chiA) is an important enzyme of the flavonoid biosynthesis pathway. In order to enhance the apigenin production, the petunia chi A gene was transformed for Astragalus trigonus. Bialaphos survived plants were screened by PCR, dot blot hybridization and RT-PCR analysis. Also, jasmonic acid, salicylic acid, chitosan and yeast extract were tested to evaluate their capacity to work as elicitors for apigenin. Results showed that yeast extract was the best elicitor for induction of apigenin with an increase of 3.458 and 3.9 fold of the control for calli and cell suspension culture, respectively. Transformed cell suspension showed high apigenin content with a 20.17 fold increase compared to the control and 6.88 fold more than the yeast extract treatment. While, transformed T1 calli derived expressing chiA gene produced apigenin 4.2 fold more than the yeast extract treatment. It can be concluded that the highest accumulation of apigenin was obtained with chiA transgenic cell suspension system and it can be utilized to enhancement apigenin production in Astragalus trigonus.

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