• Journal of Microbiology and Biotechnology
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• v.7 no.6
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• pp.373-377
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• 1997

The gene for isopenicillin N synthase (cyclase; IPNS) was cloned from Lysobacter lactamgenus using DNA probe amplified with primers based on the consensus sequences of isopenicillin N synthase genes of other ${\beta}$-lactam-producing microorganisms. The genomic library of L. lactamgenus using pUC18 plasmid cloned at the SacI site were screened with the PCR-generated DNA probe and three positive clones were isolated. Enzyme activities in E. coli clones were confirmed by bioassay and HPLC assay. Throughout the functional mapping, it was observed that the gene for isopenicillin N synthase is located at the 1.3-kb XhoI-BamHI fragment of insert of positive clones. Nucleotide sequencing at both ends of the XhoI-BamHI fragment revealed that IPNS of L. lactamgenus has the common amino acid sequences at amino- and carboxy-termini.

• Animal cells and systems
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• v.3 no.2
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• pp.229-236
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• 1999

The recA gene plays a central role in genetic recombination and SOS DNA repair in Escherichia coli (E. coli). We have previously identified a 42 kDa RecA-like protein inducible by a variety of DNA damages from a fluorescent Pseudomonas strain sp. and characterized its inducible kinetics. In the present study, we cloned and characterized the gene encoding the RecA-like protein by immunological screening of Pseudomonas genomic expression library using polyclonal E. coli anti-RecA antibodies as a probe. From 10$^{5}$ plaques screened, five putative clones were finally isolated. Southern blot analysis indicated that four clones had the same DNA inserts and the recA-like gene was located within the 3.2 kb EcoRI fragment of Pseudomonas chromosomal DNA. In addition, the cloned recA-like gene was transcribed into an RNA transcript approximately 1.1 kb in size, as judged by Northern blot analysis. The cellular level of RNA transcript of the cloned recA-like gene was increased to an average of 5.15- fold upon treatment with DNA damaging agents such as ultraviolet (UV)- light, nalidixic acid (NA), methyl methanesulfonate (MMS), and mitomycin-C (MMC). These results suggest that the cloned gene is inducible by DNA damage similarly to the recA gene in E. coli. However, the cloned gene did not restore the DNA damage sensitivity of the E. coli recA-mutant.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.38 no.3
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• pp.275-282
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• 1993

The objective of this study was to clone a partial fragment of $\alpha$-amylase from Korean maize. We designed and synthesized an oligonucleotide probe and two kinds of PCR primers based on cDNA conserved region of $\alpha$-amylase sequences from other plants. Total RNA from 3-day-old maize seedling was used as template for 1st strand cDNA synthesis and RNA-DNA hybrid was used as template for polymerase chain reaction(PCR). The product of PCR was about 0.5 kb long and inserted into pUC19. We named this recombinant plasmid as pZM$\alpha$'. The cloned fragment was certified by Southern blot analysis using labeled synthetic oligonucleotide as probe.

• The Korean Journal of Zoology
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• v.35 no.2
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• pp.203-210
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• 1992

Hsp70, a 70 kDa protein, is the maior protein expressed when cells are heat-shocked. A cDNA library from mouse ID13 cells was screened with the human hsp70 gene as a probe, and a positive clone was obtained. The positive clone was subcloned into puc19 and the precise restriction was obtained. The CDNA was sequenced by the Sanger's dideoxv termination method. Single open reading frame that codes for a protein of 70 kDa was found. The DNA sequence of the cloned mouse DNA shows great homology (66-90%) with other mouse hsp70 genes and somewhat less homology (50",) with E. coli hsp70 gene (dnak). With the exception of one amino acid, the protein sequence deduced from the CDNA is identical to the mouse that shock cognate protein 70 (hsc70) that is constitutivelv expressed at normal temperature. The result suggests that the cloned CDNA encodes a hsc70 family rather than a heatinducible family.mily.

• The Korean Journal of Zoology
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• v.39 no.3
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• pp.248-256
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• 1996

The present study was performed to clone and express human poly (ADP-ribose) synthetase (PARS) cDNA in E coli. For these purposes, the CDNA for human poly (ADP-ribose) synthetase, encoding the entire protein, was cloned into pGEM-7Zf(+). The resulting recombinant plasmid pPARS6.1 was restriction enzyme mapped and its identity was confirmed by Southern blot analysis. The pPARS6. 1 contained full-length CDNA of human PARS and the nudeotide sequences were identical with those reported previously. The recombinant protein which migrated as a unique 120 kDa band on 10% SDS-polyacrylamide gels, was identified as PARS by Southwestern blots using nick-translated DNA probes and by activity gels and activity blots using 32 P-NAD as a substrate for poly (ADP-ribose) synthetase (PARS). The signals corresponding to 120 and 98 kDa proteins were obtained following IPTG (0.4 mM) induction of the PARS cDNA cloned into Xba I-digested pGEM-7Zf(+) vector. Nonspecific signals corresponding to 45 and 38 kDa proteins were also shown in both IPTG-induced and noninduced cells. The nonspecific proteins may be products of incomplete translation or proteolytic products of intact PARS.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.323-331
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• 1996

Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

• The Korean Journal of Mycology
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• v.27 no.2 s.89
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• pp.132-137
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• 1999

This study was carried out to develop the molecular marker for the detection of Pseudomonas tolaasii, a causative agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). When several primers designed from repetitive sequences and pectin lyase genes of bacteria were used to produce DNA polymorphism from different Pseudomonas spp. isolated from edible mushrooms, PEU1 primer derived from pectin lyase gene produced polymorphic bands differentiating P. tolaasii strains from other Pseudomonas species. Two bands, 1.0kb and 0.4kb, found commonly in 6 isolates of P. tolaasii were cloned into pGEM-T vector which were designated as pPTOP1 and pPTOP2, respectively, to use as probe. The 0.4 kb insert of pPTOP2 hybridized to only 6 isolates of P. tolaasii, but did not to the other Pseudomonas species. As few as $1.5{\times}10^3$ colony forming unit (cfu) of P. tolaasii could be detected by dot blot hybridization with the cloned 0.4kb DNA in pPTOP2.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.264-270
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• 1994

A gene coding for a novel putative $\sigma$ factor of RNA polymerase has been identified from Streptomyces coelicolor A3(2) using Escherichia coli rpoS gene fragment as a probe. The 486 bp rpoS gene fragment was amplified from E. coli genomic DNA by PCR with two synthetic oligonucleotides, the sequences of which were deduced from the amino acid sequences in the regions 2.3 and 4.2 conserved among various bacterial factors. When E. coli genomic DNA fragments were hybridized with cloned rpoS probe, only one band corresponding to rpoS gene (3.2 kb PvuII fragment or 2.3 kb KpnI fragment) was detected. In S. coelicolor, however, two bands were detected both in PvuII digested DNA and SalI digested DNA. 3.5 kb PvuII fragment which binds the rpoS gene probe was cloned (pMS1) from the sublibrary, and the nucleotide sequences of 1.0 kb BamH'/HincII subclone (pBH2) was partially determined. The nucleotide sequences revealed extensive similarity to other $\sigma$ factor genes of S. coelicolor (hrdA, hrdB, hrdC, hrdD), S. aureofaciens (hrdA, hrdB, hrdC, hrdD), Synechococcus species, Pseudomonas aeruginosa, Stigmatella aurantiaca, and Anabaena species. The nucleotide sequences in regions 1.2 and 4 were compared with the corresponding regions of 5 known ${\sigma}$ factor genes of S. coelicolor by multiple alignment. It turned out that the cloned gene is most closely related to hrdA showing 88% amino acid similarity in region 1.2 and 75% in region 4.

• Mycobiology
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• v.28 no.4
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• pp.171-176
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• 2000

We have cloned a ${\alpha}-COP$ homolog, ancop, from Aspergillus nidulans by colony hybridization of chromosome specific library using ${\alpha}-COP$ homologous fragment as a probe. The probe DNA was amplified with degenerated primers designed by comparison of conserved region of the amino acid sequences of Saccharomyces cerevisiae ${\alpha}-COP$, Homo sapiens HEP-COP, and Drosophila melanogaster ${\alpha}-COP$. Full length cDNA clone was also amplified by RT-PCR. Comparison of genomic DNA sequence with cDNA sequence obtained by RT-PCR revealed 7 introns. Amino acid sequence similarity search of the anCop with other ${\alpha}-COPs$ gave an overall identity of 52% with S. cerevisiae, 47% with human and bovine, 45% with Drosophila and Arabidopsis. In upstream region from the transcription start site, a putative TATA and CAAT motif were also identified.

• Restorative Dentistry and Endodontics
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• v.21 no.1
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• pp.1-18
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• 1996

There are many advantages when using IIF and DNA probe methods over anaerobic culture method in that they are time-and effort-saving, more precise and more sensitive. Furthermore, in IIF and DNA probe methods, the detection is possible only with small amount of bacteria, the quantitative analysis is possible, and the cell viability is not necessary. The purpose of this study is to observe the incidence of P.endodontalis by carrying out anaerobic culture, IIF and colony lift using DNA probe method respectively, and to compare these 3 methods in terms of effectiveness and sensitivity in order to identify the most effective detection method. 30 teeth with at least one clinical symptoms, with single canal, and with pulp necrosis were sampled. For sampling bacteria, access cavity was prepared after disinfecting tooth and its surroundings. Then the paper point was inserted up to the periapical area, leave there for a while, and finally it was placed into PRAS Ringer's sol. and PBS sol. In anaerobic culture method, P.endodontalis was identified by biochemical tests after subculturing black and brown colonies which were produced after 7 days of incubation on BAP and Brucella BAP in anaerobic chamber. To identify P.endodontalis in IIF method, species-specific polyclonal rabbit-antisera of P.endodontalis(ATCC 35406) was reacted with sampled PBS sol. dispensed onto glass slide, and then P.endodontalis was examined by phase contrast microscopy after incubating with Goat anti-rabbit lgG conjugated to Fluorescein isothiocyanate. For colony lift using DNA probe method, membranes were laid over colonies on the surface of BAP and were hybridized with cloned DNA probe of P.endodontalis. The existence of P.endodontalis was then identified by the methods of chemiluminescent detection and color metric detection. Black colony was found in 11 teeth out of 30 teeth and P.endodontalis was detected in 6 teeth (20 %) by anaerobic culture method, 16 teeth (53 %) by IIF method, and 7 teeth (23 %) by DNA probe method. IIF method is significantly better in detecting P.endodontalis than DNA probe method and anaerobic culture method. There was no significant differences between DNA probe method and anaerobic culture method. There was significant correlation between the formation of black colony and the existence of P.endodontalis. The probability of detecting P.endodontalis when black colony being present is 2.89 times higher than when not being present. There was significant relationship between the foul odor of clinical symptoms and P.endodontalis. The sensitivity of existing P.endodontalis when foul odor being present was 93.75 %, while the specificity of not existing P.endodontalis when foul odor not being present was 28.57 %. These results suggested that the probes of P.endodontalis will be used to decide the method and prognosis in endodontic treatments.