KISEP Original Articles 생물정신의학 Vol. 6, No. 1, June 1999 PC12 세포와 A123.7 세포에서차별적으로발현되는유전자의검색 * 백승연 양병환 * 채영규 ** Screening of Differentially Expres

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1 KISEP Original Articles 생물정신의학 Vol. 6, No. 1, June 1999 PC12 세포와 A123.7 세포에서차별적으로발현되는유전자의검색 * 백승연 ** 양병환 *** 채영규 ** Screening of Differentially Expressed Genes between PC12 Cells and A123.7 Cells* Seung-Youn Baik, M.Sc.,** Byung-Hwan Yang, M.D.,*** Young-Gyu Chai, Ph.D.** ABSTRACT T he camp-dependent protein kinasepka is an intracellular enzyme with serine-threonine kinase activity that plays a key role in cell growth, differentiation, and apoptosis in eukaryotes. In order to understand the PKA signal transduction pathway regulating cell life cycle and identify its role, we focused on the characterization of up-/down-regulated genes by PKA using the differential display polymerase chain reaction. Seven differentially expressed sequence tagsdest have been obtained. Among these DESTs, 2 DESTs were homologous to the sequence of genes from BLAST search result. KC15 DEST that was up-regulated in A123.7 cells was highly corresponded to mouse apoptosis-related genema-3 or mouse mrna for topoisomerase inhibitor suppressedtis. MA3 was induced in various types of apoptosis, specially in NGF-deprived apoptotic PC12 cells. TIS was down-regulated in the RVC lymphoma cells incubated with topoisomerase inhibitor that induces DNA strand breakages. PG11 DEST that was highly expressed in PC12 cells was corresponded to transposon Tn10 3 -end. Tnansposon Tn10 was up-regulated in differentiated myeloblastic ML1 cells by 12Otetradecanoylphorbol13acetate. This study illuminates that MA3/TIS was down-regulated by PKA activity, and transposon Tn10 was up-regulated by it. KEY WORDScAMP-dependent protein kinasepkaa123.7 cellpc12 celldd-pcr. 서 This study was supported by grant from the Industrial Technology Development Supply Plan of Ansan City. Department of Biochemistry and Molecular Biology, College of Science, Hanyang University, Ansan, Korea Department of Neuropsychiatry, College of Medicine, Hanyang University, Seoul, Korea 론

2 1. 세포배양 실험방법 2. RNA 의분리및정제 3. RNase free-dnase I 처리 4. Formaldehyde 겔전기영동 5. Reverse transcription

3 Table 1. Sequences of the primer for DD-PCR Primer name Primer sequence H-AP1 5 -AAGCTTGATTGCC-3 H-AP2 5 -AAGCTTCGACTGT-3 H-AP3 5 -AAGCTTTGGTCAG-3 H-T12MA 5 -AAGCTTTTTTTTTTTTMA-3 H-T12MG 5 -AAGCTTTTTTTTTTTTMG-3 H-T12MC 5 -AAGCTTTTTTTTTTTTMC-3 6. Differential display-polymerase chain reaction (DD- PCR) 7. Polyacrylamide 겔전기영동 (PAGE) 8. DEST의획득과재증폭 9. 증폭된 DEST의클로닝과 transformants 검색 10. DNA 염기서열결정및분석 11. Northern analysis

결과 1. DD-PCR과 DEST의선택 2. DEST의획득과재증폭 3. DEST의클로닝과염기서열결정및분석 Fig. 1. Differentially expressed mrnas between in PC12 cells and A123.7 cells. The total RNAs were purified from A123.7 cell and PC12 cell.

in panel E, and H-T 12 A and H-AP3 in panel F. The radio-labelled DD-PCR products were electrophoresised on 6% polyacrylamide gel. K srands for A123.7 cell and P for PC12 cell.

4 결과 1. DD-PCR과 DEST의선택 2. DEST의획득과재증폭 3. DEST의클로닝과염기서열결정및분석 Fig. 1. Differentially expressed mrnas between in PC12 cells and A123.7 cells. The total RNAs were purified from A123.7 cell and PC12 cell. DD-PCR reaction was done with a- 35 SdATP and primers of H-T 12 C and H-AP1 in panel A, H-T 12 C and H-AP3 in panel B, H-T 12 G and H-AP1 in panel C, H-T 12 A and H-AP1 in panel D, H-T 12 C and H-AP2 in panel E, and H-T 12 A and H-AP3 in panel F. The radio-labelled DD-PCR products were electrophoresised on 6% polyacrylamide gel. K srands for A123.7 cell and P for PC12 cell. Arrow heads indicate differentially expressed mrnas. Fig. 2. Reamplification of differentially expressed cdna fragments. Purified cdna fragments out of polyacrylamide gel were reamplified by PCR using same primer sets that used in DD-PCR. Lane 1KC2-1, lane 2PG1-1, lane 3KC1-2, lane 4PG1-2, lane 5KC1-3, lane 6KC3-1, lane 7molecular weight standard 174 DNA was digested with HaeIII, lane 8KC1-1, lane 9KA1-1, lane 10KC1-5, lane 11KA3-1, lane 12KC1-4, and lane 13KC

5 Table 2. Result of DESTs and BLAST search DEST Sizebp BLAST result KC Mouse mrna for topoisomerase-inhibitor suppressedtis KC Mouse apopotosis-related genema-3 KC Unknown gene KC Unknown gene KA Unknown gene KC Unknown gene KC Unknown gene PG Transposon Tn10 DNA Fig. 4. Northern analysis of KC1-5 DEST. RNA were isolated from PC12 cells and A123.7 cells. KC1-5 DEST was differentially expressed in A123.7 cells. P stands for PC12 cells, K for A123.7 cells. Fig. 3. BLAST search result of KC1-5 DEST. KC1-5 DEST was differentially expressed in A123.7 cells. KC1-5 DEST was identical two genes, mouse apoptosis-related gene, MA-3 and mouse mrna for topoisomerase inhibitor suppressed, TIS. 4. Northern analysis를통한 DEST의확인 고찰

6 결론

7 중심단어 : 감사의글 참고문헌 Altschul SF, Madden TL, Schfer AA, Zhang J, Zhang Z, Miller W, Lipman DJ(1997):Gapped BLAST and PSI-BLAST:A new generation of protein database search programs. Nucleic Acids Res 25: Bramson HN, Kaiser ET, Mildvan AS(1983):Mechanistic studies of camp-dependent protein kinase actions. Crit Rev Biochem 15: Cho-Chung YS, Pepe S, Clair T, Budillon A, Nesterova M(1995): camp-dependent protein kinase:role in normal and malignant growth. Crit Rev Oncol Hematol 21:33-61 Chomczynski P, Sacchi N(1987):Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: Ciardiello F, Tortora G(1998):Nteractions between the epidermal growth factor receptor and type I protein kinase A:Biological significance and therapeutic implications. Clin Cancer Res 4: Garcia-Bermejo L, Perez C, Vilaboa NE, de Blas E, Aller P(1998): camp increasing agents attenuate the generation of apoptosis by etoposide in promonocytic leukemia cells. J Cell Sci 111: Ginty DD, Glowacka D, DeFrano C, Wagner JA(1991):Nerve growth factor-induced neuronal differentiation after dominant repression of both type I and type Ⅱ camp-dependent protein kinase activities. J Biol Chem 266: Greene LA, Tischler AS(1976):Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73: Hsiang YH, Hertzberg R, Heeht S, Liu LP(1985):Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260: Inoue H, Li YP, Wagner JA, Hersh LB(1995):Expression of the choline acetyltransferase gene depends on protein kinase A activity. J Neurochem 64: Kannan S, De Santis M, Lohmeyer M, Riese DJ, Smith GH, Hynes NE, Seno M, Brandt R, Bianco C, Persico MG, Kenney N, Normanno N, Martinez-Lacaci I, Ciardiello F, Stern DF, Gullick WJ, Salomon DS(1997):Cripto enhances the tyrosine phosphorylation of Shc and activates mitogen-activated protein kinase (MAPK) in mammary epithelial cells. J Biol Chem 272: Liang P, Pardee AB(1992):Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: Madhok TC, Matta SF, Sharp BM(1995):Nicotine regulates nicotinic cholinergic receptors and subunit mrnas in PC12 cells through protein kinase A. Brain Res Mol Brain Res 32: Montminy M(1997):Transcriptional regulation by cyclic AMP. Annu Rev Biochem 66: Nankova B, Hiremagalur B, Menezes A, Zeman R, Sabban E(1996): Promoter elements and second messenger pathways involved in trascriptional activation of tyrosine hydroxylase by ionomycin. Brain Res Mol Brain Res 35: Onishi Y, Hashimoto S, Kizaki H(1998):Cloning of the TIS gene suppressed by topoisomerase inhibitors. Gene 215: Onishi Y, Kizaki H(1996):Molecular cloning of the genes suppressed in RVC lymphoma cells by topoisomerase inhibitors. Biochem Biophys Res Commun 228:7-13 Salomon DS, Brandt R, Ciardiello F, Normanno N(1995):Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 19: Sanger F, Coulson AR(1975):A rapid method for determining sequences in DNA by primed synthesis with polymerase. J Mol Biol 94: Scheibe RJ, Moeller-Runge I, Mueller WH(1991):Retinoic acid induces the expression of alkaline phosphatase in P19 teratocarcinoma Cell. J Biol Chem 266: Schubert D, Klier FG(1977):Storage and release of acetylcholine by a clonal cell line. Proc Natl Acad Sci USA 74: Shibahara K, Asano M, Ishida Y, Aoki T, Koike T, Honjo T(1995): Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death. Gene 166: Shimojo M, Wu D, Hersh LB(1998):The cholinergic gene locus is coordinately regulated by protein kinase A Ⅱ in PC12 cells. J Neurochem 71: Taylor SS, Buechler JA, Yonemoto W(1990):cAMP-dependent protein kinase:framework for a diverse family of regulatory enzymes. Annu Rev Biochem 59: Wang Y, Gong B, Dai W, Lu L(1998):Identification of immediate early genes during TPA-induced human myeloblastic leukemia ML- 1 cell differentiation. Gene 216: Wu N, Armstrong I, Wagner J(1984):Genetic evidence that chloroadenosine increases the specific activity of choline acetyltransferase in PC12 via modulation of an adenosine-dependent adenylate cyclase. Neuroscience 13: Yao H, York RD, Misra-Press A, Carr DW, Stork PJS(1998):The cyclic adenosine monophosphate-dependent protein kinsae(pka) is required for the sustained activation of mitogen-activated kinase and gene expression by nerve growth factor. J Biol Chem 273:

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untitled 3. 농업환경연구과 과제구분 기본연구 수행시기 전반기 연구과제 및 세부과제 수행 기간 소 속 책임자 농가에 적합한 부식성곤충 대량 사육기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 1) 부식성 곤충 먹이 제조 기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 색인용어 부식성곤충, 장수풍뎅이, 계통, 먹이제조 ABSTRACT In first check,