• Journal of Veterinary Clinics
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• v.27 no.5
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• pp.508-513
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• 2010

Cytokines are important mediators of the immune response, and quantitating cytokine mRNA is a highly sensitive and attractive method for measuring cytokine production. The objective of the current study was to develop and validate a SYBR green quantitative real-time reverse transcriptase PCR (qRT-PCR) assay for measuring canine cytokine mRNA. The optimal annealing temperatures ($T_a$) of the designed primers were $62^{\circ}C$ for interleukin (IL)-$1{\beta}$, IL-6 and IL-10; $60^{\circ}C$ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tumor necrosis factor (TNF)-${\alpha}$; and $58^{\circ}C$ for high mobility group box 1 (HMGB1). Primer efficiencies of all primers calculated for standard curve samples were between 97.1% and 102.6%. No evidence of secondary structure or primer-dimer formation was seen via melt-curve analysis or gel electrophoresis. The developed qRT-PCR assays are highly specific and sensitive and can be used to quantify gene expression levels of canine cytokines.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.280-283
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• 1998

We have cloned the RNA polymerase sigma-like factors from a wide range of actinomycetes by using specific primers with the polymerase chain reaction (PCR). The specific oligonucleotide primers were designed on the basis of amino acid sequences of conserved regions from HrdA, B, D of Streptomyces griseus as well as from the rpoD box of many eubacteria. The consensus sequences were from the rpoD box and helix-turn-helix motif involved in -35 recognition. The designed primers were successfully applied to amplify the DNA fragments of the hrd homolog genes from 8 different strains of actinomycetes which produce a wide variety of important antibiotics. The 480 bp of the DNA fragment was amplified from all 8 strains, and it was identified as a part of hrdA and hrdB as we designed. The deduced amino acid sequence of PCR-amplified DNA fragments were highly homologous to those of other known RNA polymerase sigma factors of S. griseus and Streptomyces aureofaciens. Therefore, this study with specifically designed primers will support rapid cloning of the RNA polymerase sigma factors which recognize different classes of promoters from actinomycetes, and it will also be helpful in understanding the relationship of promoters and sigma factors leading to heterogeneity of RNA polymerases in actinomycetes.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1101-1108
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• 2011

The rise in global energy demand has prompted researches on developing strategies for transforming coal into a cleaner fuel. This requires isolation of microbes with the capability to degrade complex coal into simpler substrates to support methanogenesis in the coal beds. In this study, aerobic bacteria were isolated from an Indian coal bed that can solubilize and utilize coal as the sole source of carbon. The six bacterial isolates capable of growing on coal agar medium were identified on the basis of their 16S rRNA gene sequences, which clustered into two groups; Group I isolates belonged to the genus Rhizobium, whereas Group II isolates were identified as Chelatococcus species. Out of the 4 methods of whole genome fingerprinting (ERIC-PCR, REP-PCR, BOX-PCR, and RAPD), REP-PCR showed maximum differentiation among strains within each group. Only Chelatococcus strains showed the ability to solubilize and utilize coal as the sole source of carbon. On the basis of 16S rRNA gene sequence and the ability to utilize different carbon sources, the Chelatococcus strains showed maximum similarity to C. daeguensis. This is the first report showing occurrence of Rhizobium and Chelatococcus strains in an Indian coal bed, and the ability of Chelatococcus isolates to solubilize and utilize coal as a sole source of carbon for their growth.

• The Plant Pathology Journal
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• v.37 no.3
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• pp.243-257
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• 2021

Bacillus pumilus is the causal agent of trunk bulges disease affecting rubber and rubberwood quality and yield production. In this study, B. pumilus and other closely related species were included in B. pumilus group, as they shared over 99.5% similarity from 16S rRNA analysis. Multilocus sequence analysis (MLSA) of five housekeeping genes and repetitive elements-based polymerase chain reaction (rep-PCR) using REP, ERIC, and BOX primers conducted to analyze the diversity and systematic relationships of 20 isolates of B. pumilus group from four rubber tree plantations in Peninsular Malaysia (Serdang, Tanah Merah, Baling, and Rawang). Multi rep-PCR results revealed the genetic profiling among the B. pumilus group isolates, while MLSA results showed 98-100% similarity across the 20 isolates of B. pumilus group species. These 20 isolates, formerly established as B. pumilus, were found not to be grouped with B. pumilus. However, being distributed within distinctive groups of the B. pumilus group comprising of two clusters, A and B. Cluster A contained of 17 isolates close to B. altitudinis, whereas Cluster B consisted of three isolates attributed to B. safensis. This is the first MLSA and rep-PCR study on B. pumilus group, which provides an in-depth understanding of the diversity of these rubber-pathogenic isolates in Malaysia.

• The Plant Pathology Journal
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• v.28 no.4
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• pp.423-427
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• 2012

Pseudomonas syringae pv. actinidiae strains, the causal agents of bacterial canker on kiwifruit, were isolated from Korea and Italy in 2011. Among 87 isolates, a total of six representative strains, three from Korea and three from Italy, were identified on the basis of biochemical and physiological tests. Identities were confirmed by PCR using P. syringae pv. actinidiae-specific primers PsaF1/R2, which amplified a 280-bp DNA fragment. The strains isolated from Korea in this study displayed BOX-PCR patterns similar to those isolated from Italy but different from those isolated previously in Korea or the pathotype P. syringae pv. actinidiae strain. The effector hopA1 and hopH1 genes, which are known to be present in strains isolated recently from France and Italy, were also present in P. syringae pv. actinidiae strains, SYS1, SYS2 and SYS4, isolated from Korea in this work. However, no amplicons of the expected size were obtained from strains previously isolated from Korea and Japan. In addition, the Korean strains isolated in this work belonged to haplotype I for the cts gene identical to those strains isolated from recent outbreaks in Italy. These results suggest that P. syringae pv. actinidiae strains isolated from Korea and examined in this work are a new type of strain similar to those found from recent outbreaks in Italy. This is the first report on the occurrence of cts haplotype I strains of P. syringae pv. actinidiae affecting kiwifruit plants in Korea.

• Biomedical Science Letters
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• v.23 no.4
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• pp.416-420
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• 2017

Endoplasmic reticulum (ER) stress induces unfolded protein response (UPR) via inositol-requiring enzyme 1 (IRE1) activation, which sends a molecular signal for X box-binding protein 1 (XBP1) mRNA splicing in the cytosol. IRE1 endoribonuclease activity induces cleavage of XBP1 mRNA. The XBP1 mRNA is then ligated by an uncharacterized RNA ligase and translated to produce spliced XBP1 by 23 nt removed in which contains the PstI restriction enzyme site. The splicing of XBP1 mRNA can be detected by semiquantitative RT-PCR, and then splicing of XBP1 is a useful tool to measure the genetic variability in ER stress. In this study, we have estimated IRE1-dependent splicing of XBP1 mRNA under conditions of various hypothermia. The results indicated that hypothermia regulated ER stress. This study demonstrated that hypothermia is closely related to ER stress and may be useful for early diagnosis of ER-associated disease.

• Journal of Life Science
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• v.28 no.9
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• pp.1030-1041
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• 2018

Abiotic stresses limit the growth and productivity of plants. Cellular adaptation to abiotic stresses requires coordinated regulation in gene expression directed by complex mechanisms. This study used the activation tagging system to identify novel salt stress-responsive genes. The study selected 9 activation tagging lines that showed salt stress-tolerant phenotypes during their germination stages. Thermal asymmetric interlaced-PCR (TAIL-PCR) was used to identify the T-DNA tagging sites on the Arabidopsis genome in selected activation tagging lines, including AT7508, AT7512, AT7527, AT7544, AT7548, and AT7556. RT-PCR analysis showed that ClpC2/HSP93-III (At3g48870), plant thionin family (At2g20605), anti-muellerian hormone type-2 receptor (At3g50685), vacuolar iron transporter family protein (At4g27870), and microtubule-associated protein (At5g16730) were activated in AT7508, AT7512, AT7527, AT7544, and AT7556, respectively. Interestingly, in AT7548, both the genes adjacent to the T-DNA insertion site were activated: Arabinogalactan protein 13 (AGP13) (At4g26320) and F-box/RNI-like/FBD-like domains-containing protein (At4g26340). All of the seven genes were newly identified as salt stress-responsive genes from this study. Among them, the expression of ClpC2/HSP93-III, AGP13, F-box/RNI-like/FBD-like domains-containing protein gene, and microtubule-associated protein gene were increased under salt-stress condition. In addition, AT7508, AT7527, and AT7544 were more tolerant to salt stress than wild type at seedling development stage, functionally validating the screening results of the activation tagging lines. Taken together, our results demonstrate that the activation tagging system is useful for identifying novel stress-responsive genes.

• The Plant Pathology Journal
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• v.28 no.1
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• pp.60-67
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• 2012

For the genetic differentiation of $Pseudomonas$ $syringae$ pathovar $tomato$, a total of 51 $P.$ $syringae$ pv. strains infecting 33 different host plants were analyzed using repetitive element PCR(REP-PCR) and universal rice primer PCR(URP-PCR). The entire DNA fingerprint profiles were analyzed using unweighted pair-group method with arithmetic averages (UPGMA). The 51 $P.$ $syringae$ pv. strains could be divided into five clusters based on 65% similarity by Rep-PCR using BOX, ERIC, and REP primers. $P.$ $syringae$ pv. $tomato$ cluster was well separated from other 31 $P.$ $syringae$ pathovars. $P.$ $syringae$ pv. $tomato$ cluster included only $P.$ $syringae$ pv. $maculicola$ and $P.$ $syringae$ pv. $tomato$. $P.$ $syringae$ pv. $tomato$ strains could be divided into two genetic groups. Meanwhile, the Pseudomonas pv. strains could be divided into four clusters based on 63% similarity by URP-PCR using 2F, 9F, and 17R primers. $P.$ $syringae$ pv. $tomato$ cluster was also well separated from 30 other $P.$ $syringae$ pathovars. In this case, $P.$ $syringae$ pv. $tomato$ cluster included $P.$ $syringae$ pv. $maculicola$, $P.$ $syringae$ pv. $berberidi$, and $P.$ $syringae$ pv. $tomato$. $P.$ $syringae$ pv. $tomato$ strains was also separated into two genetic groups by URP-PCR analysis. Overall, our work revealed that $P.$ $syringae$ pv. $tomato$ can be genetically differentiated from other $P.$ $syringae$ pathovars by the DNA fingerprint profiles of REP-PCR and URP-PCR. We first report that there are two genetically diverged groups in $P.$ $syringae$ pv. $tomato$ strains.

• Journal of Plant Biotechnology
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• v.43 no.2
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• pp.204-212
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• 2016

To understand the responses of grapevines in response to cold stress causing the limited growth and development, differentially expressed genes (DEGs) were screened through transcriptome analysis of shoots from 2 grapevine cultivars ('Campbell Early' and 'Muscat Baily A') kept at -$2^{\circ}C$ for 4 days. In gene ontology analysis of DEGs from 'Campbell Early', there were 17,424 clones related with biological process, 28,954 with cellular component, and 6,972 with molecular function genes in response to freezing temperature. The major induced genes included dehydrin xero 1, K-box region and MADS-box transcription factor family protein, and MYB domain protein 36, and inhibited genes included light-harvesting chlorophyll B-binding protein 3, FASCICLIN-like arabinoogalactan 9, and pectin methylesterase 61 in 'Campbell Early' grapevines. In gene ontology analysis of DEGs from 'Muscat Baily A', there were 1,157 clones related with biological process, 1,350 with cellular component, and 431 with molecular function gene. The major induced genes of 'Muscat Baily A' included NB-ARC domain-containing disease resistance protein, fatty acid hydrozylase superfamily, and isopentenyltransferase 3, and inhibited genes included binding, IAP-like protein 1, and pentatricopeptide repeat superfamily protein. All major DEGs were shown to be expressed differentially by freezing temperature in real time-PCR analysis. Protein domain analysis using InterPro Scan revealed that ubiquitin-protein ligase was redundant in both tested grapevines. Transcriptome profile of shoots exposed to cold can provide new insights into the molecular basis of tolerance to low-temperature in grapevines, and can be used as resources for development new grapevines tolerant to coldness.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.4
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• pp.253-261
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• 2023

Objective: Azoospermia (the total absence of sperm in the ejaculate) affects approximately 10% of infertile males. Despite diagnostic advances, azoospermia remains the most challenging issue associated with infertility treatment. Our study evaluated transition nuclear protein 2 (TNP2) and synaptonemal complex protein 3 (SYCP3) polymorphisms, azoospermia factor a (AZFa) microdeletion, and gene expression levels in 100 patients with azoospermia. Methods: We investigated a TNP2 single-nucleotide polymorphism through polymerase chain reaction (PCR) restriction fragment length polymorphism analysis using a particular endonuclease. An allele-specific PCR assay for SYCP3 was performed utilizing two forward primers and a common reverse primer in two PCR reactions. Based on the European Academy of Andrology guidelines, AZFa microdeletions were evaluated by multiplex PCR. TNP2, SYCP3, and the AZFa region main gene (DEAD-box helicase 3 and Y-linked [DDX3Y]) expression levels were assessed via quantitative PCR, and receiver operating characteristic curve analysis was used to determine the diagnostic capability of these genes. Results: The TNP2 genotyping and allelic frequency in infertile males did not differ significantly from fertile volunteers. In participants with azoospermia, the allelic frequency of the SYCP3 mutant allele (C allele) was significantly altered. Deletion of sY84 and sY86 was discovered in patients with azoospermia and oligozoospermia. Moreover, SYCP3 and DDX3Y showed decreased expression levels in the azoospermia group, and they exhibited potential as biomarkers for diagnosing azoospermia (area under the curve, 0.722 and 0.720, respectively). Conclusion: These results suggest that reduced SYCP3 and DDX3Y mRNA expression profiles in testicular tissue are associated with a higher likelihood of retrieving spermatozoa in individuals with azoospermia. The homozygous genotype TT of the SYCP3 polymorphism was significantly associated with azoospermia.