scholarly journals Differences in Virulence and Genomic Features of Strains of ‘Candidatus Phytoplasma mali’, the Apple Proliferation Agent

2007 ◽  
Vol 97 (8) ◽  
pp. 964-970 ◽  
Author(s):  
Erich Seemüller ◽  
Bernd Schneider
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Southern Blot Hybridization ◽  
Apple Proliferation ◽  
Candidatus Phytoplasma Mali ◽  
Polymerase Chain

Root and shoot samples from 24 symptomatic or nonsymptomatic apple trees infected with ‘Candidatus Phytoplasma mali’ were collected at different locations in Germany and France and used to inoculate rootstock M11 top grafted with cv. Golden Delicious. Inoculated trees were monitored over a 12-year period for apple proliferation (AP) symptoms and categorized as not or slightly, moderately, or severely affected. Based on symptomatology, the phytoplasma strains were defined as being avirulent to mildly, moderately, or highly virulent. Determination of phytoplasma titers by quantitative polymerase chain reaction (PCR) with DNA from roots revealed similar phytoplasma concentrations in all virulence groups. Molecular characterization of the strains by differential PCR amplification with five sets of primers resulted in 13 profiles. Six strains that were maintained in periwinkle and tobacco were molecularly characterized in more detail. The genome sizes of these strains as determined by pulsed-field gel electrophoresis using yeast chromosomes as size references ranged between 640 and 680 kb. Cleavage of the chromosome with the rare cutting restriction enzymes ApaI, BamHI, BssHII, MluI, and SmaI resulted in macro fragment patterns distinctly different in all strains. Similar results were obtained by Southern blot hybridization with three probes derived from strain AT. Differential PCR amplification at an annealing temperature of 52°C using eight primer pairs derived from strain AT revealed heterogeneity of target sequences among all strains. Based on these results, there is considerable variability in virulence and genomic traits in ‘Ca. P. mali’. However, correlations between molecular markers and virulence or phytoplasma titer could not be identified.

Characterization of the Opisthorchis viverrini genome

1991 ◽  
Vol 65 (1) ◽  
pp. 51-54 ◽  
Author(s):  
R. Sermswan ◽  
S. Mongkolsuk ◽  
S. Sirisinha
Keyword(s):  
Southern Blot ◽  
Genomic Dna ◽  
Blot Hybridization ◽  
Restriction Enzymes ◽  
Southern Blot Hybridization ◽  
Fasciola Gigantica ◽  
Opisthorchis Viverrini ◽  
Repeated Dna ◽  
Dna Elements

ABSTRACTThe methylations of trematode genomic DNA were analyzed using restriction enzymes and Southern blot hybridization. Restriction enzymes MspI, HpaII, HhaI were used to probe CpG methylation while MboI, Sau3A, DpnI were used for A methylation. The results revealed that Opisthorchis viverrini, Fasciola gigantica and Gigantocotyle siamensis had neither CpG nor A methylations. The presence of highly repeated DNA elements was also demonstrated in O. viverrini genomic DNA.

scholarly journals Characterization of plasmid-mediated quinolone resistance by the qnrS gene in Escherichia coli isolated from healthy chickens and pigs

2009 ◽  
Vol 54 (No. 10) ◽  
pp. 473-482 ◽  
Author(s):  
H.-C. Kuo ◽  
C.-C. Chou ◽  
C. Tu ◽  
S.-R. Gong ◽  
C.-L. Han ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Quinolone Resistance ◽  
E Coli ◽  
Sequence Variations ◽  
Southern Blot Hybridization Analysis ◽  
Ciprofloxacin Resistance ◽  
Polymerase Chain

The prevalence of <I>qnr</I> and <I>qepA</I> genes in 660 <I>Escherichia coli</I> isolates was investigated in healthy animals from 30 pig farms and 30 chicken farms in Taiwan from January 2005 to February 2006 by the polymerase chain reaction. The <I>qnrS</I> gene, but not <I>qnrA, qnrB, </I> and <I>qepA</I> were detected in 12/360 pig isolates (3.33%) and in 6/300 chicken isolates (2%). Southern blot hybridization analysis indicated that <I>qnrS</I> was located on plasmids ranging in size from 50–165 kb. Eleven of the 18 <I>qnrS</I> positive isolates which showed a high ciprofloxacin resistance phenotype (minimum inhibitory concentration ≥ 8 mg/l) also had amino acid sequence variations in chromosomal quinolone resistance-determining regions of <I>gyrA</I> and <I>parC</I>. Only two <I>qnrS</I>-positive isolates carried the <I>aac(6’)-Ib-cr</I>variant that mediates FQ acetylation. For the high percentage resistance of cephalosporins, the<I> bla</I><sub>CTX-M</sub> gene was also examined in <I>qnrS</I>-positive isolates. The <I>bla</I><sub>CTX-M</sub> gene was detected in fifteen isolates (15/18, 83.3%) of which 12 isolates were <I>bla</I><sub>CTX-M-1</sub> and three isolates were <I>bl</I><sub>CTX-M-15</sub>. This study demonstrated a close linkage between the <I>qnrS</I> gene and <I>bla</I><sub>CTX-M-1</sub>, suggesting CTX-M and Qnr-based mechanisms might be co-emerging in <I>E. coli</I> strains isolated from healthy chickens and pigs under selective pressure of quinolone and cephalosporine administration.

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

scholarly journals Identification and Functional Characterization of a Cold-Related Protein, BcHHP5, in Pak-Choi (Brassica rapa ssp. chinensis)

2018 ◽  
Vol 20 (1) ◽  
pp. 93
Author(s):  
Jin Wang ◽  
Feiyi Huang ◽  
Xiong You ◽  
Xilin Hou
Keyword(s):  
Brassica Rapa ◽  
Abiotic Stresses ◽  
Quantitative Polymerase Chain Reaction ◽  
Functional Characterization ◽  
Regulation Mechanism ◽  
Pak Choi ◽  
Negative Effect ◽  
Polymerase Chain ◽  
Related Proteins

In plants, heptahelical proteins (HHPs) have been shown to respond to a variety of abiotic stresses, including cold stress. Up to the present, the regulation mechanism of HHP5 under low temperature stress remains unclear. In this study, BcHHP5 was isolated from Pak-choi (Brassica rapa ssp. chinensis cv. Suzhouqing). Sequence analysis and phylogenetic analysis indicated that BcHHP5 in Pak-choi is similar to AtHHP5 in Arabidopsis thaliana. Structure analysis showed that the structure of the BcHHP5 protein is relatively stable and highly conservative. Subcellular localization indicated that BcHHP5 was localized on the cell membrane and nuclear membrane. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that BcHHP5 was induced to express by cold and other abiotic stresses. In Pak-choi, BcHHP5-silenced assay, inhibiting the action of endogenous BcHHP5, indicated that BcHHP5-silenced might have a negative effect on cold tolerance, which was further confirmed. All of these results indicate that BcHHP5 might play a role in abiotic response. This work can serve as a reference for the functional analysis of other cold-related proteins from Pak-choi in the future.

scholarly journals Antioxidant or Apoptosis Inhibitor Supplementation in Culture Media Improves Post-Thaw Recovery of Murine Spermatogonial Stem Cells

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 754
Author(s):  
Sang-Eun Jung ◽  
Hui-Jo Oh ◽  
Jin-Seop Ahn ◽  
Yong-Hee Kim ◽  
Bang-Jin Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Functional Activity ◽  
Culture Media ◽  
Quantitative Polymerase Chain Reaction ◽  
Spermatogonial Stem Cells ◽  
Ros Generation ◽  
Apoptosis Assay ◽  
Polymerase Chain ◽  
Apoptosis Inhibitor

We postulated that supplementation of antioxidant or apoptosis inhibitor in post-thaw culture media of spermatogonial stem cells (SSCs) alleviates reactive oxygen species (ROS) generation and apoptosis. Our aim was to develop an effective culture media for improving post-thaw recovery of SSCs. To determine the efficacy of supplementation with hypotaurine (HTU), α-tocopherol (α-TCP), and Z-DEVD-FMK (ZDF), we assessed the relative proliferation rate and SSC functional activity and performed a ROS generation assay, apoptosis assay, and western blotting for determination of the Bax/Bcl-xL ratio, as well as immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization. The relative proliferation rates with HTU 400 μM (133.7 ± 3.2%), α-TCP 400 μM (158.9 ± 3.6%), and ZDF 200 μM (133.1 ± 7.6%) supplementation were higher than that in the DMSO control (100 ± 3.6%). ROS generation was reduced with α-TCP 400 μM (0.8-fold) supplementation in comparison with the control (1.0-fold). Early apoptosis and Bax/Bcl-xL were lower with α-TCP 400 μM (2.4 ± 0.4% and 0.5-fold) and ZDF 200 μM (1.8 ± 0.4% and 0.3-fold) supplementation in comparison with the control (5.3 ± 1.4% and 1.0-fold) with normal characterization and functional activity. Supplementation of post-thaw culture media with α-TCP 400 μM and ZDF 200 μM improved post-thaw recovery of frozen SSCs via protection from ROS generation and apoptosis after cryo-thawing.

scholarly journals First Isolation and Molecular Typing of Pathogenic and Intermediate Leptospira Species from Urine of Symptomatic Dogs

2021 ◽  
Vol 8 (12) ◽  
pp. 304
Author(s):  
Ivana Piredda ◽  
Loris Bertoldi ◽  
Giuseppe Benvenuto ◽  
Bruna Palmas ◽  
Aureliana Pedditzi ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Zoonotic Risk ◽  
Leptospira Spp ◽  
Blood And Urine Samples ◽  
Healthy Dogs ◽  
Polymerase Chain ◽  
Sequence Types ◽  
Leptospira Species

Aim of this study was to evaluate, the presence and diversity of Leptospira spp. in blood and urine samples collected from 175 owned-dogs from Sardinia, Italy. After determination of leptospiral infection by microscopic agglutination test (MAT), urine from MAT-positive dogs were examined by real-time polymerase chain reaction (lipL32 rt-PCR) and then isolated by culture. In order to characterize obtained serovars, positive cultures were then subjected to 16S rRNA and secY sequencing, phylogenetic analysis and Multilocus Sequence Typing (MLST). Results showed that seven dogs (4%; 95% CI: 0–55) had Leptospira DNAs in their urine and five strains were isolated from urine cultures. The three different sequence types (ST17, ST198 and ST24) belonging to Leptospira interrogans genomospecies identified by MLST analyses in this study, confirmed that the leptospiral infection was widespread in Sardinian dogs. We also reported the first characterization of a new Leptospira spp. isolated from urine of one dog living in the study area. Whole genome sequencing and phylogenetic analysis, confirmed that this genospecies was closely related to Leptospira hovindhougenii, an intermediate Leptospira spp. with unknown pathogenicity previously isolated from a rat in Denmark. Further studies are required to clarify whether healthy dogs that shed leptospires in their urine could represent a zoonotic risk for humans in this region.

Characterization of nematode mitochondrial genomes.

2021 ◽  
pp. 250-264
Author(s):  
Danny A. Humphreys-Pereira ◽  
Taeho Kim ◽  
Joong-Ki Park
Keyword(s):  
Polymerase Chain Reaction ◽  
Mitochondrial Genome ◽  
Genome Annotation ◽  
Pcr Amplification ◽  
Gene Identification ◽  
Mitochondrial Genomes ◽  
Pcr Cloning ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.

scholarly journals Characterization of two BHV-4 strains isolated in the Czech Republic

2010 ◽  
Vol 55 (No. 3) ◽  
pp. 106-112 ◽  
Author(s):  
V. Fichtelova ◽  
K. Kovarcik
Keyword(s):  
Reference Strain ◽  
Fragment Size ◽  
Blot Hybridization ◽  
Size Variation ◽  
Southern Blot Hybridization ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
The Czech Republic ◽  
Herpesvirus 4

This study describes the isolation of bovine herpesvirus 4 (BHV-4) from the respiratory tract of animals suffering from respiratory disease. DNA of new isolates, CH and Ni, was cleaved with <I>Bam</I>HI, <I>Eco</I>RI and <I>Hind</I>III in restriction enzyme analysis and the fragments were identified by co-migration with the restriction profile of the reference strain Movar 33/63 cleaved with the appropriate endonuclease. Typical profiles with polyrepetitive DNA (prDNA) fragments were detected. In order to localize the size variation within the obtained digestion fragments, Southern blot hybridization was performed. Differences between the isolates CH, Ni were localized in both the prDNAs and the unique central part of the genome and were restricted to fragment size variation.

Sign in / Sign up

Export Citation Format

Share Document