From Splicing to Editing. ls RNA Processing Evolved with lnside Out Guide Evolution?

최용천 한국분자생물학회 1991 분자생물학뉴스 Vol.3 No.2

핵소체 비대와 암생성

최용천 암연구센터 1994 암연구의최신지견 Vol.1994 No.-

건조 혈액 여과지에서의 β-Globin DNA와 RNA의 추출 및 그 안전성에 관한 검토

김덕인,김인후,김정만,한진영,오성용,최용천 東亞大學校附設遺傳工學硏究所 1996 遺傳工學硏究 Vol.- No.3

연구배경: Guthrie spot는 1963년부터 신생아 유전 질환의 선별검사에 사용되기 시작한 이후로 최근에는 DNA와 RNA를 이용한 분석에도 응용이 시도되고 있다. 저자들은 일반 여과지에 채취된 건조 혈액으로부터 β-globin 유전자의 DNA와 RNA를 추출하는 데에 있어서 여과지의 전처리 조건이 미치는 영향과 특히 장기 보관에 따른 RNA의 안정성을 살펴보고자 하였다. 방법: 전처리를 하지 않은 경우, 5% HCI로 전처리를 한 경우, 고압 멸균을 한 경우, 그리고 5% HCI 전처리 후 고압 멸균을 한 경우의 네가지로 나누어서 정상인의 혈액을 떨어뜨린 후 상온에서 2주 동안 보관하였다가 각각 DNA와 RNA를 추출하여 PCR과 RT-PCR을 실시하였다. 그리고 2년간 보관하였던 전처리를 하지 않은 건조 혈액 여과지에서 RNA를 추출하여 RT-PCR을 시행하고 염기서열 분석을 하여 β-globin 유전자인지를 확인하였다. 결과: 여과지의 전처리 여부에 관계없이 DNA와 RNA는 PCR에 의해 기대된 크기의 증폭 산물을 나타내었고, 2년간 장기 보관하였던 여과지에서도 RNA가 안정하게 보존되어 있어서 RT-PCR 생성물을 염기서열 분석한 결과, β-globin 유전자의 일부임을 확인할 수 있었다. 결론: 건조 혈액 여과지를 이용한 β-globin 유전자의 DNA와 RNA 분석은 검체의 채취와 보관이 용이하고 안정성이 높아서 혈색소 이상증을 포함한 유전 질환의 대규모 연구나 선별 검사에 매우 유용할 것으로 사료된다. Background: Since newborn screening efforts were facilitated by the use of Guthrie spots in 1963, the development of a routine procedure for microextraction of DNA and recently even RNA from these specimens would allow direct screening at the molecular level. The purpose of the current investigation was to optimize pretreatment condition of filter paper for improvement of microextraction and stability of β-globin DNA and RNA from dried blood specimens on Whatman filter papers. Methods: Normal whole blood was spotted on filter papers pretreated by four different ways, respectively ; untreated, 5% HCI treated, autoclaved, and 5% HCI treated and autoclaved. The samples were then stored at room temperature for two weeks until processed. DNA and RNA were extracted from each filter paper and amplified by PCR(polymerase chain reaction) and RT(reverse transcriptase)-PCR. We also extracted RNA from untreated dried blood spot stored at room temperature for two years for cDNA synthesis and did nucleotide sequencing to identify that the PCR products were part of β-globin gene. Results: DNA and RNA were stable in filter papers, and could be microextracted for PCR and RT-PCR regardless of pretreatment conditions. It also has been demonstrated that RNA could be obtained from dried blood spots stored for two years in sufficient quality and quantity for amplification and sequencing. Conclusion: The prolonged stability of β-globin DNA and RNA in these specimens will be very helpful for conducting a mass screening and research in genetic diseases including hemoglobinopathies principally because of the ease of sample collection, storage, handling, and shipment.

Relux를 이용한 가로등 배열에 따른 배광 시뮬레이션

최용천(Yong-Chun Choi),윤아영(A-Young Yun),어익수(Ik-Soo,Eo) 대한전기학회 2011 대한전기학회 학술대회 논문집 Vol.2011 No.7

건조 혈액 여과지에서의 β-Globin DNA와 RNA의 추출 및 그 안정성에 관한 검토

한진영,김정만,오성용,김덕인,최용천,김인후,한진영,최용천,김인후 大韓血液學會 1995 大韓血液學會誌 Vol.30 No.2

A Simple Histochemical Method to Characterize Nucleolar Components using Isolated Nuclei

Choi,Yong Chun,Lee,Hye Jeong,Jeong,Jin Sook,Kim,In Hoo 東亞大學校附設遺傳工學硏究所 1994 遺傳工學硏究 Vol.- No.1

핵소체의 형태를 연구할 때 그 재료로는 조직, 세포 또는 분리한 단백질 등이 사용되며 광학 현미경상 1,000배의 고배율이 필요된다. 본 연구에서는 면역조직화학(immunohistochemistry) 방법으로써 분리한 핵을 이용하여 200 배의 배율로써 핵소체를 관찰할 수 있는 간단한 기법을 개발하였다. 이 방법의 장점은 조직 절편을 만드는 불편함이 없고, 분리한 핵에 간단한 조작만이 필요하므로 시간도 적게 걸린다. 그러므로 핵소체의 형태변화를 연구하는데 많은 도움이 될 것으로 사료된다. A simple method was described bo characterize nucleolar structures using isolated nuclei. This method is to apply immunohistochemistry to isolated nuclei using various monoclonal antibodies as probe for study of nucleolar morphology. The results indicate that the simple method offers advantages over other methods and can be used as a routine standard method for observation of isolated nuclei.

핵소체 단백 B23과 세포질 단백 p80의 유사성에 관한 연구

이혜정,윤상인,최용천,안영수,Lee, Hye-Jeong,Yoon, Sang-In,Choi, Yong-Chun,Ahn, Young-Soo 대한약리학회 1995 대한약리학잡지 Vol.31 No.2

Protein B23 is one of the major nucleolar phosphoproteins associated with pre-ribosomal particles, and is localized in the granular region of the nucleolus. Recent studies suggest that protein B23 shuttles between nucleus and cytoplasm and also interacts with HIV Rev. These findings indicate that protein B23 is important in nucleocytoplasmic relationship and viral replication. However, the exact function of protein B23 is not clear yet. In acute nucleolar hypertrophy of rat liver, treated with thioacetamide, there was observed an increase of not only protein B23 but also B23-like protein p45 when anti-B23 monoclonal antibody (MAb) was used for identification. On the basis of the large B23 specific epitope structure composed of 68 amino acids, a hypothesis was formulated to examine that p45 is the pre-B23 resulting from excessive production of B23. In an attempt to investigate the precursor of B23, we analyzed the subcellular fractions and microsomal subfractions. Subsequently, we analyzed the finger printings of B23-like proteins using the tryptic peptide mapping. The results are summarized: 1) Using B23 MAb, we observed the presence of B23-like proteins in nucleolar fraction, nucleoplasmic fraction and microsomal fraction. 2) In the further microsomal subfractionation, we could partially purify B23-like protein in 2M layer of sucrose gradient. 3) When ion exchange chromatography was employed, there were protein species 80kDa(p80), 65kDa(p65) and 60kDa(p60). 4) Based on the tryptic map analysis of $^{125}I$ labeled proteins, the similarity between B23 and p80 was found only in 9 out of 14(B23) and 21(p80) peptides, and difference was found in the remaining peptides. p80 and p60 had 18 common peptides, and all the peptides of p60 were similar to those of p80. From these results, it is proposed that p45 is an abnormal metabolite resulting from carcinogenesis by thioacetamide, and it is not the precursor of B23. In addition, we suggest that p80 may be a precursor of p45.

Peculiarity of Osteogenic Sarcoma Cell Membrane

LEE, Hye-Jeong,KIM, Yi-Tae,CHOI, Yong-Chun 동아대학교의과대학 1997 동아의대학술지 Vol.9 No.1

Types of Nucleolar Hypertrophy in Rat Liver

Jeong,Jin Sook,Kang,Hong Ja,Lee,Hye Jeong,Choi,Yong Chun 東亞大學校附設遺傳工學硏究所 1995 遺傳工學硏究 Vol.- No.2

The mechanism of nucleolar hypertrophy was investigated in various hypertrophogenic conditions of rat liver. Induction of nucleolar enlargement was made by partial hepatectomy, thioacetamide treatment or partial hepatectomy after thioacetamide treatment. In those experimental conditions, the nucleolar structures were characterized by staining technology for morphology and also by immunological analysis for molecular characterization. Based on the immunological studies using a proliferative antigen p120, the nucleolar hypertrophy could be classified into the proliferative hypertrophy type and the non-proliferative type. The most prominent enlargement was observed in the thioacetamide treated hepatocytes where B23 increased at the highest levels among other nucleolar proteins.

In Vitro Regeneration of Carcinogen Thioacetamide Treated Rat Hepatocytes

유소영,김규원,이혜정,최용천,Yoo, So-Young,Kim, Kyu-Won,Lee, Hye-Jeong,Choi, Yong-Chun The Korean Society of Pharmacology 1996 대한약리학잡지 Vol.32 No.3

Thioacetamide는 non-genotoxic 발암제로서 세포단백의 변형을 초래하는 것으로 알려져 있으며 이것을 단기간 처리하면 핵소체의 비대를 초래하게 된다. 본 연구에서는 thioacetamide를 처리한 간세포를 in vivo와 in vitro 상태에서 관찰하였다. In vivo상태로서 thioacetamide를 쥐의 복강에 7일간 주사하면 (50mg/kg), 핵소체 비대와 B23 및 MAP kinase와 같은 신호전달분자들이 증가하는 것을 관찰할 수 있었다. In vitro 상태로서 쥐의 간장을 collagenase로 분리하여 유전자 치료에 사용될 수 있는 배양조건으로 간세포를 배양하여 핵소체를 관찰한 결과 핵소체 비대가 현저하였으며, B23의 양도 증가하였다. 본 실험의 결과로 미루어 볼 때, 간세포는 핵소체 비대 수용능력이 약 100배 이상이라고 할 수 있으며, thioacetamide 처리에 의하여 라이보좀 생성과 핵소체 증가 능력이 증폭되어 나타나는 것으로 사료된다. Thioacetamide is a non-genotoxic carcinogen, a protein modifying agent. It causes nucleolar hypertrophy in short term treatment. In the present work, thioacetamide treated hepatocytes were observed in vivo and in vitro conditions. After 7 day treatment of rat liver with thioacetamide, the hepatocyte nucleoli were enlarged and their signalling molecules such as B23 and p38 MAPK were increased. When these hepatocytes were released by collagenases and were grown under the conditions of gene therapy grade tissue culture system, the enlarged nucleoli were further enlarged. The B23 content was again increased under in vitro conditions. From these experiments, it is clear that the hepatocytes possess approximately 100 fold flexibility of nucleolar capacity. It is suggested that thioacetamide enhances the ribosome genesis and exaggerates the nucleologenesis ability.