臺灣博碩士論文加值系統

Yeast SMT3(Suppressor of Mif2 temperature sensitive mutant 3)為一個與Mif2(Mitotic fidelity of chromosome transmission protein 2)有關蛋白，最初由supression Mif2mutant中發現。 目前在較高等的動物中，已發現3種SMT3蛋白異構脢(A, B, C,)存在。人類的SMT3A已定位於第21染色體。果蠅的smt3基因序列與酵母菌、人類Hsmt3A，Hsmt3B，Hsmt3C有55%，73%，70%及52%的類似度。

我們分析了smt3十株p element插入位置於chromosome 2L 之27C6-8（smt3 基因位置） 區段的果蠅，找到一接近smt3基因位置的p element 突變株P997，利用P element target 技術建立果蠅smt3 mutation stock ,並以PCR的技術來快速且大量的篩選突變株的果蠅，藉以觀察smt3在果蠅發育中的表現。
stock 11378 之P element插入點位於smt3 start codon 之前約300 b.p位置，利用stock 11378 與w1118雜交後取得smt3-null mutation ，經觀察後發現smt3-null mutation是一株隱性致死突變株，死亡於胚胎發育第四期，大約細胞核分裂約10到13次，也就是胚胎產生後約90至120分鐘，smt3-null mutation 的死亡胚胎有兩個重要的特徵，其一：胚胎內DNA的分佈散亂，無法濃縮與集中於細胞核內。第二個特徵則是在胚胎內觀察到大型且分佈異常的異泡產生。

Yeast SMT3 (Suppressor of Mif2 temperature sensitive mutant 3) is a Mif-related prortein, and is first found in suppression Mif2 (Mitotic fidelity of chromosome transmission protein 2) mutant. So far, among animals of higher level, we have found the existence of 3 SMT3 isozymes (A,B,C). It’s known that SMT3A is located on chromosome 21. And smt3 sequence of Drosophila is similar to those of yeast or human in a percentage of 55%, 73%, and 52%.

We analyze 10 strains of flies with p element inserted into position 27C6-8 on chromosome 21 and found a p element mutant, P997, near the position of smt3 gene. Then we built fly’s smt3 mutation stock by using P element technology, and by using PCR, we can sieve out mutant fly fast and massively. Thus we can observe the expression of smt3 during the development of fly.

The P element of stock 11378 is inserted 300bp before smt3 start codon. After stock 11378’s crossing with W1118, we get smt3-null mutation. Then we found that smt3-null mutation is a recessive lethal mutant, fly will die in the forth period of embryo development. At that time, the cell nucleus has divided for about 10 to 13 times, also means 90 to 120 minutes after the embryo is produced. We also found two important characteristics about dead embryo of smt3-null mutation: (1) The DNA in the embryo is scattered and couldn’t be condensed in the nucleus. (2) Huge and abnormally-dispersed vacuoles are observed in the embryo.

感言 ……………………………………………………………3
中文摘要 ………………………………………………………...….4
英文摘要 ……………………………………………………...…….5
序論 …………………………………………………...……….6
材料與方法………………………………………………………..…14
Drosophila stock………………………………………………….14

P element target ……………………...…………………………..15

Isolation of fly genomic DNA……………………………………16

PCR .……………………………………………………16
收集smt3-null mutation embryo ………………………………...17
胚胎固定與觀察……………………………………...………….18
胚胎細胞核的染色………………………………………………18
共軛焦顯微鏡的操作……………………………………………19
結果 ………………………………………………………………20
1 果蠅smt3基因定位…………………………………………..21
2 果蠅P element突變株的建立………………………………..21
3 果蠅smt3突變株之胚胎發育………………………………..22
討論…………………………………………………………………..26
1 P997 local jump………………….……………..………………26
2 smt3-null mutation 性狀觀察…………………………………26
3 further experiment design………………………………………27
summary………………………………………………………………28
參考文獻………………………………………………..…………….29
圖表…………………………………………………..……………….33
表1 11378相關資訊………………………………………………..33
表2 實驗設計流程…………………………………...……………34
圖……………………………………………...……………………….35
附錄………………………………………………………………….63

Akam,M. (1987). The molecular basis for metameric pattern in the Drosophila embryo. Development 101, 1-22.
Bier,E., Vaessin,H., Shepherd,S., Lee,K., McCall,K., Barbel,S., Ackerman,L., Carretto,R., Uemura,T., Grell,E., and . (1989). Searching for pattern and mutation in the Drosophila genome with a P- lacZ vector. Genes Dev. 3, 1273-1287.
Bryant,P.J., Watson,K.L., Justice,R.W., and Woods,D.F. (1993). Tumor suppressor genes encoding proteins required for cell interactions and signal transduction in Drosophila. Dev. Suppl 239-249.
Driever,W. and Nusslein-Volhard,C. (1988). A gradient of bicoid protein in Drosophila embryos. Cell 54, 83-93.
Epps,J.L. and Tanda,S. (1998). The Drosophila semushi mutation blocks nuclear import of bicoid during embryogenesis. Curr. Biol. 8, 1277-1280.
Fasano,L., Roder,L., Core,N., Alexandre,E., Vola,C., Jacq,B., and Kerridge,S. (1991). The gene teashirt is required for the development of Drosophila embryonic trunk segments and encodes a protein with widely spaced zinc finger motifs. Cell 64, 63-79.
Huang,H.W., Tsoi,S.C., Sun,Y.H., and Li,S.S. (1998). Identification and characterization of the SMT3 cDNA and gene encoding ubiquitin-like protein from Drosophila melanogaster. Biochem. Mol. Biol. Int. 46 , 775-785.
Ingham,P.W. (1988). The molecular genetics of embryonic pattern formation in Drosophila. Nature 335, 25-34.
Johnson,E.S., Schwienhorst,I., Dohmen,R.J., and Blobel,G. (1997). The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J. 16, 5509-5519.
Kim,Y.H., Choi,C.Y., and Kim,Y. (1999). Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1. Proc. Natl. Acad. Sci. U. S. A 96, 12350-12355.
Lapenta,V., Chiurazzi,P., van der,S.P., Pizzuti,A., Hanaoka,F., and Brahe,C. (1997). SMT3A, a human homologue of the S. cerevisiae SMT3 gene, maps to chromosome 21qter and defines a novel gene family. Genomics 40, 362-366.
Lehembre,F., Badenhorst,P., Muller,S., Travers,A., Schweisguth,F., and Dejean,A. (2000). Covalent modification of the transcriptional repressor tramtrack by the ubiquitin-related protein Smt3 in Drosophila flies. Mol. Cell Biol. 20, 1072-1082.
Liu,Q., Jin,C., Liao,X., Shen,Z., Chen,D.J., and Chen,Y. (1999). The binding interface between an E2 (UBC9) and a ubiquitin homologue (UBL1). J. Biol. Chem. 274, 16979-16987.
Mahajan,R., Delphin,C., Guan,T., Gerace,L., and Melchior,F. (1997). A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97-107.
Mannen,H., Tseng,H.M., Cho,C.L., and Li,S.S. (1996). Cloning and expression of human homolog HSMT3 to yeast SMT3 suppressor of MIF2 mutations in a centromere protein gene. Biochem. Biophys. Res. Commun. 222, 178-180.
Matunis,M.J., Coutavas,E., and Blobel,G. (1996). A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135, 1457-1470.
Meluh,P.B. and Koshland,D. (1995). Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol. Biol. Cell 6, 793-807.
Okura,T., Gong,L., Kamitani,T., Wada,T., Okura,I., Wei,C.F., Chang,H.M., and Yeh,E.T. (1996). Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. J. Immunol. 157, 4277-4281.
Pirrotta,V., Steller,H., and Bozzetti,M.P. (1985). Multiple upstream regulatory elements control the expression of the Drosophila white gene. EMBO J. 4, 3501-3508.
Pluta,A.F., Mackay,A.M., Ainsztein,A.M., Goldberg,I.G., and Earnshaw,W.C. (1995). The centromere: hub of chromosomal activities. Science 270, 1591-1594.
Ready,D.F., Hanson,T.E., and Benzer,S. (1976). Development of the Drosophila retina, a neurocrystalline lattice. Dev. Biol. 53, 217-240.
Rodriguez,M.S., Desterro,J.M., Lain,S., Midgley,C.A., Lane,D.P., and Hay,R.T. (1999). SUMO-1 modification activates the transcriptional response of p53. EMBO J. 18, 6455-6461.
Sun,Y.H., Tsai,C.J., Green,M.M., Chao,J.L., Yu,C.T., Jaw,T.J., Yeh,J.Y., and Bolshakov,V.N. (1995). White as a reporter gene to detect transcriptional silencers specifying position-specific gene expression during Drosophila melanogaster eye development. Genetics 141, 1075-1086.
Takahashi,Y., Iwase,M., Konishi,M., Tanaka,M., Toh-e A, and Kikuchi,Y. (1999). Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component of septin rings at the mother-bud neck in budding yeast. Biochem. Biophys. Res. Commun. 259, 582-587.
Williams,J.A., Paddock,S.W., and Carroll,S.B. (1993). Pattern formation in a secondary field: a hierarchy of regulatory genes subdivides the developing Drosophila wing disc into discrete subregions. Development 117, 571-584.