• 식물조직배양학회지
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• 제25권1호
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• pp.45-50
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• 1998

꽃양배추의 하배축 조직을 proteinase inhibitor II 유전자가 도입된 Agrobacterium tumefaciens LBA 4404와 2일간 pH 5.5로 조절된 MS 액체배지에서 공동배양후 carbenicillin 500mg/L kanamycin 20mg/L와 BA 1mg/L가 함유된 MS 재분화배지에 옮겼다. 이들 조직을 매 2주마다 계대배양하였으며 약 4주후에 kanamycin 저항성 개체를 얻었다. 형질전환된 것으로 추정되는 식물체는 kanamycin 30mg/L가 함유된 선발배지에서 생존하였다. PCR 분석결과, PI-II 유전자가 형질전환체의 게놈상에 삽입되어 있음을 확인하였다. 형질전환체의 Southern blot 분석을 통하여 ECL-labelling된 PI-II 유전자와 동일한 것으로 판단되는 약 500bp 위치에서 밴드를 확인할 수 있었다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제29권2호
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• pp.86-94
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• 2003

It has been known that transglutaminase C (TGase C, TGase II) is directly participated in the DNA organization of chromosome, and affects the cellular processes such as proliferation, differentiation, and apoptosis of cells, but still not known what mechanism is working on. In this study, the cytogenetic and the immunohistochemical methods were used to observe the TGase C expression in the nuclear chromosome of the proliferating cells, especially in mitotic stage. The human gastric adenocarcinoma (SNU-1) cell line was used for immunohistochemistry and antisense inhibition study in vitro. The present study was also aimed to disclose the efficiency of antisense inhibition by using antisense oligonucleotide DNA labeled with fluorescence, and found that anti-TGase C probe was diffusely infiltrated into the cytoplasm and the nucleus of the cell. By the antisense inhibition the nuclei of SNU-1 cells became rough nuclear shape, as they were greatly reduced in TGase C immunoreactivity both for the normal and apoptotic SNU-1 cells. However, it is clearly presumed that the TGase C directly interacts with the chromosome of SNU-1 cells and it may play an important role in the division and organization of the chromosome during the mitotic stage.

• BMB Reports
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• 제35권6호
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• pp.562-567
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• 2002

In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed. Then VP39 was used as bait. The host actin gene was isolated from the cDNA library with the yeast two-hybrid system. This demonstrated that VP39 could interact with its host actin in yeast. In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39. The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter. The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin. Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39. Red actin cables were also formed in the cytoplasm at the same time. Actin was aggregated in the nucleus 9 h after the transfection. The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin. Such a combination would result in the actin skeleton rearrangement.

• BMB Reports
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• 제37권5호
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• pp.546-551
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• 2004

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.310-320
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• 2005

A membrane-based oligonucleotide array was used to detect predominant bacterial species in human fecal samples. Digoxygenin-labeled 16S rDNA probes were generated by PCR from DNA that had been extracted from fecal samples or slurries. These probes were hybridized to an array of 120 oligonucleotides with sequences specific for 40 different bacterial species commonly found in human feces, followed by color development using an alkaline phosphatase-conjugated antibody and NBT /BCIP. Twenty of the species were detected by this method, but E. coli, which was present at $\~$1 $\times 10$^5$ CFU per gram feces, was not detected. To improve the sensitivity of this assay, reverse transcriptase-PCR was used to generate probes from RNA extracted from fecal cultures. Coupled with a chemiluminescence detection method, this approach lowered the detection limit for E. coli from $\~1$ $\times 10$^6$ to ${\leq}$ 1 $\times 10$^5$ These results indicate that the membrane-array method with reverse transcriptase-PCR and chemiluminescence detection can simultaneously identify bacterial species present in fecal samples at cell concentrations as low as${\leq}$ 1 $\times 10$^5$ CFU per gram.

• Applied Biological Chemistry
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• 제38권6호
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• pp.512-515
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• 1995

동결건조후 재수화 방법이 Streptomyces aureofaciens 생존도에 미치는 영향을 실험하기 위해 $^3H-adenine$을 DNA에 주입시킨후 유출되는 방사선량과 균을 전자현미경으로 관찰하였다. 그 결과 Streptomyces aureofaciens의 손상은 동결건조 과정에서 일어나는것 보다 재수화 과정에서 공기를 허용한 상태에서 재수화시 크게 일어났으며, 그 생존도는 약 20%이고, 공기가 없는 밀봉된 진공상태의 이중 용기에서 재수화시 약 91%로 나타났다. 공기를 허용한 개봉된 이중 용기에서 증류수에 잠긴채 재수화한 생존도는 약 36%이며, 이중 용기에 $N_2-gas$를 주입하여 밀봉된 상태에서 재수화한 경우 약 83% 생존도를 나타냈다. 따라서 생존도는 재수화 과정에서 산소의 영향을 매우 크게 받는 것으로 나타났다.

• Parasites, Hosts and Diseases
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• 제60권3호
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• pp.201-205
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• 2022

Babesia microti is one of the most common causative agents of babesiosis. A sensitive and rapid detection is necessary for screening potentially infected individuals. In this study, B. microti cytochrome c oxidase subunit I (cox1) was selected as the target gene, multiple primers were designed, and optimized by a recombinase-aided amplification (RAA) assay. The optimal primers and probe were labeled with fluorescein. The sensitivity of fluorescent RAA (fRAA) was evaluated using gradient diluents of the cox1 recombinant plasmid and genomic DNA extracted from whole blood of B. microti infected mice. The specificity of fRAA was assessed by other transfusion transmitted parasites. The analytical sensitivity of the fRAA assay was 10 copies of recombinant plasmid per reaction and 10 fg/µl B. microti genomic DNA. No cross-reaction with any other blood-transmitted parasites was observed. Our results demonstrated that the fRAA assay would be rapid, sensitive, and specific for the detection of B. microti.

• 핵의학기술
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• 제18권1호
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• pp.153-157
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• 2014

Anti double-stranded DNA (Anti-ds DNA)항체의 검출은 SLE의 진단에 중요하며 American College of Rheumatologists의 SLE 진단기준에 포함되어 있다. 또한 SLE 질병의 활성도와 Anti-ds DNA 항체 수준의 상관성이 보고되어 있으며 Anti-ds DNA 항체 정량검사는 SLE의 치료 전, 후 추적에 매우 유용하다. Anti-ds DNA 항체의 검출을 위한 검사 방법으로 방사면역측정법(radioimmunoassay, RIA)을 이용한 Farr assay, Crithidia luciliae를 이용한 면역형광 측정법(immunofluorescence Test, CLIFT), 효소면역측정법(enzyme-linked immunosorbent assay, ELISA), 화학발광면역 측정법(chemiluminescence immunoassay, CLIA)이 있다. 본원에서 방사면역측정법(radioimmunoassay, RIA)으로 Anti-ds DNA 항체 검사 과정에서 lipemic한 검체의 경우 침전물의 형성이 원활하지 않거나 침전물도 같이 흡입되는 상황이 발생 하였고, 이러한 문제점을 해결하기 위해 lipemic 검체가 분석 결과에 미치는 영향 정도를 평가하였다. 2012년 9월부터 2013년 2월까지 Anti-ds DNA 항체 검사가 의뢰된 검체 중 lipemic한 검체(n=81)를 선택하여 마이크로 원심분리기로 전처리(고속 원심분리: 14,000 rpm 5 mins)한 후 동시에 Anti-ds DNA 항체 검사(Anti-ds DNA kit, Trinity Biotech, Ireland)를 시행하였다. 실험군 1 (lipemic 검체의 Anti-ds DNA 항체 농도 ${\leq}7IU/mL$)에서 y=0.3636x+4.7322, $R^2=0.0238$, Pearson 상관계수는 0.154, paired t-test (P=0.003), Difference (%) mean 65.7의 결과를 얻었으며 통계적으로 유의한 차이를 보였다. 그러나 실험군 2 (lipemic 검체의 Anti-ds DNA 항체 농도 ${\geq}8IU/mL$)에서 y=0.9837x+0.2982, $R^2=0.994$, Pearson 상관계수는 0.997, paired t-test (P=0.181), Difference (%) mean -5.53을 보였고 통계적으로 유의한 차이가 없음을 확인할 수 있었다. 임상에서 SLE (systemic lupus erythematosus)의 진단에 중요한 역할을 하는 Anti-ds DNA 항체 검사는 lipemic 검체의 영향을 배제하기 위해서 반드시 전처리(고속 원심분리: 14,000 rpm 5 mins) 과정을 통해 혈청 내 지질 성분을 제거한 후 검사를 시행하여야 한다.

• Journal of Periodontal and Implant Science
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• 제26권4호
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• pp.841-858
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• 1996

Epidermal growth factor(EGF) is one of polypeptide growth factors. EGF has been reported as a biological mediator which regulates activities of wound healing process including the cell proliferation, migration and metabolism. The purposes of this study is to evaluate the effects of EGF on the human periodontal ligament cells and human gingival fibroblast cells that promote regeneration of periodntal tissue. The mitogenic effects of epidermal growth factor on human periodontal ligament cells and human gingival fibroblasts were evaluated by determining the incorporation of 5-Bromo-2'-deoxy-uridine into DNA of the cells in a dose dependent manner. The prepared cells were the primary cultured gingival fibroblast and periodontal ligament cells from humans, the fourth or sixth subpassages were used in the experiments. Cells were seeded in DMEM containing 10% FBS. 1, 10, 50, 100, $200{\eta}g/ml$ and epidermal growth factor were added to the quiescent cells for 24 hours, 48 hours and 72 hours. They were labeled with $10\{mu}l/200{\mu}l$ 5-Bromo-2'-deoxy-uridine for the last 6 hours of each culture. The results of the five determinants were presented as mean and S.D.. The results were as follows : The DNA synthetic activity of human gingival fibroblasts were increased dose dependently by epidermal growth factor at 24 hours, 48 hours and 72 hours. The mitogenic effects were similar at the 24 and 48 hours of epidermal growth factor, but the DNA synthetic activity of human gingival fibroblasts generally decreased at 72 hours. The DNA synthetic activity of human periodontal ligament cells were increased dose dependently by epidermal growth factor at 24 hours but the DNA synthetic activity decreased at $200{\eta}g/ml$ of each hour. Generally the maximum mitogenic effects were observed at the 48 hours application of epidermal growth factor. The DNA synthetic activity of human periodontal ligament cells generally decreased lower at 24, 72 hours than at 48 hours the application of epidermal growth factor. In the comparison of DNA synthetic activity between human gingival fibroblasts and human periodontal ligament cells, human periodontal ligament cells had slightly higher proliferation activity than human gingival fibroblasts for a longer time at the high dosage of the epidermal growth factor. In conclusion, epidermal growth factor have important roles in the stimulation of DNA synthesis in human periodontal ligament cells and human gingival fibroblasts, and thus may be useful for clinical applications in periodontal regenerative procedures.

• Bulletin of the Korean Chemical Society
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• 제31권7호
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• pp.2011-2014
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• 2010

The fluorescence intensity of a single-stranded oligonucleotide containing a fluorene-labeled deoxyuridine $(U^{Fl})$ unit increases by only 1.5-fold upon formation of its perfectly matched duplex. To increase the fluorescence signal during hybridization, we positioned a quencher strand containing a deoxyguanine (dG) nucleobase, functioning as an internal quencher, opposite to the $U^{Fl}$ unit to reduce the intrinsic fluorescence upon hybridization with a probe. From an investigation of the optimal length of the quencher strand and the effect of the neighboring base sequence, we found that a short strand (five-nucleotide) containing all natural nucleotides and dG as an internal quencher was effective at reducing the intrinsic fluorescence of a linear beacon; it also exhibited high total discrimination factors for the formation of perfectly matched and single base-mismatched duplexes. Such assays that function based on clear changes in fluorescence in response to single-base nucleotide mutations would be useful tools for accelerating diagnoses related to various diseases.