• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.1125-1131
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• 2006

A kan'::luxCDABE fusion strain that was both highly bioluminescent and responsive to benzoic acid was constructed by transforming E. coli strain W3110 with the plasmid pUCDK, which was constructed by digesting and removing the 7-kb KpnI fragment from the promoterless luxCDABE plasmid pUCD615. Experiments using buffered media showed that this induction was dependent on the pH of the media, which influences the degree of benzoic acid protonation, and the expression levels seen are likely due to acidification of the cytoplasm by uncoupling of benzoic acid. Consequently, the sensitivity of this strain for benzoic acid was increased by nearly 20-fold when the pH was shifted from 8.0 to 6.5. Benzoic acid derivatives and several phenolics also resulted in significantly increased bioluminescent signals. Although these compounds are known to damage membranes and induce the heat-shock response within E. coli, bacterial strains harboring mutations in the fadR and rpoH genes, which are responsible for fatty acid biosynthesis during membrane stress and induction of the heat-shock response, respectively, showed that these mutations had no effect on the responses observed.

• Korean Journal of Microbiology
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• v.24 no.4
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• pp.357-363
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• 1986

Synthesis of threonine and methionine in yeast, Saccharomyces cerevisiae shares a common pathway from aspartate via homoserine. HOM6 gene encodes homoserine dehydrogenase (HSDH) which catalyzes the inter-conversion of beta-aspartate semialdehyde and homoserine. The level of HSDH is under methionine specific control. A recombinant plasmid (pEK1: 13.3kb), containing HOM6 gene, has been isolated and cloned into E. coli by complenemtary transformation of a homoserine auxotrophic yeast strain M-20-20D (hom6, trp1, ura3) to a prototrophic M20-20D/pEK1, using a library of yeast genomic DNA fragments in a yeast centromeric plasmid, YCp50(8.0kb). Isolation of HOM6has been primarily confirmed by retransformation of the original yeast strain M20-20D, using the recombinant plasmid DNA which was extracted from M20-20D/pEK1 and subsequently amplified in E. coli. Eleven cleavage sites in the insery (5.3kb) have been localized through fragment analysis for 8 restriction endonucleases; Bgl II(2 site), Bgl II(1), Cla I(3), Eco RI(1), Hind III(2), Kpn I (1), Pvu II(1) and Xho I(1).

• BMB Reports
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• v.33 no.3
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• pp.208-212
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• 2000

In order to develop a specific genetic marker for the Korean native cattle (Hanwoo), an arbitrarily-primed polymerase chain reaction (AP-PCR) analysis of 6 different cattle breeds was attempted. Eight different arbitrary primers, each longer than 20-mer nucleotides, were used. In comparison to the AP-PCR patterns, several distinctive DNA bands that are specific for a certain breed were detected. When the primer Kpn-X was employed, a 280bp DNA fragment was found to be specific only for Hanwoo. In an individual analysis of Hanwoo, this AP-PCR marker was observed in 123 head of cattle among the 153 that were tested (80.4%). Nucleotide sequencing revealed that this fragment has a short microsatellite sequence of tandem repeat, $A(G)_{1-2}\;(C)_{1-3}AGAG$. According to the analysis of AP-PCR band patterns, Hanwoo was discovered to be genetically most closely-related with Holstein among the various cattle breeds.

• Parasites, Hosts and Diseases
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• v.33 no.4
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• pp.331-340
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• 1995

Interstrain polymorphisms of isoenzyme profiles and mitochondrial (Mt) DNA fingerprints were observed among seven strains of Acnnthnmoeba isolated from different sources and morphologically assigned to A. polvphngn. Mt DNA ringerprints by eight restriction endonucleases (Bgl II, Sca I, Cla I, EcoR I, Xbo I, Kpn I, Sal I, and Sst I) revealed considerable interstrain polymorphisms . Isoenzyme profiles revealed considerable interstrain polymorphisms for acid phosphatase, lactate dehydrogenase, and glucose-6- phosphate dehydrogenase while those for glucose phosphate isomerase , leucine aminopeptidase , and malate dehydrogenase showed similarity Despite of the interstrain polymorphisms, the isoengyme profiles and Mt DNA fingerprints of the strain Ap were found to be identical with those of the strain .tones . Mt DNA fingerprinting was found to be highly applicable for the strain identification, characterization, and differentiation. Key words: Acanthnmoebn polyphcga, interstrain polymorphism, isoenzyme profiles , Mt DNA fingerprints, strain differentiation, strain identification.

• Journal of Microbiology
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• v.37 no.3
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• pp.168-174
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• 1999

Escherichia coli O157 is an important pathogenic organism which causes diarrhea, haemorrhagic colitis, and haemolytic ureamic syndrome (HUS) in human. E. coli O157 KNIH317 was isolated form patients suffering with HUS in Korea. We designed a primer set for cloning shiga-like toxin (slt) gene. The amplified PCR product was used to Southern and colony hybridization as a probe. As a result, we cloned 4.5-kb KpnI fragment containing the slt gene encoding shiga-like toxin from chromosomal DNA of E. coli O157 KNIH317. This recombinant plasmid was named pOVT45. E. coli XL1-Blue harboring pOVT45 showed cytotoxicity in Vero cells. We sequenced the slt gene of this strain. The A-subunit gene of the slt was composed of 960 base pairs with ATG initiation codon and TAA terminationcodon. The B-subunit was composed of 270 base paris with ATG initiation codon and TGA termination codon. Nucleotide sequence comparison of the slt gene exhibited 100%, 98.4%, 93.7%, and 93.7% identity with that of shiga-like toxin type II (sltII) of E. coli bacteriophage 933W, variant slt of E. coli, slt of E. coli, and variant sltII of E. coli, respectively. From these results, it was concluded that the cloned slt gene belongs to SltII family and that the strain used in this study may be a lysogeny of E. coli bcteriphage 933W.

• The Journal of Korean Medicine
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• v.29 no.3
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• pp.88-99
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• 2008

Objectives: In order to identify the anti-inflammatory and analgesic properties of Salvia miltiorrhiza (Dan-Sam), widely used in Korean traditional medicine, an in vitro screening system was designed using pGL3, a luciferase reporter vector, and the tumor necrosis factor (TNF)-${\alpha}$ and cyclooxygenase (COX)-2 as target genes. Methods: The promoter regions of each gene were generated by PCR using the human chromosome as template DNA, and inserted into pGL3 vector with Kpn I and Hind III. The final construct was transfected into human myelomonocytic leukemia cells (U-937) that could be differentiated and activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). Using this system, the anti-inflammatory and analgesic effects of several herbal extracts regarded to have the medicinal effects of diminishing body heat and complementing Qi were tested. The chemicals PD98059 and berberine chloride were used as controls of the transcriptional inhibitors of TNF-${\alpha}$ and COX-2, respectively. Results: Salvia miltiorrhiza (Dan-Sam) demonstrated significant decrease of TNF-${\alpha}$ and COX-2 mRNA in the in vitro assay system. In MTT assay, Salvia miltiorrhiza (Dan-Sam) did not significantly inhibit the survival and proliferation of human myelomonocytic leukemia cells (U-937). Conclusions: Salvia miltiorrhiza (Dan-Sam) was found to exhibit the significant medicinal properties of anti-inflammatory and analgesic effects.

• BMB Reports
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• v.35 no.6
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• pp.637-641
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• 2002

Only one natural promoter that interacts with bacteriophage K11 RNA polymerase has so far been identified. To identify more, in the present study restriction fragments of the phage genome were individually assayed for transcription activity in vitro. The K11 genome was digested with two 4-bp-recognizing restriction enzymes, and the fragments cloned in pUC119 were assayed with purified K11 RNA polymerase. Eight K11 promoter-bearing fragments were isolated and sequenced. We report that the nine K11 promoter sequences (including the one previously identified) were highly homologous from -17 to +4, relative to the initiation site at +1. Interestingly, five had -10G and -8A, while the other four had -10A and -8C. The consensus sequences with the natural -10G/-8A and -10A/-8C, and their variants with -10G/-8C and -10A/-8A, showed nearly equal transcription activity, suggesting residues at -10 and -8 do not regulate promoter activity. Using hybridization methods, physical positions of the cloned promoter-bearing sequences were mapped on SalI-and KpnI-restriction maps of the K11 genome. The flanking sequences of six cloned K11 promoters were found to be orthologous with T7 or T3 genomic sequences.

• The Korean Journal of Malacology
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• v.10 no.1
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• pp.27-37
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• 1994

한국산 담수 또아리물달팽이과(Planorbidae)에 속하는 또아리물달팽이(Gyraulus convexiusculus),수정또아리물달팽이(Hippeutis cantori) 및 배꼽또아리물달팽이(Segmentina hemisphaerula)3종에 대한 종간 유전적 변이와 이들 상호간의 분류학적 유연 관계를 생화학적 측면에서 밝히고자 하였다. 즉, 모계유전으로 자손에 유전되고 있는 미토콘드리아 DNA(mitochondrial DNA; mt DNA)의 변이를 보기위하여 제한효소(restriction enzyme)를 처리하고 잘라진 mtDNA절편들을 상호 비교하는 restriction fragmint length polymorphism(RFLP)기법을 응용하였다. 본 실험에서 10개의 제한효소 중 CIa I, Dra I, Eco RI, Hin dIII, Kpn I및 pst I의 6개 제한효소에서 좋은 결과를 얻어 종간의 공통절편(shated fragmints)을 비교하였고, 염기분화율(nucleotide divergince rate)을 각각 측정하였다. 미토콘드리아 DNA 크기(genome size)는 또아리물달팽이가 12.08 kb, 수정또아리물달팽이가 14.4 kb, 그리고 배꼽또아리물달팽이가 12.93 kb로 관찰되었다. 염기분화율(p)는 또아리물달팽이/수정또아리물달팽이 군에서 p=12.7%, 배꼽또아리물달팽이와 상기 2종군 사이의 염기분화율은 P=56.6%여서 배꼽 또아리물달팽이류는 타 2종보다 그 분화율이 매우 높음을 알 수 있었다. 이상의 결과로 보아 분류군(taxa)의 mtDNA 변이에 의한 RELP기법이 앞으로 한국산 담수 패류 연구에 널리 응용될 수 있음이 확인되었다.

• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.1
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• pp.129-141
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• 2001

When oral microorganisms were sampled from occlusal surfaces of caries and non-caries teeth, $3.43\times10^5$ CFU and $3.47\times10^3$ CFU of bacteria were counted on MSB agar plates, respectively. All the 20 colonies isolated from a caries surface were Streptococcus mutans but, only two of 20 colonies were identified as Streptococcus mutans by API test. S. mutans SM1 from caries tooth and S. mutans SM2 from non-caries tooth showed the same results except for $\alpha-galactosidase$ activity on sugar fermentation tests and biochemical tests. For the bacterial replication, both SM1 and SM2 were actively multiplicated at pH 5.5. And the viability of SM1 was high at 20% of sucrose, while that of SM2 was high at 5% of sucrose in the media. SM1 actively replicated at 16mM of $CaCl_2$, 160mM of KCl, and 6.4mM of $MgCl_2$, and the replication of SM2 was increased at 16mM of $CaCl_2$, 40mM of KCl, 6.4mM of $MgCl_2$. At 1mM of sodium bicarbonate and sodium phosphate, both bacteria were actively multiplicated. SM1 and SM2 were actively replicated at 1mM and 10mM of Tris, respectively. For potassium phosphate buffer, SM1 grew well proportionally to the concentration up to 100mM, while the growth of SM2 were inhibited by the increase of concentration. The 4.6 kb of gtf gene was amplified with a pair of primer, gtfB-F961 and gtfC-R5574 by polymerase chain reaction from the chromosomal DNA of SM1 and SM2. When 4.6kb bands were eluted from gel and were treated with restriction enzyme, EcoR I produced the same RFLP like 0.8kb and 3.8kb of DNA fragments for S. mutans GS-5, SM1 and SM2. By Hind III, the PCR products weren't digested for S. mutans GS-5 and SM1, but 3 fragments such as 2.4kb, 1.8kb and 400bp were examined for SM2. These results indicated the difference between gtf genes of SM1 and SM2. BamH I treatment showed 4 fragments for SM1 and SM2, while the 3 fragments for S. mutans GS-5. The PCR products were not digested by Kpn I, Sma I, Xho I and Pst I.

• Journal of Embryo Transfer
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• v.32 no.1
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• pp.1-8
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• 2017

Cryopreservation of germ cells from genetically proven animals could be a source of restoration tools from the risk of extinction or disappearance of wanted characteristics. Using frozen semen, the genetic gains of Korean native cattle have been increased greatly for 70 years. The preservation of genetic resources as a form of frozen semen straw has limited availability due to the numbers. To circumvent this weakness of frozen semen, we tested two re-freezing methods with different initial thawing temperatures using frozen Korean proven semen and rare breed semen from albino, black and chikso breeders. It has been known that human sperm could resist to cryo-damages by repeated freeze-thaw cycles, but not for Korean proven bulls number (KPN) or for rare breeds. Total 7 frozen semem from brindled(2), black(1), Korean Albino(2) and KPN(1) bulls were used for our research. After thawing straws under $5^{\circ}C/2min$ or $37^{\circ}C/40sec$ with low temperature water bath and thermo jug, spermatozoa were re-diluted with triladyl diluents after first thawing and re-frozen. Sperm motilities were compared between animals and treated groups after re-thawing. Mean values of motility and viability of refrozen/thawed sperm for expansion of the number of straws were significantly higher in $5^{\circ}C$ than in $37^{\circ}C$ (P < 0.05). However, the activity of viable sperm thawed at $5^{\circ}C$ was significantly decreased before refreezing. It is estimated that re-freezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa.