RT-PCR 기법을 이용한 mRNA의 정량

예성수 인제대학교 백병원 2002 仁濟醫學 Vol.23 No.2

Although steady state levels of individual RNA transcripts have traditionally been measured by northern blotting, in situ hybridization and nuclease protection assays, these techniques are limited by their sensitivity. For analysis of mRNA expression, RT-PCR is very sensitive technique. It is capable of detecting moderately expressed transcripts from a single cell. Though mainly used for qualitative studies, several modifications of this method have been developed that allow quantitative analyses to be performed. Quantification of transcription via RT-PCR can be approached using either relative RT-PCR or competitive RT-PCR strategy. Quantification by relative RT-PCR involves normalization to a housekeeping gene that is amplified within the same reaction as the target gene. Competitive RT-PCR, as the name implies, utilizes synthetic competitor RNA within the same reaction as the sample RNA. Recently the introduction of the new procedure based on fluorescence-kinetic RT-PCR enables quantification of the PCR product in real-time. The newest form of quantitative RT-PCR is termed real-time RT-PCR, as measurements are taken throughout the reaction due to oligonucleotide cleavage by DNA polymerase that release a fluorescent dye. The purpose of this review is to summarize the RT-PCR-based quantification methods of mRNA expression for useful clinical applications.

마우스 Hepa-1c1c7 세포주에서 B형 간염 바이러스에 의한 tumor necrosis factor-a의 발현 유도

예성수,장원희,양영일,이연재,김미성,석대현,박영홍,백계형,Yea Sung Su,Jang Won Hee,Yang Young-Il,Lee Youn Jae,Kim Mi Seong,Seog Dae-Hyun,Park Yeong-Hong,Paik Kye-Hyung 한국생명과학회 2005 생명과학회지 Vol.15 No.1

B형 간염 바이러스(HBV)에 의한 감염은 인류의 보건에 대단히 중요한 문제이며, 따라서 HBV에 대한 많은 연구가 수행되어져 왔다. 그러나 HBV 연구에 있어서의 주된 장애요인은 그 감염이 사람과 일부 영장류에 국한된다는 점이다. 본 연구에서는 마우스 간암 세포주인 Hepa-1c1c7 세포를 이용하여 HBV의 감염성 및 그에 따른 염증성 사이토카인인 TNF-a의 발현의 변화를 측정하였다. HBV의 표면항원(HBsAg)분비는 microparticle enzyme immunoassay를 사용하여 측정하였고, TNF-a mRNA 발현 측정에는 quantitative competitive RT-PCR 방법을 사용하였다. HBV 발현 벡터를 Hepa-1clc7 세포에 도입시켰을 경우뿐만 아니라 HBV 비리온을 갖고 있는 혈청을 사용하여 Hepa-lc1c7 세포를 감염시켰을 때에도 HBV mRNA 발현 및 HBsAg 분비가 측정되었다. 또한 두 상황 모두에서 TNF-a mRNA발현이 증가됨을 알 수 있었다. 이러한 결과는 사람과 영장류에 특이적인 HBV가 마우스 간암 세포주인 Hepa-lclc7 세포도 감염시킬 수 있다는 가능성을 제시한다. 또한 마우스 기원의 Hepa-lclc7 세포에서도 HBV의 유전자 발현에 필요한 여러 인자들이 존재하며, TNF-a와 같은 사이토카인 유전자 발현을 조절하는 세포 내 기전에 HBV가 영향을 미친다고 할 수 있다. 따라서 마우스 간암 세포주인 Hepa-lclc7 세포는 HBV 연구를 위한 시험 관내 모델로서 사용되어질 수 있을 것으로 보인다. Infection with hepatitis B virus (HBV) is a major health problem worldwide. Although a tremendous amount has been known about HBV, there have been obstacles in the study of HBV due to the narrow host range of HBV limited to humans and primates. In the present study, we investigated the susceptibility to HBV infection of mouse hepatoma cell line, Hepa-1c1c7. In addition, based on that human hepatocytes infected by HBV increase the expression of the pro-inflammatory cytokine TNF-a, the inducibility of TNF-a expression by HBV in the cells was determined. HBV surface antigen (HBsAg) secretion was measured by the microparticle enzyme immunoassay and steady state mRNA expression was analyzed by quantitative competitive RT-PCR. Transient transfection of Hepa-1c1c7 cells with HBV expression vector resulted in a dose-dependent induction of TNF-a expression. Infection of Hepa-1c1c7 cells with the serum of HBV carrier also increased TNF-a mRNA expression. Both in the transfected and infected cells, HBV mRNA was expressed and significant HBsAg secretion was detected. There was no significant variation in $\beta-actin$ mRNA expression by HBV. These results demonstrate that HBV is infectious to Hepa-lc1c7 in vitro and the viral infection induces TNF-a expression, which suggests that Hepa-lc1c7, a mouse hepatoma cell line, may be a possible model system for analysis of various molecular aspects of HBV infection.

마우스 간세포암 기원의 Hepa-1c1c7 세포주에서의 B형 간염 바이러스에 의한 TNF-α발현의 유도

예성수,장원희,양영일,이연재,김미성,백계형 인제대학교 2000 仁濟醫學 Vol.21 No.2

Infection with hepatitis B virus (HBV) is a major health problem worldwide. Although a tremendous amount has been known about HBV. there have been obstacles in the study of HBV due to the narrow host range of HBV limited to humans and primates. In the present study, we investigated the susceptibility to HBV infection of mouse hepatoma cell line, Hepa-Iclc7. In addition, based on that human hepatocytes infected by HBV increase the expression of the pro-inflammatory cytokine TNF-α, the inducibility of TNF-α expression by HBV in the cells was determined. HBV surface antigen (HBsAg) secretion was measured by the microparticle enzyme immunoassay and steady state mRNA expression was analyzed by quantitative competitive RT-PCR. Transient transfection of Hepa-1c1c7 cells with HBV expression vector resulted in a dose-dependent induction of TNF-α expression. Infection of Hepa-lclc7 cells with the serum of HBV carrier also increased TNF-α mRNA expression. Both in the transfected and infected cells, HBV mRNA was expressed and significant HBsAg secretion was detected. There was no significant variation in β-actin mRNA expression by HBV. These results demonstrate that HBV is infectious to Hepa-1c1c7 in nitro and the viral infection induces TNF-α expression, which suggests that Hepa- 1c1c7. a mouse hepatoma cell line, may be a possible model system for analysis of various molecular aspects of HBV infection.

Single Nucleotide Primer Extension 기법을 이용한 SNP 연구

안영욱,예성수,박영홍 인제대학교 백병원 2002 仁濟醫學 Vol.23 No.5

Single nucleotide polymorphisms(SNPs) are the most frequent type of DNA sequence variation of individuals. They are defined by the presence of two alternative bases at a particular position in a DNA sequence and occur about one per 500-1000bp in the human genome. The recent completion of the first reference sequence of the human genome has provided a basis for comprehensive analysis of sequence variation in man. The identification and dense mapping of SNPs is of considerable significance for association studies of complex diseases, pharmacogenetics, population genetics and physical mapping. Their use as genetic markers is favored by their high abundance, low mutation rate and the easy automation of typing. With the development of ABI Prism 3100 or 3700 system, single nucleotide primer extension, we car get a straightforward and large-scale method for validation of or comparative genotyping of known SNPs and point mutations. In the mini sequencing primer extension reaction, a DNA polymerase is used specifically to extend a primer that anneals immediately adjacent to the nucleotide position to be analyzed with a single labeled nucleoside triphosphate complementary to the nucleotide at the variant site. The reactor allows highly specific detection of point mutations and single nucleotide polymorphisms(SNPs) without direct sequencing. Because all SNPs can be analyzed with high specificity at the same reaction conditions, mini sequencing is a promising reaction principle for multiplex high-throughput genotyping assays. It is also a useful tool for accurate quantitative PCR-based analysis. This review discusses the advanced biotechnique.

Kinesin Superfamily KIF1A Protein Binds toSynaptotagmin XI

박혜영,예성수,장원희,정준용,이상경,김상진,양영일,김주영,박영홍,석대현 대한해부학회 2005 Anatomy & Cell Biology Vol.38 No.5

The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF1A is a monomeric motor that conveys synaptic vesicle precursors and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein (s) that interacts with the tail region of KIF1A and found a specific interaction with synaptotagmin XI. The amino acid residues between 830 and 1300 of KIF1A are required for the interaction with synaptotagmin XI. KIF1A also bound to the tail region of synaptotagmin IV but not to other synaptotagmin in the yeast two-hybrid assay. KIF1A interacted with GST-synaptotagim XI fusion proteins, but not with GST alone. An antibody to synaptotagmin XI specifically co-immunoprecipitated KIF1A associated with synaptotagimin from mouse brain extracts. These results suggest that KIF1A motor protein transports of synaptotagmin XI-containing synaptic vesicle precursors along microtubule. 분자 motor로서 큰 superfamily를 형성하는 Kinesin 단백질은 분비소포, 단백질 복합체, 세포 내 각소기관을 운반한다. KIF1A는 단량체의 motor 단백질로서 신경세포의 시냅스 소포 전구체를 시냅스 말단으로 이동시키는 역할이 밝혀졌다. 본 연구에서 우리는 효모 two-hybrid system을 사용하여 KIF1A와 결합하는 신경세포 유래의 단백질을 분리하였다. 결과 KIF1A와 특이적으로 결합하는 synaptotagmin XI을 확인하였다. KIF1A의 830에서 1300 아미노산 부위가 synaptotagmin XI과의 결합에 관여하였다. 또한 효모 two-hybrid assay에서 KIF1A는 synaptotagmin IV, XI와 결합하지만 다른 종류의 synaptotagmin과는 결합하지 않았다. 그리고 단백질간의 결합을 pull-down assay로 확인한 결과 KIF1A는 GST와는 결합하지 않으나 GST결합 synaptotagmin XI 단백질과는 결합하였다. 또한 생쥐의 뇌 파쇄 액에 synaptotagmin XI 항체로 면역침강을 행하여 KIF1A를 확인한 결과 synaptotagmin XI과 같이 침강하였다. 이러한 결과들은 KIF1A는 synaptotagmin XI와 결합하여 synaptotagmin XI이 포함된 시냅스 소포 전구체를 미세소관을 따라 이동시킴을 시사한다.

무증상 B형 간염 보유자에서 한국 야생형 HBV DNA 염기서열의 추론

박영홍,예성수,박기호 인제대학교 백병원 2002 仁濟醫學 Vol.23 No.5

Objective: The aim of this study were to investigate the whole sequence of Korean wild type of HBV including X, C and S regions from asymtomatic HBV carriers and to compare it with other wild types reported previously. Methods and Materials: Sera from 500 healthy person were analysed by HBV-PCR in which positive carriers were subjected to direct sequencing for X, C and S regions. Analysis of regional sequences revealed the tendency of mutations and the sequence of wild type, which were collected and produced the whole sequence. Korean type of HBV was compared with other wild types reported previously. Results: From the PCR analysis, we confirmed 16 carriers(3.2%) all of who had point mutations. The deletion mutations was detected at 4 case of which 2 case had at pre-S1, 1 case at pre-S1 and 1 case at surface gene. Compared with the subtype for Kobayashi, Ono-1, Ono-2, Rho and Sugauchi, sequence variations at 27, 39, 63, 83, 46 sites were found, respectively. Some locations including nt7 at pre-S2, nt705 at S gene and nt2651 upstream to pre-S1 had high frequency of mutations. Conclusion: These observations suggested that Korean wild type of HBV was the most similar to the subtype of Kobayashi and very different from the subtype of Rho which were suspected to the mutants. The high frequency of mutation at some locations including nt7 at pre-S2, nt705 at S gene and nt265l upstream to Pre-S1 might suggest the variants rather than the mutants.

Myoclonic Epilepsy with Ragged-Red Fibers(MERRF) 증후군 환자의 미토콘드리아 tRNALys 유전자에서 점돌연변이의 검출

박영홍,예성수,장원희,박기호 인제대학교 백병원 2002 仁濟醫學 Vol.23 No.5

The author reports a case of myoclonal epilepsy with ragged red fibers(MERRF) syndrome in a patient with confirmed mitochondrial DNA mutation. Diagnosis was made on the basis of the clinical finding, but pathologic finding of muscle tissue didn't reveal the ragged red fiber. The mismatched primer-PCR followed by Nae-IRFLP analysis showed a point mutation from A to G at the 8344th nucleotide located in the mitochondrial tRNALysgene and the heteroplasmy containing wild type and mutant type band. The present report supports the notation that the mitochondrial DNA point mutation at the 8344th nucleotide position is the most common cause of MERRF syndrome. This is the first report of MERRF syndrome analyzed biochemically in Korea and it is necessary to get more detection of MERRF syndrome and the construction of characteristics of pedigree.

만성 B형 간염 환자에서 HBV X, core promoter, precore 변이종의 임상적 중요성

장원희,예성수,백계형,양영일,이연재 대한간학회 2000 Clinical and Molecular Hepatology(대한간학회지) Vol.6 No.4

Background/Aims: There have been much debates on the effects of HBV mutants on the clinical course of HBV-associated chronic liver diseases. The purpose of this study was to define the relationship among HBV mutants, severity of hepatitis and expression patterns of HBcAg Methods: HBV DNA was extracted from the liver tissue of 31 patients who had been HBsAg positive for more than 6 months. The amplification of X was performed as well as core promoter/precore region of HBV DNA by polymerase chain reaction and then direct sequencing of them. Pathologic severity was classified utilizing Scheuer's scoring system and immunohistochemical staing for HBcAg in hepatocytes was performed. The expression patterns of HBcAg were divided into four types according to expression location of HBcAg: Type Ⅰas a nuclear predominant expression of HBcAg; Type II as mixed patterns, combined expression of cytoplasmic and nuclear localization of HBcAg; Type III as a diffuse cytoplasmic expression of HBcAg; and Type Ⅱ as an inclusive dy-like expression in cytoplasm. Results: In investigating the relationship between HBV mutants and clinical findings, ALT, HBV DNA and hepatitis activity index (HAI) in hepatitis with wild HBV were normal to high but those in hepatitis with core promoter or precore mutants were high . There were no statistically significant differences (p=0.062). In terms of the relationship between HBV mutants and the expression pattern of HBcAg, type Ⅰ, Ⅱ, Ⅳ were noticed in hepatitis with wild HBV but in almost all mutants cases type Ⅲ, Ⅱ were noticed (p$lt;0.01). The score of HAI increased as the number of the expression pattern of HBcAg increased from type Ⅰto type Ⅲ or Ⅳ(p$lt;0.05). No relationships among the mutation in X region, the mutations in other regions and clinicopathological severity could be found. Conclusion: The mutation in X, core promoter and precore region had little association with the severity of hepatitis. And the relationship did not exist between precore mutants and X mutants. The expression pa HBcAg could be a useful indicator in determining what stage of chronic hepatitis B is in and whether mutant strains exist(Korean J hepatol 2000;6;425-440)

마그네슘에 의한 혈소판 응집의 억제에 관한 생화학적 기전

박기호,장원희,예성수,박영홍 인제대학교 백병원 2002 仁濟醫學 Vol.23 No.5

Magnesium ion, with high concentration(2mM), is associated with inhibition of platelet activity in several molecular moiety, but biochemical mechanism are not well known. Since the property of a platelet agonist is to potentiate the response by another agonist in synergistic manner and reduce the response by antagonist, combination test between magnesium ion and the classical agonist was performed. Epinephrine showed irreversible aggregation profile, and with magnesium ion, caused a little inhibitory effect on platelet aggregation: this inhibition was synergistically done by indomethacin and ASA: ADP only showed reversible aggregation profile, and combination with magnesium ion also showed inhibitory effects on platelet aggregation. This result may suggest magnesium ion induce inhibition of platelet aggregation not by direct action as inhibitor on each agonist, but by certain mechanism of simultaneous action on several agonists.

Kinesin Superfamily KIF5 Proteins Bind to βIII Spectrin

백재은,김나리,예성수,장원희,정준영,이상경,박영홍,한진,석대현 대한약리학회 2004 The Korean Journal of Physiology & Pharmacology Vol.8 No.3

The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF5 is a heterotetrameric motor that conveys vesicles and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein(s) that interacts with the tail region of KIF5 and found a specific interaction with βIII spectrin. The amino acid residues between 1394 and 1774 of βIII spectrin were required for the interaction with KIF5C. βIII spectrin also bound to the tail region of neuronal KIF5A and ubiquitous KIF5B but not to other kinesin family members in the yeast two-hybrid assay. In addition, these proteins showed specific interactions, confirmed by GST pull-down assay and co-immunoprecipitation. βIII spectrin interacted with GST-KIF5 fusion proteins, but not with GST alone. An antibody to βIII spectrin specifically co-immunoprecipitated KIF5s associated with βIII spectrin from mouse brain extracts. These results suggest that KIF5 motor proteins transport vesicles or organelles that are coated with βIII spectrin.