在T細胞急性白血病( T cell acute lymphoblastic leukemia , T-ALL)的病人中發現，其癌細胞有染色體缺陷的現象，造成TAL1基因在T細胞內不正常的被活化，干擾T細胞的發育，而促使腫瘤的形成。TAL1蛋白帶有basic helix-loop-helix (bHLH)功能區的轉錄調節因子，其bHLH為形成蛋白質雙體及與DNA結合所必需具備的的功能區。在T-ALL細胞內可以檢測到兩種TAL1蛋白，分別為全長蛋白pp42(1-331胺基酸)及部分蛋白pp22(176-331胺基酸)，兩者都帶有bHLH功能區；且在in vitro及in vivo情形下都會與免疫球蛋白基因促進子結合蛋白E12/E47的bHLH功能區形成DNA結合異雙體，此異雙體會與特定的E box DNA序列AACAGATGGT結合。
TAL1致癌蛋白具有許多的絲胺酸磷酸化位點；其中S122為胞外訊號調節激脢(ERK1)之磷酸化位點，其磷酸化與TAL1蛋白質的轉錄活性有關；而S172為蛋白質激脢A (PKA) 磷酸化位點，在in vivo情形下，S172並不被磷酸化，但加入PKA的活化劑forskolin，則可促進其磷酸化，其磷酸化會增加E12蛋白與TAL1蛋白之異雙體與DNA結合的能力。本實驗旨在以定點突變的方式探討TAL1致癌蛋白於176-331區域可能存在之絲胺酸磷酸化位點是否與bHLH之功能有相關。利用細胞轉染方法，將S194，S224定點突變後之表達質體分別與E12 表達載體一起轉染到COS-1細胞內，先以西方墨點法確定表現載體可在細胞內表達，再以電泳位移分析法(EMSA)檢測其E-box DNA結合能力。結果發現將TAL1基因 S194，S224兩位點不論是個別或一起定點突變後，皆會影響E12-Tal1異雙體與DNA結合能力，顯示S194及S224兩位點，對bHLH之功能為必須的。至於S194，S224之in vivo磷酸化情況及其磷酸化激脢之種類為何，則有待進一步研究加以釐清。

The genetic defects that results in TAL1 oncogene activation are commonly seen in leukemic cells of the patient with T-cell Acute Lymphoblastic Leukemia ( T-ALL ). The ectopic expression of TAL1 oncoprotein perturbs the development of T-cell, hence promotes the formation of leukemia. TAL1 gene encodes proteins with basic helix-loop-helix ( bHLH ) domain, a protein dimerization and DNA binding domain. In T-ALL cells, two Tal1 proteins, pp42(1-331 amino acids) and pp22(176-331 amino acids) are produced that both contain bHLH domain. Both proteins interact with immunoglobulin gene enhancer binding protein, E12/E47 to form DNA-binding heterodimers, that can bind to consensus E-box DNA sequence AACAGATGGT. Phosphorylation of S122 residue modulates the trans-activation potential of Tal1 protein. In addition, S172 is an inducible c-AMP dependent protein kinase (PKA) phosphorylation site in vivo. The phosphorylation of TAL1 S172 upon stimulation by forskolin can increase the DNA binding of E12-Tal1 heterodimer. We used site-directed mutagenesis to investigate the effect of S194,S224 mutation on the function of truncated Tal1 oncoprotein.Mutant Tal1 and E12 proteins expression plasmids were constructed and introduced into COS-1 cells by cotransfection. Tal1 and E12 protein expression in transfected cell were evaluated by Western blotting. The protein-DNA interaction were evaluated by electrophorectic mobility shift assay. The mutation of S194 and S224 of Tal1 protein all resulted in the loss of DNA-binding complex formation. This data indicated that these serine residues are essential for bHLH function. However, the phosphorylation status of these two residues in vivo, and what kinase is responsible for the phosphorylation of these residues, await further investigation.

前言 1
材料與方法 7
一、TAL1基因定點突變質體之建立 7
二、定點突變質體之分子選殖和純化 8
三、聚合酵素連鎖反應分析 14
四、細胞株及細胞培養 19
五、以定點突變質體轉染COS-1細胞 20
六、轉染細胞內蛋白質表達之分析 21
七、轉染蛋白質與DNA結合能力之測試 26
結果 28
一、TAL1定點突變表現載體之構築與選殖 28
二、聚合酵素連鎖反應之分析 29
三、以重組質體轉染COS-1細胞及轉染細胞內蛋白質表現之分析 30
四、轉染蛋白質與DNA結合能力之測試 31
討論 35
參考文獻圖表 40
表一、TAL1基因與其引子之PCR最佳反應溫度及時間 49
表二、TAL1基因之定點突變與其引子之PCR最佳反應溫度及時間 50
表三、TAL1基因定點突變質體核酸序列分析其引子之PCR最佳反應溫度及時間 51
圖一，TAL1致癌蛋白表達重組質體之組成及其限制酵素圖譜 52
圖二、bHLH蛋白之胺基酸序列 53
圖三、E12抗體及TAL1抗體抗原表位之位置 54
圖四、TAL1第194胺基酸絲胺酸點突變為丙胺酸之南方墨點法分析 55
圖五、TAL1第224胺基酸絲胺酸點突變為丙胺酸之南方墨點法分析 56
圖六、TAL1第194及第224胺基酸絲胺酸點突變為丙胺酸之南方墨墨點法分析 57
圖七、TAL1定點突變之重組質體其PCR產物於0.8%洋菜膠之電泳圖 58
圖八、TAL1第194絲胺酸突變為丙胺酸之序列分析 59
圖九、TAL1第224絲胺酸突變為丙胺酸之序列分析 60
圖十、TAL1第194及第224絲胺酸突變為丙胺酸之序列分析 61
圖十一、轉染細胞內E12蛋白質之西方墨點法分析 62
圖十二、轉染細胞內Tal1蛋白質之西方墨點法分析 63
圖十三、轉染重組質體之蛋白質與DNA結合能力之EMSA分析 64

1. Champlin, R., and Gale, R. P. 1989. Acute lymphoblastic
leukemia: recent advances in biology and therapy. Blood.
73:2051-2066.
2.Jonsson, O. G., Kitchens, R. L., Bear, R. J., Buchanan, G. R., and Smith, R. G. 1991. Rearrangements of the tal-1 locus as clonal markers for T cell acute lymphoblastic leukemia. J. Clin. Invest. 87:2029-2035.
3.Yunis, J. J. 1983. The chromosomal basis of human neoplasia. Science.221:227-236.
4.Heim, S., and Mitelman, F. 1987. Cancer cytogenetics. New York: A . R. Liss , Inc.
5.Boehm, T., and Rabbitts, T.H. The human T cell receptor gene
are targets for chromosomal abnormalities in T cell tumors. The Faseb Journal. 3:2344-2360.
6.Cheng, J-T., Yang, C. Y-C., Hernandez, A. D., and Bear, R. 1990. The chromosome translocation (11;14)(p13;q11) associated with T cell acute leukemia. J. Exp. Med. 171:489-501.
7.Carrol, A.J., Crist, W.M., Link, M. P., Amylon, M. D., Pullen, D. J., Ragab, A. H., Buchanan, G. R., Wimmer, R. S., Vietti, T. J. 1990. The t(1;14)(p34;q11) is nonrandom and restricted to T-cell acute lymphoblastic leukemia: a pedeatic oncology group study. Blood. 76:1120-1124.
8.Chen, Q., Cheng, J-T., Tsai,L-H., Scheider, N., Buchanan, G., Carroll, A., Crist, W., Ozanne, B., Sicillano, M. J., and Bear, R. 1990. The tal gene undergoes chromosome translocation in T cell leukemia and potentially encodes a helix-loop-helix protein. EMBO. 9:415-424.
9.Chen, Q., Yang, C. Y-C., Tsou, J. T., Xia, T., Ragab, A. h., Peiper, S. T., Carrol, A., and Bear, R. 1990. Coding sequences of the tal-1 gene are disrupted by chromosome translocation in human T cell leukemia. J. Exp. Med. 172:1403-1408.
10.Bret, T., Mol, E. J., Wolvers-Trttero, L. M., Ludwing, W-D., Wering, E. R., and Dongen, J. M. 1993. Site-specific deletion involving the tal-1 and sil genes are restricted to cell of the T cell receptor a/b lineage: T cell receptor d gene deletion mechanism affects multiple gene. J. Exp. Med. 177:965-977.
11.Brown, L., Cheng, J-T., Chen, Q., Sicillon, M. J., Crist, W., Buchanan, G., and Bear, R. 1990. Site-specific recombination of tal-1 gene is a common occurrence in human T cell leukemia. EMBO J. 9:3343-3351.
12.Jonsson, O. G., Kitchens, R. L., Bear, R., Buchanan, G., Smith, R. G. 1991. Rearrangment of tal-1 locus as clonal markers for t cell acute lymphoblastic leukemia. J. Clin. Invest. 87:2029-2035.
13.Korsmeyer, S. J. 1992. Chromosome translocation in lymphoid malignancies reveal novel proto-oncogene. Annu. Rev. Immunol. 10:785-870.
14.Bernard, O., Lecointe, N., Jonveaux, p., Souyri, M., Mauchauffe, M., Bear, R., Laraen, C. J., Mathieu-Mahul, D. 1991.Two site-specific deletion and t(1;14) translocation restricted to human T cell acute leukemia disrupt the 5' part of the tal-1 gene. Oncogene. 6:1477-1488.
15.Bernard, O., Gugliemi, P., Jonveaux, P., Cherif, D., Gisselbrecht, S., Mauchauffe, m., Berger, R., Larsen, C-J., and Mathieu-Mahul, D. 1990. Two distinct mechanisms for the SCL gene activation in the t(1;14) translocation of T-cell leukemia. Gene, Chromosome & Cancer. 1:194-208.
16.Elwood, N. J., Cook, W. D., Matcalf, D., and Begley, G. 1993. SCL, the gene implicated in human T-cell leukemia is oncogenic in a murine T-lymphocyte cell line. Oncogene. 8:3093-3101.
17.Green, A. R., Salvaris, P. S., and Begley, C. G. Erythroid expression of the helix-loop-helix gene, SCL. Oncogene. 6:475-479.
18.Murre, C., McCaw, P. S., and Baltimore, D. 1989. A new DNA binding and dimerization in immunoglobulin enhancer binding, daughterless, MyoD, and myc protein. Cell. 56:777-783.
19. Murre, C., McCaw, P. S., Vaessin, H., Caudy, M., Jan, Y. N., Cabrera, C. V., Buskin, J. N., Cabera, C. V., and Baltimore, D. 1989. Interaction between heterologous helix-loop-helix protein generate complexes that bind specifically to a common DNA sequence. Cell. 58:537-544.
20.Voronva, A., and Baltimore, D. 1990. Mutation that disrupt DNA binding and dimerformation in the E47 helix-loop-helix protein map to distinct domain. Proc. Natl. Acad. Sci. U.S.A. 87:4722-4726.
21.Hu, J. S., Olson, E. N., and Kingston, R. E. 1992. HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability to myogenic regulatory factors. Mol. Cell. Biol. 12:1031-1042.
22.Henthorn, P., Kiledjian, M., and Kadesch, T. 1990. Two transcription factors that bind the immunoglobulin enhancer
μE5/κE2 motif. Science. 247:467-470.
23.Lassar, A. B., Murre, C., Davis, R. L., Voronova, A., Wright, W. E., Baltimore, D., Kadesch, T., and Weintraub, H. 1991. Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47 like protein in vivo. Cell. 66:305-315.
24.Hsu, H-L., Cheng, J-T., Chen, Q., and Bear, R. 1991. Enhancer-binding activity of tal-1 oncoprotein in association with the E47/E12 helix-loop-helix proteins. Mol.and Cell. Biol. 11:3037-3042.
25.Hsu, H-L., Wadman, I., and Bear, R. 1994. Formation of in vivo complexes between the TAL-1 and E2A polypeptides of leukemic T cells. Mole. Cell. Biol. 91:3181-318
26.Hsu, H-L., Huang, L., Tasa, J. T., Funk, W., Wright, W., Hu, J-S., Kingston, R. E., and Bear, R. 1994. Preferred sequences for DNA recognition by the TAL-1 helix-loop-helix proteins. Mol. and Cell. Biol. 14:1256-1265.
27.Weintraub, H., Davis, R., Tapacott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y., and Lassar, A. 1991. The myoD genefamily: nodal point during specification of the muscle cell lineage. Science. 251:761-766.
28.Mouthon, M-A., Bernard, O., Mitjavlia, M-T., Romeo, P-H., Vainchenker, W., Mathieu-Mahul, D. 1993. Expression of tal-1 and GATA-binding protein human hematopoiesis. Blood. 91:647-655.
29.Cheng, J-T., Hsu, H-L., Hwang, L-Y., and Bear, R. 1993. Products of the TAL-1 oncogene: basic helix-loop-helix proteins phosphorylated at serine residues. Oncogene. 8:667-683.
30.Cheng, J-T., Cobb, M. H., and Bear, R. 1993. Phosphorylation of TAL1 oncoprotein by the extracellular-signal-regulated protein kinase ERK1. Mol. And Cell. Biol. 13:801-808.
31.Wadman, I. A., Hsu, H-L., Cobb, M. H., and Bear, R. 1994. The MAP kinase phosphorylation site of TAL1 occurs within a translation activation domain. Oncogene. 9:3713-3716.
32.Cheng, J-T. et. al., unpublished data.
33.王俊凱. 1997. TAL1致癌蛋白DNA結合能力之研究. 國立中山大
學生物科學研究所碩士論文.
34.Cheng, J-T. , unpublished data.
35.Bain, G. Gruenwald, S., and Murre, C. 1993. E2A and E2-2 are subunits of B-cell-specific E2-box DNA-binding proteins. Mol. Cell. Biol. 13:3522-3529.
36.Kadesch, T. 1992. Helix-loop-Helix proteins in the regulation of immunoglobulin gene transcription. Immunol. Today. 13:31-36.
37.Hsu, H-L., Huang, L., Tasa, J. T., Funk, W., Wright, W., Hu, J-S., Kingston, R. E., and Bear, R. 1994. Preferred sequences for DNA recognition by the TAL1 helix-loop-helix proteins. Moil. Cell. Biol. 14:1256-1265.
38.Hunter, T., and Karin, M. 1992. The regulation transcription by phosphorylation. Cell. 70:375-387.
39.Li, L., J. Zhou, G James, R. Heller-harrison, M. P. Czech, and E. N. Olson. 1992. FGF in activates myogenic helix-loop-helix proteins through phosphorylation of conserved protein kinase C site in their DNA-binding domain. Cell. 71:1181-1194.
40.Zhou, J., and Olson, E. 1994. Dimerization through the Helix-loop-Helix motif enhance phosphorylation of the transcription activation domains of myogenin. Mol. Cell. Biol. 9:6232-6243.
41.Porcher C., Swat W., Rockwell K., Fujiwara Y., Alt F. W., and Orkin S. H. 1996. The T cell leukemia oncoprotein SCL/TAL is essential for development of all hematopoietic lineages. Cell. 86:47-57.
42.Prasad K. S.S., and Brandt S. J. 1997. Target-depentent effect of phosphorylation on the DNA binding activity of the TAL1/SCL oncoprotein. J. Biol. Chem. 272:11457-11462.
43.Goldfarb, A. N., Lewandowska, K., and Shoham, M. 1996. Determinants of Helix-loop-Helix dimerization affinity. J. Biol. Chem. 271:2683-2688.