• 한국식물병리학회지
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• 제10권3호
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• pp.228-234
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• 1994

Genomic RNA was extracted from Cy strain of odontoglossum ringspot tobamovirus (ORSV-Cy) isolated from infected leaves of tobacco cv. Samsun. Size of the genomic RNA was about 6.6 kb in length. The genomic RNA was fractionated using Sephadex G-50 column chromatography into 2 fractions. They were polyadenylated at their 3'-end using E. coli poly(A) polymerase. Polyadenylated viral RNA was recovered by oligo (dT) primer adapter containing NotI restriction site and Moloney murine leukemia virus SuperScript reverse transcriptase (RNase H-). Second-strand cDNA was synthesized by using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H. Recombinant plasmids containing cDNAs for ORSV-Cy RNA ranged from about 800 bp to 3,000 bp. Among the selected 238 recombinants, pORCY-124 clone was the largest one covering 3'-terminal half of the viral RNA. This clone contained two restriction sites for EcoRI and XbaI and one site for AccI, AvaI, BglII, BstXI, HindIII, PstI, and TthIII 1. respectively. The clone contained partial viral replicase, a full-length movement protein and a complete coat protein genes followed by a 3' untranslated region of 414 nucleotides based on restriction mapping and nucleotide sequencing analyses. Clones pORCY-028, -068, -072, -187 and -224 were overlapped with the pORCY-124. Clones pORCY-014 and -095 covered 5' half upstream from the middle region of the viral RNA, which was estimated based on restriction mapping and partial sequence analysis. Constructed cDNA library covered more than 90% of the viral genome.

• 미생물학회지
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• 제30권4호
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• pp.246-251
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• 1992

대두 (Glycine max) 뿌리혹의 질소고정 공생균주 Bradyrhizobium japonicum SNU001 의 nod 유전자를 클로닝하였다. Rhizobium meliloti 의 4.5 kb EcoRI/ HindIII 절편을 탐침으로 한 게놈 혼성화 반응을 분리균주의 게놈상에 nod 유전자가 존재함을 확인하고, lambda EMBL3-BamHI vector 를 이용하여 genomic library 를 작성하였다. 작성된 library 로부터 1, 2 차 선별과정을 통해 nod 유전자가 있는 클론 1-5 를 선별하고, 클론 2로부터 nod DABC 탐침과 lambda DNA 탐침을 사용한 혼성화반응을 수행하여 삽입된 genomic DNA 에 대한 부분적인 제한효소 지도를 작성하였다. nod DABC 탐침과 가장 강한 혼성화반응을 보인 phage 클론 lambda CNS-1 의 3.9 kb BamHI 절편을 pBS KS(+) vector 에 subcloning 하고 동일한 탐침을 이용한 혼성화반응을 통해 subclone pBjCNS-1 을 선별하였다. 이 subclone 에 대한 부분적인 제한효소 지도를 작성하여 nod DABC 가 1.8 KpnI/SacI 절편에 존재함을 확인하였다.

• Journal of Microbiology
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• 제39권1호
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• pp.37-41
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• 2001

The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

• Journal of Microbiology
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• 제44권6호
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• pp.671-673
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• 2006

A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminogly-coside phosphotransferase gene (aph) from Tn903, a lacZ' gene for $\alpha$-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DHS${\alpha}$ and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.

• Journal of Microbiology and Biotechnology
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• 제7권5호
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• pp.293-298
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• 1997

A 4.6-kb HindIII fragment encompassing the complete 140-kDa ${\alpha}$-amylase gene of Lactobacillus amylovorus B 4540 was cloned into pBR322 by the shot gun method. Southern blotting and restriction mapping for the insert were performed. The recombinant 9.0-kb plasmid, pFML1, conferred ${\alpha}$-amylase activity to E. coli and Lactococcus lactis hosts when introduced by electroporation. SDS-PAGE and zymography confirmed the production of 140-kDa ${\alpha}$-amylase and its proteolytic degradation products with enzyme activity in transformants. Total ${\alpha}$-amylase activity of E. coli $DH5{\alpha}$ cells harboring pFML1 was 1.8 units and most activity was detected from cell pellets. Total enzyme activity of L. lactis subsp. lactis MG1363 transformant was five to ten-fold lower than that of E. coli cell but more than half of the activity was detected in the culture supernatant.

• 한국임상수의학회지
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• 제24권4호
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• pp.636-639
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• 2007

Septicemic polyserositis and navel ill associated with Escherichia coli were reported in a 14-day-old male thoroughbred foal. The horse died after showing 12-day history of anorexia, lethargy, lameness and endophthalmus. Grossly, milky yellow abscesses were occupied in umbilicus, umbilical vein and artery. Large amounts of turbid pale yellow fluids were seen in pericardial sac, thoracic and abdominal cavity. Yellowish fibrinous materials were also presented in thoracic and abdominal cavity. Sticky pale yellow fluid and fibrinous materials were filled in stifle joint cavities of both hind limbs. Histologically, fibrino-purulent polyserositis and arthritis were observed. Severe omphalophlebitis with intra-lesional Gram negative bacterial colonies were noted in umbilical vein. Most of mesothelial cells in serosal cavities were severely hypertrophied. Pathogenic E. coli was purely isolated from ascites, thoracic and synovial fluids. Based on the results, the septicemic polyserositis may be originated from the umbilical cord infected with E. coli in this foal.

• Journal of Periodontal and Implant Science
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• 제29권3호
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• pp.663-676
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• 1999

Porphyromonas gingivalis, a Gram negative, anaerobic, asaccharolytic rod, is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated has recently been purified and characterized in this oral pathogen. This study has identified a hemin-binding P. gingivalis protein by expression of a P. gingivalis genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. A library of genomic DNA fragments from P. gingivalis was constructed in plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$ , and screened for recombinant clones with hemin-binding activity by plating onto hemin-containing agar. Of approximately 10,000 recombinant E. coli colonies screened on LB-amp-hemin agar, 10 exhibited a clearly pigmented phenotype. Each clone contained various insert DNA. The Hind III fragment transferred to the T7 RNA polymerase/promoter expression vector system produced a sligltly smaller (21 kDa) protein, a precursor form, immunoreactive to the antibody against the 24 kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 24 kDa hemin-binding protein.

• 약학회지
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• 제50권1호
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• pp.26-32
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• 2006

Osteocalcin is a vitamin K dependent and bone specific protein which plays an important role in the regulation of bone and calcium metabolism. In this study, we evaluated the relationship between the C298T polymorphism in the osteocalcin gene and bone mineral density (BMD) in Korean young men and their interaction with physical activity. BMDs of the femoral neck and lumbar spine were measured using dual energy X-ray absorptiometry, and the C298T polymorphism in the osteocalcin gene determined using polymerase chain reaction (PCR)-HindIII restriction fragment length polymorphism (RFLP) method. We did not observe any significant differences in the femoral neck and lumbar spine BMDs across genotypes of this polymorphism in controls, athletes or combined groups, respectively (P>0.05). Therefore, our data suggest that the C298T polymorphism in the osteocalcin gene is not a suitable genetic marker for the susceptibility to BMD.

• 한국미생물·생명공학회지
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• 제25권1호
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• pp.30-36
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• 1997

To genetically breed powerful multifunctional antagonistic bacteria, the urease gene of alkalophilic Bacillus pasteurii was transferred into Bacillus subtilis YB-70 which had been selected as a powerful biocontrol agent against root-rotting fungus Fusarium solani. Urease gene was inserted into the HindIII site of pGB215-110 and designated pGU266. The plasmid pGU266 containing urease gene was introduced into the B. subtilis YB-70 by alkali cation transformation system and the urease gene was very stably expressed in the transformant of B. subtilis YB-70(pGU266). The optimal conditions for the transfomation were also evaluated. From the in vitro antibiosis tests against F. solani, the antifungal activity of B. subtilis YB-70 containing urease gene was much efficient than that of the non-transformed strain. Genetic improvement of B. subtilis YB-70 by transfer of urease gene for the efficient control seemed to be responsible for enhanced plant growth and biocontrol efficacy by combining its astibiotic action and ammonia producing ability.

• Journal of Periodontal and Implant Science
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• 제32권2호
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• pp.269-280
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• 2002

The purpose of this study is to develop species-specific DNA probes and polymerase chain reaction (PCR) primers for detection and identification of Prevotella nigrescens (P. nigrescens) 9336. This study procedure includes (1) whole-genomic DNA extraction of P. nigrescens 9336 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse Dot Hybridization method, (4) confirmation of strain-specific DNA probe by Southern blot analysis, (5) determination of nucleotide sequences of strain-specific DNA probe. Thirty-five restriction fragments of P. nigrescens 9336 genomic DNA digested with the Hind III were obtained. Reverse dot hybridization and Southern blot analysis data showed that three of them, Pn10, Pn23, and Pn35, could be P. nigrescens 9336-specific DNA probes. These data indicated that these DNA probes could be useful in detection and identification of the P. nigrescens 9336.