• 한국식품영양학회지
• /
• 제9권4호
• /
• pp.459-465
• /
• 1996

지금까지 많은 연구가 되어 있지 않던 Bacillus thuringiensis serovar. darmstadiensis의 내독소를 Renografin-76 단계적 기울기 원심분리로 분리하여 전자 현미경으로 관찰하여 이중피라미드 구조를 가진 독소 단백질을 확인하였으며 B. thuringiensis serovar. kurstaki HD1의 독소 생성유전자와 B. thuringiensis serovar. darmstadiensis의 유전자가 유사성이 있다는 보고를 근거로 하여 B. thuringiensis serovar. HD1의 독소 생성유전자를 가진 프로브(pUYBT 9044)로 이용하여 colony hybridization 및 Southern hybridization 한 결과 2.6Kb EcoRI 단편 및 3.6Kb Hind III 단편을 선발할 수 있었다. 이들 단편들은 B. thuringiensis serovar. kurstaki HD1 독소 유전자와 hybridization시 유사성이 있었다. 특히 3.5Kb HindIII 단편은 2.6Kb EcoR I 단편에 클로닝되어 있는 1.8Kb의 HD1 독소 유전자와 유사성이 있는 부분을 공유하고 있었으며 1.0Kb 정도의 EcoR I-HindIII 부분이 더 삽입한 것을 알 수 있었다.

• Journal of Microbiology and Biotechnology
• /
• 제10권5호
• /
• pp.700-706
• /
• 2000

Iron- and zinc-containing superoxide dismutase (FeZnSOD) and nickel-containing superoxide dismutase (NiSOD) are cytoplamic enzymes in Streptomyces griseus. The sodF gene coding for FeZnSOD was cloned from genomic Southern hybridization analysis with a 0.5-kb DNA probe, which was PCR-amplified with facing primers corresponding to the N-terminal amino acid of the purified FeZnSOD of S. griseus and a C-terminal region which is conserved among bacterial FeSODs and MnSODs. The sodF open reading frame (ORF) was comprised of 213 amino acid (22,430 Da), and the deduced sequence of the protein was highly homologous (86% identity) to that of FeZnSOD of Streptomyces coelicolor. The FeZnSOD expression of exponentially growing S. griseus cell was approximately doubled as the cell growth reached the early stationary phase. The growth-associated expression of FeZnSOD was mainly controlled at the transcriptional level, and the regulation was exerted through the 110 bp regulatory DNA upstream from the ATG initiation codon of the sodF gene.

• 한국치위생학회지
• /
• 제6권4호
• /
• pp.311-324
• /
• 2006

The purpose of this investigation was to evaluate of the specificity of Fusobacterium nucleatum subspecies-specific DNA probes using dot blot hybridization. To confirm whether the clinical isolates were F. nucleatum or not, 16S rDNA of them were cloned and sequenced. The sequencing data were used in homology search with database of GenBank. When the homology was above 98% compared with the nucleotide sequence of a certain bacteria, it was judged as the same species with the bacteria. 23 strains of F. nucleatum were isolates from subgingival plaque of periodontitis patient. The clinical isolates of F. nucleatum were classified into 10 groups using phylogenetic analysis of 16S rDNA sequence. F. nucleatum subspecies nucleatum-specific DNA probe Fu4(1.3 kb) reacted with genomic DNAs from 8 type strains of F. nucleatum and it reacted strongly with those from 8 clinical isolates. The Fp4(0.8 kb) reacted with F. nucleatum subsp. polymorphum ATCC 10953 and one clinical isolates. Fv35(1.9 kb) and Fs17(8.2 kb) probes reacted with genomic DNAs from F. nucleatum subsp. vincentii ATCC 49256 and F. nucleatum subsp. fusiform ATCC 51190, respectively. Our results showed that it is not enough to evaluate the specificity of F. nucleatum subspecies-specific DNA probes with only dot blot hybridization. Therefore, Southern blot analysis will be necessary to confirm the specificity of F. nucleatum subspecies-specific DNA probes.

• Applied Biological Chemistry
• /
• 제41권2호
• /
• pp.125-129
• /
• 1998

한국 토양에서 분리된 Xanthomonas sp.는 Candida albicans에 대한 용균성을 나타내며 분비효소로서 chitinase를 분비하는 것으로 사료되었다. 특히 chitinase활성은 chitin배지에서 배양했을 때 3일 배양에서 최대치를 나타내었다. 이러한 특성이 있는 Xanthomonas의 chitinase 유전자를 cloning하기 위하여 cosmid vector를 이용한 genomic library를 작성하였으며, 다른 박테리아 chitinase 유전자와 homology를 가진 지역의 DNA sequence를 oligonucleotide로 합성하여 probe로 사용한 결과 4개의 독립된 positive clone을 cloning 하였다. 이중 pXCHl(1.2 kb insert) 이라고 명명한 clone에 대해 해석한 결과 이 크론의 전사산물은 chitin 배지에서만 유도됨을 확인하였으며 대장균 발현 vector를 이용한 이 유전자의 대장균에서의 발현에 대한 실험의 결과 약 35 kDa의 단백질을 생산하는 것으로 확인하였다. 또한 이 산물의 chitinase활성을 측정한 결과 유전자가 포함되지 않은 산물에 비해 약 10배의 활성을 나타내어 이 유전자를 Xanthomonas sp.의 chitinase유전자임을 증명하였다.

• 한국미생물·생명공학회지
• /
• 제22권1호
• /
• pp.37-44
• /
• 1994

The homology among the genes coding for degradation of bipheny(BP) and 4-chlorobiphenyl(4CB) was comparatively analyzed by Southern hybridization in several BP/4CB degrading bacterial strains. As the hybridization results of their genomic DNAs with pcbABCD as the DNA probe, the group of Pseudomonas sp. DJ-12. P08 and P27 strain was separated by the group of P20 and P1242 strains. The P. pseudoalcaligenes KF707 showed the hybidization signal which was homologous to the group of DJ-12, but they had different restriction endonuclease sites. The pcbAB genes in pCUl recombinant plasmid from Pseudomonas sp. DJ-12 appeared to be homologous to pchAB genes in pKTF20 cloned from P. pseudoalcaligenes KF707, but the C genes in both strains were not homologous. The bphABC in pKTF20 showed the signals homologous to the cbp ACB in pAW6194 cloned from P. putida OU83, but homologous signal was not found botween the pcbABCD genes in pCUl and the cbpADCB genes in pAW6194 recombbinant plasmid.

• 한국미생물·생명공학회지
• /
• 제18권3호
• /
• pp.305-308
• /
• 1990

Shuttle plasmid vector인 pHN114를 이용하여 Kluyveromyces fragilis의 3-isopropylmalate dehydrogenase 유전자를 cloning 하였다. 그 결과 Saccharomyces cerevisiae의 leu2변이와 E.coli의 leuB변이를 상보하는 두가지 clone 체 pJK104와 pJK106을 얻었다. Restriction mapping 결과 이들은 서로 반대방향으로 삽입되어 있었으며 expression activity는 pJK104가 높았다. pJK104에 삽입된 유전자를 BglII와 SalI으로 끊은 1.6kb fragment를 probe 로 하여 Southern Hybridization 한 결과 유전자의 유래가 Kluyveromyces fragilis 임을 확인하였다.

• 환경위생공학
• /
• 제23권2호
• /
• pp.1-18
• /
• 2008

The tetrachloroethylene (PCE) dehalogenase of Clostridium bifermentans DPH-1 (a halorespiring organism) was purified, cloned, and sequenced. This enzyme is a homodimer with a molecular mass of ca. 70 kDa and exhibits dehalogenation of dichloroethylene isomers along with PCE and trichloroethylene (TCE). Broad range of substrate specificity for chlorinated aliphatic compounds (PCE, TCE, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropene, and 1,1,2-trichloroethane) for this enzyme was also observed. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. A partial sequence (81 bp) of the encoding gene for PCE dehalogenase was amplified and sequenced with degenerateprimers designed from the N-terminal sequence (27 amino acid residues). Southern analysis of C. bifermentans genomic DNA using the polymerase chain reaction product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase. So, C. bifermentans could play some important role in the initial breakdown of PCE and other chlorinated aliphatic compounds in sites contaminated with mixtures of halogenated substances.

• 한국잠사곤충학회지
• /
• 제34권2호
• /
• pp.20-25
• /
• 1992

유용물질을 생산할 수 있는 곤충 baculovirus expression vector system의 국내 개발을 위하여, Bm-NPV의 다각체 단백질 유전자를 탐색, 클로닝 하고 그 구조를 분석한 결과는 다음과 같다. 1. AcNPV의 다각체 단백질 유전자를 포함하고 있는 EcoRI-Ⅰ fragment내의 0.93kb를 probe로 하여 southern 분석한 결과, BmNPV의 다각체 단백질 유전자는 PstⅠ-F fragment(7.4Kb)에 위치하였다. 2. BmNPV의 다각체 단백질 유전자를 포함하는 PstⅠ-F fragment를 E. coli에 클로닝시켜서 pBmP-F라 명명하고, 다시 southern분석을 통해 subcloning하여 pBmP-H라 명명하였으며 pBmP-H의 제한효소 지도를 작성하였다. 3. pBmP-H의 염기서열분석결과 구조유전자를 포함한 1,259 bp의 염기서열이 결정되었다. Iatrou 등이 보고한 BmNPV 다각체 단백질 유전자의 염기서열과 비교한 결과 10개의 염기서열에서 차이를 보였으며, 74 Val이 Ile로, 76 Asn이 Ser으로 155 Met이 Val로 아미노산 변경된 결과를 보였다.

• 한국초지조사료학회지
• /
• 제17권3호
• /
• pp.285-292
• /
• 1997

This study was conducted to obtain the transgenic Brnssica napus plants with tobacco Apase gene using the binary vector system of Agrobacteriurn fumefociens. The results obtained were summarized as follows: A repressible acid phosphatase gene of Saccharon~yces cerevisiae, pho105 was used for screening of tobacco Apase cDNA. In order to identify Apase gene in tobacco genome, Southern blot analysis was pcrformed and the Apase gcnc may be present as a single copy, or at most two or three copies, in tobacco genome. To isolate the tobacco Apase gene, tobacco cDNA library was constructed using purifed mRNA from -Pi treated tobacco root and the plaque forming unit of the library was 2.8 x $10^5$ pfu/m${\ell}$, therefore the library might cover all expressed mRNAs. Using pho5 as a probe. tobacco Apase cDNA was cloned, and restriction mapping and Southern blot analysis of cDNA insert were revealed that the 3.6 kb cDNA contained tobacco acid phosphatase cDNA. Plasmid pGA695 -tcAPl was constructed by subcloning tobacco Apase cDNA into the Hind site of pGA695 with 35s promoter which can be expressed constitutively in plants. The Brassica napus cotyledonary petioles were cocultivated with the ,4 grobacteriunz and transferred to the selection medium. The transformed and regenerated plants were transplanted to soil medium. Southern blot analysis was done on the transformed plants, and it was confirmed that a foregin gene was stably integrated into the genonies of B. nnpus plants.

• 대한소아치과학회지
• /
• 제25권2호
• /
• pp.292-303
• /
• 1998

The arbitrary primer polymerase chain reaction(AP-PCR) and Southern blot restriction fragment length polymorphism(RFLP) were used to genotype the cariogenic pathogen S. mutans in children. Following the morphologic chracteristics of colony on selective medium for S. mutans, total genomic DNA from 155 strains was extracted by conventional methods. Among 155 strains, 143 strains (92.3%) were confirmed S. mutans by PCR with dexA gene and 114 strains were used in this study. Three random sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 2% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI and BamHI, separated on a 0.7 % agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterised 1.6 kilobases (kb) DNA fragment cloned from gtf B gene of S. mutans. The probe was labeled with isotope[$^{32}P-{\alpha}CTP$], and hybridized fragments were detected with intensifying screen. AP-PCR produced 4-8 DNA bands in the 0.25-10 kb regions and distinguished 9, 10 or 12 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 2 hybridization patterns consisting of 1 DNA fragments 450, 500 bp. These results indicate that AP-PCR is more discriminative method for genotyping of S. mutans.