• Parasites, Hosts and Diseases
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• v.62 no.2
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• pp.226-237
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• 2024

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase's role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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• pp.1-35
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• 1997

Fertilized egg, by successive cell divisions, differentiates into different tissues and organs with various structures and functions. Different cells and tissues contain different proteins, products of selective gene expression. Not all the genes in any genomes are equally active, temporal and spatial gene expression being the general rule. Present paper attempts to review the tanscriptional mechanisms or the initiations of transcription from several angles. In some of the organisms the genes in the process of transcription or the genes in the inactive state can be seen under the light microscope. Some bands of Drosophila polytene chromosomes may exhibit a swollen or puff appearance under certain conditions. A puff, unfolded or decondensed form of chromomere, represents sets of intense transcriptional activity or RNA synthesis. The heterochromatic X chromosome whose genes remain inactive in the female mammals can be visualized as a dark staining structure called Barr body, Configuration of chromatin differs between transcribed and nontranscribed chromatin. Modification to the chromatin facilitates RNA synthesis. The movement of large polymerase molecule along the DNA would probably be facilitated if some modifications of the chromatin configuration is effected. Methylation of cytosines in CG sequences is associated with inactive genes. Methylation can play a role in determination of mammalian cells during embryogenesis. Demethylation is necessary for the gene to be expressed during development A histone modification that is also known to be correlated with transcriptional capacity of chromatin is acetylation of the lysine residues of the core histones. Chromatin containing a high level of histone acetylation is very sensitive to DNase 1. For the transcription to occur TBP must first bind to the TATA box. Another TF, TF IIB, then binds to the promoter-TBP complex, facilitating the access of RNA polymerase to the transcription initiation site. As recently as eight years ago researchers assumed that histones were irrelevant to the regulation of gene expression. Histones combine with the DNA to form nucleosome of the chromatin. Histones are vital participant in gene regulation. Histone and basal factors compete for access to TATA box. When DNA is exposed to basal factors before histones are introduced, the basal factors assemble on TATA boxes preventing the access of histones, allowing transcription to occur, for transcription to begin, activator protein at the upstream activation sequence or enhancer must interact with the tail of histone H4 at TATA box and cause the histone role particle to dissociate from the TATA box leading to partial breakup of the histone core particle and allowing the basal factors to bind to the TATA box. New concept of genomic flux in contrast to the old concept of static genome has been developed based on the powerful new molecular techniques. Genomic changes such as repetitive DNAs and transposable elements, it is assumed but not yet proved, may affect some of the developmental patterns that characterize particular cells, tissues, organs, and organisms. In the last decade or so remarkable achievement have been made in the researches of the structures and functions of TFs and the specific target sequences located in promoters or enhancers where these TFs bind. TFs have independent domains that bind DNA and that activate transcription. DNA binding domain of TFs serves to bring the protein into the right location. There are many types of DNA binding domains. Common types of motifs can be found that are responsible for binding to DNA. The motifs are usually quite short and comprise only a small part of the protein structure. Steroid receptors have domains for hormone binding, DNA binding, and activating transcription. The zinc finger motif comprises a DNA binding domain. Leucine zipper consist of a stretch of amino acids with a leucine residue in every seventh position Two proteins form a dimer because they interact by means of leucine zippers on similar α-helical domain. This positions their DNA binding basic domains for interaction with the two halves of a DNA sequence with dyad symmetry of TGACTCA, ACTGAGT.

• Journal of Plant Biotechnology
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• v.33 no.1
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• pp.11-18
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• 2006

Salt stress is one of major environmental factors influencing plant growth and development. To identify salt tolerance determinants in higher plants, a large-scale screen was conducted with a bialaphos marker-based T-DNA insertional collection of Arabidopsis ecotype C24 mutants. One line for salt stress-sensitive mutant (referred to as ssm1) exhibited increased sensitivity to both ionic (NaCl) and nonionic (mannitol) osmotic stress in a root growth assay. This result suggests that ssm1 mutant is involved in ion homeostasis and osmotic compensation in plant. Molecular cloning of the genomic DNA flanking T-DNA insert of ssm1 mutant was achieved by mutant genomic DNA library screening. T-DNA insertion appeared in the first exon of an open reading frame on F3M18.7, which is the same as AtSYP61. SSM1 is SYP61/OSM1 that is a member of the SNARE superfamily of proteins required for vesicular/target membrane fusions and factor related to abiotic stress.

• Journal of Life Science
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• v.25 no.8
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• pp.903-909
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• 2015

Loop-mediated isothermal amplification (LAMP), a PCR-based diagnostic method, is based on autocycling strand displacement DNA synthesis in the presence of exonuclease-negative Bst DNA polymerase under isothermal conditions. With the help of four specific primers that recognize six different sequences of a target DNA, LAMP has high specificity in pathogenic identification in a short time. Hence, in the present study, LAMP is used as a diagnostic tool in the identification of the most dreadful aquatic pathogenic species, Vibrio alginolyticus, and to develop species-specific LAMP primers and optimization of LAMP reaction conditions such as annealing temperature, elongation time, and other PCR chemical concentrations, including MgSO₄, dNTPs, Betaine, and Bst polymerase. The optimized LAMP primers were also checked for specificity with other Vibrio species, which showed that the designed primers were very specific to V. alginolyticus After the first introduction of a species name like this one, the first part (“Vibrio” in this case) should be abbreviated to only the first letter.only. These are usually the most harmful pathogens of the Vibrio species that appear in shrimp and crabs. The results also revealed that the LAMP assay could be 10-fold more sensitive than conventional PCR in detecting V. alginolyticus. This could be the first report on using a rapid and highly sensitive technique, the LAMP assay, in the effective diagnosis of the pathogenic bacteria V. alginolyticus, which could help in the early detection of diseases, particularly in aquaculture.

• Journal of Genetic Medicine
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• v.6 no.2
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• pp.146-154
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• 2009

Multiplex ligation dependent probe amplification (MLPA) is a PCR-based method to detect gene dosage. Since its introduction, MLPA has been used to test a large number of genes for major deletions or duplications. Genetic testing, as a diagnostic tool for genetic disease, has been used primarily to identify point mutations, including base substitutions and small insertions/deletions, using PCR and sequence analysis. However, it is difficult to identify large deletions or duplications using routine PCR- gel based assays, especially in heterozygotes. The MLPA is a more feasible method for identification of gene dosage than another routine PCR-based methods, and better able to detect deleterious deletions or duplications. In addition to detection of gene dosage, MLPA can be applied to identify methylation patterns of target genes, aneuploidy during prenatal diagnoses, and large deletions or duplications that may be associated with various cancers. The MLPA method offers numerous advantages, as it requires only a small amount of template DNA, is applicable to a wide variety of applications, and is high-throughput. On the other hand, this method suffers from disadvantages including the possibility of false positive results affected by template DNA quality, difficulties identifying SNPs located in probe sequences, and analytical complications in quantitative aspects.

• Korean Journal of Microbiology
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• v.43 no.2
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• pp.91-99
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• 2007

For the detection of Human Immunodeficiency Virus (HIV), multiple and ultra-rapid real-time PCR methods were developed. The target DNA sequences were deduced from HIV-1 specific 495bp partial env gene (gi_1184090) and from HIV-2 specific 294 bp partial env gene (gi_1332355), and were synthesized by using PCR-based gene synthesis on the reason of safety. Ultra-rapid real-time PCR was performed by $Genspector^{TM}$ using microchip-based, $1\;{\mu}l$ of reaction volume with extremely short time in each 3 step in PCR. The detection including DNA-amplification and melting temperature analysis was completed inner 15 minutes. The HIV-1 specific 117 bp-long and HIV-2 specific 119 bp-long PCR products were successfully amplified from minimum of template,2.3 molecules of each env gene. This kind of real-time PCR was designated as ultra-rapid real-time PCR in this study and it could be applied not only an alternative detection method against HIV, but also other pathogens using PCR-based detection.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.110-115
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• 2004

This study was undertaken to develop PCR primers for the simultaneous detection of Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans, using two species-specific reverse primers in combination with a single conserved forward primer. These primers target the variable and conserved regions of the 16S rDNA. The primer specificity was tested against (i) four F. nucleatum and three A. actinomycetemcomitans strains and (ii) seven representatives of the different species of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of F. nucleatum and A. actinomycetemcomitans. The data indicate that species-specific amplicons could be obtained for all the F. nucleatum and A. actinomycetemcomitans strains tested, which were not found in the seven other species. The multiplex PCR could detect as little as 4 fg of chromosomal DNA of F. nucleatum and A. actinomycetemcomitans simultaneously. These findings suggest that these PCR primers are highly sensitive and are suitable for applications in epidemiological studies, diagnosis, and monitoring F. nucleatum and A. actinomycetemcomitans after the treatment of periodontitis.

• Korean Journal of Food Science and Technology
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• v.37 no.4
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• pp.611-617
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• 2005

Dispersed repetitive DNA elements in genomes of microorganisms differ among and within species. Because distances between repetitive sequences vary depending on bacterial strains, genomic fingerprinting with interspersed repetitive sequence-based probes can be used to distinguish unrelated organisms. Among well-known bacterial repetitive sequences, Repetitive Extragenic Palindromic (REP) sequence has been used to identify environmental bacterial species and strains. We applied REP-PCR to detect and differentiate four major Gram-negative food-borne bacterial pathogens, E. coli, Salmonella, Shigella, and Vibrio. Target DNA fragments of these pathogens were amplified by REP-PCR method. PCR-generated DNA fragments were separated on 1.5% agarose gel. Dendrograms for PCR products of each strain were constructed using photo-documentation system. REP-PCR reactions with primer pairs REP1R-I and REP2-I revealed distinct REP-PCR-derived genomic fingerprinting patterns from E. coli, Salmonella, Shigella, and Vibrio. REP-PCR method provided clear distinctions among different bacterial species containing REP-repetitive elements and can be widely used for typing food-borne Gram-negative strains. Results showed established REP-PCR reaction conditions and generated dendrograms could be used with other supplementary genotyping or phenotyping methods to identify isolates from outbreak and to estimate relative degrees of genetic similarities among isolates from different outbreaks to determine whether they are clonally related.

• The Journal of the Korean Society for Microbiology
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• v.17 no.1
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• pp.7-14
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• 1982

A model of induction of neoplasia by viruses has develpoed from experimental studies in animals and in cultured cells and oncogenic transformation of cells is the result of integration of viral genetic information into the cellular DNA. The evidence for these associations was derived primarily from seroepidemiologic investigation. However, data indicating that the relation between HSV-2 and cervical cancer fits the model derived from experimental animal studies are not yet sufficient to draw conclusion with regard to the etiologic role the virus in the development of the neoplasms. In other hand, the K562 tumor cell is highly susceptible target for natural killer cell lysis by the lymphocytes of human and murine periperal blood. The characteristics of this effector cell type has been investigated. A study on natural killer cell mediated cytotoxicity(NKMC) against $^{51}Cr$-K562 as target cell was studed in HSV-2 infected ICR mouse. We have studied for susceptibility of HSV-2 against mouse embryo fibroblast(MEF) cells and NKMC from HSV-2 infected mouse. The results obtained that the mouse embryo fibroblast cells culture, the number and size of the cells were markedly increased and formed a monolayers relatively rapid, and become complete monolayer sheet around 72 hrs. Duration of cytopathic effect on MEF cells was rapid by serial passing of HSV-2. The morphology of the HSV-2 infected cells appear to be mainly round, ovium, spindle form and some of them was forming large giant cells. The NKMC was decrease in mouse with HSV-2 and comparison between effector/target cells ratio as 25:1 and 50:1 respectively, the NKMC was found to be more significantly decreased than normal control we have concluded that the natural killer cell activity of the viral infected mouse was shown as a suppressed during the HSV-2 infection, day 7th and 14th.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10299-10306
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• 2015

The esophageal squamous cell carcinoma (ESCC) is thought to develop through a multi-stage process. Epigenetic gene silencing constitutes an alternative or complementary mechanism to mutational events in tumorigenesis. Posttranscriptional regulation of human leukocyte antigen class I (HLA-I) and antigen processing machinery (APM) proteins expression may be associated with novel epigenetic modifications in cancer development. In the present study, we determined the expression levels of HLA-I antigen and APM components by immunohistochemistry. Then by a bisulfite-sequencing PCR (BSP) approach, we identified target CpG islands methylated at the gene promoter region of APM family genes in a ESCC cell line (ECa109), and further quantitative analysis of CpG site specific methylation of these genes in cases of Kazakh primary ESCCs with corresponding non-cancerous esophageal tissues using the Sequenom MassARRAY platform. Here we showed that the development of ESCCs was accompanied by partial or total loss of protein expression of HLA-B, TAP2, LMP7, tapasin and ERp57. The results demonstrated that although no statistical significance was found of global target CpG fragment methylation level sof HLA-B, TAP2, tapasin and ERp57 genes between ESCC and corresponding non-cancerous esophageal tissues, there was significant differences in the methylation level of several single sites between the two groups. Of thesse only the global methylation level of LMP7 gene target fragments was statistically higher ($0.0517{\pm}0.0357$) in Kazakh esophageal cancer than in neighboring normal tissues ($0.0380{\pm}0.0214$, p<0.05). Our results suggest that multiple CpG sites, but not methylation of every site leads to down regulation or deletion of gene expression. Only some of them result in genetic transcription, and silencing of HLA-B, ERp57, and LMP7 expression through hypermethylation of the promoters or other mechanisms may contribute to mechanisms of tumor escape from immune surveillance in Kazakh esophageal carcinogenesis.