• Asian Pacific Journal of Cancer Prevention
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• 제16권4호
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• pp.1599-1603
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• 2015

Background: Epigenetic silencing of tumor suppressor genes due to promoter hypermethylation is one of the frequent mechanisms observed in cancers. Hypermethylation of several tumor suppressor genes involved in cell cycle regulation has been reported in many types of tumors including oral squamous cell carcinomas. LATS1 (Large Tumor Suppressor, isoform 1) is a novel tumor suppressor gene that regulates cell cycle progression by forming complexes with the cyclin dependent kinase, CDK1. Promoter hypermethylation of the LATS1 gene has been observed in several carcinomas and also has been linked with prognosis. However, the methylation status of LATS1 in oral squamous cell carcinomas is not known. As oral cancer is one of the most prevalent forms of cancer in India, the present study was designed to investigate the methylation status of LATS1 promoter and associate it with histopathological findings in order to determine any associations of the genetic status with stage of differentiation. Materials and Methods: Tumor chromosomal DNA isolated from biopsy tissues of thirteen oral squamous cell carcinoma biopsy tissues were subjected to digestion with methylation sensitive HpaII enzyme followed by amplification with primers flanking CCGG motifs in promoter region of LATS1 gene. The PCR amplicons were subsequently subjected to agarose gel electrophoresis along with undigested amplification control. Results: HpaII enzyme based methylation sensitive PCR identified LATS1 promoter hypermethylation in seven out of thirteen oral squamous cell carcinoma samples. Conclusions: The identification of LATS1 promoter hypermethylation in seven oral squamous cell carcinoma samples (54%), which included one sample with epithelial dysplasia, two early invasive and one moderately differentiated lesions indicates that the hypermethylation of this gene may be one of the early event during carcinogenesis. To the best of our knowledge, this is the first study to have explored and identified positive association between LATS1 promoter hypermethylation with histopathological features in oral squamous cell carcinomas.

• 한국동물위생학회지
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• 제28권3호
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• pp.259-265
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• 2005

The melanocortin 1 receptor (MC1R) encoded by the coat color extension gene (E) plays a key role in the signaling pathway of melanin synthesis. The primers for the amplification of bovine MC1R gene were designed based on a bovine MC1R gene sequence (GenBank accession no. Y19103). A size of 483bp (482bp for Hanwoo) was amplified by PCR, digested with Hpa II restriction enzyme and electrophoresed in $1.5\%$ agarose gel. When the amplified DNA product (483 bp) was digested with Hpa II restriction enzyme, Hanwoo meat showed a single band of 482bp, whereas two fragments of 325bp and 158bp were detected in Holstein, Angus and meat of Hanwoo / Holstein cross cow having back coat color phenotype, respectively. The results of this experiment Indicate that new designed primers of bovine MCIR gene may be useful for identification of Hanwoo meat from Holstein, Black Angus and Hanwoo / Holstein cross cow meat.

• 한국균학회지
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• 제28권2호
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• pp.112-117
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• 2000

야생에 서식하고 있는 번데기 및 성충에 감염된 기주의 자실체로부터 동충하초속균 3속 14균주를 분리하였고, RFLP에 의한 유전정보를 이용하여 ITS 영역에 대한 유연관계를 분석하였다. 수집된 각 균주의 rDNA ITS I과 II 부위를 primer ITS 1과 ITS 4를 사용하여 PCR에 의해 증폭한 결과 증폭된 산물은 500 bp크기로 잘 보존되어 종간 또는 속간 서로 구분되지 않았다. 따라서 증폭산물을 7종의 제한효소로 절단하여 밴드상을 관찰한 결과 수집된 Paecilomyces tenuipes 4균주와 Beauveria bassiana 2균주, JB3 균주를 제외한 Cordyceps militaris 6균주는 각각 동일 종으로 분류되었다. 한편 C. scarabaeicola 균주는 7종의 제한효소에서 모두 B. bassiana의 밴드와 일치하여 C. scarabaeicola의 불완전세대는 B. bassiana로 간주되었다. 제한 효소 중에 Paecilomyces속과 Cordyceps 속간 구분에 용이한 제한효소는 CfoI, HpaII이었으며, 그중 CfoI는 Paecilomyces, Cordyceps, Beauveria속간 구분에 용이하였다. UPGMA 분석결과 P. tenuipes, C. militaris, C. scarabaeicola와 B. bassiana, JB4 균주 등 4개의 군으로 그룹화되었으며, 그룹간에는 100% 유의수준을 나타냈다. 따라서 rDNA-RFLP 분석에서 속간 유연관계는 적었으나 종간 유연관계는 가까운 결과를 얻었다.

• The Plant Pathology Journal
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• 제23권2호
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• pp.106-108
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• 2007

The Symptoms of witches' broom disease caused by phytoplasma including general stunting and yellowing, were observed in leafy lespedeza (Lespedeza cyrtobotrya M.) on Doam-myeon, Pyeongchang-gun, in 2006. Based on the sequence analysis of PCR-amplified 16S ribosomal DNA and 16S-23S spacer region DNA products using universal phytoplasma primers, the phytoplasma associated with leafy lespedeza witches' broom (LLWB) disease was identified as a member of Candidatus Pytoplasma trifolii. It was most closely related to alsike clover proliferation phytoplasma (99.8% similarity, accession no. AY390261), Candidatus Pytoplasma trifolii strain. RFLP patterns generated with AluI, HpaII clearly differentiated LLWB phytoplasma from the referenced phytoplasma strains, water dropwort witches' broom, mulberry dwarf, glehni aster yellow dwarf and jujube witches' broom. This paper is the first report on Candidatus Phytoplasma trifolii in leafy lespedeza identified at a molecular level.

• Plant Biotechnology Reports
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• 제3권2호
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• pp.139-145
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• 2009

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we isolated NtMET1 from Nicotiana tabacum cv. Havana (SR1) and obtain transgenic plants that reduced MET1 expression level with the double-strand RNA (dsRNA) MET1 gene. Transgenic tobacco plants showed dwarf and abnormal flower development when compared with the wild type. Using methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in transformed plants and the wild type were compared. MseI/HpaII selection primers showed an interesting polymorphism, and 153 DNA bands of interest were detected. Among these, 30 selective fragments were sequenced and analyzed with a BLAST search by successful MSAP modifications. The homology search showed that the transposons and tandem repeated sequences were related to the phenotypes. These results suggested that the decreased degree of methylation by dsRNA strategy caused abnormal growth and development in N. tabacum.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1472-1476
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• 2006

Previous study demonstrated that the restriction barrier of Streptomyces griseus is almost completely bypassed by the Streptomyces-E. coli shuttle vectors passed through the E. coli GM161 strain and methylated with AluI and HpaII methyltransferases. The same DNA methylation of the genomic DNA fragments cloned the nonreplicative vectors generated integrative transformation and gene disruption of their chromosomal counterparts at high efficiencies in S. griseus. This result indicated that the efficiency of gene disruption depends on the efficient transfer of the incoming DNA into bacterial hosts.

• 대한미생물학회지
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• 제35권2호
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• pp.191-201
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• 2000

The viruses of Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2) and Varicella-Zoster virus (VZV) which belong to the alpha herpes subfamily are important human pathogens. When eruptions were not fully developed from these viral infections, clinical diagnosis was not always easy and required virological confirmation test. The above viruses were reactivated in individuals who were compromised in immune competence for one reason or another. Polymerase chain reaction (PCR) enables rapid and sensitive detection of HSV and VZV DNAs. Its sensitivity was largely influenced by choice of primers. Authors conducted a study to detect of those three viruses in human specimens including vesicle fluid and joint fluid and serum using PCR methods. Primers used for this study were the general primer pair GPHV-RU which was known to amplify within the genes enjoying the highest degree of homology between UL15 of HSV and UL42 of VZV. PCR with primers hybridized pair GPHV-RU amplifies a 396 bp with THP-1 and HSV-2 standard strain DNA and 405 bp with VZV standard strain DNA. Restriction enzyme cleavage with HpaII and DdeI were used to detect and distinguish DNAs of THP-1 and HSV-2 and VZV. The purpose of this study was a rapid and easy detection of VZV and THP-1 or HSV-2 from various clinical specimens (vesicle fluid, serum and joint fluid) by PCR method. Used methods were: HSV PCR with primer 1, 2 and HpaII RE digestion; VZV nested PCR; HSV PCR with primer A, Band BssHII RE digestion. 1) In 33 cases (33/42, 78.6%) VZV was detected single or mixed infection from 42 clinical specimens which included vesicle fluid (5), serum form respiratory infected children (10), serum from immune suppressed adult cancer patients (7) and joint fluid from arthritis patients (20). 2) In 20 cases (20/42, 47.6%) HSV was detected singly or mixed infection and 19 of those cases were HSV-2 and 1 case was THP-1. 3) In 19 cases (19/42, 45.2%) VZV was singly detected which included serum from respiratory infected children (6 cases), joint fluid from arthritis patients (9 cases), vesicle fluid (2 cases) and serum form immunosuppressed cancer patients (2 cases). 4) HSV was singly detected in 6 cases (6/42, 14.3%) which included joint fluid from arthritis patients (5 cases) and serum form respiratory infected children (1 cases). 5) 14 cases of VZV and HSV mixed infection (14/42, 33.3%) were detected. They included vesicle fluid (3 cases), serum form immunosuppressed cancer patients (4 cases), serum from respiratory infected children (2 cases) and joint fluid from arthritis patients (5 cases). 6) HSV-1 and HSV-2 detection and typing by HSV PCR with primer A, Band BssHII RE digestion method was more sensitive and the results were easier to detect than on other method.

• 대한수의학회지
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• 제45권3호
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• pp.335-339
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• 2005

The melanocortin 1 receptor (MC1R) plays an important role in regulation of melanin pigment synthesis within mammalian melanocytes. Mutations within the gene encoding MC1R have been shown to explain coat color variations within several mammalian species including cattle. To develope a rapid and accurate method for the identification of Hanwoo, we performed a modified PCR-RFLP analysis of MC1R gene using single nucleotide polymorphism (SNP) within MC1R as a target. A size of 538 bp (537 bp for Hanwoo) was amplified by PCR, digested with Hpa II, and electrophoresed on a 1.5% agarose gel. A PCR product from Hanwoo showed a single band of 537 bp, whereas two fragments of 328 bp and 210 bp were detected in both Holstein and Black angus. The current result suggests that the PCR-RFLP using our primers and enzyme digestion system would be very accurate, easy and reproducible method to discriminate between Hanwoo and Holstein/Black angus meat.

• The Plant Pathology Journal
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• 제38권3호
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• pp.254-260
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• 2022

Fusarium incarnatum-equiseti species complex (FIESC) contain over 40 members. The primer pair Smibo1FM/Semi1RM on the RPB2 partial gene has been reported to be able to identify Fusarium semitectum. The F. fujikuroi species complex (FFSC) contains more than 50 members. The F. verticillioides as a member of this complex can be identified by using VER1/VER2 primer pair on the CaM partial gene. In this research, the Smibo1FM/Semi1RM can amplify F. sulawesiense, F. hainanense, F. bubalinum, and F. tanahbumbuense, members of FIESC at 424 bp. The VER1/VER2 can amplify F. verticillioides, F. andiyazi, and F. pseudocircinatum, members of FFSC at 578 bp. Polymerase chain reaction-restriction fragment length polymorphism by using the combination of three restriction enzymes EcoRV, MspI, and HpyAV can differentiate each species of FIESC used. The two restriction enzymes HpaII and NspI can distinguish each species of FFSC used. The proper identification process is required for pathogen control in the field in order to reduce crop yield loss.

• BMB Reports
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• 제34권2호
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• pp.114-117
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• 2001

Cervi Parvum Cornu is widely used as a hemopoietic, tonifying, growth-promoting, cardiotonic, and immuno-modulating agent in Korea. In order to develop the quality control method of Cervi Parvum Cornu by the identification of the biological source or origin, the molecular approach was applied using PCR (polymerase chain reaction) and PCR-RFLF (PCR-restriction fragment length polymorphism) analysis. In the PCR analysis of the mitochondrial 12S rRNA gene and cytochrome b gene regions, no distinctive DNA bands from Cervidae (deer) antlers and Rangifer (reindeer) antlers were observed. However, when the amplified products in the mitochondrial cytochrome b gene region were subjected to restriction digestion with TaqI, Cervidae antlers showed an undigested state of 380 by band, differently from two bands of 230 by and 1S0 by from Rangifer antlers. Based on this finding, the base sequences of amplified PCR products in the range of mitochondria) cytochrome b gene from Cervidae antlers and Rangifer antlers were determined and subjected to restriction analysis by various endonucleases. The results showed that antlers from Rangifer species could be simply discriminated with other antlers from 8 Cervidae species (Chinese deer, Russian deer, Hong Kong deer, New Zealand deer, Kazakhstan deer, elk, red deer and Sika deer) by PCR-RFLP analysis using AtuI, HaeIII, HpaII or Sau3AI(MboI) as well as TaqI in the range of the mitochondrial cytochrome b gene.