• 제목/요약/키워드: genomic pattern

검색결과 170건 처리시간 0.031초

초위성체 DNA를 이용한 제주마 집단의 품종특성 및 개체식별 체계설정 (Establishment of Genetic Characteristics and Individual Identification System Using Microsatellite Loci in Jeju Native Horse)

  • 조병욱;정지혜;김상욱;김희수;이학교;조길재;송기덕
    • 생명과학회지
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    • 제17권10호
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    • pp.1441-1446
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    • 2007
  • 본 연구는 서로 다른 상염색체에 위치하고 있는 초위성체유전 표지를 활용하여 제주마 집단의 타 품종과의 차별적 유전특성분석과 제주마 집단에서 활용할 수 있는 효율적인 개체 식별 시스템 설정을 위해 수행되었다. 공시재료로서는 5품종에서 총 191두가 사용되었으며 13종의 좌위에 대한 개체별 유전자형을 분석하였다. 이들 13종에서 출현된 총 대립 유전자수는 제주마의 경우 155종이 나타났다. 한국 제주마 집단에서 나타난 평균 이형접합도는 0.317-0.902였으며 marker 다형성 정보량은 0.498-0.799로 나타났다. 제주마 집단에서 나타난 대립 유전자 발현 특성은 다른 외래 품종 집단과 매우 상이한 결과를 나타냈다. ATH4 좌위의 경우 제주마 집단에서는 5종의 대립 유전자가 고른 분포를 나타낸 반면 QUA종의 경우와 THO종집단에서 특정 좌위의 극단적 발현 빈도를 나타냈다. 품종특이성을 나타내는 분자표지의 대립유전자 발현양상이 확인되었으며 이러한 품종특이성 발현 분자표지는 집단 내 개체에 대한 품종식별 유전자표지로 활용이 가능한 것으로 나타났다. 9종의 초위성체 유전 표지를 활용할 경우 누적 개체 식별력은 99.9%를 나타냈으며 두 마리의 서로 다른 개체가 서로 같은 유전자형을 가질 짝확률은 $0.60\;{\times}\;10^{10}$으로 추정되었다. 따라서 9종의 선정된 유전표지는 제주마 품종 집단에서 적정 신뢰도를 제공할 수 있는 개체 식별 시스템을 설정할 수 있을 것으로 생각된다.

Comparative Study of Anti-Apoptotic Genes, Bcl-2 and P35 for the Suppression of Apoptosis Induced in Suspension Culture of Transformed Trichoplusia ni BTI Tn 5B1-4 Cells

  • Lee, Jong-Min;Sohn, Bong-Hee;Kang, Pil-Don;Lee, Sang-Uk;Chung, In-Sik
    • International Journal of Industrial Entomology and Biomaterials
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    • 제9권2호
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    • pp.173-181
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    • 2004
  • To delay the onset of apoptosis in the culture, transformed Tn 5B1-4 cells harboring anti-apoptotic genes, bcl-2 and baculovirus p35, have been established and analyzed for their anti-apoptotic ability in suspension culture using spinner flasks. In the suspension culture at agitation speeds of 100 rpm and 200 rpm, the cell growth of cell clone expressing Bcl-2 protein was much higher than other two clones and the maximum cell density of the clone was 6.0 ${\times}$ 10$^{6}$ cells/ml and 6.2 ${\times}$ 10$^{6}$ cells/ml at day three of the incubation. On the other hand, the cell growth of cell clone expressing baculovirus protein P35 was much higher than other two clones in suspension culture at agitation speed of 300 rpm and the maximum cell density of the clone was 6.1 ${\times}$ 10$^{6}$ cells/ml at day three of the incubation. Based on the pattern of genomic DNA laddering and the microscopic observation of apoptotic bodies, the more apoptotic bodies are induced in Tn 5B1-4 control cell clone at higher agitation speed. This result shows that the shear stress can be a main factor in inducing apoptosis in spinner flask culture. At low agitation speed, cell clone expressing Bcl-2 was more effective in delaying the onset of apoptosis than the cell clone expressing P35. On the other hand, at high agitation speed, cell clones expressing baculovirus P35 was more effective in delaying the onset of apoptosis than the cell clone expressing Bcl-2. Therefore, anti-apoptotic genes, bcl-2 and baculovirus p35, can playa distinct role depending on agitation speed in the suspension culture.

Southern hybridization에 의한 질편모충의 유전학적 다양성 (Genetic variance of Tuchomonns uaginclis isolates by Southern hybridization)

  • 류재숙;민득영
    • Parasites, Hosts and Diseases
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    • 제36권3호
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    • pp.207-212
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    • 1998
  • 질편모충 7개 분리주 (국내분리주 UT8, KT6, UT-Kim 및 fr-Lee, 외국 분리주 CDC85, IR78 및 NYH286주)의 유전학적 차이점을 관찰하고자 Southemhybridization을 하였다. 탐침 (probe) 은 질편모충 DNA에 있는 반복적인 염기서열을 기초로 하여 337 bp의 탐침을 제작하였다. 질편모충 각 분리주를 클로닝하고 각각의 원충을 따로 배양하여 DNA를 분리하여 제한효소를 처리한 후 전기영동하고 Southemhybridization을 하였다. 질편모충 분리주에 관계엄이 11개 내외의 븐회이 관찰되었다. 콜로니가 2개 형성된 KT8, IR78 및 KT-Lee 분리주에서는 클로닝하기 전의 분리주와 클로닝하여 형성된 콜로니를 배양한 질편모충에서 같은 bandpattem이 관찰되었다. 사용된 모든 질편모충을 bandpattem에 따라 3군으로 나눌 수 있었는데, KT8 분리주는 국내 분리주인 KT6, KT-Kim 분리주와 같은 band pattern을 보여 1 kb, 1,2 kb, 1.6 kb, 1.9 kb, 2.3 kb, 2.7 kb, 3.2 kb, 3.4 kb, 3.8 kb, 4.9 kb 및 6.0 kb의 11개의 공통 분획을 보였다. 외국분리주로 metronidazole에 내성인 IR78 분리주는 국내 분리주인 fr-Lee 분리주와 같은 분획을 보여 1 kb, 1.2 kb, 1.6 kb, 1.8 kb, 2.1 kb, 2.5 kb, 2.7 kb, 2.9 kb, 3.4 kb, 5.0 kb 및 6.0 kb의 분획을 보였으며 IR78과 같이 약제 내성이 있다고 알려진 CDC85의 경우 IR78, KT-Lee 분리주와 비슷한 분획을 보였으나 2.9 kb가 없고 3.2 kb의 분획이 관찰되었다. 세번째 군에 해당되는 NYH286주는 12개의 분획을 보였는데 IR78, KT-Lee 분리주와 유사한 분획을 보였으나 그 차이점 은 6.0 kb 대신 6.2 kb를, 2. 1 kb 대신 2.0 kb와 2.2 kb를 나타냈다. 이상의 결과로 질편모충 여러 분리주의 유전학적 다양성이 관찰되었다.

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소 c-KIT Receptor 유전자의 다형성에 관한 연구 (A Study on DNA Polymorphism of the Bovine c-KIT Receptor Gene)

  • 장요순;김태헌;윤두학;박응우;이혜원;이학교;정일정
    • Journal of Animal Science and Technology
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    • 제44권6호
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    • pp.653-660
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    • 2002
  • 소의 흰 반점 관련 후보유전자로 c-KIT receptor 유전자를 선정하여, c-KIT receptor 유전자내의 변이를 탐색하고 변이가 흰반점 표현형과 연관성이 있는지를 분석하였다. 한우, Angus, Brown Swiss, Charolais, Hereford, Hols- tein, Limousin 및 Simmental 등 8개 품종의 DNA 시료를 사용하여 c-KIT receptor 유전자의 intron 6번 영역에서 다형성을 조사하고 분석하였다. c-KIT receptor 유전자의 intron 6번 영역에서는 4개의 염기치환이 발견되어, MspⅠ, BsrBⅠ 및 NdeⅠ 제한효소를 이용하여 PCR- RFLP 분석을 실시하였다. Intron 6번을 포함하는 영역의 PCR 산물 크기는 2,440 bp 이었다. MspⅠ다형성은 PCR-RFLP 분석 결과 3개의 대립유전자가 존재하였으며, 한우품종에서는 3개의 대립유전자 모두가 발견되었고, CC 형태의 유전자형을 제외한 5개의 유전자형 (AA, AB, AC, BC 및 BB)을 확인하였다. Angus, Brown Swiss, Hereford, Holstein 및 Simmental 품종에서는 A 대립유전자만을 갖는 것으로 조사되었고, 한우는 44%만 AA 유전자형을 나타내었다. BsrBⅠ 다형성은 2개의 대립유전자로서 3개의 유전자형이 나타나는 것을 확인하였으며, Charo- lais 및 Hereford 품종이 다른 소 품종에 비하여 A 대립유전자의 빈도가 높게 나타났다. NdeⅠ다형성을 분석한 결과 Brown Swiss 품종에서는 NdeⅠ에 의해 절단되는 형태인 A 대립유전자만 관찰되었으며, Holstein 품종은 92%, Simmental 품종은 72%가 절단되는 형태를 나타내어, 모색이 흰색을 띠는 소 품종에서 절단되는 형태가 많았다. 소 c-KIT receptor 유전자의 intron 6번 영역에서 확인된 4개의 염기치환은 품종에 따라 다른 빈도를 보였으나, 이들 염기치환과 흰 반점과의 연관성에 대한 증거는 발견하지 못하였다. 그러므로 소의 흰 반점과 c-KIT receptor 유전자 내의 변이와의 관련성은 다른 영역에 대한 추가적인 분석과, 이미 보고된 다른 모색관련 유전자의 다형성과의 연관성 분석 등과 같은 연구가 필요한 것으로 판단된다.

Studies of Molecular Breeding Technique Using Genome Information on Edible Mushrooms

  • Kong, Won-Sik;Woo, Sung-I;Jang, Kab-Yeul;Shin, Pyung-Gyun;Oh, Youn-Lee;Kim, Eun-sun;Oh, Min-Jee;Park, Young-Jin;Lee, Chang-Soo;Kim, Jong-Guk
    • 한국균학회소식:학술대회논문집
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    • 한국균학회 2015년도 춘계학술대회 및 임시총회
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    • pp.53-53
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    • 2015
  • Agrobacterium tumefaciens-mediated transformation(ATMT) of Flammulina velutipes was used to produce a diverse number of transformants to discover the functions of gene that is vital for its variation color, spore pattern and cellulolytic activity. Futhermore, the transformant pool will be used as a good genetic resource for studying gene functions. Agrobacterium-mediated transformation was conducted in order to generate intentional mutants of F. velutipes strain KACC42777. Then Agrobacterium tumefaciens AGL-1 harboring pBGgHg was transformed into F. velutipes. This method is use to determine the functional gene of F. velutipes. Inverse PCR was used to insert T-DNA into the tagged chromosomal DNA segments and conducting sequence analysis of the F. velutipes. But this experiment had trouble in diverse morphological mutants because of dikaryotic nature of mushroom. It needed to make monokaryotic fruiting varients which introduced genes of compatible mating types. In this study, next generation sequencing data was generated from 28 strains of Flammulina velutipes with different phenotypes using Illumina Hiseq platform. Filtered short reads were initially aligned to the reference genome (KACC42780) to construct a SNP matrix. And then we built a phylogenetic tree based on the validated SNPs. The inferred tree represented that white- and brown- fruitbody forming strains were generally separated although three brown strains, 4103, 4028, and 4195, were grouped with white ones. This topological relationship was consistently reappeared even when we used randomly selected SNPs. Group I containing 4062, 4148, and 4195 strains and group II containing 4188, 4190, and 4194 strains formed early-divergent lineages with robust nodal supports, suggesting that they are independent groups from the members in main clades. To elucidate the distinction between white-fruitbody forming strains isolated from Korea and Japan, phylogenetic analysis was performed using their SNP data with group I members as outgroup. However, no significant genetic variation was noticed in this study. A total of 28 strains of Flammulina velutipes were analyzed to identify the genomic regions responsible for producing white-fruiting body. NGS data was yielded by using Illumina Hiseq platform. Short reads were filtered by quality score and read length were mapped on the reference genome (KACC42780). Between the white- and brown fruitbody forming strains. There is a high possibility that SNPs can be detected among the white strains as homozygous because white phenotype is recessive in F. velutipes. Thus, we constructed SNP matrix within 8 white strains. SNPs discovered between mono3 and mono19, the parental monokaryotic strains of 4210 strain (white), were excluded from the candidate. If the genotypes of SNPs detected between white and brown strains were identical with those in mono3 and mono19 strains, they were included in candidate as a priority. As a result, if more than 5 candidates SNPs were localized in single gene, we regarded as they are possibly related to the white color. In F. velutipes genome, chr01, chr04, chr07,chr11 regions were identified to be associated with white fruitbody forming. White and Brown Fruitbody strains can be used as an identification marker for F. veluipes. We can develop some molecular markers to identify colored strains and discriminate national white varieties against Japanese ones.

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Evolutionary Explanation for Beauveria bassiana Being a Potent Biological Control Agent Against Agricultural Pests

  • Han, Jae-Gu
    • 한국균학회소식:학술대회논문집
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    • 한국균학회 2014년도 춘계학술대회 및 임시총회
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    • pp.27-28
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    • 2014
  • Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

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급성전골수성백혈병 HL-60 세포주에서 방사선조사에 의한 세포고사기전 (A Study on Apoptotic Signaling Pathway in HL-60 Cells Induced by Radiation)

  • 김혜정;문성근;이재훈;문성록
    • Radiation Oncology Journal
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    • 제19권2호
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    • pp.153-162
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    • 2001
  • 목적 : 방사선 조사에 의하여 유발되는 세포고사의 신호전달기전, 특히 caspase계 cysteine protease의 활성화, Bcl2 및 Bax 단백질, cytochrome c의 세포질내로의 방출, Fas 와 Fas-L 단백질의 발현양상 등의 조사를 통하여 방사선 조사에 의하여 유발되는 세포고사기전을 규명하고자 하였다. 대상 및 방법 : HL-60 세포주에 6 MV의 X-선을 조사하고 세포생존율, Caspase의 활성도, $Bcl_2$ 및 Bax 단백질, cytochrome c의 세포질내로의 방출여부, 및 Fas 와 Fas-L 단백질의 발현양상을 조사하였다. 결과 : 방사선조사 후 세포의 생존율은 조사선량과 조사 후 시간경과에 따라 감소되었다. 세포고사의 특징인 사다리형 DNA 분절은 방사선조사 4시간 후부터 시간경과에 따라 증가하였으며, 조사선량이 증가할수록 더욱 현저하였다. 방사선조사 후 caspase계 cysteine proteases 중 caspase-2, 3, 6, 8 및 9의 활성화가 시간경과에 따라 증가하였으며, 16 Gy의 방사선량조사 4시간 후에 poly (ADP-ribosyl) polymerase (PARP)의 분절과 Western blot을 이용한 procaspase-3의 분절을 확인함으로서 caspase-3의 활성을 간접적으로 증명할 수 있었다. $Bcl_2$ 단백질은 방사선조사 후 시간경과에 따라 감소하였으며, Bax 단백질은 시간경과에 따라 발현이 증가하는 양상을 관찰할 수 있었다. 방사선조사 후 cytochrome c의 세포질내로의 방출을 확인하였다. 또한 Fas 및 Fas-L 단백질 모두 방사선조사 후 발현이 증가하는 양상을 관찰할 수 있었다. 결론 : HL-60 세포주에서 방사선 조사에 의해 유발되는 세포사멸이 세포고사기전에 의해서 매개됨을 확인하였으며, 이는 세포내 caspase계 cysteine proteases, $Bcl_2$, Bax, 세포질내로의 cytochrome c 방출 그리고 Fas, Fas-L가 관여하는 신호전달경로의 활성화에 의한 것임을 의미하였다.

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구강 편평세포암종에서 $P16^{ink4}$ 유전자의 Methylation에 대한 연구 (($P16^{ink4}$ Methylation in Squamous Cell Carcinoma of the Oral Cavity.)

  • 강진원;김경욱;류진우;김창진
    • Maxillofacial Plastic and Reconstructive Surgery
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    • 제22권2호
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    • pp.164-173
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    • 2000
  • The p16 protein is a cyclin dependent kinase inhibitor that inhibits cell cycle progression from $G_1$ phase to S phase in cell cycle. Many p16 gene mutations have been noted in many cancer-cell lines and in some primary cancers, and alterations of p16 gene function by DNA methylation have been noticed in various kinds of cancer tissues and cell-lines. There have been a large body of literature has accumulated indicating that abnormal patterns of DNA methylation (both hypomethylation and hypermethylation) occur in a wide variety of human neoplasma and that these aberrations of DNA methylation may play an important epigenetic role in the development and progression of neoplasia. DNA methylation is a part of the inheritable epigenetic system that influences expression or silencing of genes necessary for normal differentiation and proliferation. Gene activity may be silenced by methylation of up steream regulatory regions. Reactivation is associated with demethylation. Although evidence or a high incidence of p16 alterations in a variety of cell lines and primary tumors has been reported, that has been contested by other investigators. The precise mechanisms by which abnormal methylation might contribute to carcinogenesis are still not fully elucidated, but conceivably could involve the modulation of oncogene and other important regulatory gene expression, in addition to creating areas of genetic instability, thus predisposing to mutational events causing neoplasia. There have been many variable results of studies of head and neck squamous cell carcinoma(HNSCC). This investigation was studied on 13 primary HNSCC for p16 gene status by protein expression in immunohistochemistry, and DNA genetic/epigenetic analyzed to determine the incidence, the mechanisms, and the potential biological significance of its Inactivation. As methylation detection method of p16 gene, the methylation specific PCR(MSP) is sensitive and specific for methylation of any block of CpG sites in a CpG islands using bisulfite-modified DNA. The genomic DNA is modified by treatment with sodium bisulfate, which converts all unmethylated cytosines to uracil(thymidine). The primers designed for MSP were chosen for regions containing frequent cytosines (to distinguish unmodified from modified DNA), and CpG pairs near the 5' end of the primers (to provide maximal discrimination in the PCR between methylated and unmethylated DNA). The two strands of DNA are no longer complementary after bisulfite treatment, primers can be designed for either modified strand. In this study, 13 paraffin embedded block tissues were used, so the fragment of DNA to be amplified was intentionally small, to allow the assessment of methylation pattern in a limited region and to facilitate the application of this technique to samlples. In this 13 primary HNSCC tissues, there was no methylation of p16 promoter gene (detected by MSP and automatic sequencing). The p16 protein-specific immunohistochemical staining was performed on 13 paraffin embedded primary HNSCC tissue samples. Twelve cases among the 13 showed altered expression of p16 proteins (negative expression). In this study, The author suggested that low expression of p16 protein may play an important role in human HNSCC, and this study suggested that many kinds of genetic mechanisms including DNA methylation may play the role in carcinogenesis.

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백화사설초(白花蛇舌草) 메탄올 추출물(抽出物)의 항종양(抗腫瘍) 효과(效果) 및 항암(抗癌) 기전(機轉)에 관(關)한 연구(硏究) (Study of Hedyotis Diffusa Methanol Extract on Anti-tumoral Effect and Mechanism)

  • 노훈정;문구;문석재;원진희;문영호;박래길
    • 대한한방종양학회지
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    • 제6권1호
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    • pp.81-97
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    • 2000
  • Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.

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콩(Glycine max)에서 콩위축바이러스(Soybean dwarf virus)의 최초 발생보고 (First Report of Soybean Dwarf Virus on Soybean(Glycine max) in Korea)

  • 김상목;이재봉;이영훈;최세훈;최홍수;박진우;이준성;이관석;문중경;문제선;이기운;이수헌
    • 식물병연구
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    • 제12권3호
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    • pp.213-220
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    • 2006
  • 2003년 청송지역 농가포장에서 심한 위축증상을 나타내는 콩 시료를 채집하였다. 이 시료를 RT-PCR로 진단한 결과 콩위축바이러스, Soybean dwarf virus(SbDV-L81)에 감염된 것으로 확인되었다. 본 연구는 우리나라에서 콩위축바이러스의 발생에 관한 최초 보고이다. 이 바이러스의 추가적인 특징을 알아보기 위하여 종 특이적 프라이머를 이용한 RT-PCR로 게놈의 부분 염기서열을 결정하였다. 결정된 염기서열은 병징 발현 유형과 매개충 특이성에 기초하여 이전에 조사된 SbDV 계통들과 비교분석하였다. ORF 2와 ORF 3 유전자사이 영역(intergenic region)과 ORF 2, ORF 3 및 ORF 4의 코팅영역에서 SbDV-L81은 황화계통(SbDV-YS and YP) 보다 위축계통(SbDV-DS and DP)과 유사한 특징을 보였다. 한편, ORF 5의 5'쪽 코딩영역은 완두수염진딧물(Acyrthosiphon pisum)이 매개충인 계통(SbDV-YP and DP)과 밀접한 관련이 있은 것으로 나타났다. SbDV-L81은 자연상태에서의 병정과 5가지 영역의 염기서열 비교결과에서 SbDV-DP 계통과 밀접한 관련이 있는 것으로 생각된다.