• Archives of Pharmacal Research
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• v.15 no.4
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• pp.360-364
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• 1992

Methylcatechol 2, 3-dioxygenase encoded in pWWO megaplasmid of Pseudomonas putida mt-2 has been cloned and overexpressed in Escherichia coli. This enzyme gene has been localized inside 2. 3-kb XhoI fragment derived from the pWWO megaplasmid. Analysis of enzyme activity and SDS-PAGE showed that the cloned methylcatechol 2, 3-dioxygenase gene in E. coli was about 100 fold overexpressed compared with the parental gene in P. putida mt-2 (pWWO). The cloned enzyme exhibited higher ring-fission activity to catechol than catechol derivatives including 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol.

• KSBB Journal
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• v.11 no.1
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• pp.115-123
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• 1996

Two kinds of mutants were isolated to clone a cluster of genes essential for zooglan biosynthesis. Zoogloea ramigera 115 strains produce capsular polysaccharide. To achieve conjugation in strain 115 and to facilitate recovery of product, a capsule non-forming strain was isolated via successive centrifugation and screening. The other kind of mutants devoid of or producing altered exopolysaccharides were obtained using classical transposon(Tn5) technique and screened for altered colony morphology and celluflour binding properties. Complementation of these mutants was achieved with Z. ramigera 115 slime gene library constructed in a broad host range cosmid vector and helper plasmid by triparental conjugation.

• Korean Journal of Microbiology
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• v.26 no.1
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• pp.32-36
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• 1988

The yeast gene HOM3 encodes aspartokinase, which catalyses the first step (aspartate to and from beta-aspartyl phosphate) of common pathway to threonine and methionine. The yeast HOM3 gene expression is known to be regulated by threonine and methionine specific control, and also by general control of amino acid biosynthesis. Isolation and characterization of the HOM3 gene are essential for the molecular genetic study on its regulation of expression. A recombinant plasmid pSC3 (15.5kb, vector YCp50) has been cloned into E. coli HB101 from yeast genomic library through their complementing activity of HOM3 mutation in a yeast recipient strain M34-24B. Organization of the plasmid was characterized by delineation of restriction cleavage sites in the insert fragment.

• Journal of Microbiology
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• v.35 no.3
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• pp.222-227
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• 1997

DNA fragments homologous to chitin synthase gene were amplified from the genomic DNA of Beauveria brongniartii by PCR using degenerate primers. Cloning and sequencing of the PCR-amplified fragments led to the identification of a gene, designated BbCHSl. Comparison of the deduced amino acid sequence of BbCHSl with those of other Euascomycetes revealed that BbCHSl is a gene for class II chitin synthase. The Blastp search of the deduced amino acid sequence of BbCHSl displayed the highest rate of similarity, 95.8%, with CHS2 of Metarhizium unisopliae. Phylogenetic analysis of the amino acid sequences confirmed the taxonomic and evolutionary position of B. brongniartii, which was previously derived by traditional fungal classification based on morphological features.

• KSBB Journal
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• v.16 no.1
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• pp.36-40
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• 2001

In order to study the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in plants, we cloned and sequenced a partial glutamate decarboxylase (GAD) cDNA from the Arabidopsis thaliana cDNA library, using primers targeted at highly conserved sequences of the petunia GAD gene. The cDNA fragment was inserted into TA cloning vector with T7 promoter and the recombinant plasmid obtained was used to transform E. coli. The plasmid DNA purified from the transformed E. coli was digested with EcoRI and the presence of the insert was confirmed. Nucleotide sequence analysis showed that the fragment is a partial Arabidopsis thaliana GAD gene and that the sequence showed 98% and 78% identity to the region of the putative Arabidopsis thaliana GAD sequences deposited in GenBank, Accession nos: U46665 and U10034, respectively. The amino acid sequence deduced from the partial Arabidopsis thaliana GAD gene showed 99% and 91% identities to the GAD sequences deduced from the genes of the U46665 and U10034, respectively. The partial cDNA sequence determined may facilitate the study of the molecular mechanism of GABA metabolism in plants.

• Journal of Microbiology and Biotechnology
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• v.2 no.4
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• pp.237-242
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• 1992

In order to clone the gene coding for alkaline phosphatase in the yeast Kluyveromyces fragilis, a genomic library was constructed using the yeast-E. coli shuttle vector pHN114 as a cloning vector. From the genomic library, a clone carrying the gene was isolated and the plasmid was designated as pSKH101. A restriction enzyme map was made using this plasmid. Subcloning experiments and complementation studies showed that alkaline phosphatase was active only in the original 3.1 kb insert. Southern hybridization analysis confirmed that the cloned DNA fragment was derived from K. fragilis genomic DNA. Using a minicell experiment, the product of the cloned gene was identified as a protein with a molecular weight of 63 KDa. A 0.6 kb HindIII fragment, which showed promoter activity, was isolated using the E. coli promoter-probe vector pKO-1.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.647-652
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• 1990

The production of delta-endotoxin crystal and the cloning of endotoxin protein gene in Bscillus thuringiensis subsp. kurstaki HD1 strain were studied. The strain produced bipyramidal crystals ($2.9\times 1.0 \mu m$) in their cells during sporulation. The B. thuringiensis contained about 10 plasmid DNA elements ranging from 2.1 to 80 kilobases. The 73 kb plasmid DNA, the 29 kb BamHI fragment and the 7.9 kb Pstl DNA fragment hybridized to the pHL probe. The 7.9 kb fragment was eluted and cloned in the PstI site of pBR322 vector and transformed into E. coli HB101, which produced insecticidal proteins killing Bornbyx mori larvae.

• Applied Biological Chemistry
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• v.31 no.3
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• pp.219-225
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• 1988

Reverse transcriptase gene of Avian sarcoma virus(ASV) was cloned with a thermoinducible expression vector, pPL-lambda. E. coli N4830 which carries temperature sensitive cI857 la mbda repressor, was transformed with this pPL-pol plasmid DNA. The RNA transcribed by those tranoformants was isolated and analyzed. It was shown that the inserted reverse transcriptase gene of ASV was transcribed at high-level when cells were grown at high temperature.

• Korean Journal of Veterinary Research
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• v.32 no.4
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• pp.555-567
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• 1992

In this study gene encoding structural proteins of a CPV isolate was cloned and sequenced to elucidate the molecular genetical properties of the canine parvoviruses isolated from the field. Six recombinant plasmids of pEP3, p1471, p2070, pEP069, pEP338 and p14711p were constructed from the map positions 22 to 98 of RF DNA to clone the VP1 and VP2 genes of CPV-V20. Sequentialy the gene comprising 3780 nucleotides were sequenced by dideoxy chain termination method. When nucleotide sequence of gene encoding the structural proteins of CPV-V20 was compared with those of other strains, CPV-N, CPV-d and CPV-780929 published previously, DNA, homologies to CPV-V20 were 99.87% with CPV-N, 99.73% with CPV-d, 96.85% with CPV-780929 and 98.4% with FPLV-Carl, respectively. The DNA sequence data of CPV-V20 showed seven point mutations and also deletion of 135 nucleotides from the nucleotide position 4745 to 4879 located in the 3'-noncoding region of CPV-N.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 1998.05a
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• pp.12-28
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• 1998

The technology of creating transgenic animals has a potential value in improving productivity and disease resistance of animals, gene therapy, drug pharming and production of model animals for certain diseases. Up to date, fairly low success rate of production of transgenic animals and a pronounced variability with respect to the expression of transgenes have been much observed. The mechanisms how to integrate the injected genes with a certain part of the genomes are unknown yet. Many techniques in gene transfer, beside microinjection, have been introduced and explored thus to improve the production efficiency of transgenic animals. In this article, the methods and efficiency of gene-transfer techniques, the detection and preselection of transgenes in embryos by PCR- and GFP-screenings and cloning of preselected transgenic embryos by nuclear transplantation are described and discussed. Some experimental results showed that the early screening and selection of integration of the injected gene with embryonic genome by polymerase chain reaction(PCR) and green fluorecence protein(GFP) were promising methods. Further, the application of nuclear transplantation technology to cloning and multiplication of the positively integrated genes in the cleaving embryos and embryonic cells will be beneficially used for the mass production of transgenic embryos and consequently improving the production efficiency in transgenic animals.