• Korean Journal of Microbiology
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• v.38 no.3
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• pp.213-220
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• 2002

Inducible expression system for the three structural proteins, capsid (C), precursor membrane (prM/M), and envelop (E) of Japanese encephalitis virus (JEV) was established in BHK-21 cells. Doxycycline, a tetracycline analog, was utilized as an inducer. Transfectants BHK-21/IV (vector only), BHK-21/IC (for C), BHK-21/IP3 (for prM), and BHK-21/IE1 (for E) were selected and cloned in the presence of G4l8 or hygromycin. Transcribed mRNAs for the corresponding genes were observed after doxycycline induction. Effects by the JEV structural gene expression on the transfectants were monitored via cell growth, chromatin condensation, internucleosomal DNA fragmentation, and DNA contents analyses. Clear cell growth retardation and chromatin condensation were observed in all three transfectants while only BHK-2/IC corresponded to the induction status in the DNA fragmentation and DNA content analyses. Combined results, therefore, suggested that JEV capsid protein should be one of the direct and independent factors in apoptotic cell death induced by IEV infection.

• Korean Journal of Microbiology
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• v.25 no.4
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• pp.282-289
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• 1987

The characteristics of the plasmid pKU10 isolated from Pseudomonas putida KU816 were investigated and its restriction map was constructed. The pKU10 plasmid was a small R plasmid carrying genes for resistance to ampicillin, tetracyclin, and chloramphenicol, and cured by treatment with mitomycin C. The molecular size of pKU10 was estimated to be 9.4Kb. Pseudomonas strains and E. coli cells could be transformed for antibiotic resistance characters specified by pKU10 plasmid DNA. By incompatibility test with other plasmids, pKU10 is grouped into IncP-1. EcoRI, XhoI, SalI, BglII, and SmaI cleaved pKU10 once, while PstI cleaved at two sites, and HindIII cleaved at six sites. The restriction map was constructed by partial and complete digestion of the purified plasmid DNA with single, double, or triple restriction enzymes. Thus, pKU10 is expected to be used for a cloning vector in Pseudomonas cells.

• Journal of Environmental Science International
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• v.28 no.1
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• pp.75-84
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• 2019

Cytochrome c oxidase subunit II gene(CO II gene) is subunit of cytochrome oxidase, which is complex IV of mitochondria electron transport system. It has been frequently used in molecular phylogenetic studies because the speed of its DNA variation is faster than that of nucleus. It is especially useful in phylogenetic study of molecular biology in insects. In this study, we cloned and sequenced CO II gene of mitochondria DNA from Rhabditidae family nematode. Our results showed that this gene is comprised of 696 base pairs(bp). In the analysis of similarity of this gene with other known genes of 14 species of nematodes in Rhabditida order, we identified that this gene has high similarity with that of Caenorhabditis briggsae(86.0%) and C. elegans(85.6%) in Rhabditidae family. On the meanwhile, it has very low similarity with that of Angiostrongylus cantonensis(31.8%) in Angiostrongylidae family and Metastrongylus salmi(31.6%) in Metastrongylidae family. Based on the results of this study, we suggest that this nematode is closely related with that of Caenorhabditis genus in Rhabditidae family.

• Microbiology and Biotechnology Letters
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• v.50 no.4
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• pp.508-511
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• 2022

In this study, Phaseolus vulgaris (kidney bean) leghemoglobin a (PhLba) gene was cloned into pPICZαA and expressed in Pichia pastoris to sustainably produce a heme-carrying protein for organoleptic use in plant-based meat. The recombinant PhLba protein was secreted into the culture medium in a solubilized form, and the molecular weight of the purified PhLba was estimated to be 16.5 kDa using SDS-PAGE. In addtion, the yield of recombinant PhLba holoprotein was enhanced by supplementation of the cultivation medium with hemin. This result indicates that the apo-forms of PhLba can be effectively saturated with cofactor.

• Horticultural Science & Technology
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• v.28 no.4
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• pp.648-655
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• 2010

This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$ $japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2000.11a
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• pp.59-75
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• 2000

This study was carried out to develop a DNA marker for identifying between Korean cattle (Hanwoo) and other breeds. First experiment was performed to isolate Hanwoo specific DNA marker at sequence characterized amplified regions (SCARs). Five breeds of cattle including Hanwoo, Holstein, Hereford, Angus and Charolais were represented with the from 8 to 20 individuals. Fourteen primers of 300 arbitrary primers of 10 nucleotides showed reproducible polymorphism across the breeds. An amplified band of 0.9 kb in the primer MG-3 showed the specificity to Holstein breed. And MG-6 and MG-12 detected the Hereford and Hanwoo specific markers at the size of 2.0 kb and 1.0 kb, respectively. A 1.0 kb band of MG-12 was cloned and sequenced. A SCAR primer was designed based on the obtained sequences. It was possible to identify the Hanwoo from Holstein breed. Second experiment was carried out to observe the genotype frequencies of MC1R in 1,044 samples of imported beef and eight different cattle breeds including Hanwoo, Holstein, Angus, Brown-Swiss, Charolais, Limousin, Simmental and Hereford. The primers for the amplification of bovine MC1R gene were designed based on a bovine MC1R gene sequence (GenBank accession no.Y19103). A size of 350 bp was amplified by polymerase chain reaction(PCR), digested with two different restriction enzyme, BsrFI and MspA II, and electrophoresed in 2.5% Metaphore agarose gel for determination of genotypes. Genotype frequencies of Hanwoo were 0.10 in E+e and 0.90 in ee. Allele ED was shown in all of Holstein and Angus breeds tested which have black coat color phenotypes. We suggested that SCAR marker and the bovine MC1R gene could be used as a DNA marker for distinguishing beef between Hanwoo and Holstein.

• Journal of Plant Biotechnology
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• v.29 no.3
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• pp.151-159
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• 2002

Genetic map and molecular marker have a great importance in improving and facilitating crop breeding program as well as in genome analysis and map-based cloning of genes representing desirable characters. This study aimed at developing RAPD markers and constructing a genetic linkage map using 82 BC$_1$F$_1$individuals originated from the cross between '835' and B$_2$in radish (Raphanus sativus L.). One of the parents for genetic linkage map construction, '835'(P$_1$) of egg type is susceptible to Fusarium wilt and have medium resistance to virus infection and the other parent, B$_2$(P$_2$) of round type, is susceptible to Fusarium wilt and virus, Screening of 394 RAPD primers in BC$_1$F$_1$) population resulted in selecting 128 polymorphic markers which displayed 1:1 segregation pattern. Two markers failed to display 1:1 segregation and showed the segregation ratio skewed to maternal genotype. Selected markers were categorized into 14 linkage group based on LOD score represented by MAPMAKER/EXP program. Five groups composed of single marker among them were excluded from the linkage map, and consequently, the remaining groups are well matched with the number of radish chromosome (n＝9). The linkage map constructed with 128 markers covers 1,688.3 cM and the average distance between markers was 13.8 cM. For developing STS marker, we determined the partial nucleotide sequence of OPE10 marker at both ends and designed a oligonucleotide primer pair based on this sequence. STS PCR using the primer pair displayed a single, clear band of which segregation is perfectly matched with that of OPE10 marker. This implies that RAPD markers could readily convert into clear and reliable STS markers.

• Tuberculosis and Respiratory Diseases
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• v.61 no.3
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• pp.239-247
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• 2006

Background: Culture filtrate proteins secreted by mycobacteria are thought to play an important role in inducing protective immunity and to develop new methods for diagnosing tuberculosis. Methods: A culture filtrate protein of M. avium that was strongly reactive with goat antiserum against M. intracellulare was constructed. Its homologous protein (TB-14) in M. tuberculosis was cloned, expressed and purified. The inductions of IFN-${\gamma}$ stimulated with $10{\mu}g$ of TB-14 recombinant protein and $10{\mu}g$ PPD were estimated by using whole bloods from seven PPD (-) subjects, seven PPD (+) healthy volunteers and nine tuberculosis patients. Results: M. avium culture filtrate protein was confirmed as a hypothetical protein that was termed contig 116. A novel 14-kDa recombinant protein (TB-14) of M. tuberculosis was composed of 148 amino acids, including 30 amino acids of the signal peptide, and it showed 78% homology with M. avium. In the PPD (+) healthy volunteers, recombinant TB-14 protein strongly induced the secretion of IFN-${\gamma}$ in whole blood cultures. Conclusion: These results suggest that TB-14 recombinant protein might play an important role in inducing cell-mediated immunity against tuberculosis. Furthermore, TB-14 protein antigen and its antiserum will be available for the development of new diagnostic tools for tuberculosis.

• Journal of Life Science
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• v.24 no.4
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• pp.343-351
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• 2014

Phosphoinositide 3-kinase (PI3K) plays a central role in cell signaling and leads to cell proliferation, survival, motility, exocytosis, and cytoskeletal rearrangements, as well as specialized cell responses, superoxide production, and cardiac myocyte growth. PI3K is divided into three classes; type I PI3K is preferentially expressed in leukocytes and activated by ${\beta}{\gamma}$ subunits of heterotrimeric G-proteins. In this study, the cDNAs encoding the $PI3K{\gamma}$ gene were isolated from a brain cDNA library constructed using the flounder (Paralichthys olivaceus). The sequence of the isolated $PI3K{\gamma}$ was 1341 bp, encoding 447 amino acids. The nucleotide sequence of the $PI3K{\gamma}$ gene was analyzed with that of other species, including Oreochromis niloticus and Danio rerio, and it turned out to be well conserved during evolution. The $PI3K{\gamma}$ gene was subcloned into the expression vector pET-44a(+), and expressed in the E. coli BL21 (DE3) codon plus cell. The resulting protein was expressed as a fusion protein of approximately 49 kDa containing a C-terminal six-histidine extension that was derived from the expression vector. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to $PI3K{\gamma}$. The binding of wortmannin to $PI3K{\gamma}$, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. The results obtained from this study will provide a wider base of knowledge on the primary structure and characterization of the $PI3K{\gamma}$ at the molecular level.

• Tuberculosis and Respiratory Diseases
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• v.58 no.2
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• pp.142-152
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• 2005

Background : Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. Method : M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and $IFN-{\gamma}$ after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. Result : Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and $IFN-{\gamma}$ responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. Conclusion : Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant.