• Environmental Analysis Health and Toxicology
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• v.20 no.1
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• pp.67-73
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• 2005

오염물질의 노출에 의해 발현이 변화되는 유전자의 발굴은 외부환경 자극에 대한 적응이나 반응의 메커니즘을 알아내는뎨 중요한 정보를 제공하며, 오염물질에 반응하는 유전자는 환경오염을 감지하는 분자 마커로 개발될 수 있다. DD-PCR 기법은 차등 발현 유전자들을 발굴해내기 위한 유용한 방법으로 사용되어 왔고, 본 연구는 이 방법을 이용하여 벤조피렌에 반응하는 조피볼락 유전자들의 발굴을 목적으로 진행되었다. 간조직에서 추출한 RNA로부터 벤조피렌의 노출에 의해 발현 양이 달라진 12개의 클론을 발굴하였고, 그 염기서 열을 분석하였다. 또한 벤조피렌의 노출시간을 각각 6, 12, 24시간으로 달리한 조피볼락에서 12개의 클론 중 4개의 클론에 대해 northern blot 분석이 실시되었으며, 이들 모두 노출시간에 따 라 발현양이 증가 또는 감소하는 것이 확인되었다. 본 연구결과는 오염물질의 영향에 의한 유전자들의 발현에 관한 전반적인 지식을 제공하였고, 나아가 환경오염이나 외부 스트레스를 감지해 낼 수 있는 바이오마커의 개발을 위한 첫 단계로서의 정보를 제공할 수 있을 것으로 생각된다.

• Korean Journal of Breeding Science
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• v.42 no.5
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• pp.534-539
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• 2010

'Hongro' is early-mid maturing cultivar with good quality like 'Tsugaru' and it has not preharvest drop. The CAX1 gene was introduced into Korean apple cultivar 'Hongro' by Agrobacterium tumefaciens LBA4404 harboring pBI121 to obtain transgenic apple with enhanced Ca level. The CAX1 gene playing the role of $H^+/Ca^{2+}$ transporter from Arabidopsis thaliana increases Ca concentration in several plants. Regenerated transgenic lines were confirmed by polymerase chain reaction (PCR) analysis and Southern blot analysis of genomic DNA for the existence of CAX1 gene. Southern blot analysis of 'Hongro' transformants showed that two putative transgenic lines were integrated with CAX1 gene in genomic DNA. The CAX1 comparative expression levels of two transgenic lines were higher than that of non-transformant evaluated by comparative quantification analysis using a real-time PCR. These two lines were multiplied in vitro, and micro-grafted on apple rootstocks 'M.9' in the isolated greenhouse. Since two years after micro-grafting, the fruits came into bearing. Compared to Ca level of the non-transgenic 'Hongro', that of the CAX1 transgenic 'Hongro' in the flesh and leaves was higher.

• Fisheries and Aquatic Sciences
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• v.18 no.1
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• pp.73-80
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• 2015

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring ${\beta}$-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

• Journal of Mushroom
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• v.9 no.3
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• pp.91-95
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• 2011

Pleurotus ostreatus, the oyster mushroom, is one of the most important edible mushrooms. It is especially susceptible to bacterial blotch disease, which is caused by Pseudomonas tolaasii. In order to develop bacterial blotch disease-resistant transgenic mushroom, NUECIN cDNA, a gene for an antibacterial peptide cloned from Bombyx mori, was overexpressed in Pleurotus ostreatus. NUECIN cDNA was fused to the ${\beta}$-TUBULIN promoter of oyster mushroom and co-transformed with the pTRura3-2 vector into the uracil auxotrophic mutant strain. Twelve transformants containing the NUECIN gene were identified by genomic PCR and Southern blot analysis. NUECIN gene expression was confirmed by Northern blot analysis. Three transformants showed the transcriptional expression of the gene. However, we could not detect expression of the protein in the transformants. This study showed the possibility of transgenic mushroom development for disease resistance.

• Biomedical Science Letters
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• v.6 no.1
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• pp.65-72
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• 2000

In order to improve the early diagnosis of Johne's disease in ruminants, duplex polymerase chain reaction system for the detection of the etiologic agent of M. paratuberculosis and for the differentiation of other mycobacterial animal pathogens, such as M. bovis and M. avium, was applied. Genomic DNAs were purified from peripheral blood monocytes or milk macrophages and were used as templates in the duplex PCR. Detection of Mycobacterium spp. in the specimen was carried out by PCR using primer set specific to the mycobacterial 16S rDNA. And then, mycobacterial DNA-positive specimens were further differentiated with duplex PCR system which was composed of primer sets specific to 16S rDNA of M. avium complex and Is900 gene of M. paratuberculosis. The results were re-confirmed by Southern blot hybridization with oligonucleotide specific to the internal sequence of IS900 PCR amplicons. The applicability of this duplex PCR system was evaluated with DNAs extracted from clinical specimens of peripheral blood monocytes and milk macrophages. In summary, the duplex PCR amplification system described in this experiment is promising molecular technique for the early diagnosis of Johne's disease in ruminants.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2004.10a
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• pp.62-65
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• 2004

To protect the watermelon against soil-borne pathogens, we are currently producing disease-resistant transgenic root stock for the growth of watermelon, A defensin gene (J1-1) from Capsicum annum, a ACC deaminase gene from Pseudomonas syringae, a galactinol synthase (CsGolS) gene from Cucumis sativus, and a WRKY (CvWRKY2) gene from Citullus vulgaris were used as transgenes for disease resistance. The gene were transformed into a inbred line (6-2-2) of watermelon, Kong-dae watermelon and a inbred line (GO702S) of gourd, respectively, by Agrobacterium-mediated transformation. Putative transgenic plants were selected in medium containing 100mg/L kanamycin, and then integration of the genes into the genomic DNA were demonstrated by PCR analysis. Successful integration of the gene in regenerated plants was also confirmed by PCR (Figf 1), genomic Southern blot (Fig 2), RT-PCR (Fig 3), and Northern blot analysis(Fig 4). Several T1 lines having different transgene were produced, and disease resistance of the T1 lines are under estimation.

• BMB Reports
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• v.31 no.5
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• pp.475-483
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• 1998

Many antibiotics contain partially deoxygenated sugar components that are usually essential for biological activity, affinity, structural stability, and solubility of antibiotics. Gene probes of the biosynthetic genes related with the deoxysugar were obtained from PCR. Primers were designed from the conserved peptide sequences of the known dTDP-D-glucose 4,6-dehydratases, which are the key step enzymes in the biosynthesis of deoxysugar. The primers were applied to amplify parts of dehydratase genes to 27 actinomycetes that produce the metabolites containing deoxysugar as structural constituents. About 180 and 340 bp DNA fragments from all of the actinomycetes were produced by PCR and analyzed by Southern blot and DNA sequencing. The PCR products were used as gene probes to clone the biosynthetic gene clusters for the antibiotic mithramycin, rubradirin, spectinomycin, and elaiophyrin. This method should allow for detecting of the biosynthetic gene clusters of a vast array of secondary metabolites isolated from actinomycetes because of the widespread existence of deoxysugar constituents in secondary metabolites.

• The Korean Journal of Mycology
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• v.25 no.3 s.82
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• pp.167-175
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• 1997

The PCR fragments of two distinct chitin synthase genes, PdCHSl and PdCHS2, were cloned from Penicillium diversum KCTC 6786. The nucleotide sequences of PdCHSl and PdCHS2 contained uninterrupted open reading frames (ORFs) of 570 bp excluding the primer sequence. The similarity analysis of the deduced amino acid sequences using BLASTP indicated that the possible evolutionary relationship between P. diversum and ascomycetous fungi. Multialignment of the deduced amino acid sequences of PdCHSs using CLASTAL W revealed that the PdCHSs fell into two different classes: PdCHSl into Class I and PdCHS2 into Class II of chitin synthase defined by Bowen et al. (1992). By Southern blot analysis, it was shown that each of the two genes is present as a single copy in the genome of P. diversum KCTC 6786.

• Korean Journal of Veterinary Research
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• v.36 no.1
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• pp.187-193
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• 1996

The T sergenti DNA fragments used as probes of KTS1(2.4kb) and KTS3(1.5kb) were labeled with digoxigenin-11-dUTP for the Southern hybridization. T sergenti DNAs from different geographic locations(Korea; Chonbuk, Kyungbuk, Chungnam, Kangwon, Cheju island, Japan; Shintoku, Shintoku 9209, Shintoku 9201, Shintoku 9202, Shintoku 9102) which had been digested with Pst I and EcoR I were probed by the digoxigenin-11-dUTP-labeled KTS1 and KTS3. As the results, the samples from Chonbuk, Kyungbuk, Cheju island in Korea and Shintoku, Shintoku 9209, Shintoku 9201, Shintoku 9102 in Japan were positively reacted, but the others from the other locations not reacted. In the comformation test of T sergenti DNA from different geographic locations, all of the samples were positively detected by PCR amplification.

• Journal of the Korean Academy of Child and Adolescent Psychiatry
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• v.11 no.1
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• pp.3-15
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• 2000

Objectives：There has been a rapid expansion of studies aimed at elucidating the genetic basis of autistic disorder, especially it’ relationship to fragile-X syndrome. The detection of fragile X chromosome(Xq27.3) by cytogenetic analysis has revealed many difficulties in testing. Therefore, to explore the relationship between autistic disorder and fragile X syndrome, this study administered molecular biologic methods which examined an unstable CGG repeat within the fragile X mental retardation-1(FMR-1) gene. Methods：Ninety nine autistic children and eight normal control children were tested. The number of CGG repeats within FMR-1 gene was measured after amplification by PCR, and cytogenetic analysis was also carried out to detect fragile site Xq27.3. Southern blot hybridization, using StB12.3 and/or Pfxa3 probe, was done for the patients showing expansion of more than 50 CGG repeats (premutation). Results：All but two autistic patients had no expansion in CGG repeats by PCR and there was no significant statistical difference in number of CGG repeat in comparison with normal control. Two autistic patients, considered as premutation by PCR analysis, had no full mutation or premutation by Southern blot hybridization. All autistic children tested did not have any abnormal karyotype or fragile site Xq27.3. Conclusions：These results suggest that autistic patients may not have abnormality in FMR-1 gene or abnormal expansion in CGG repeat. In conclusion, fragile X syndrome may not be antecedent of autistic disorder.