• Parasites, Hosts and Diseases
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• v.34 no.2
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• pp.135-142
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• 1996

Antigenic domain of jai or surface protein (p30) of Toxoplosmc Sondii was analyzed after polymerase chain reaction (PCR) of its gene fragments. Hydrophilic or hydrophobic moiety of amino acid sequences were expressed as glutathione S-transferase (G57) fusion proteins. Fragments of p30 gene were as follows: 737, total p30 open reading frame (ORF) ; S28, total ORF excluding N-terminal signal sequence and C-terminal hydrophobic sequence; Al9, N-terminal 2/3 parts of A28; A19, N-terminal 2/3 of S28; P9, C-terminal 2/3 part of S28; Z9. middle 1/3 of S28; and 29, C-terminal 1/3 of S28. respectively. Primer of each fragment was synthesized to include clamp sequence of EcoR I restriction site. PCR amplified DNA was inserted info GST (26 kDa) expression vector, PGEX-47-1 to transform into Escheri,hia coei (.JM105 strain). G57 fusion proteins were expressed with IPTG induction as 63. 54, 45, 45, 35, 36. and 35 kDa proteins measured by SDS-PAGE. Each fusion protein was confirmed with G57 detection kit. Western blot analysis with the serum of a toxoplasmosis patient revealed antigenicity in proteins expressed by T37. S28, and Al9 but not those by Pl8. X9, Y10, and Z9. Antigenicity of p30 seems to be located either in N-terminal 115 part in the presence of middle 1/3 part or in the oligopeptides between margins of the first and second 1/3 parts.

• Journal of Plant Biotechnology
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• v.34 no.1
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• pp.55-59
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• 2007

The study was carried out to develop transformants resisting to Phyophthora blight disease in the domestic pepper cultivar Subicho. In transforming of syn600 promoter with elicitin gene using Agrobacterium (LBA4404/pBI101 syn600-syn${\alpha}$-elicitin) to cotyledons of pepper, rate of shoot formation in 'Subicho' was 11.1% in medium containing 3 mg/L zeatin and 0.05 mg/L NAA, and also 12.8% in medium containing combination of 4 mg/L zeatin and 0.05 mg/L MAA. For PCR reaction using elicitin gene primer of transformants regenerated from cotyledons, we detected a specific band of 536 bp, and also showed strong signal at position of 536 bp in accordance with NPTII gene used as probe in Southern blot. Transformants pepper shown resistance to blight fungus was inoculated to seedlings of the $T_{1}\;and\;T_{2}$ transformants by concentration (density: zoo spore $10^{3}/mL$).

• Development and Reproduction
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• v.7 no.2
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• pp.127-136
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• 2003

The present study was conducted to investigate gene expression profile of mouse ovaries during the primordial-primary follicle transition. We isolated total RNA from mouse ovaries at day1(contains only primordial follicles) and day5(contains both primordial and primary follicles) and synthesized cDNA using annealing control primers(ACP, Seegene, Inc., Seoul, Korea). Using 80 different ACPs for PCR, we cloned, sequenced, and analyzed identities of 41 differentially expressed genes(DEGs). According to BLAST analysis, sequences of 33 clones significantly matched database entries, 4 clones were novel, and 4 clones were ESTs. We selected 8 DEGs with interesting functions, Anx11 and Pepp2-Pending highly expressed in day1 ovary, while Apg3/Autlp-like, BPOZ, Ches1, Kcmf1, NHE3, Nid2, Ninj1, SENP3, Suil-rsl, and TIAP/m-survivin highly expressed in days ovary, and confirmed their different expression between day1 ovaries and days ovaries using semi-quantitative RT-PCR. There was no false positive result. Using in situ hybridization, we found that almost all of genes studied were expressed in the oocyte from primordial follicle stage but expression decreased from primary follicle stage. Meanwhile their expression was increased in cuboidal granulosa cells. Different expression of BPOZ and TIAP/m-survivin between primordial and primary follicles was confirmed by using laser capture microdissection followed by real-time PCR BPOZ and TIAP/m-survivin expressed 4.5 and 3.4 fold higher in primary than primordial follicles, respectively. List of genes obtained from the present study will provide insights for the study of mechanism regulating primordial-primary follicle transition.

• Korean Journal of Animal Reproduction
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• v.27 no.2
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• pp.179-185
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• 2003

To determine whether the diphtheria toxin-A (DT) gene disrupts development of thymocytes in transgenic animal, the DT-A gene was used for the production of transgenic mice directed by proximal Ick promoter sequences. Two transgenic founder mice that contained several copies of transgene were produced by DNA microinjection and integration of transgene in transgenic mice was confirmed by PCR and Southern blotting analysis. Transgenic $F_1$ and $F_2$ mice were produced by outbreeding of founder and $F_1$ mice to investigate expression of transgene and phenotypes in transgneic mice. Expression of the diphtheria toxin gene was confirmed in thymus, spleen and liver of transgenic mice by RT-PCR. In circulating blood of transgenic mice, lower number of circulating white blood cells and platelets were observed compared with that of normal mice. In addition, transgneic mice had reduced number of circulating peripheral T-cells analyzed by FACS with anti-CD3 antibody. The data in these transgenic mice indicate that DT gene can play a disruptive role in developing thymocytes of transgenic mice resulted in lower number of T-cells that can be applicable to a wide range of tissues in other animals.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.199-205
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• 1998

The present study was performed to analyze the expression of LH genes in the rat ovary. Expression of LH subunit genes in the rat ovary was demonstrated by amplification of ovarian RNA by RT-PCR. The ovarian $LH_\beta$ transcripts contained at least two parts of the published cDNA structure, the pituitary exons 1, 2 and 3 and the part of testicular ex on 1 in the major trancripts form in rat testis. Using RIA, significant amount of LH-like molecules were detected in crude ovarian extracts, and the competition curves with increasing amount of tissue extracts were parallel with those of standard peptide, indicating that the ovarian immunoreactive LH-like material is similar to authentic pituitary LH molecule. The administration of PMSG to immature rats resulted in a sharp decrease of the ovarian LH contents after 24 h post-injection. In conclusion, these findings demonstrate that genes for LH subunits are expressed in the rat ovary, and suggest that LH can playa central role in regulation of female reproduction with both endocrine (by pituitary LH) and auto- and/or para-crine (by ovarian LH) manner.

• Korean Journal of Agricultural Science
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• v.28 no.2
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• pp.78-84
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• 2001

This study was analyzed by PCR technique with specific primer in order to investigate the characteristics of ${\beta}$-lactoglobulin (${\beta}$-LG) gene 5'flanking region in Korean native goat. This work confirmed amplified product of 1,077 bp fragments obtained from the amplification of ${\beta}$-LG promoter from genomic DNA using PCR in Korean native goat. The nucleotide sequence of ${\beta}$-LG gene 5'flanking region in Korean native goat as compared with that of sheep ${\beta}$-LG were different at 46 base of 897 nucleotides, and showed high homology as about 94.9% each breed. Especially we confirmed that the difference of nucleotide sequences between Korean native goat and sheep were consisted of $T{\rightarrow}C$ substitution and $C{\rightarrow}T$ substitution reversely. As a consequences, the sequences of ${\beta}$-LG gene 5'flanking region showed a high homology between Korean native goat and sheep. Furthermore we should be studied that relationships between the control of gene expression and nucleotide sequences of transcription factor in Korean native goat.

• Journal of Life Science
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• v.20 no.12
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• pp.1896-1901
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• 2010

This study was conducted using quantitative real-time PCR using Lactobacilli as probiotics. Quantitative real-time PCR (RT PCR) was conducted via a method involving SYBR Green 1 and a probe. Plasmid DNA was cloned using the 16S-23S rRNA intergenic species region. Gene clones were diluted from $10^2$ to $10^{10}$. Standard curves were constructed via Ct values obtained from the results of Real-time PCR via the aforementioned SYBR Green 1 and probe method. Plasmid DNA was also cloned using the 16S-23S rRNA intergenic species region and the gene clones were diluted from $10^2$ to $10^{10}$ copy numbers via the probe method. Using RT PCR, a standard curve of plasmid DNA copy numbers was also determined. The slope value for the Y-axis intercept and $R^2$ value were measured as -3.346, 33.18, and 0.993, respectively, via the first method. For the second method, the slope value for the Y-axis intercept and $R^2$ were -3.321, 31.10 and 0.995, respectively. The PCR inhibitor could not express the detection curve at a copy number over $10^{10}$ via either method, owing to high DNA density. The DNA extract from probiotics was diluted without pre-culturing, and 16 products were amplified via both methods. The Ct value was 11.06~18.12 in the first method and 16.74~22.11 in the second method. Measured probiotics and log copy values were largely similar among the methods used. It was concluded that both methods are effective for analysis, but further research will be required to verify the optimal method.

• Korean journal of applied entomology
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• v.48 no.4
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• pp.509-517
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• 2009

Bacillus thuringiensis subsp. aizawai CAB109 isolated in Korea is known active against Spodoptera sp.. Especially, B. thuringiensis aizawai CAB109 isolates showed 100% mortality against Spodoptera litura and Spodoptera exigua. To screen highly active B. thuringiensis, the pathogenicity of B. thuringiensis CAB109 was compared with that of commercialized B. thuringiensis products. $LC_{50}$ values of CAB109, product TB-WP and product SC strains of B. thuringiensis were $1.3{\times}10^5$, $2.3{\times}10^6$ and $5.2{\times}10^5\;cfu/ml$ against the 2nd larva of S. litura and $1.8{\times}10^4$, $1.3{\times}10^6$ and $1.5{\times}10^6\;cfu/ml$ against the 2nd larva S. exigua, respectively. To determine new gene's existence and absence, the plasmid DNA was extracted, and compared to that of B.t. aizawai HD-133. Both B. thuringiensis were not like plasmid DNA pattern. PCR technique was used to predict both plasmid DNA's cry gene. PCR products analysis showed that B.t. CAB109 harbor Cry1Aa, Cry1Ab, Cry1C and Cry1D and B.t. HD-133 has Cry1Aa and Cry1Ab, respectively.

• Tuberculosis and Respiratory Diseases
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• v.40 no.6
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• pp.653-658
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• 1993

Background: Recent advancement of molecular genetics has revealed that malignant transformation of a cell may be a complex multistep process and this process is grouped, in general, into two distinct categories, activation of protooncogenes and inactivation of tumor suppressor genes. This study was focused on the mutation of p53 tumor suppressor gene, because p53 gene mutation is now generally accepted to be one of the most frequent genetic changes in a variety of human cancers. Although lung cancer is one of the common cancers in Korea, the genetic change in the carcinogenesis process is not yet known clearly. To investigate the role of p53 gene mutation in lung cancer, we examined the mutations of exon 4-8 of the p53 gene in humna lung cancer cell lines, because most of the mutations of p53 gene have been reported to develop in exon 4-8. Method: Genomic DNA was obtained by the digestion of proteinase K and the extraction by phenol-chloroform-ethanol method from two human pulmonary adenocarcinoma cell lines, PC-9 and PC-14, and one human small cell lung cancer cell line, H69. To detect the mutations of exon 4-8 of the p53 gene, polymerase chain reaction single-strand conformation polymorphism(PCR-SSCP) analysis was performed with the DNA extracted from the cells. Results: The mutation of p53 gene was present in all three cell lines tested. In PC-9, PC-14 and H69, the altered mobility was detected in exon 7, 7 and 5, respectively. Conclusion: These results suggest that p53 gene mutation plays an important role in certain steps of the carcinogenesis of human non-small cell and small cell lung cancer.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.624-630
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• 2008

Terminal restriction fragment length polymorphism (T-RFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct T-RFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.