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인체 노로바이러스의 한국분리주 Hu/NLV/Gunpo/2006/KO의 분자생물학적 특성
정아용,윤상임,지영미,강윤성,이영민,Jeong, Ah-Yong,Yun, Sang-Im,Jee, Young-Mee,Kang, Yoon-Sung,Lee, Young-Min 한국미생물학회 2009 미생물학회지 Vol.45 No.2
노로바이러스는 급성 위장염을 일으키는 Caliciviridae 과(family)에 속하는 바이러스로 유전자형이 매우 다양하다. 본 연구에서는 노로바이러스 국내분리주의 게놈 RNA로부터 3개의 open reading frame (ORF) 모두의 염기서열을 분석하고, 유전학적 계통분석을 통하여 분자생물학적 특성을 분석하였다. 본 연구에 사용된 노로바이러스(Hu/NLV/Gunpo/2006/KO)는 바이러스성 식중독, 장염 증세를 보이는 2세 여아 가검물로부터 분리되었다. 역전사반응과 PCR 증폭을 통해서 바이러스의 게놈 RNA를 3개의 중첩되는 cDNA 단편으로 합성하였으며, 합성된 cDNA를 염기서열 분석에 직접 사용하였다. 시퀀싱 결과 Hu/NLV/Gunpo/2006/KO는 3개의 ORF (ORF1, 5,100 bp; ORF2, 1,647 bp; ORF3, 765 bp)로 구성되어 있음을 알 수 있었다. 35개의 노로바이러스 국외 분리주와 비교한 결과, ORF1은 ORF2 또는 ORF3에 비해서 상대적으로 염기의 변이율이 낮았으며, 특히 ORF2와 ORF3의 C-말단 부위에서 높은 변이율을 관찰하였다. 유전학적 계통도를 분석한 결과, Hu/NLV/Gunpo/2006/KO는 genogroup II 에 속하며, Saitama U1, Gifu'96, Mc37, Vietnam 026과 같은 클러스터를 형성하는 것을 알 수 있었다. 본 연구를 통하여 노로바이러스 Hu/NLV/Gunpo/2006/KO의 3개의 ORF 염기서열을 모두 밝힘으로써, 앞으로 노로바이러스의 검출법 개발과 유전학적 상관관계뿐 아니라, 유전자의 기능 분석과 관련된 기초연구에 중요한 기초자료를 제공할 수 있을 것으로 기대한다. Norovirus (NV) with a variety of genotypes, a member of the family Caliciviridae, causes acute nonbacterial gastroenteritis in humans. We determined the nucleotide sequence of three open reading frames (ORFs) of a NV Korean strain and characterized the genetic relationship with others. The Korean strain designated Hu/NLV/Gunpo/2006/KO was isolated from the stool specimen of a 2-year-old female suffering from gastroenteritis. By performing reverse transcription and PCR amplification, three overlapping cDNAs were synthesized and used for direct sequencing. We found that like other NVs, this strain contains three ORFs: ORF1, 5,100 bp; ORF2, 1,647 bp; ORF3, 765 bp. Of 35 NVs, ORF1 had a level of genetic diversity lower than ORF2 and ORF3, of which the C-termini of the ORF2 and ORF3 showed a relatively high degree of genetic diversity. Phylogenetic analyses indicated that the Korean strain belonged to genogroup II, with Saitama U1, Gifu'96, Mc37, and Vietnam 026 being formed a single genetic cluster. The nucleotide sequence information of three ORFs of a NV Korean isolate will be useful not only for the development of a diagnostic tool and understanding of genetic relationship, but also provide important basic information for the functional analysis of their gene products.
염색체 위치 특이적 삽입과 안정적인 유전자 발현을 유도하는 플라스미드 백터의 제작
문영준,강윤성,최지혜,손진숙,민나영,이광호 中央大學校 基礎科學硏究所 2002 基礎科學硏究所 論文集 Vol.16 No.-
Insertion of reporter constructs into the mammalian genome leads to variable gene expression due to position effects at the site of integration. This random integration has limited the gene therapy of human genetic disorders by its undesirable effects. We report here the newly constructed plasmid vector(pIRES-neo-YJ) based on the concepts of homologous recombination and position-independent promoter enhancing of beta-globin matrix attachment region(Glb-MAR). chromosome 7 centromere-specific alpha satellite(alphoid) DNA sequence was cloned into pIRES-neo-YJ for homologous recombination of the cloned gene with the centromeric region of chromosome 7, which is genetically silent. Beta Glb-MAR sequence that allows high levels of transcription independent of the chromosomal site of integration was also inserted into pIRES-neo-YJ to ensure the stable and higher expression of the cloned genes. We expect that pIRES-neo-YJ would provide a valuable tool to eliminate random integration of cloned genes into the undesirable chromosomal region and their short-lived expression which often encounters during the construction of transgenic animals and human gene therapy.
사람과 설치류간의 체세포 융합세포 개발과 사람유전자 지도작성을 위한 융합세포의 응용
진미욱,강윤성,이광호 中央大學校 基礎科學硏究所 1998 基礎科學硏究所 論文集 Vol.12 No.-
Human-rodent somatic cell hybrids have proven very useful in mapping genes and DNA sequences to specific chromosomes. Our experiment was directed to construct a human chromosome panel which consists of hybrid cell lines containing only a human chromosome or a few. Forty seven hybrid clones were analyzed for contained human chromosomes by G-, Q-banding but rearranged fragments of human chromosome could not be characterized by these methods. To overcome these problems, we performed IRS-PCR-FISH(interspersed repetitive sequence-PCR-FISH). To amplify the human specific DNA sequences in human-rodent somatic cell hybrids, primers directed at human-specfic regions of Alu-517(SINE) and Ll-Hs(LINE) were used. FISH using the biotin-labelled products made it possible to identify individual human chromosomes and rearranged human materials in hybrids, which were not identifiable by conventional cytogenetic methods. Thus, IRS-RCR-FISH allowed us to characterize human chromosomes in hybrid cells to generate the human chromosome panel. Unknown genes could be mapped to specific chromosmes by biochemical and molecular techniques such as enzyme and southern blot analysis combining with this panel. This panel will serve as a useful resource for mapping and cloning of novel genes.