• Microbiology and Biotechnology Letters
• /
• v.43 no.1
• /
• pp.1-8
• /
• 2015

In this study, a DNA-launched reverse genetics system was developed from a type 2 porcine reproductive and respiratory syndrome virus (PRRSV) strain, KNU-12. The complete genome of 15,412 nucleotides was assembled as a single cDNA clone and placed under the eukaryotic CMV promoter. Upon transfection of BHK-tailless pCD163 cells with a full-length cDNA clone, viable and infectious type 2 progeny PRRSV were rescued. The reconstituted virus was found to maintain growth properties similar to those of the parental virus in porcine alveolar macrophage (PAM) cells. With the availability of this type 2 PRRSV infectious clone, we first explored the biological relevance of ORF5a in the PRRSV replication cycle. Therefore, we used a PRRSV reverse genetics system to generate an ORF5a knockout mutant clone by changing the ORF5a translation start codon and introducing a stop codon at the 7^th codon of ORF5a. The ORF5a knockout mutant was found to exhibit a lack of infectivity in both BHK-tailless pCD163 and PAM-pCD163 cells, suggesting that inactivation of ORF5a expression is lethal for infectious virus production. In order to restore the ORF5a gene-deleted PRRSV, complementing cell lines were established to stably express the ORF5a protein of PRRSV. ORF5a-expressing cells were capable of supporting the production of the replicationdefective virus, indicating complementation of the impaired ORF5a gene function of PRRSV in trans.

• The Microorganisms and Industry
• /
• v.14 no.1
• /
• pp.22-28
• /
• 1988

DNA 클로닝과 조작기술의 발전은 어떤 유전자의 특정한 위치에 선택적으로 돌연변이를 도입할 수 있는 site-specific mutagenesis 기술을 창출해 내었다. 이 기술로 DAN 염기의 치환, 결실, 삽입등을 클론된 유전자에 직접 도입할 수가 있게 되어 생체의 유전자 조작이나 유전자의 산물인 단백질의 구조와 기능을 의도적으로 변화시키는 protein engineering에 광범위하게 이용되고 있다. Protein engineering은 주로 단백질의 촉매 및 생리활성의 증가, 효소의 특성및 기질 특이성의 변화, 단백질 구조의 안정화 및 내염성 증가, 분자량의 감소, 효소및 생리활성 단백질의 구조의 안정화및 내열성 증가 등에 활용되고 있으며 산업적 유용성이 큰새로운 단백질의 창조에도 기여할 것으로 기대를 모으고 있다. Site-specific mutagenesis 기술로 현재 가장 널리 이용되는 것이 in vitro상에서 수행하는 oligonucleotide-directed site specific mutagenesis이다. 이 방법은 생화학적으로 합성한 특정한 염기서열을 가진 oligonucleotide들을 일종의 mutagen으로 사용하거나 효소적 DNA 합성을 위한 primer로 사용하여 클론된 DNA의 염기서열을 선택적으로 개조하거나 혹은 다른 조작을 하는 것이다. 여기서는 돌연변이율을 높이는 여러가지 개량된 방법들이 나왔으며 그중의 몇가지를 소개하였다.

• The Korean Journal of Mycology
• /
• v.28 no.1
• /
• pp.26-31
• /
• 2000

We isolated genomic clone of FVFD16 and FVFD30 gene specifically expressed during fruit body formation of Flammulina velutipes [(Curt: Fr.) Sing] and determinated the sequences. The FVFD16 gene is including two introns in open reading frame, and FVFD30 gene is including four introns. The introns were matched GT/AG rule. The FVFD16 and FVFD30 genes contained CAAT box with similarity arrange and TATA box. CT-rich region was presented before the transcription start point. FVFD30 gene is investigated that expected the most activity of CCACC arrange. The result of FVFD16 gene analysis showed 80% homology by cDNA clone that is gene family. From the results of genomic southern blot analysis, we presumed more than two copy number gene family of FVFD16 and FVFD30 gene.

• Korean Journal of Microbiology
• /
• v.51 no.2
• /
• pp.148-155
• /
• 2015

The diversity of endobacteria in the edible part (cap and stipe) king oyster mushroom (Pleurotus eryngii) was investigated using 16S rRNA sequence analysis. The bacterial 16S rRNA libraries were constructed from the body cap (BC) and the body stipe (BS) of the king oyster mushroom. The twenty sequenced BC clones were divided into four groups and the largest group was affiliated with the Firmicutes (40% of clones). While, the twenty sequenced BS clones could be divided into six groups and the largest group was affiliated with the Actinobacteria (40% of clones). The predominant bacterial family from both the cap and stipe of the mushroom was corresponded with the Gram positive bacteria (62.5%).

• Microbiology and Biotechnology Letters
• /
• v.16 no.5
• /
• pp.363-368
• /
• 1988

Molecular cloning of gene for $\alpha$-acylamino-$\beta$-lactam acylhydrolase (ALAH) III from Acetobacter turbidans has been attempted by immunochemical detection method, in which polyclonal antibody from mouse Balb/c against this enzyme was employed as a probe. As a cloning vector, λ gtll was chosen for this purpose. Two positive clones has been selected from genomic libraries of A. turbidans, which had somewhat different binding affinities on anti-ALAH III umm and anti-$\beta$-galactosidase. By restriction analysis, both clones has been turned out to lose one of EeoRI sites. From these results, it concluded that deletion of DNA between lacZ gene and inserted DNA has occurred during replication of these clones in host cells.

• Korean Journal of Agricultural and Forest Meteorology
• /
• v.13 no.3
• /
• pp.140-147
• /
• 2011

We studied the influence of livestock waste leachate on oxidative damage and antioxidative responses in poplar clones in August which increase the demand of antioxidants because of high temperature and high light during this period. We measured ion leakage, antioxidant enzyme activities (APX, GR), and carotenoid contents. Oxidative damage and antioxidative responses by treated livestock waste leachate in poplar clones showed various results. We divided poplar clones into three groups using the criteria based on ion leakage which represent cell damage induced oxidative stress. Eco 28, 62-10, Bonghwa1 and Dorskamp belonged to the first group in which the cell damaged level was lower than that of the control. The results suggest that this group augmented for demand of antioxidative in summer because high concentration of nitrogen induced by treatment of live stock wastes acted as environmental stress. Consequently, they failed to keep up the homeostasis of reactive oxygen species. The second group in which the cell damaged level was similar to that of the control was Suwon, 72-30 and 72-31 clones. Finally, 97-18 clone belonged to the third group in which the cell damaged level was lower than that of the control group. In this case, nitrogen treated by livestock waste leakage decreased oxidative stress. 97-18 clone was the clones with the least damage in summer oxidative stresses treated by livestock waste leakage. These results suggest that the high concentration nitrogen due to the livestock waste leakage can act differently upon the clones. We speculate that the added oxidation damage in the summer (growing season) may have an effect on the total fresh weight and also influence the purification ability for livestock waste leakage. However, further studies are needed for the confirmation.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
• /
• v.15 no.1
• /
• pp.25-35
• /
• 2010

The biodiversity of eukaryotic plankton has commonly been used to evaluate the status of aquatic ecosystems. Therefore, an accurate and rapid method for species identification is needed to reveal the biodiversity of environmental water samples. To date, molecular methods have provided a great deal of information that has enabled identification of the hidden biodiversity in environmental samples. In this study, we utilized environmental polymerase chain reaction (PCR) and constructed the 18S nuclear ribosomal RNA clone library from environmental water samples in order to develop more efficient methods for species identification. For the molecular analysis, water samples were collected from the Seonakdong River (Gimhae Bridge) and the coast of Namhae，(Namhaedo). Colony PCR and restriction fragment length polymorphism of PCR (PCR-RFLP) were then adopted to isolate unique clones from the 18S rDNA clone library. Restriction fragment length polymorphism pattern analysis of the Gimhae Bridge sample revealed 44 unique clones from a total of 60 randomly selected clones, while analysis of the Namhae sample revealed 27 unique clones from 150 clones selected at random. A BLAST search and subsequent phylogenetic analysis conducted using the sequences of these clones revealed hidden biodiversity containing a wide range of taxonomic groups (Heterokontophyta (7), Ciliophora (23), Dinophyta (1), Chytridiomycota (1), Rotifera (1) and Arthropoda (11) in the Gimhae Bridge samples Ciliophora (4), Dinophyta (3), Cryptophyta (1), Arthropoda (19) in the Namhae samples). Therefore, the molecular monitoring method developed here can provide additional information regarding the biodiversity and community structure of eukaryotic plankton in environmental samples and helps construct a useful database of biodiversity for aquatic ecosystems.

• Applied Biological Chemistry
• /
• v.50 no.4
• /
• pp.268-275
• /
• 2007

Diversity of the mud flat microbial population in Suncheon Bay was investigated by studying extracellular enzyme activities and 16S rDNA sequences. Four culturable bacterial strains with CMCase, xylanase and protease activities were isolated from the wetland and the mud flat. All the strains produced more xylanase activity than CMCase or protease activity, and the properties of the isolate enzymes from the wetland were similar to those from the mud flat. About 2,000 clones were obtained with the 16S rDNA amplified from the metagenomic DNA isolated from the mud samples. Based on the restriction pattern(s), seventeen clones were selected for base sequence analysis. Of the 17 clones, only 35% (6 clones) were found to be cultured strains and 65% (11 clones) to be uncultured strains. The similarities in the base sequences of the clones ranged from 91.0% to 99.9% with an average similarity of 97.3%. The clones could be divided into 7 groups, Proteobacteria (9 clones, 52.9%), Firmicutes (3 clones, 17.6%), Bacteroidetes (1 clone), Flavobacteria (1 clone), Verrucomicrobia (1 clone), Acidobacteria (1 clone), and Chloroflexi (1 clone). Most of the Proteobacteria clones were gamma Proteobacteria associated with oxidation-reduction of sulfur.

• Proceedings of the Korean Society of Fisheries Technology Conference
• /
• 1998.06a
• /
• pp.138-138
• /
• 1998

• Proceedings of the Korean Radioactive Waste Society Conference
• /
• 2005.06a
• /
• pp.74-75
• /
• 2005