Cloning and Characterization of Novel p53-Regulated Genes

• Main Authors: Pang-Kuo Lo, 駱邦國
• Other Authors: Fung-Fang Wang
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/61123204931547812228

博士 === 國立陽明大學 === 生物化學研究所 === 88 === p53 is a tumor suppressor gene that functions as a guardian to maintain the integrity of the genome. It is the most frequently mutated and disrupted target in human cancers. Activation of p53 triggers cells into cell cycle arrest, apoptosis and differentiation, depending on the cell type, cellular environment and extracellular stimuli. p53 acts through direct interaction with other proteins or as a transcription factor regulating the expression of its downstream effector genes. A key approach to elucidating the biological function of p53 is to look for its direct target genes. By mRNA differential display analysis on murine IW32 erythroleukemia cells containing a temperature sensitive p53 allele (tsp53val-135) cultured at 32.5°C and 38.5°C, two novel p53-regulated genes, designated mDDA1 and mDDA3, have been identified. Induction of mDDA1 and mDDA3 occurred in all IW32 sublines expressing tsp53val-135 cultured at permissive temperature. Elevated levels of mDDA1 and mDDA3 transcripts were detected within 1 h and 2 h, respectively, after down-shifting the temperature from 38.5℃ to 32.5℃. Moreover, actinomycin D, but not cycloheximide, inhibited the p53-dependent induction of these genes, suggesting that their activation was through transcriptional regulation and did not require de novo protein synthesis. DDA1 transcript was predominantly expressed in mouse liver and human skeletal muscle, while that of DDA3 was found in multiple mouse and human tissues. Using 5''-RACE and a PCR-based genome walking method, full-length cDNAs and genomic DNAs of mDDA1 and mDDA3 were cloned. The mDDA1 cDNA encodes a putative protein of 498 amino acids containing 12 transmembrane domains. The genomic DNA of mDDA1 is 18 kb in length, consisting of six exons and five introns. Four putative p53 recognition motifs are found in intron 1; at least one of these sites was demonstrated to support the responsiveness to wild-type, but not mutant p53, in a transient transfection assay. Sequence comparison revealed that mDDA1 shares 73% and 90% identity in its nucleotide and protein sequences, respectively, to the newly identified human thiamine transporter gene (hTHTR-1). The gene structures of mDDA1 and hTHTR-1 are similar; both contain identical numbers of exon and intron, and the RNA splicing joint sites are also conserved. Therefore, mDDA1 is very likely to be the mouse homologue of the hTHTR-1. Based on these analyses, mDDA1 was hereafter named mouse THTR-1 (mTHTR-1). mTHTR-1 also shares 40% identity in its protein sequence to the reduced folate carrier-1 (RFC-1) from human and mouse. However, We have shown that mTHTR-1 exhibited very low methotrexate uptake activity when compared to that of RFC-1. The mDDA3 gene is composed of eight exons and seven introns, and a putative p53 recognition motif was found in its intron 3. Sequence analysis of the cloned mDDA3 cDNAs indicated that there were at least seven types of transcripts, differed only in their 5''-termini. Results from primer extension and RNase protection assays suggest that the 5''-heterogeneity of mDDA3 mRNAs may result from multiple transcriptional initiations as well as alternative splicing of the transcripts. Three of these mDDA3 cDNAs contain uninterrupted open reading frames; two of them encode a protein of 329 amino acids (mDDA3S) and the third, 344 amino acids (mDDA3L). Except for a 15 amino acid-extension at the N-terminus of mDDA3L, the two proteins are identical in sequence. The mDDA3 protein is rich in serine and proline; it contains one coiled-coil domain and six "PXXP" motifs capable of interacting SH3 containing proteins. Full-length human DDA3 cDNA has been obtained by homology searches of a human EST database; sequence analysis indicates that hDDA3 encodes a protein of 333 amino acids that shares 68% identity to mDDA3S. Overexpression of both mTHTR-1 and mDDA3 in H1299 non-small cell lung carcinoma cells partially suppressed colony formation. In summary, we have cloned and characterized two p53 transcriptional target genes mTHTR-1 and DDA3. Our analyses have implicated mTHTR-1 in thiamine homeostasis and suggested a role of DDA3 in the p53-mediated growth suppression. 英文摘要--------------------------------------------------------------------------------------- 3 (壹)‧緒論 1.1 細胞的生長與死亡之調控------------------------------------------------------- 5 1.2 p53 tumor suppressor ------------------------------------------------------------- 7 1.3 p53蛋白的結構與功能----------------------------------------------------------- 7 1.3.1 Transactivation domain ------------------------------------------------------- 7 1.3.2 Proline-rich domain ------------------------------------------------------------ 8 1.3.3 Central DNA-binding core domain ----------------------------------------- 8 1.3.4 C端oligomerization domain -------------------------------------------------- 9 1.3.5 C端multi-functional basic domain ------------------------------------------- 9 1.4 影響p53的上游訊息(Signals to p53) ------------------------------------------ 10 1.4.1 Translational regulation ------------------------------------------------------- 10 1.4.2 Post-translational modification ----------------------------------------------- 11 (1) Phosphorylation ----------------------------------------------------------------- 11 (2) Dephosphorylation ------------------------------------------------------------- 12 (3) Acetylation ----------------------------------------------------------------------- 12 1.4.3 Oncogenic regulation ---------------------------------------------------------- 13 1.4.4 Telomere shortening ----------------------------------------------------------- 13 1.5 p53所引發的下游訊息(Signaling out) ---------------------------------------- 14 1.5.1 p53對Cell Cycle的調控------------------------------------------------------- 14 1.5.2 p53對apoptosis的調控-------------------------------------------------------- 15 1.6 p53在維持基因體穩定所扮演的角色---------------------------------------- 18 1.7 p53 kingdom ------------------------------------------------------------------------ 19 1.7.1 The roles in DNA damaging signals ---------------------------------------- 19 1.7.2 The roles in development of embryo --------------------------------------- 20 1.7.3 Involvement in tumor suppression ------------------------------------------ 20 (貳)‧本論文研究的目的------------------------------------------------------------------ 22 (參)‧實驗材料與方法 3.1 材料------------------------------------------------------------------------------------ 23 3.1.1 化學藥品和實驗材料---------------------------------------------------------- 23 3.1.2 酵素和試劑----------------------------------------------------------------------- 23 3.1.3 質體DNA ------------------------------------------------------------------------- 24 3.1.4 cDNA ----------------------------------------------------------------------------- 24 3.1.5 細胞株---------------------------------------------------------------------------- 25 3.1.6 放射性物質---------------------------------------------------------------------- 25 3.1.7 選殖cDNA及genomic DNA的引子----------------------------------------- 25 (1) 選殖cDNA的引子--------------------------------------------------------------- 25 (2) 選殖genomic DNA的引子----------------------------------------------------- 26 3.2 方法------------------------------------------------------------------------------------ 28 3.2.1 細胞培養------------------------------------------------------------------------- 28 3.2.2 RNA的製備--------------------------------------------------------------------- 28 3.2.3 北方墨點轉漬分析(Northern blot analysis) ------------------------------ 30 3.2.4 質體製備------------------------------------------------------------------------- 32 3.2.5 DNA片段的選殖(cloning) -------------------------------------------------- 34 3.2.6 mRNA差異展現分析法(mRNA Differential Display) ----------------- 36 3.2.7 Rapid Amplification of cDNA 5''-Ends (5''RACE) ------------------------ 37 3.2.8 RNase protection assay ------------------------------------------------------- 37 3.2.9 PCR-based genome walking method --------------------------------------- 38 3.2.10 Primer extension method ---------------------------------------------------- 39 3.2.11 質體的構築--------------------------------------------------------------------- 40 3.2.12 細胞群落形成分析(Colony formation assay) --------------------------- 42 3.2.13 MTX uptake -------------------------------------------------------------------- 42 3.2.14 Luciferase assay --------------------------------------------------------------- 43 (肆)‧實驗結果與討論 4.1 p53下游基因之選殖 4.1.1 實驗結果------------------------------------------------------------------------- 45 4.1.2 討論------------------------------------------------------------------------------- 47 4.1.3 圖表------------------------------------------------------------------------------- 49 4.2 mDDA1基因之選殖與功能分析及受p53誘導之機制分析 4.2.1 實驗結果 (1) mDDA1 cDNA之選殖-------------------------------------------------------- 54 (2) mDDA1 genomic DNA之選殖---------------------------------------------- 56 (3) p53活化mDDA1表現之機制----------------------------------------------- 57 (4) mDDA1的生物功能分析---------------------------------------------------- 58 (5) mDDA1為thiamine transporter基因---------------------------------------- 59 4.2.2 討論 (1) p53誘導mTHTR-1表現之機制--------------------------------------------- 61 (2) mTHTR-1基因的生物功能角色------------------------------------------- 62 4.2.3 圖表------------------------------------------------------------------------------- 67 4.3 mDDA3基因之選殖與功能分析及受p53誘導之機制分析 4.3.1 實驗結果 (1) mDDA3 cDNA之選殖-------------------------------------------------------- 82 (2) mDDA3基因的genomic DNA之選殖------------------------------------- 83 (3) mDDA3 transcripts的5''-end heterogeneity成因之分析---------------- 83 (4) Heterogeneous mDDA3 cDNAs之分析----------------------------------- 84 (5) DDA3 transcript在老鼠和人類組織之表現分析----------------------- 85 (6) 以RNase protection和primer extension方法印證 mDDA3 mRNA的5''-end heterogeneity ------------------------------------ 85 (7) p53誘導mDDA3表現之機制----------------------------------------------- 87 (8) mDDA3的生物功能分析---------------------------------------------------- 88 (9) 人類DDA3 cDNA之選殖----------------------------------------------------- 89 4.3.2 討論------------------------------------------------------------------------------- 90 (1) Differential transcription initiation及alternative RNA splicing調控mDDA3表現之探討------------------------------------ 90 (2) mDDA3基因表現之調控---------------------------------------------------- 92 (3) mDDA3的生物功能角色---------------------------------------------------- 94 4.3.3 圖表------------------------------------------------------------------------------- 97 (伍)‧結論----------------------------------------------------------------------------------- 119 (陸)‧參考文獻----------------------------------------------------------------------------- 120 附錄 (一) 附圖------------------------------------------------------------------------------------ 139 (二) 英文論文 1. Identification of a novel mouse p53 target gene DDA3---------------------- 140 2. Transcriptional induction of a thiamine transporter gene by p53----------------------------------------------------------------------------------- 150