臺灣博碩士論文加值系統

線虫(C. elegans)為生長於土壤中，大小約1mm長圓柱形如針線般的小蟲；1963年開始被應用在生物學的研究上，是一種被廣泛使用的模式生物。其生活史約3 ~ 4天一個循環，包括卵、胚胎、L1、L2、L3、L4、成體、dauer 幼蟲等幾個階段。基因組DNA約97 Mb，有6對染色體。我們以線虫為材料，尋找hsmt3 gene family相似的基因，結果發現一與hsmt3b相似的基因，命名為cesmt3b，較之hsmt3b僅有三個胺基酸不同，分別是threonine 12變為alanine 12，glutamic acid 69變為aspartic acid 69，isoleucine 84變成leucine 84。目前已知smt3蛋白在人類、豬、牛、小鼠、大鼠其序列皆相同。而在較低等的線虫也僅只有三個胺基酸的差異；這一點與泛素 (ubiquitin) 相似。cesmt3b基因的大小為285 bp，deduced 95aa.，與hsmt3a、hsmt3b、hsmt3c 相似度分別為91%、96%、45%。此基因的轉錄長度為2.0 kb，表現出之蛋白質約14 kDa，於L2、L3、成體及dauer幼蟲皆有表現，此外北方墨點分析顯示另有3.4 kb、8.0 kb的信號可能為cesmt3b融合基因。此基因的表現在dauer幼蟲期會受到藥物taxol抑制，而北方轉漬分析中3.4 kb訊號則會受到熱休克處理及藥物DMSO、taxol、pheylarsenate oxide、nocodazole、vinblastine等抑制。我們也以hsmt3a蛋白序列為基礎設計引子，結果找到一類似ETS致癌蛋白DNA結合區域的片段，推測cesmt3b及與ETS相似的基因，兩者和細胞分裂、細胞內微管組合有關。

Caenorhabditis elegans is a 1-mm long, cylinder shape, soil-living nematode. It has been used as a model organism for biological research since 1963. Its life cycle take about 3 ~ 4 days, and consists of six stages include eggs, embryo, first-stage larvae (L1), second-stage larvae (L2), third-stage larvae (L3), fourth-stage larvae (L4), adults and dauer larvae. The size of genomic DNA of C. elegans is approximately 97 Mega base pair nucelotides within its six chromosomes. We used C. elegans as an experimental organism and try to identify hsmt3 family gene homologues. Using RT-PCR strategy, we obtained a hsmt3b homologue of C. elegans and termed cesmt3b. There were 3 different residues between hsmt3b and cesmt3b through alignment of the amino acid sequence. The different amino acids are threonine , glutamic acid 69, and leucine 84 instead of Ala12, Asp69, and Leu84, respectively. The smt3b protein was identical in human, bovine, porcine, mouse and rat. The residued difference between hsmt3b and cesmt3b revealed that this gene highly conserved. The situation is the same as ubiquitin. The protein-coding region of cesmt3b was 285 bp and deduced 95 amino acids. The amino acid identity of cesmt3b with hsmt3a, hsmt3b, hsmt3c were 91%, 96%, 45%, respectively. The transcribed size was 2.0 kb and was expressed in L2, L3, adult, dauer larvae stages. Northern blotting data showed additional 3.4, 8.0 kb hybridization signals were that supposed to be mRNA expression signals of cesmt3b fusion genes. The molecular weight of cesmt3b expressed fusion protein was 14 kDa. The expression of cesmt3b was inhibited by 12 hours taxol treatment at dauer larvae stage. This 3.4 kb signal also was disappeared after DMSO, heat shock, taxol, PAsO, nocodazole, vinblastine treatment. Another fragment was amplified by PCR method based on the sequence data of hsmt3a. The DNA sequence was submitted to BLAST search program and shown identity to a gene that is similar to ETS family oncoprotein DNA binding domain. Both cesmt3b and ETS-like fragment might play important roles in mitosis, microtubule integrity, centromere / kinetochore structure maintenance mechanisms.

目錄....................................................p.2
中文摘要................................................p.3
英文摘要..................... ..........................p.4 ~5
緒論....................................................p.6 ~12
材料與方法...... ...... ..............................p.13 ~29
結果....................................................p.30 ~33
討論....................................................p.34 ~38
參考文獻...... .........................................p.39 ~44
附圖....................................................p.45 ~56
附表....................................................p.57 ~60

Berlethk, E. S., Kasperek, E. M., Grill, S. P., Braunscheidel, J. A., Graziani, L. A. and Pickart, C. M.: Inhibition of ubiquitin-protein ligase (E3) by mono- and bifuctional phenylarsenoxides. J. Biol. Chem. 267 (1992) 16403-16411.
Brabander, M. D., Geuens, G., Nuydens, R., Willebroads, R. And Mey, J. D.: Taxol induces the assembly of free microtubules in living cells and blocks the organizing capacity of the centrosomes and kinetochores. Proc. Natl. Acad. Sci. USA 78 (1981) 5608-5612.
Brown, M. T., Goetsch, L. and Hartwell, L. H.: MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisae. J. Cell Biol. 123 (1993) 387-403.
Brown, M. T.: Sequenece similarities between the yeast chromosome segregation protein Mif2 and the mammalian centromere protein CENP-C. Gene 160 (1995) 111-116.
Chen, A., Mannen, A. and Lee, S. S.: Characterization of mouse ubiquitin-like SMT3A and SMT3B cDNAs and gene/pseudogenes. Biochem. Mol. Biol. Int. 46 (1998) 1161-1174.
Choudhury, B. K. and Li, S. S.: Identificaiton and characterization of the SMT3 cDNA and gene from Nematode Caenorhabditis elegans. Biochem. Biophys. Res. Commun. 234 (1997) 788-791.
Graham, R. W., Doren, K. V., Bektesh, S. and Candido, E. P. M.: Muturation of major ubiquitin gene transcript in Caenorhabditis elegans involves the acquisition of a trans-spliced leader. J. Biol. Chem. 263 (1998) 10415-10419.
Huang, H. W., Tsoi, S. C. M., Sun, Y. H. and Li, S. S. L.: Identification and characterization of the SMT3 cDNA and gene encoding ubiquitin-like protein from Drosophila melanogaster. Biochem. Mol. Biol. Int. 46 (1998) 775-785.
Johnson, E. S, Schwienhorst, I., Dohmen, R. J. and Blobel, G.: The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J. 16 (1997) 5509-5519.
Johnson, E. S. and Blobel, G.: Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p. J. Biol. Chem. 272 (1997) 26799-26802.
Jones, D. and Candido, E. P. M.: Novel ubiquitin-like ribosomal protein fusion genes from the nematodes Caenorhabditis elegans and Caenorhabdidtis briggsae. J. Biol. Chem. 268 (1993) 19545-19551.
Klemperer, N. S., Berleth, E. S., and Pickart, C. M.: A novel, arsenite-sensitive E2 of ubiquitin pathway: purification and properties. Biochemistry 28 (1989) 6035-6041.
Lapenta, V., Chiurazzi, P., van der Spek, P., Pizzuti, A., Hanooka, F. and Brahe, C.: SMT3A, a human homologue of the S. cerevisiae SMT3 gene, maps to chromosome 21qter and defines a novel gene family. Genomics 40 (1997) 362-366.
Lee, G. W., Melchior, F., Matunis, M. J., Mahajan, R., Tian, Q. and Anderson P.: Modificaiton of Ran GTPase-activating protein by the small ubiquitin-related modifier SUMO-1 requires Ubc9, an E2-type ubiquitin-conjugating enzyme homologue. J. Biol. Chem. 273 (1998) 6503-6507.
Leggett, D. S. and Candido, E. P. M.: Biochemical characterization of Caenorhabditis elegans UBC-1: self-association and auto-ubiquitination of a RAD6-like ubiquitin-conjugating enzyme in vitro. Biochem. J. 327 (1997) 357-361.
Leggett, D. S., Jones, D. and Candido, E. P. M. Caenorhabditis elegans UBC-1, a ubiquitin-conjugating enzyme homologous to yeast RAD6/UBC2, contains a novel carboxy-terminal extension that is conserved in nematodes. DNA Cell Biol. 14 (1995) 883-891.
Li, S. J., Hochstrasser, M.: New protease required for cell-cycle progression in yeast. Nature 398 (1999) 246-251.
Liou, M. L., Liou, H. C.: The ubiquitin-homology protein, DAP-1, associates with tumor necrosis factor receptor (p60) death domain and induces apoptosis. J. Biol. Chem. 274 (1999) 10145-10153.
Mahajan, R. Gerace, L. and Melchior, F.: Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association. J. Cell Biol. 140 (1998) 259-269.
Mahajan, R., Delphin, C., Guan, T., Gerace, L. and Melchior, F.: A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88 (1997) 97-107.
Mannen, H., Iseng, H.-M., Cho, C.-L., and Li, S.S.-L.: Cloning and expression of human homolog HSMT3 to yeast smt3 suppressor of MIF2 mutation in a centromere protein gene. Biochem. Biophys. Res. Common. 222 (1996) 178-180.
Matunis, M. J., Wu, J. and Blobel, G.: SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex. J. Cell Biol. 140 (1998) 499-508.
Meluh, P.B., and Koshland, D.: Evidence that the MIF2 gene of Saccharomyces cerevisae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol. Biol. Cell 6 (1995) 793-807.
Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M. and Yeh, E. T.H.: Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. J. Immunol. 157(1996) 4277-81.
Pluta, A. F., Mackay, A. M., Ainsztein, A. M., Goldberg, I. G. And Earnshaw, W. C.: The centromere: hub of chromosomal activities. Science 270 (1995) 1591-1594.
Qi, F., Ridpath, J. F. and Berry, E. S.: Insertion of a bovine SMT3B gene in NS4B and duplication of NS3 in a bovine viral diarrhea virus genome correlate with the cytopathogenicity of the virus. Virus Res. 57(1998) 1-9.
Saitoh, H., Tomkiel, J., Cooke, C.A., Ratrie, H. III, Maurer, N., Rothfield, N.F., and Earnshaw, W.C.: CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate. Cell 70 (1992) 115 -125.
Saitoh, H., Pu, R. T., Dasso, M.: SUMO-1: wrestling with a new ubiquitin-related modifier. Trends Biochem. Sci. 22 (1997) 374 -376. Review.
Schwarz, S. E., Matuschewski, K., Liakopoulos, D., Scheffner, M. and Jentsch, S.: The ubiquitin-like proteins SMT3 and SUMO-1 are conjugated by the UBC9 E2 enzyme. Proc. Natl. Acad. Sci. U S A. 95 (1998) 560-564.
Shen, Z., Pardington-Purtymun, P.S., Comeaux, J.C., Moyzis, R.K. and Chen, D.J.: UBL1, a human ubiquitin-like protein associating with human RAD51/RAD52 proteins. Genomics 36 (1996) 271-279.
Sorger, P. K., Doheny, K. F., Hieter, P., Kopski, K. M., Huffaker, T. C. and Hyman, A. A. Two genes required for the binding of an essential Saccharomyces cerevisae kinetochore complex to DNA. Proc. Natl. Acad. Sci. USA 92 (1995) 12026-12030.
Sternsdorf, T., Jensen, K. and Will, H.: Evidence for covalent modification of the nuclear dot-associated proteins PML and Sp100 by PIC1/SUMO-1. J. Cell Biol. 139 (1997) 1621-1634.
Tan P. B., Lackner M. R. and Kim S. K.: MAP kinase signaling specificity mediated by the LIN-1 Ets/LIN-31 WH transcription factor complex during C. elegans vulval induction. Cell 93 (1998) 569-580.
Wang, Y. and Burke, D.: Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisae. Mol. Cell Biol. 15 (1995) 6838-6844.
Wendell, K. L., Wilson, L. and Jordan, M. A.: Mitotic block in HeLa cells by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes. J. Cell Sci. 104 (1993) 261-274.
Zhen, M., Heinlein, R., Jones, D., Jentsch, S. and Candido, E. P. M.: The ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective prrotein degradation. Mol. Cell Biol. 13 (1993) 1371-1377.
Zhen, M., Schein, E. J., Baillie, D. L. and Candido, E. P. M.: An essential ubiquitin-conjugating enzyme with tissue and developmental specificity in the nematode Caenorhabditis elegans. EMBO J. 15 (1996) 3229-3237.