• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1993년도 제1회 추계심포지움 and 제2회 생리분자과학연구센터워크숍
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• pp.17-20
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• 1993

As a step toward a more complete understanding of the molecular actions of TNF, we prepared a cDNA library from TNF-treated human FS-4 fibroblasts and used differential hybridization to identify cDNA clones corresponding to mRNAs enriched in TNF-treated eells. In Quiescent FS-4 cells n induces an increase in the level of some mRNAs within 20 to 30 min. Some of these immediate-early response mRNAs are elevated only transiently for about 30 to 120 min, e. g., c-fos and c-myc (Lin and Vilcek,1987) or the transcription factor IRF-1 (Fujita et al.1989). Such immediate-early gene products may be important for the activation of other genes, but their transient induction suggests that they are not the actual effector molecules responsible for the phenotypic changes induced by TNF. We chose a 3-h incubation with W because we were seeking cDNAs corresponding to messages that are more stably elevated after TNF treatment. Indeed, the results shown in Figure 8 and 9 indicate that all of the mRNAs corresponding to the eight TSG cDNAs isolated remained significantly elevated after 16h of continuous treatment with TNF, and their kinetics of induction were clearly different from those of the immediate-early response mRNAs such as c-fos, c-myc or IRF-1. Nevertheless, only the induction of TSG-21 (collagenase) and TSG-27 (stromelysin) nNAs was completely inhibited by cycloheximide and the induction of TSG-37 (metallothionein-II) was reduced in the presence of this inhibitor of protein synthesis. Induction of the other five TSG mRNAs by TNF was completelyresistant to cycloheximide, suggest ins that no protein intermediate is needed for the upregulation of these mRNAs.

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2001년도 제44회 춘계 학술연구 발표자료
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• pp.54-54
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• 2001

No Abstract. See Full-text

• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.544-550
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• 2005

To develop a highly efficient mammalian expression vector, a series of vectors were constructed based on the murine cytomegalovirus (MCMV) immediate-early (IE) promoter and human ${\beta}$-globin second intron. The resulting MCMV promoter was several-fold stronger than the HCMV promoter in various mammalian cell lines, such as the NIH3T3, Neuro-2a, 293T, and HT1080 cell lines, and was only slightly weaker than the HCMV promoter in HeLa and CHO cells. The inclusion of the human ${\beta}$-globin second intron behind the MCMV promoter or HCMV promoter markedly enhanced the promoter activity in various mammalian cell lines, and the resultant MCMV/Glo-I expression system was stronger than the HCMV promoter from 4.7- to 11.2-fold in every cell line tested. Also, the MCMV/Glo-I promoter induced a higher level of the VSV-G protein in a transiently transfected 293T cell line, which is useful for the production of recombinant retrovirus and lentivirus vectors.

• 한국동물학회지
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• 제36권4호
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• pp.487-495
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• 1993

TRH (Thvrotropin-Releasing Hormone) known to regulate the transcription of the TSH (Thyroid-Stimulating Hormones gene in pituitary cells, but little is understood about the mechanism(sl involved. re present study was attempted to elucidate the role of Ca2+ movement through the voltage-gated channels in the regulation of TSH gene transcription. The c-fos is one of immediate early genes and used as model system for the investigation of signaling pathwavs involved in various stimuli. The changes of c-fos mRNA levels were determined after treatment of various agents using Northern and slot hybridization analysis. The c-fos mRNA was rapidly and transiently induced by TRH (about 3-fold) in GH3 cells and this induction was repressed by calcium chelating agent (EGTA), calcium channel blocker (verapamil) anti protein kinase C inhibitor (aminoacridine). The abilities of forskolin (adenvlate cvclase activators, PMA (protein kinase C activator), and A23187 (calcium ionophore) to affect c-ios gene transcription, either alone or in combination with TRH were tested in the same cells. All of them significantly increased the level of c-fos mRUA. However, no additive relationship was observed in all combined treatments except forskolin. These results suggest that TRH action on the c-fos gene activation is mediated by calcium influx as well as through protein kinase C.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.180-180
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• 1996

Previous studies have shown that kainic acid (KA) causes an elevation of hippocampal proenkephalin mRNA level. However, the role of proto-oncogene products, such as c-Fos, c-Jun and Fra proteins in the regulation of KA-induced proenkephalin mRNA increase in the hippocampus has not been well characterized. Thus, in the present study, the effect of cycloheximide (CHX) on KA-induced proenkephalin mRNA and immediate early gene products induction was examined. After pretreating with either vehicle or CHX (20 mg/kg, s.c.) for 30 min, KA (10 mg/kg) was administered s.c. The animals were sacrificed 1,2, or 8 hrs after KA administration. Total RNA and were isolated for Northern blot assay, and proteins were isolated for Western and electrophoretic gel-shift assays. First, we found that CHX inhibited KA-induced proenkephalin mRNA increase without altering intracellular proenkephalin protein level. Secondly, Western blot assays showed that KA increased c-Fos, c-Jun and Fra proteins at 1,2, and 8 hrs and CHX inhibited these immediate early gene products. Finally, electrophoretic gel shift assays revealed that KA increased both AP-1 and ENKCRE-2 DNA binding activities. Furthermore, CHX attenuated KA-induced AP-1 and ENKCRE-2 DNA binding activities. Both AP-1 and ENKCRE-2 DNA binding activities were abolished by cold AP-1 or ENKCRE-2 oligonucleotides, and further reduced by antibodies against c-Fos or c-Jun. Antibody against CREB reduced ENKCRE-2, but not AP-1, DNA binding activity. Our results suggest that on-going protein synthesis is required for elevation of hippocampal proenkephalin mRNA level induced by KA. All c-Fos, c-Jun, and Fra proteins appears to be involved in the regulation of hippocampal proenkephalin mRNA level induced by KA (This study was supported by a grant from KOSEF).

• 한국미생물·생명공학회지
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• 제29권1호
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• pp.18-24
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• 2001

누에 핵다각체병 바이러스의 gp64 프로모터를 이용한 transient 발현 벡터를 제작하기 위해서 gp64 유전자의 구조를 분석하였다. Southern blotting 분석을 통해 genome DNA에서 gp64 유전자를 탐색하기 gp64 구조유전자를 포함하는 2,277 nucleotide의 염기를 분석하였으며 gp64의 early, late 프로모터 발현을 조절하는 인자들을 확인하였다. gp64프로모터를 이용한 transient 발현 벡터를 제작하고 외래유전자로써 lacZ 유전자를 Bm5 세포주에서 transient 발현시켰다. 세포주 내에 도입된 플라스미드 DNA의 안정성을 확인하였으며, gp64 프로모터의 외래유전자 발현성 여부를 조사하기 위하여 gp64 프로모터 하에 laxZ 유전자를 가지는 재조합 바이러스를 제작하고 $\beta$-galactosidase in 냐셔 staining을 수행한 결과 전체적인 발현량은 매우 약한 것으로 판단되었다. BmNPV-K1의 gp64 프로모터를 이용한 벡터는 더욱 민감한 표지 유전자를 발현시켜 재조합 바이러스의 분리에 이용하거나 숙주세포에 독성을 보이는 유전자 산물의 소량 발현에 더욱 유용할 것으로 판단된다.

• 대한바이러스학회지
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• 제28권3호
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• pp.267-274
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• 1998

Human cytomegalovirus (HCMV) has the ability to activate the expression of many viral and cellular genes. Among various viral proteins, the immediate early proteins (IE1-72kDa, IE2-86kDa) have been known to be potent transactivators. The product of c-jun proto-oncogene is important in cell activation and differentiation. Here, we tried to find out if the IE could activate the c-jun promoter and also tried to identify the responsible sequence elements in the c-jun activation by IE1-72kDa. We found HCMV IE expression transactivated the c-jun promoter in human embryonal lung fibroblasts (HEL). The activation fold by IE1-72kDa, IE2-86kDa and IE2-55kDa was 23, 35, and 5, respectively. When the expression of each IE was combined, it showed synergism. Expression of (IE1-72kDa + IE2-86kDa) and (IE1-72kDa + IE2-86kDa + IE2-55kDa) resulted in 131 and 162 fold increase, respectively. The c-jun promoter region between -117 and -59 contains binding sites for the transcription factors Spl, CAAT, AP-l like (ATF/CREB), and MEF2. Transient expression assays were performed using various reporter plasmids containing the c-jun promoter-regulatory region linked to the luciferase gene and a plasmid expressing HCMV IE1 gene. Deletional and point mutational analysis showed that the sequence between -225 to -160 and the CTF binding site were involved in the up-regulation of c-jun promoter.

• 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.

• Journal of Microbiology
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• 제39권4호
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• pp.314-320
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• 2001

Macrophages stimulated by lipopolysaccharide(LPS) from gram negative bacteria undergo activation of a group of immediate early genes including Rantes. The mouse Rantes gene promoter region contains an LPS rsponsive element(LPE) We detected 3 specific bands termed B1, B2 and 3 formed by the interaction of the LPE and proteins found in LPS-stimulated RAW 367.7 cells. An additional band B4 was determined to be an Ap-1 binding protein. The B1 band appears within 1 hour of LPS nuclear extracts from LPS-stimulation, and this protein kinase enhances B1 and formation. The B1 band can be converted to band B2/B3 by adding specific heparin column fraction purified Ser/Thr specific protein phosphatases PP-1 and PP-2A can stimulate the same conversion to about the same extent. Thus, the formation of the LRE sequence binding complex appears to be regulated by Ser/Thr protein kinase and one or more Ser/Thr specific phosphatases. At least four proteins are involved in the trgulation of the LRE-dependent Rants experssion: two binding factors that bind directly to the target sequences. and two factors that control their binding. The future purification and characterization of these binding pro-teins will reveal in detail the mechanism of Rantes gene activation after LPS stimulation.

• 대한바이러스학회지
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• 제29권2호
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• pp.137-144
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• 1999

Transformation efficiency, virus multiplication and foreign gene expression were characterized in the insect cells transformed with Autographa californica nuclear polyhedrosis virus (AcNPV) immediate early 1 gene (IE1). Transformation efficiency of insect cells by AcNPV IE1 gene vector horboring foreign gene was approximately 8-fold higher in the Sf9 cells transformed previously with AcNPV IE1 gene than in the normal Sf9 cells. Virus multiplication and foreign gene expression of recombinant baculovirus in the Sf9 cells transformed with AcNPV IE1 gene were similar to those of the normal Sf9 cells. These results suggest that transformed cells displaying foreign gene product by using AcNPV IE1 gene promoter will be useful for the diverse applications of insect cells.