• 미생물학회지
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• 제45권2호
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• pp.105-111
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• 2009

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.

• BMB Reports
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• 제47권10호
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• pp.563-568
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• 2014

Human genome projects have enabled whole genome mapping and improved our understanding of the genes in humans. However, many unknown genes remain to be functionally characterized. In this study, we characterized human chromosome 4 open reading frame 34 gene (hC4orf34). hC4orf34 was highly conserved from invertebrate to mammalian cells and ubiquitously expressed in the organs of mice, including the heart and brain. Interestingly, hC4orf34 is a novel ER-resident, type I transmembrane protein. Mutant analysis showed that the transmembrane domain (TMD) of hC4orf34 was involved in ER retention. Overall, our results indicate that hC4orf34 is an ER-resident type I transmembrane protein, and might play a role in ER functions including $Ca^{2+}$ homeostasis and ER stress.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1033-1039
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• 2008

A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of otfl was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.415-422
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• 1996

A gene coding for a pyruvyl transferase enzyme involved in exopolysaccharide biosynthesis of Zoogloea ramigera 115SLR was isolated and sequenced. A 4.5 kb of BamHI DNA fragment was isolated from chromosomal DNA using a probe derived from ketal pyruvyl transferase gene of Xanthomonas campestris. The nucleotide sequence of 2.66 kb Pst1/HindIII DNA fragment which was homology with a probe revealed the existence of two complete open reading frames (ORF2 and ORF3) and two partial open reading frames (ORFI and ORF4). The deduced amino acid sequence of ORF3 was homologous to the ketalase (GumL product) of X campestris with 49.5% of similarity and 21.6% of identity. ORF2 on the other hand showed the higher identity with the ketalase (ExoV product) of Rhizobium meliloti (36%) as well as the ketalase of X campestris (23%) than that of ORF3. A gene product of ORF2 was determined with a bacteriophage T7 RNA polymerase/promoter system in E. coli. The molecular weight of protein was 33,500 dalton.

• Genomics & Informatics
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• 제7권4호
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• pp.217-219
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• 2009

The primary clue for locating protein-coding regions is the open reading frame and the determination of ORFs (Open Reading Frames) is the first step toward the gene prediction, especially for prokaryotes. In this respect, we have developed a web-based ORF search tool called ORF Miner. The ORF Miner is a graphical analysis utility which determines all possible open reading frames of a selectable minimum size in an input sequence. This tool identifies all open reading frames using alternative genetic codes as well as the standard one and reports a list of ORFs with corresponding deduced amino acid sequences. The ORF Miner can be employed for sequence annotation and give a crucial clue to determination of actual protein-coding regions.

• ALGAE
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• 제18권3호
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• pp.183-190
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• 2003

Except for a group I intron in trnL-uaa occuring in eubacteria and plastids, group I introns are rarely documented in plastid genomes. Here, we report that a green alga, Caulerpa sertularioides, contains three group IA3 introns in the 16S gene (cpSSU), CS-cpSSU.i1, CS-cpSSU.i2 and CS-cpSSU.i3. Each intron has an open reading frame with LAGLIDADG motifs. CS-cpSSU.i1orf and CS-cpSSU.i3orf occur at Loop 6 in the intron secondary structure and CScpSSU. i2orf at Loop 8. CS-cpSSU.i1orf and CS-cpSSU.i2orf contain both LAGLI-DADG motifs but CS-cpSSU.i3orf has only one. CS-cpSSU.i1 and CS-cpSSU.i2 share the insetion sites and the ORFs at Loop 6 and 8 with CpSSU·1 and CpSSU·2 introns of Chlamydomonas pallidostigmatica (Chlorophyceae). In contrast, CS-cpSSU.i3, containing 28 copies of GAAATAT at Loop 6, is a novel intron found only in Caulerpa sertularioides. Possible scenarios of the evolution of the three introns and their possible use in systematic research are discussed.

• 한국동물위생학회지
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• 제32권1호
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• pp.19-25
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• 2009

Porcine reproductive and respiratory syndrome (PRRS) virus is the etiological agent of diseases characterized by reproductive losses in sows and respiratory disorders in piglets. The PRRS virus is a small enveloped virus containing a positive-sense, single-stranded RNA genome. In the present study, ORF6 gene of Korean PRRS virus isolate, CNV, was cloned and expressed in baculovirus expression system. The ORF6 gene and expressed protein in the recombinant virus were confirmed by PCR/indirect fluorescence antibody (IFA) test and Western blotting, respectively. The recombinant protein with a molecular weight of approximately 24KDa was confirmed by Western blotting using His6 and PRRS virus-specific antiserum. Expressed ORF6 protein was applied for IFA to detect antibody against PRRS virus using field porcine sera. However, the sensitivity and specificity of developed IFA using expressed ORF6 protein were considerably low compared to those of commercial ELISA kit. This results suggest that IFA using expressed ORF6 protein could not be used as a diagnostic test for PRRS virus infection without further improvements.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.229-233
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• 2010

Pluripotency and self-renewal capacity of human embryonic stem cells (hESCs) are retained by hESCs related genes as OCT4, SOX2 and NANOG. These genes are shown high expression level in diverse cancer cells and have potential role in the carcinogenesis. On the contrary to this, several genes which are up-regulated in the differentiated hESCs are involved to suppress the carcinogenesis or proliferation of cells. We discovered several genes in immortalized lung fibroblast (WI-38 VA13) by suppression subtractive hybridization. Among them, we focused chromosome 6 open reading frame 62 (C6orf62) which is uncharacterized, mapped to 6p22.3 and generated to Hepatitis B virus X-transactivated proteins (HBVx-transactivated proteins, XTP). Aim of this study was to characterize C6orf62 through analyzing of expression pattern in various cell lines. Expression of C6orf62 was significantly upregulated in diverse normal cell lines than cancer cell lines. And C6orf62 was up-regulated in differentiated hESCs (endothelial cells, neural cells) compared to those of undifferentiated hESCs. Also, C6orf62 in WI-38 cells was highly up-regulated during G1/S transition of the cell cycle. Taken together, C6orf62 is shown expression pattern similar to differentiated hESCs-associated genes which down-regulated in cancer cells. Therefore, we assume that C6orf62 may participate to suppress the proliferation and to induce differentiation through regulating the cell cycle.

• 한국동물위생학회지
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• 제37권3호
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• pp.143-150
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• 2014

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiological agent of PRRS characterized by reproductive losses in sows and respiratory disorders in piglets. The PRRSV is a small enveloped virus containing a positive-sense, single-stranded RNA genome and divided into two genotype, type 1 (European) and type 2 (North American), respectively, by nucleotide identity. In this study, ORF7 gene of the type 1 and type 2 PRRSV was cloned and expressed in Baculovirus expression system. Also, monoclonal antibodies (MAbs) against ORF7 were produced and characterized. The expressed ORF7 proteins in the recombinant virus were confirmed by indirect fluorescence antibody (IFA) test using His6 and PRRSV-specific antiserum. A total of eight MAbs were produced and characterized. One (3G12) MAb was type 1 PRRSV ORF7-specific and two (6B10 and 16H8) were type 2 PRRSV ORF7-specific. Other five (1A1, 2A4, 4B4, 12C4 and 13F11) MAbs reacted with both type 1 and type 2 PRRSV. Some PRRSV ORF7-specific MAbs recognized the porcine tissues infected with PRRSV by IFA or immunohistochemistry (IHC) assay. From this experiment, it was confirmed that MAbs produced in this study were PRRSV ORF7-specific and could be used as reliable reagents for type 1/type 2 PRRSV detection.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.749-754
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• 2000

알려진 dTDP-D-glucose 4,6-dehydratase의 amino acid 서열로 부터 primer를 제작하여 내열성 균주인 Thermus caldophilus GK24에서 colony hybridization 과정을 거쳐 dTDP-D-glucose 4,6-dehydratase를 포함하는 cosmid DNA를 얻었다. 유전자 분석을 위해 cosmid DNA를 subclone 하여 작은 크기로 분리하였다. 분리된 cosmid를 pSMTC-1 으로 명명하고 pSMTC-1를 BamHI으로 반응시켜 BamHI 단편 모두를 pGEM 7(+)를 이용하여 subclone 하였다. 각각의 이름은 크기에 따라 pKCB10(1.2kb-BamHI), pKCB20(1.6kb-BamHI), pKCB30(2.Ikb-BamHI), pKCB40(2.5kb-BamHI), pKCB50(2.5kb-BamHI), pKCB60(2.7kb-BamHI), pKCB70(3.4kb-BamHI), pKCB80(4.4kb-BamHI), pKCB90(7.0kb-BomHI) 으로 명명하였다. 각각의 subclone된 유전자를 분석하기 위해 Erase-a-base 방법을 이용하여 template를 준비하였고 이를 자동 염기서열 분석기를 이용하여 염기서열을 분석하였다. 염기서열분석 결과 pKCB80(4.2kb)에 dTDP-D-glucose synthase(orfA) 유전자를 비롯하여 dTDP-D-glucose 4,6-dehydratase(orfB), orfC (dTDP-4-keto-L-rhamnose reductase) 그리고 orfD(dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase)와 유사한 유전자들이 있음이 확인 되었고 dTDP-L-rhamnose의 생합성 과정을 예상할 수 있었다.