• 대한수의학회지
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• 제35권3호
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• pp.515-530
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• 1995

Coronaviridae에 속하는 transmissible gastroenteritis virus(TGEV)와 porcine epidemic diarrhea virus(PEDV)를 specific하게 detection할 수 있는 방법을 개발하고자 본 연구를 수행하였다. 두 바이러스 모두 RNA 바이러스이기 때문에 reverse transcription-polymerase chain reaction(RT-PCR)으로 nucleocapsid(N) protein gene의 cDNA를 증폭시켰다. SmaI으로 처리한 pTZ19R에 ligation시킨 후 염기서열을 밝히고자 sequencing하였다. 각각의 prototype virus와 비교하여 상동성을 밝혔다. 두 바이러스에 대한 cDNA probe를 제작하여 Southern blot hybridization을 실시하였다. TGEV의 경우 백신주인 P45와 병독주인 Miller strain을 사용하였다. cDNA를 증폭시키기 위해 N1/N1R과 N2/N2R 두 가지 primer를 이용한 결과, N1/N1R primer의 경우 586bp 크기의 PCR product를 얻을 수 있었고, N2/N2R primers로 582bp의 cDNA를 증폭시킬 수 있었다. PEDV 실험을 위하여 PED 임상 증상을 나타내는 분변을 이용하여 RT-PCR을 실시하였다. P2/P2R primer로 753bp의 PCR product를 얻을 수 있었다. TGEV의 두 가지 strain의 N protein gene을 sequencing하여 prototype인 Purdue strain과 염기서열 상동성을 조사한 결과, 97%이상의 높은 homology를 나타내었다. PED-V 역시 N protein gene을 sequencing하여 CV777과 염기서열 상동성을 조사한 결과 97%이상의 homology로 PEDV임을 알 수 있었다. TGEV와 PEDV의 염기서열을 비교한 결과 29%의 낮은 homology를 관찰할 수 있었다. 두 가지 바이러스의 N protein gene에 대한 cDNA probe를 제작하여 Southern blot hybridization을 한 결과, 각 바이러스에 매우 특이적 반응을 나타내었다.

• 한국작물학회지
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• 제41권5호
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• pp.608-618
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• 1996

Glycine속의 야생 다년생종인 G. tomentella가 가지고 있는 내병성, 고단백 및 다년생 유전자등 유용형질들을 재배콩(G. max)에 도입하기 위한 종간교잡시 염색체수가 다른 4개의 G. tomentella (2n=38, 40, 78, 80) 계통들과 재배콩과의 교잡친화성 검정, 정역간 교잡효과, 교잡엽의 발육비교, 종간 교배적기 등에 대해 시험한 결과를 요약하면 다음과 같다. 1. G. tomentella의 염색체수가 2n=38, 40, 78, 80인 4개 계통과 재배콩인 울산콩, 뱀콩, 단엽콩과의 종간 교잡에서 G. tomentella의 염색체수가 2n=38인 계통과 교배된 것이 교배율 및 교배후 경과일수에 따른 협생존률이 가장 양호하여 2n=38계통이 재배콩과의 교잡친화성이 가장 높은 것으로 나타났다. 2. G. max(cv. Baemkong)와 G. tomentella(2n=38)와 종간교잡에서 정역교잡 효과는 G. max를 모본으로 했을 때가 G. tomentella를모본으로 했을 때보다 교배율이 더 높았고, 엽생존기간도 더 길어 정역교잡의 효과가 인정되었다. 3. G. max(cv. Baemkong)와 G. tomentella(2n=38)와의 종간교잡에서 교잡협의 발육상태를 자가수정된 모본의 협과 비교했을 때 G. max를 모본으로 한 것이 G. tomentella를 모본으로 한 것보다 협당 배주수의 감소율이 적었으나, 교배후 경과일수에 따른 협의 장, 폭 및 무게는 G. max를 모본으로 한 경우가 생육이 저조하였다. 4. 자연조건에서 G. max와 G. tomentella(2n=38)와의 종간 교잡을 할 경우 6월 이나 7월에 하는 것보다 대체로 온도와 습도가 높고 단일조건인 8월 중하구에 하는 것이 교배율이 높았다.

• Parasites, Hosts and Diseases
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• 제35권3호
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• pp.203-210
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• 1997

이질아메바 병원성 분리주에서 특이적으로 발현되는 mRNA를 동정하고자 differential display reverse transcription-polymerase chain reaction(DDRT-PCR)을 수행하여 병원성 특이 증폭산물을 확인하였다. 한국인에서 검출한 이질아메바 병원성 분리주 YS-27과 Entamoeba dispar분리주인 S 16으로부터 정제한 mRAN를 주형으로 11개의 arbitrary primer와 3개의 one base anchored $oligo-dT_{11}M$(M: A, C 또는 G)의 조합을 이용, DDRT-PCR을 실시한 결과 31개의 분획이 YS-27주에서만 증폭된 것으로 확인되었다. 이 331개 DNA 중 21개는 cysteine proteinase 유전자와 상동성을 나타내었다. YS-27주로부터 제작된 cDNA library를 나머지 DNA를 탐침으로 사용, 검색하여 최종 4개의 clone을 얻었다. 이 4개의 clone을 이용, immunoscreening을 수행한 결과, 이 clone들은 이질아메바 감염자 혈청과 양성반응을 나타내고 있었다.

• 한국수의병리학회:학술대회논문집
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• 한국수의병리학회 2003년도 추계학술대회초록집
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• pp.27-27
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• 2003

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) infections are considered difficult to distinguish clinically and histopathologically. Prompt differentiation between PEDV- and TGEV-associated enteritis would greatly facilitate the management of disease in countries where PEDV and TGEV are epizootic. Rapid differential diagnosis and treatment are crucial to reducing mortality and morbidity from PEDV- and TGEV-induced enteritis in piglets. The objective for this study was to develop a protocol to differentiate between PEDV and TGEV directly from formalin-fixed, paraffin-embedded tissue, using a multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) assay. (omitted)

• 한국연초학회지
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• 제15권2호
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• pp.161-166
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• 1993

A double - stranded cDNA fragment (436bp) encoding coat protein of tobacco mosaic virus(TMV) was derived from the total 480nucleotides gene after reverse transcription of TMV RNA, and subclorled into a plant expression vector pBl 121, resulting in pBL 430. The plasmid DNA containing this chimeric gene was moved from E. cofi to Agrobacterium tumefaciens strain A28l, and was introduced in시 the tobacco plant by the Agrobocterium Ti - mediated transformtion system. The transformants were selected on a selection media containing kanamycin. The shoots add roots could be differentiated from the explants and whole plants were obtained. From Southern blot hybridization analysis, DNA extracted from transformants, it could be conformed that the chimeric gene fragment was inserted into the genomic DNA of tobacco plant.

• Animal cells and systems
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• 제3권3호
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• pp.311-317
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• 1999

Prolactin (PRL) was reported to be locally synthesized in many brain areas including the hypothalamus, thalamus (TH) and hippocampus (HIP). In the pituitary lactotrophs, PRL synthesis is dependent upon a pituitary-specific transcription factor, Pit-1. In the present study, we attempted to identify Pit-1 or Pit-1-like protein in brain areas known as the synthetic sites of PRL. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis showed the same Pit-1 transcripts in brain areas such as the medial basal hypothalamus (MBH), preoptic area (POA), TH, and HIP with the Pit-1 transcripts in the anterior pituitary (AP). Electrophoretic mobility shift assay (EMSA) was run with nuclear protein extracts from brain tissues using a double strand oligomer probe containing a putative Pit-1 binding domain. Shifted bands were found in EMSA results with nuclear proteins from MBH, POA, TH and HIP. Specific binding of the Pit-1-like protein was further confirmed by competition with an unlabeled cold probe. Antisense Pit-1 oligodeoxynucleotide (Pit-1 ODN), which was designed to bind to the Pit-1 translation initiation site and block Pit-1 biosynthesis, was used to test Pit-1 dependent brain PRL transcription. Two nmol of Pit-1 ODN was introduced into the lateral ventricle of a 60-day old male rat brain. RNA blot hybridization and in situ hybridization indicated a decrease of PRL mRNA signals by the treatment of Pit-1 ODN. Taken together, the present study suggests that Pit-1 may play an important role in the transcriptional regulation of local PRL synthesis in the brain.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2003년도 춘계학술대회
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• pp.107-107
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• 2003

The heavy metal cadmium is a xenobiotic toxicant of environmental and occupational concern and it has been classified as a human carcinogen. Inhalation of cadmium has been implicated in the development of emphysema and pulmonary fibrosis, but, the detailed mechanism by which cadmium induces adverse biological effects is not yet known. Therefore, we undertook the investigation of genes that are induced after cadmium exposure to illustrate the mechanism of cadmium toxicity For this purpose, we employed the polymerase chain reaction-based suppression subtractive hybridization technique. We identified 29 different cadmium-inducible genes in human peripheral mononuclear cells, such as macrophage migration inhibitory factor, lysophosphatidic acid acyltransferase-${\alpha}$, enolase-1${\alpha}$, VEGF, Bax, neuron-derived orphan receptor-1, and Nur77, which are known to be associated with inflammation, cell survival, and apoptosis. Induction of these genes by cadmium treatment was further confirmed by semi-quantitative reverse-transcription polymerase chain reaction. Further, we found that these genes were also induced after cadmium exposure in normal human lung fibroblast cell line, WI-38, suggesting potential use of this induction profile to monitor cadmium toxicity in the lung. Next, Nur77, one of cadmium-inducible genes, was further studied since the products of Nur77 are known to be involved in the apoptotic process of lung cells. Following cadmium treatment, Nur77 gene expression was increased at protein-level in A549 cells. Consistently, the reporter containing Nur77 binding sequence was activated by 2.5-fold after exposure to cadmium in reporter gene analysis by transient transfection experiments. When the plasmid encoding dominant negative Nur77 that represses the transcriptional function of wild-type Nur77 was transfected into A549 cells, the expression of Bax was significantly reduced, suggesting that induction of Nur77 was an important process in cadmium-induced apoptosis in the cells. Cadmium induced the expression of Nur77 in vivo, confirming the relevance of the data obtained in viro. Together our results suggest that Nur77 gene expression in exposure to cadmium leads apoptosis of lung cells which may cause pathological changes in lung.

• Restorative Dentistry and Endodontics
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• 제29권4호
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• pp.399-408
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• 2004

Odontoblasts are responsible for the formation and maintenance of dentin. They are known to synthesize unique gene products including dentin sialophosphoprotein (DSPP). Another unique genes of the cells remain unclear. OD314 was isolated from the odontoblasts/pulp cells of rats and partially characterized as an odontoblast-enriched gene (Dey et al., 2001). This study aimed to elucidate the biological function of OD314, relating to odontoblast differentiation and dentinogenesis. After determining the open reading frame (ORP) of OD314 by transient transfection analysis using green fluorescent protein (GPP) expression vector, mRNA in-situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western analysis were performed. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were expressed in odontoblasts of developing coronal and root pulp. 2. OD314 was a novel protein encoding 154 amino acids, and the protein was mainly expressed in cytoplasm by transient transfection analysis. 3. Mineralized nodules were associated with multilayer cell nodules in the culture of human dental pulp cells and first detected from day 21 using alizarin-red S staining. 4. In RT-PCR analysis, OD314, osteocalcin (OC) and DSPP strongly expressed throughout 28 days of culture. Whereas, osteonectin (ON) mRNA expression stayed low up to day 14, and then gradually decreased from day 21. 5. Western blots showed an approximately 17 kDa band. OD314 protein was expressed from the start of culture and then increased greatly from day 21. In conclusion, OD314 is considered as an odontoblast-enriched gene and may play important roles in odontoblast differentiation and dentin mineralization.

• 한국수정란이식학회지
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• 제24권2호
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• pp.77-87
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• 2009

To understand molecular and cellular mechanisms of many gene products in the female reproductive organs including the ovary and uterine endometrium as well as during embryo development, researchers have developed and utilized many effective methodologies to analyze gene expression in cells, tissues and animals over the last several decades. For example, blotting techniques have helped to understand molecular functions at DNA, RNA and protein levels, and the reverse transcription-polymerase chain reaction (RT-PCR) method has been widely used in gene expression analysis. However, some conventional methods are not sufficient to understand regulation and function of genes expressed in very complex patterns in many organs. Thus, it is required to adopt more high-throughput and reliable techniques. Here, we describe several techniques used widely recently to analyze gene expression, including annealing control based-PCR, differential display-PCR, expressed sequence tag, suppression subtractive hybridization and microarray techniques. Use of these techniques will help to analyze expression pattern of many genes from small scale to large scale and to compare expression patterns of genes in one sample to another. In this review, we described principles of these methodologies and summarized examples of comparative analysis of gene expression in female reproductive organs with help of those methodologies.

• Animal cells and systems
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• 제1권1호
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• pp.143-150
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• 1997

Transcription factor CP2 was identified initially to bind the promoter region of the murine a-globin gene and its activity was shown to increase 2 to 3 fold during the induced differentiation of murine erythroleukemia (MEL) cells. To get further insight into the role of CP2 during development and differentiation, steady-state levels of CP2 message were monitored by using reverse transcriptase (RT)-PCR and in situ hybridization assays in the cultured MEL cells and differentiating embryonic stem (ES) cells in vitro, and in fetal and adult mouse tissues. The amount of CP2 messages increased 3 to 5 fold during induced differentiation of MEL cells, suggesting that the increment of CP2 activity during induced differentiation of MEL cells is originated from the increase of transcription initiation. On the other hand, CP2 expression is not restricted to the erythroid lineage cells; CP2 expressed ubiquitously from the undifferentiated ES cells to adult tissue cells. CP2 transcript was observed even in the undifferentiated ES cells and the level of expression increased from day 8 of the differentiating embryoid bodies. RT-PCR assay in the total RNAs prepared from several tissues of the adult mouse also showed ubiquitous expression profile, although the levels of expression were variable among tissues. When non-radioactive in situ hybridization assay was performed to the paraffin-sectioned whole body mouse embryos at days 11.5, 13.5, and 16.5 after fertilization, variable amounts of positive signals were also detected in different tissues.