• Microbiology and Biotechnology Letters
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• v.37 no.1
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• pp.17-23
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• 2009

The gene encoding Thermus thermophilus HJ6 DNA polymerase (Tod) was cloned and sequenced. The open reading frame (ORF) of the Tod gene was composed of 2,505 nucleotides and encoded a protein (843 amino acids) with a predicted molecular weight of 93,795 Da. The deduced amino acid sequence of Tod showed 98% and 86% identities to the Thermus thermophilus HB8 DNA pol and Thermus aquaticus DNA pol, respectively, The Tod gene was expressed under the control of the bacteriophage $\lambda$ promoters PR and PL on the expression vector pJLA503 in Escherichia coli strain BL21 (DE3) codon plus. The expressed enzyme was purified by heat treatment, $HiTrap^{TM}$ Q column, and $HiPrep^{TM}$ Sephacryl S-200 HR 26/60 column chromatographies. The optimal temperature and pH for DNA polymerase activity were found to be $75{\sim}80^{\circ}C$ and 9.0, respectively. The optimal concentrations of $Mg^{2+}$ and $Mn^{2+}$ were 2.5 mM and 1 mM, respectively. The enzyme activity was activated by divalent cations, and was inhibited by monovalent cations. The result of the PCR experiment with Tod DNA polymerase indicates that this enzyme might be useful in DNA amplification and PCR-based applications.

• BMB Reports
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• v.29 no.4
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• pp.359-364
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• 1996

By both in vitro hydroxylamine mutagenesis of the wild type 3-phosphoglycerate kinase gene (PGK) promoter DNA and insertion of the leu2-d gene, we have created yeast expression vectors potentially useful for production of eukaryotic genes in yeast. The guanine (G) to adenine (A) change at the -3 position from the ATG start codon of the PGK promoter-based vector rendered a 6~7 times elevated expression of the adjacent eukaryotic gene, and insertion of the leu2-d gene in the vector containing the mutated PGK promoter further enhanced the expression of the gene. When expression of the AIDS virus HIV1-gagP17 gene in a lacZ fusion form was examined with this new vector, a 15 times higher level of expression than that from the original PGK promoter was observed. Northern and Southern analysis showed that this elevated expression is due to the production of a high copy number of mRNA by leu2-d gene functioning and by efficient translation of the produced mRNA. Thus, the vector that contained the A at the -3 position from the ATG start codon in the promoter region and the leu2-d gene shows increased expression capability and will be potentially useful for production of eukaryotic genes in yeast.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.18-24
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• 2001

Expression of the baculovirus major envelope glycoprotein gene(gp64) is regulated by transcription from botha early and late promoters. To develop a transient expression vector under the control of gp64 gene promoter, the gp64 gene of Bombyx mori nucleopolyhedrovirus-K1(BmNPV-K1) was characterized. The gp64 gene was local-ized at EcoR I-Pst I 7.38-kb fragment of the BmNPV-K1 genome. The EcorR 1-Pst I 7.38-kb fragment was cloned and the nucleotide sequence of 2,277 bases including the coding region of gp64 gene was determined. Based on these results, transient expression vector using gp64 gene promoter was constructed and named as pBm64. E.coli lacZ gene was introduced onto pBm64 as a reporter gene and expressed transiently in B. mori 5(Bm 5) cells. The expression vector transfected into the cells was maintained stably for 1 to 5 days. In order to confirm the expression of the reporter gene by gp64 promoter, recombinant virus was constructed. The recombinant virus has two independent transcription units in opposite orientations with two promoters; gp64 and polyhedrin gene promoters each initiating transcription of $\beta$-galactosidase and polyhedrin, respectively. Polyhedra formation and expression of $\beta$-galactosidase in Bm5 cells infected with the recombinant virus were observed with phase contrast microscope and in situ staining.

• The Korea Journal of Herbology
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• v.25 no.4
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• pp.47-54
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• 2010

Objectives : The original plant species of Schisandrae Fructus (O-mi-ja) is prescribed as Schisandra chinensis $B_{AILL.}$, in Korea, but S. chinensis $B_{AILL.}$ and S. sphenanthera $R_{EHD.}$ et $W_{ILS.}$ in China. Moreover, fruit of several other species in genus Schisandra also have been used as the same herbal medicines. To develop a reliable method for correct identification of Schisandrae Fructus and to evaluate the phylogenetic relationship of S. chinensis and its related species, we analyzed internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA). Methods : Twenty-four plant samples of three Schisandra species and one Kadsura species, S. chinensis $B_{AILL.}$, S. spenanthera $R_{EHD.}$ et $W_{ILS.}$, S. nigra $M_{ax.}$ and Kadsura japonica $D_{UNAL}$ were collected from each different native habitate and farm in Korea and China. The nrDNA-ITS region of each samples were amplified using ITS1 and ITS4 primer and nucleotide sequences were determined after sub-cloning into the pGEM-Teasy vector. Authentic marker nucleotides were estimated by the analysis of ClastalW based on the entire nrDNA-ITS sequence. Results : In comparative analysis of the nrDNA-ITS sequences, we found specific nucleotide sequences including indels (insertions and deletions) and substitutions to distinguish C. chinensis, S. spenanthera, S. nigra, and K. japonica. These sequence differences at corresponding positions are avaliable nucleotide markers to determine the botanical origin of O-mi-ja. Moreover, we evaluated the phylogenetic relationship of four plant species by the analysis of nrDNA-ITS sequences. Conclusions : These marker nucleotides would be useful to identify the official herbal medicines by the providing of definitive information that can identify each plant species and distinguish it from unauthentic adulterants for O-mi-ja.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.164-167
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• 2008

Recently, rolling circle amplification (RCA) technique has been widely focused in the field of gene amplification just like PCR method. We have developed RCA-mer, which is a web-based program searching for primer candidates from a given sequence. It can be applied to find primer lists in DNA amplification experiment based on RCA method. The RCA-mer compares 8-mer primer lists with user's input sequence such as vector, mitochondria, and microbial genome sequence. After calculating 8-mers existences in a given sequence, it displays matched and no-matched primer lists with their GC-contents. In addition to it, RCA-mer can search the existence of 8-mer primer lists in two sequences whether they are co-existed or not. Users can apply candidate primer lists to their researches which use RCA techniques.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.866-871
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• 2012

A novel cholesterol-based cationic lipid containing a tri-2-hydroxyethylamine head group and ether linker (Chol-THEA) was synthesized and examined as a potent gene delivery vehicle. In the preparation of cationic liposome, the addition of DOPE as helper lipid significantly increased the transfection efficiency. To find the optimum transfection efficiency, we screened various weight ratios of DOPE and liposome/DNA (N/P). The best transfection efficiency was found at the Chol-THEA:DOPE weight ratio of 1:1 and N/P weight ratio of 10~15. Most of the plasmid DNA was retarded by this liposome at the optimum N/P weight ratio of 10. The transfection efficiency of Chol-THEA liposome was compared with DOTAP, Lipofectamine, and DMRIE-C using the luciferase assay and GFP expression. Chol-THEA liposome with low toxicity had better or similar potency of gene delivery compared with commercial liposomes in COS-7, Huh-7, and MCF-7 cells. Therefore, Chol-THEA could be a useful non-viral vector for gene delivery.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1123-1126
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• 2011

The IncP plasmid R995 has been a useful self-transmissible, broad-host-range vector for a number of applications including the recombinase/conjugation-based cloning of large genomic DNA segments. However, R995 derivatives (or related plasmids) expressing a wide range of different resistance markers and Flp recombinase target sites do not exist in the literature. In addition, documented strategies for applying such plasmids in cloning applications that take advantage of conjugation for the convenient isolation and recovery of constructs are extremely limited. Here, we report a new series of R995 plasmids with alternative markers to increase options for applications in backgrounds already expressing resistance to a particular antibiotic(s). These R995 plasmids have been engineered to contain FRT sites that can be used for recombinase-based cloning. We demonstrate the utility of this approach by cloning 20 kb regions from the Salmonella Typhimurium and Escherichia coli genomes and by cloning DNA from an exogenous plasmid source. To our knowledge, this represents the first systematic engineering of an intact, self-transmissible IncP plasmid with a series of alternative antibiotic markers and FRT sites.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.489-493
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• 1988

To obtain a new yeast strain that is able to efficiently produce ethanol from starch, the glucoamylase gene of Saccharomyces diastaticus was transformed into S. cerevisiae without a cloning vector. The competent cells of S. cerevisiae, induced by the treatment of Li$_2$SO$_4$, were transformed with the partial BamHI-digests of chromosomal DNA of S. diastaticus, and the transformants were selected by their abilities to utilize and ferment starch. The transformants, which appeared at a frequency of 8.5$\times$10$^{-7}$, were able to withstand up to 800 ppm of copper sulfate like the recipient and retained the phenotypic expression of the recipient with the exception of the acquisition of STA gene and MAL gene, as regards fermentation of carbohydrates. The enzymatic properties of glucoamylases produced by transformants were very similar to those produced by S. diastaticus as based on optimium pH and temperature.

• Journal of Microbiology and Biotechnology
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• v.18 no.10
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• pp.1735-1740
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• 2008

Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.

• Journal of Microbiology and Biotechnology
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• v.21 no.1
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• pp.93-99
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• 2011

Amongst a number of potential nonviral vectors, cationic liposomes have been actively researched, with both gemini surfactants and bola amphiphiles reported as being in possession of good structures in terms of cell viability and in vitro transfection. In this study, a cholesterol-based diquaternary ammonium gemini surfactant (Chol-GS) was synthesized and assessed as a novel nonviral gene vector. Chol-GS was synthesized from cholesterol by way of four reaction steps. The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE (1,2-dioleoyl-L-${\alpha}$- glycero-3-phosphatidylethanolamine), and at a ratio of between 10:1~15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP). The results indicate that the efficiency of Chol-GS is greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed through the use of green fluorescent protein expression. Chol-GS exhibited a moderate level of cytotoxicity, at optimum concentrations for efficient transfection, indicating cell viability. Hence, the newly synthesized Chol-GS liposome has the potential of being an excellent nonviral vector for gene delivery.