• Journal of Plant Biotechnology
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• v.36 no.4
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• pp.384-391
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• 2009

Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5'-flanking region of the CP gene and a Blastbased search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.144-151
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• 2014

Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the ${\alpha}$-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2031-2037
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• 2012

Background: Analysis of gene-gene and gene-environment interactions for complex multifactorial human disease faces challenges regarding statistical methodology. One major difficulty is partly due to the limitations of parametric-statistical methods for detection of gene effects that are dependent solely or partially on interactions with other genes or environmental exposures. Based on our previous case-control study in Chongqing of China, we have found increased risk of colorectal cancer exists in individuals carrying a novel homozygous TT at locus rs1329149 and known homozygous AA at locus rs671. Methods: In this study, we proposed statistical method-crossover analysis in combination with logistic regression model, to further analyze our data and focus on assessing gene-environmental interactions for colorectal cancer. Results: The results of the crossover analysis showed that there are possible multiplicative interactions between loci rs671 and rs1329149 with alcohol consumption. Multifactorial logistic regression analysis also validated that loci rs671 and rs1329149 both exhibited a multiplicative interaction with alcohol consumption. Moreover, we also found additive interactions between any pair of two factors (among the four risk factors: gene loci rs671, rs1329149, age and alcohol consumption) through the crossover analysis, which was not evident on logistic regression. Conclusions: In conclusion, the method based on crossover analysis-logistic regression is successful in assessing additive and multiplicative gene-environment interactions, and in revealing synergistic effects of gene loci rs671 and rs1329149 with alcohol consumption in the pathogenesis and development of colorectal cancer.

• Journal of Periodontal and Implant Science
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• v.41 no.4
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• pp.167-175
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• 2011

Purpose: Periodontal ligament (PDL) cell differentiation into osteoblasts is important in bone formation. Bone formation is a complex biological process and involves several tightly regulated gene expression patterns of bone-related proteins. The expression patterns of bone related proteins are regulated in a temporal manner both in vivo and in vitro. The aim of this study was to observe the gene expression profile in PDL cell proliferation, differentiation, and mineralization in vitro. Methods: PDL cells were grown until confluence, which were then designated as day 0, and nodule formation was induced by the addition of 50 ${\mu}g$/mL ascorbic acid, 10 mM ${\beta}$-glycerophosphate, and 100 nM dexamethasone to the medium. The dishes were stained with Alizarin Red S on days 1, 7, 14, and 21. Real-time polymerase chain reaction was performed for the detection of various genes on days 0, 1, 7, 14, and 21. Results: On day 0 with a confluent monolayer, in the active proliferative stage, c-myc gene expression was observed at its maximal level. On day 7 with a multilayer, alkaline phosphatase, bone morphogenetic protein (BMP)-2, and BMP-4 gene expression had increased and this was followed by maximal expression of osteocalcin on day 14 with the initiation of nodule mineralization. In relationship to apoptosis, c-fos gene expression peaked on day 21 and was characterized by the post-mineralization stage. Here, various genes were regulated in a temporal manner during PDL fibroblast proliferation, extracellular matrix maturation, and mineralization. The gene expression pattern was similar. Conclusions: We can speculate that the gene expression pattern occurs during PDL cell proliferation, differentiation, and mineralization. On the basis of these results, it might be possible to understand the various factors that influence PDL cell proliferation, extracellular matrix maturation, and mineralization with regard to gene expression patterns.

• Microbiology and Biotechnology Letters
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• v.31 no.4
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• pp.334-341
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• 2003

This study was carried out to investigate the cause of cholera outbreak in Gyeongbuk province in 2001.90 strains of Vibrio cholerae O1 El Tor serotype Inaba were isolated from diarrheal patients. By multiplex-PCR, all of the isolated strains revealed positive for detection ctxA, hlyA and tcpA genes. There were DNA sequence difference of the cholera-toxin subunit A gene and subunit B gene between isolated V. cholerae O1 and the strain of GenBank. In analysis of PFGE patterns, all of the isolated strains were showed the same DNA fragments. We also collected plankton samples in the east coast of Gyeongbuk to isolate V. cholerae O1 and V. cholerae O139 from August to October 2002. The samples were examined to detect the rfb gene and cholera-toxin gene by multiplex-PCR. The cholera-toxin gene was detected and then we tried to isolate V. cholerae O1 and V. cholerae O139, but they were not isolated.

• Plant Breeding and Biotechnology
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• v.6 no.4
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• pp.313-320
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• 2018

Rice is the staple food of more than 50% of the world population. Cultivated rice has the AA genome (diploid, 2n = 24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized transposable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos have been used to generate gene-tagged populations. To date T-DNA and maize transposable element systems have been utilized as main insertional mutagens in rice. The Ac/Ds system offers the advantage of generating new mutants by secondary transposition from a single tagged gene. To enhance the efficiency of gene detection, advanced gene-tagging systems (i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of insertional mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been conducted by collaborative works in Korea.

• Korean Journal of Clinical Laboratory Science
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• v.41 no.1
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• pp.11-23
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• 2009

Chromosomal rearrangements are major pathology in hematological malignancies. The detection of minimal residual disease (MRD) for these gene rearrangements helps in monitoring treatment outcomes and predicting prognosis of patients. Recently, quantification of these gene transcripts based on real-time quantitative polymerase chain reaction (RQ-PCR) has been used as MRD detection. The purpose of this study is to ensure the usefulness of the RQ-PCR technique for detecting MRD in hamatological malignancy patients. The patients had been diagnosed to AML1-ETO positive AML, PML-RARa positive AML and BCR-ABL positive MPN at Chonnam National University Hwasun Hospital from Jan. 2006 to Aug. 2008. The fusion transcript was quntified by RQ-PCR and analyzed in comparison to conventional cytogenetics, FISH and RT-PCR. The fusion gene transcript was quantified by RQ-PCR in 57 samples from 14 patients with AML1-ETO positive AML, 79 samples from 27 patients with PML-RARa positive AML and 108 samples from 36 patients with CML. At diagnosis, the quantitative fusion transcripts for AM1-ETO, PML-RARa and BCR-ABL showed the range of 0.485552651~10.82233683 (mean 3.782217131, SD 2.998052348), 0.005300395~0.29267494 (mean 0.056901315, SD 0.080131381) and 0.1293929~12.94826849 (mean 1.701935665, SD 2.200913158). The increase of AML1-ETO fusion gene transcripts preceded morphologic relapse in two patients. Quantification of fusion gene transcripts by RQ-PCR could detected MRD in samples which were negative by in cytogenetic analysis or FISH. Our findings indicated that quantitative analysis of AML1-ETO, PML-RARa and BCR-ABL transcripts by RQ-PCR might be a useful tool for the monitoring of minimal residual disease in hematological malignancies.

• Applied Biological Chemistry
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• v.48 no.4
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• pp.335-338
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• 2005

For the development of qualitative PCR detection method of genetically modified rice (Oryza sativa L.), rice species specific gene, SAMDC1 (S-adenosylmethionine decarboxylase), was selected and validated as suitable for use as an endogenous reference gene in rice. The primer pair OsSAMDC1-5'/3' with 110 bp amplicon was used for amplification of the rice endogenous gene, SAMDC1 and no amplified product was observed from 19 different plants as templates. Qualitative PCR method was assayed with 2 different GM rices (Milyang 204 and Iksan 483) developed in Korea. For the qualitative PCRs, the construct-specific detection primer pairs were constructed. Os204-5'/OsNOS-3' amplifying the junction region of GUS gene and NOS terminator introduced in Milyang 204 gave rise to an amplicon 172 bp; also, Os483-5'/OsNOS-3' amplifying the junction region of Bar gene and NOS terminator introduced in Iksan 483 gave rise to an amplicon 161 bp.

• Tuberculosis and Respiratory Diseases
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• v.69 no.6
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• pp.442-449
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• 2010

Background: Melanoma antigen genes (MAGE) are expressed in many human malignant cells and are silent in normal tissues other than in testis and in placenta. But MAGE expression in benign lung diseases, such as pulmonary tuberculosis or cases with severe inflammation, needs further evaluation to overcome false-positive findings. We evaluated detection rates of the melanoma antigen genes (MAGE) RT-nested PCR in bronchoscopic washing samples from patients with benign lung disease, as well as in patients with malignancies. Methods: Bronchial washing fluid from 122 patients was used for cytological examination and MAGE gene detection using RT-nested-PCR of common A1-6 mRNA. We compared the results from the RT-nested PCR and the pathologic or bacteriologic diagnosis. We also analyzed the expression rate and false positive rate of MAGE gene. Results: Among 122 subjects, lung cancer was diagnosed in 23 patients and benign lung disease was diagnosed in 99 patients. In patients with lung cancer, the positive rate of MAGE expression was 47.8% (11/23) and in benign lung disease group, the expression rate was 14.1% (14/99). Among benign lung disease group, the expression rate of MAGE gene (25.0%) in patients with pulmonary tuberculosis (11/44) was especially high. Conclusion: MAGE A1-6 RT-nested PCR of bronchial washing fluid can be used as a complementary method in lung cancer, but that test results in a high false positive rate in tuberculosis patients.

• Korean Journal of Veterinary Research
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• v.38 no.4
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• pp.763-770
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• 1998

Salmonella typhimurium is a causitive agent of diarrhea, fever, gastroenteritis, septicemia and sudden death in piglet. The currently used methods such as IFA, ELISA, DNA hybridization assay is needed a long-time and difficult to detect the organism in carrier animal or contaminated sample with other agents. However, it is important to detect rapidly and sensitively S typhimurium in piglet with other infectious pathogens to minimize an economic loss. Two sets of PCR primer, rfbJ forward primer(5'-AGAATATGTAATTGTCAG-3') and reverse primer(5'-TAACCGTTTCAGTAGTTC-3') were designed to amplify a 882 by fragment of Salmonella serovar type B gene. The target genomic DNA for PCR was extracted from the cultivated materials with various enrichment periods in a nonselective enrichment agar and broth with clinical specimens. The PCR is carried out here made it possible to detect the gene from two hours. Also, the amplified fragment with PCR was cloned into pGEM-T vector and digested with restrict enzyme, and sequenced for the identification of Salmonella serotype B rfbJ gene. Duplicated cultivation agar-broth followed by PCR were performed to develop a rapid and sensitive detection of S typhlmurium based on serovar type. This duplicated cultivation-PCR method provides a sensitive and rapid diagnostic tool to detect Salmonella from infected piglet with improved sensitivity.