• Journal of Life Science
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• v.12 no.1
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• pp.96-105
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• 2002

To investigate expression of the artificial gene encoding a repeated tripeptide lysyl-glutamyl-tryptophan in tobacco plant, the plant binary vector, pART404 has been constructed, which contains the duplicated CaMV 35S promoter, an artificial gene coding for repetitive polymer (Lys-Glu-Trp)$_{64}$, and nopaline synthase (nos) terminator. The recombinant expression vector was introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated trans-formation. The transgenic calli selected by kanamycin containing medium were then regenerated to whole plants. Southern blot analysis indicated that five transgenic plants (No. 1, 7, 9, 43, 45) showed the hybridizing signals at 1.1 kb of the expected size on EcoRI digestion and each of the transgenic plants contained 1 or 3 copies of the artificial gene inserted into its genome. By northern blot analysis, the size of the hybridized total RNA was estimated to be approximately 1.2 kb and the RNA appeared generally to have the integrity. Western blot indicated that the protein was detected at the position of 33 kDa and the expression level of the polypeptide in the transgenic plant (No. 45) was measured to approximately 0.1% of the total protein.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1995.06b
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• pp.77-96
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• 1995

To obtain basic informations for the improvement of seed potato production in Korea, some etiological properties of potato virus Y(PVY) distributed in the major seed potato production area(Daekwanryeong) were characterized, and the nucleotide and amino acid sequences of the coat protein gene of the PVY strains isolated were analyzed. PVY strains in Daekwonryeong, an alpine area, were identified to be two strains, PVYo and PVYN by symptoms of indicator plants, and their distribution in potato fields was similar. Major symptom on potato varieties by PVY was grouped as either mosaic alone or mosaic accompanied with veinal necrosis in the lower leaves. The symptom occurrence of the two symptoms was similar with Irish Cobbler, but Superior showed a higher rate of mosaic symptom than the other. The PVY strain which was isolated from potato cv. Superior showing typical mosaic symptoms produced symptoms of PVY-O on the indicator plants of Chenopodium amaranticolor, Nicotiana tabacum cv. Xanthi nc and Physalis floridana, but no symptom o Capsicum annum cv. Ace. Moreover, results from the enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies showed that the isolated PVY reacts strongly with PYV-O antibodies but does not react specifically with PVY-T antibodies. The purified virus particles were flexious with a size of 730$\times$11nm. On the basis of the above characteristics, the strain was identified to be a PVY-O and named as of PVY-K strain. The flight of vector aphids was observed in late May, however, the first occurrence of infected plants was in mid June with the bait plants surrounded with PVY-infected potato plants and early July with the bait plants surrounded with PVY-free potato plants. PVY infection rates by counting symptoms on bait plants (White Burley) were 1.1% with the field surrounded with PVY-free potato plants and 13.7% the fields surrounded with PVY-infected potato plants, showing the effect of infection pressure. The propagated PVY-K strain on tobacco(N. sylvestris) was purified, and the RNA of the virus was extracted by the method of phenol extraction. The size of PVY-K RNA was measured to be 9, 500 nucleotides on agarose gel electrophoresis. The double-stranded cDNAs of PVY-K coat protein(CP) gene derived by the method of polymerase chain reaction were transformed into the competent cells of E. coli JM 109, and 2 clones(pYK6 and pYK17) among 11 clones were confirmed to contain the full-length cDNA. Purified plasmids from pYK17 were cut with Sph I and Xba I were deleted with exonuclease III and were used for sequencing analysis. The PVY-K CP gene was comprised of 801 nucleotides when counted from the clevage site of CAG(Gln)-GCA(Ala) to the stop codon of TGA and encoded 267 amino acids. The molecular weight of the encoded polypeptides was calculated to be 34, 630 daltons. The base composition of the CP gene was 33.3% of adenine, 25.2% of guanine, 20.1% of cytosine and 21.4% of uracil. The polypeptide encoded by PVY-K CP gene was comprised of 22 alanines, 20 threonines, 19 glutamic acids and 18 glycines in order. The homology of nucleotide sequence of PVY-K CP gene with those of PVY-O(Japan), PVY-T(Japan), PVY-TH(Japan), PVYN(the Netherlands), and PVYN(France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. The amino acid sequence homology of the polypeptide encoded by PVY-K CP gene with those encoded by viruses was represented as 97.4%, 92.5%, 92.9%, 92.9%, and 98.5%, respectively.

• The Korean Journal of Nuclear Medicine
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• v.38 no.1
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• pp.62-73
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• 2004

Purpose: The herpes simplex virus type 1 thymidine kinase gene(HSV1-tk) is an attractive candidate as a reporter gene in noninvasive reporter gene monitoring system. The HSV1-tk gene was chosen as a reporter gene, because it has been extensively studied, and there are appropriate reporter probes, substrates of HSV1-tk gene product, to apply for HSV1-tk gene imaging. We used radiolabeled 5-iodovinyl-2'-deoxyuridine (IVDU) and 5-iodovinyl-2'-fluoro-2'-deoxyuridine (IVFRU) as reporter probes for HSV1-tk gene monitoring system. Materials and Methods: We prepared HSV1-tk gene transduced Morris hepatoma cell line using retroviral vector, MOLTEN containing HSV1-tk gene. And we confirmed the HSV1-tk gene expression by Northern blotting and Western blotting. We compared in vitro uptakes of radioiodinated IVDU and IVFRU to monitor HSV1-tk gene expression in Morris hepatoma cell line (MCA) and HSV1-tk gene tranduced MCA (MCA-tk) cells until 480 minutes. We also peformed correlation analysis between percentage of HSV1-tk gene tranduced MCA cell % (MCA-tk%) and uptakes of radiolabeled IVDU or IVFRU. Results: MCA-tk cell expressed HSV1-tk mRNA and HSV1-TK protein. Two compounds showed minimal uptake in MCA, but increased uptake was observed in MCA-tk. IVDU showed 4-fold higher accumulation than IVFRU at 480 min in MCA-tk (p<0.01). Both IVDU and IVFRU uptake were linearly correlated ($R^2>0.96$) with increasing MCA-tk%. Conclusion: The radiolabeld IVDU and IVFRU showed higher specific accumulation in retrovirally HSV1-tk gene transfected Morris hepatoma cell line. Both IVDU and IVFRU could be used as good substrates for evaluation of HSV1-tk gene expression.

• Development and Reproduction
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• v.14 no.1
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• pp.13-19
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• 2010

We developed a novel dicistronic system for the expression of target cDNA sequences in the milk of transgenic animals using goat beta-casein/hGH fusion construct, pGbc5.5hGH (Lee, 2006) and internal ribosome entry site (IRES) sequences of encephalomyocarditis virus (EMCV). Granulocyte colony-stimulating factor (hG-CSF) cDNA was linked to 3' untranslated region of hGH gene in the pGbc5.5hGH via EMCV IRES sequences. Transgenic mice were generated by microinjection and transgene expression was examined in the milk and mammary gland of transgenic mice at 10 days of lactation. Northern blot analysis showed that hGH gene and hG-CSF cDNA were transcribed as a single dicistronic mRNA. The hG-CSF and hGH proteins were independently translated from the dicistronic mRNA and secreted into the milk of transgenic mice. The highest concentration of hG-CSF and hGH in the milk of transgenic mice were $237{\mu}g/m{\ell}$ and $8,990{\mu}g/m{\ell}$, respectively. In contrast, another hG-CSF expression cassette, in which hG-CSF genomic sequences were inserted into a commercial milk-specific expression vector (pBC1), generated a lower level ($91{\mu}g/m{\ell}$) of hG-CSF expression in the milk of transgenic mice. These results demonstrated that the novel pGbc5.5hGH-based dicistronic construct could be useful for an efficient cDNA expression in the milk of transgenic animals.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.250-256
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• 2009

Acinetobacter strain $KNF2022^T$ was isolated from tobacco plant roots during the screening of antiviral substances having inhibitory effects on Tobacco mosaic virus (TMV) and examined by phenotypic, chemotaxonomic, and genetic characterization. It was a nonmotile, Gram-negative bacterium. This strain contained Q-9 as the main respiratory quinone. The major cellular fatty acids of the isolate were 16:0, 18:1 w9c, and 16:1 w7c/15 iso 2OH. The DNA base composition was 44 mol%. Phylogenetic analysis based on the 16S rRNA sequence revealed that the isolate formed an evolutionary lineage distinct from other Acinetobacter species. Based on the evaluation of morphologic, physiologic, and chemotaxonomic characteristics, DNA-DNA hybridization values, and 16S rRNA sequence comparison, we propose the new species Acinetobacter antiviralis sp. nov., the type strain of which is $KNF2022^T$ (=KCTC $0699BP^T$).

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.151-160
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• 1997

In view of the potential of replicase protein as a diagnostic reagent for human caliciviruses (HuCVs), we have cloned and over-expressed this gene from the Snow Mountain-like Korean strains in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and described the preliminary antigenic characterization of the recombinant products. Each 470bp fragment corresponding to highly conserved region of RNA-dependent RNA polymerase was generated by RT-PCR from stools of two diarrheal children, cloned in pMOSBlue T-vector, and subcloned between the EcoRI and SalI restriction sites of pGEX-4T-3, a GST gene fusion vector, yielding $pGCV_{pol}$. This construct expressed a Snow Mountain-like HuCV replicase under the control of the IPTG-inducible tac promoter. An extract prepared by sonication of the E. coli cell inclusion bodies bearing $pGCV_{pol}$ products was purified and analyzed by SDS-PAGE. After Coomassie blue staining, it was shown that the recombinant replicase migrated on the gels with an approximate molecular mass of 46.5 kDa, that was subsequently cleaved into a 26 kDa GST fragment and a 20.5 kDa replicase protein upon digestion with thrombin protease. The replicase was recognized on immunoblotting with the sera from symptomatic children with the HuCV-associated diarrhea but not by asymptomatic sera from adults. The results presented the first biological activity of individually expressed HuCV replicase subunit and provided important reagents for diagnosis of HuCV infection.

• 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.

• International Journal of Industrial Entomology and Biomaterials
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• v.23 no.1
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• pp.129-135
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• 2011

Cathepsins are well-characterized proteases that are ubiquitously expressed in lysosomes. Previous work revealed that $Bombyx$ $mori$ cathepsins B and D are expressed in the fat body and undergo decomposition during larval-pupal metamorphosis. Quantitative RT-PCR was performed to detect cathepsin gene expression at the transcription level when challenged by $B.$ $mori$ nuclear polyhedrosis virus (BmNPV), temperature and hormones (20-hydroxyecdysone (20E) and juvenile hormone analogue (JHA)). mRNAs encoding cathepsins B and D were significantly enhanced after the larvae were infected with BmNPV, and the peak of the induction appeared at 1 day before spinning. This attenuated the inducing effect on cathepsin expression caused by infection. Temperature shock induced cathepsin expression at the later stage of the $5^{th}$ instar, and transcription levels varied with development stage and temperature. Cathepsin B and D mRNA expression in the fat body were significantly induced by JHA at the day before spinning, and with 20E, the expression reached a peak at the last day of the $5^{th}$ instar. Cathepsin B and D mRNA expression exhibited detectable changes post-treatment, without significant differences between or among the hormone concentrations.

• Journal of the Korean Society of Tobacco Science
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• v.23 no.2
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• pp.123-132
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• 2001

In order to transform pokeweed antiviral protein cDNA to tobacco plant, total RNA was extracted from Phytolacca americana. PAP-II cDNA was synthesized from purified total RNA via RT-PCR and subcloned to recombinant vector pBluescript II SK-. 10 deletion mutant PAP-II cDNA fragments which were sequentially deleted from N-terminal by 90bp were synthesized from PAP-II cDNA except leading frame by PCR with primers designed in our laboratory. To select non-cytotoxic clone, pAc55M was constructed with yeast expression vector pAc55 and multicloning site(MCS). Sequentially deleted mutant PAP-II cDNAs were cloned on downstream of gall promoter of pAc55M. 6 non-cytotoxic deletion mutant PAP-II cDNA were selected. Selected cDNAs were cloned into plant expression vector pKGT101BH for transformation of these clones to plant through Agrobacterium tumefacience. After cloning, recombinant pKGT101BH carrying deleted mutant PAP-IIcDNA were transformed to Nicotiana tabacum cv. NC567. Transformed tobacco plants cultured on shooting and rooting media were transfered to green-house. About four weeks later, these plants were infected with physically infection using carborandum with PVY-VN strain. After 4 weeks, plants resistant to virus were selected , and seeds of these plants were gathered. Southern blot hybridization showed deleted fragments by 220bp and 420bp, so resistant ability of these plants is due to mutant PAP-II cDNA.

• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.190-198
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• 2021

Human Aichivirus (Aichivirus A; AiV-A) is a positive-sense single-strand RNA non-enveloped virus that has been detected worldwide in various water environments including sewage, river, surface, and ground over the past decade. To develop a method with excellent sensitivity and specificity for AiV-A diagnosis from water environments such as groundwater, a combination capable of reverse transcription (RT)-nested polymerase chain reaction (PCR) was developed based on existing reported and newly designed primers. A selective method was applied to evaluate domestic drinking groundwater samples. Thus, a procedure was devised to select and subsequently identify RT-nested PCR primer sets that can successfully detect and identify AiV-A from groundwater samples. The findings will contribute to developing a better monitoring system to detect AiV-A contamination in water environments such as groundwater.