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 - 67 -
1. 세포배양 실험방법 2. RNA 의분리및정제 3. RNase free-dnase I 처리 4. Formaldehyde 겔전기영동 5. Reverse transcription - 68 -
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 - 69 -
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 13KC3-2. - 70 -
Table 2. Result of DESTs and BLAST search DEST Sizebp BLAST result KC1-5 142 Mouse mrna for topoisomerase-inhibitor suppressedtis KC1-5 142 Mouse apopotosis-related genema-3 KC1-3 169 Unknown gene KC1-1 165 Unknown gene KA3-1 158 Unknown gene KC1-2 157 Unknown gene KC2-1 148 Unknown gene PG1-1 190 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 - 71 -
결론 - 72 -
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