中文摘要
以5’ Rapid Amplification of cDNA End (5’RACE)的方法找出黃果蠅丙酮酸鹽激脢的轉錄起始點，並以黃果蠅丙酮酸鹽激脢基因組DNA當探針篩選λEMBL-3黃果蠅基因組圖書館，分離出一個含黃果蠅丙酮酸鹽激脢基因組DNA 5’ 端的選殖體-λpk61，再以ABI 377核酸自動定序儀定出黃果蠅丙酮酸鹽激脢轉錄起始點上游 734 個鹼基對的序列，將這些序列加以分析找出了許多可能與轉錄有關及可能會與蛋白質結合的核心序列。
由北方墨漬法分析不同發育期、不同性別黃果蠅丙酮酸鹽激脢基因的表現，發現在黃果蠅蛹期丙酮酸鹽激脢的表現明顯弱於黃果蠅幼蟲期及成蟲期，而在黃果蠅成蟲期不論雌或雄成蟲，其丙酮酸鹽激脢的基因表現差異不大，顯示在黃果蠅雄蟲中丙酮酸鹽激脢的基因表現有劑量補償的現象出現。此外，北方墨漬實驗亦顯示在不同發育期、不同性別的黃果蠅中可能只有一種丙酮酸鹽激脢 mRNA 存在。
RT-PCR則是被用來決定黃果蠅成蟲丙酮酸鹽激脢轉錄物的種類，而從結果中每一個聚合脢連鎖反應僅複製出一條複製片斷，顯示在黃果蠅成蟲中可能只有一條丙酮酸鹽激脢轉錄物，綜合北方墨漬及RT-PCR的結果推測在黃果蠅中可能只有一種丙酮酸鹽激脢基因存在。

Abstract
5’ Rapid Amplification of cDNA End (5’RACE) analysis was applied to identify the transcription start point (tsp) of pyruvate kinase gene (PyK) from Drosophila melanogaster. Screening a EMBL-3 genomic library of D.melanogaster using PyK genomic DNA as a probe, a genomic DNA clone (pk61), which contains the 5’ untranscribed region of D. melanogaster PyK gene was isolated. The insert was reconfirmed to contain Pyk gene by Southern blot analyses. A sequence of 734 bp upstream from tsp of Pyk was determined, from which some core sequences that might regulate transcription or might be recognized by proteins were proposed.
The PyK expression of D. melanogaster from different developmental stages and sexes was analyzed by Northern blot analyses. The results that the amounts of PyK transcripts produced in larvae and adults was higher than those in pupae, indicates that Pyk expression is low in pupal stages. The fact that similar level of PyK expression in both male and female adults, suggests that there is a dosage compensation phenomenon in Pyk gene of D. melanogaster male adults. In addition, the results that there is a single band present in all samples of the Northern blot analyses reveals that there is only a single PyK mRNA in different developmental stages and sexes of D. melanogaster.
RT-PCR analysis was performed to determine the number of species of PyK transcript in adults of D. melanogaster. The result that only a single amplicon appeared in each amplication, suggests that there might be a single PyK transcript expressed in adults of D. melanogaster. Furthermore, this might suggest that there is only a single PyK gene in D. melanogaster.

目錄
致謝…………………………………………………………..I
中文摘要………………………………………………….…II
英文摘要……………………………………………………III
目錄………………………………………………………….IV
表、圖目錄…………………………………………………...V
一、前言……………………………………………………….1
二、研究目的…………………………………………………6
三、材料與方法………………………………………………7
四、結果……………………………………………………..16
五、討論…………………………………………………….23
六、未來展望……………………………………………….27
七、參考文獻……………………………………………….28
表1…………………………………………………………...35
圖1-11……………………………………………………….36

七.參考文獻
Arkhipova IR, YV Ilyin. 1991. Properties of promoter regions of mdg1Drosophila retrotransponson indicate that it belongs to a specific class of promoters. EMBO J. 10:1169-1177.
Arkhipova IR. 1995. Promoter elements in Drosophila melanogaster revealed by sequence analysis. Genetics. 139:1359-1369.
Ashburner M. 1973. Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster II. The effects of inhibitors of protein synthesis. Dev. Biol. 39: 141-157.
Ashburner M, C Chihara, P Meltzer, G Richards. 1974. On the temporal control of puffing activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38: 655-622.
Benoist C, K O’Hare, R Breathnach, P Chambon. 1980. The ovalbumin gene-sequence of putative control regions. Nucl. Acids Res. 8: 127-142.
Benson M, V Pirrotta. 1988. The Drosophila zeste protein binds cooperatively to sites in many gene regulatory regions: implication for transvection and gene regulation . EMBO J. 7: 3907-3915.
Benyajati C, L Mueller, N Xu, M Pappano, J Gao, M Mosammaparast, D Conklin, H Granok, C Craig, S Elgin. 1997. Multiple isoforms of GAGA factor, a critical component of chromatin structure. Nucl. Acids Res. 25(16): 3345-3353.
Biggin M and R Tjian. 1988. Transcription factors that activate the Ultrabithorax promoter in developmentally staged extracts. Cell. 53: 699-711.
Biggin MD, S Bickel, M Benson, V Pirrotta, R Tjian. 1988. Zeste encodes a sequence-specific transcription factor that activates the Ultrabithorax promoter in vitro. Cell. 53: 713-722.
Bucher P. 1990. Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements dervived from 502 unrelated promoter sequences. J. Mol. Biol. 212: 563-578.
Burke TW, JT Kadonaba. 1996. Drosophila TFII D binds to a conserved downstream basal promoter element that is present in many TATA-box-deficient promoters. Genes Dev. 10: 711-724.
Burke TW, JT Kadonaga. 1997. The downstream core promoter element DEP, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila. Genes Dev. 11: 3020-3031.
Chien Y-C, Y-J Zhu, C-M Chuen. 1999. Complemintary DNA cloning and analysis of genus structure of pyruvate kinase from Drosophila melanogaster . Zoological Studies. 38(3): accepted.
Chodosh LA, AS Baldwin, RW Carthew, PA Sharp. 1988. Human CCAAT-binding proteins have heterologous subunits. Cell. 53: 11-24.
Cognet M, YC Lone, S Vaulont, A Kahn, J Marie. 1987. Structure of the rat L-type pyruvate kinase gene. J. Mol. Biol. 196: 11-25
Collier GE, JM James.1987. Arginine kinase and pyruvate kinase are coordinately controlled by the 75B “Early” gene in Drosophila imaginal discs. Genetics. 116: S12.
Dorn A, J Bollekens, A Staub, C Benoist, D Mathis. 1987. A multiplicity of CCAAT box-binding proteins. Cell. 50: 863-872.
Efstratiadis A, JW Posakony, T Maniatis, RM Lawn, C O’Cnnell, RA Blechl, O Smithies, FE Baralle, CC Shoulders, NJ Proudfoot . 1980. The structure and evolution of the human β—globin gene family. Cell. 21: 653-668.
Garen A, L Kauvar, JA Lepesant. 1977. Roles of ecdysone in Drosophila development. Proc. Natl. Acad, Sci. (USA) 74: 5099-5103.
Gelbart WM, CT Wu. 1982. Interactions of zeste mutation with loci exhibiting transvection effects in Drosophila melanogaster. Genetics. 102: 179-189.
Gunaratne PH, A Mansukhane, SE Lipari, HC Liou. DW Martindale, ML Goldberg. 1986. Molecular cloning, germline transformation and transcriptional analysis of the zeste locus of Drosophila melanogaster. Proc. Natl. Acad. Sci. 83: 701-705.
Handler AM. 1982. Ecdysteroid titers during pupal and adult
development in Drosophila melanogaster. Dev. Biol. 93: 73-82.
Hodgetts RB, B Sage, O’connor. 1977. Ecdysone titers during postembryonic development of Drosophila melanogaster. Dev. Biol. 60: 310-317.
Imamura K, T Tanaka. 1972. Mutimolecular forms of pyruvate kinase from rat and other mammalian tissues. J. Biochem. 71: 1043-1051.
Imamura K, T Tanaka, T Nishina, K Nakashima, S Miwa. Studies on pyruvate kinase (PK) deficiency. J. Biochem. 74: 1165-1175.
James JM. 1987. Ph.D. Dissertation, Illinois State University.
James JM, GE Collier. 1988. Distribution and genetic basis of arginine kinase in wild type and flightless mutants of Drosophila melanogaster. J. Exp. Zool. 248: 185- 191.
James JM, GE Collier. 1990. Hormonally regulated expression of arginine kinase in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 198: 474-478 .
James JM, GE Collier. 1992. Early gene interaction during prepupal expression of Drosophila arginine kinase. Dev. Genent. 13: 302-305.
Kadalayil L, U-M Petersen, Y Engstrom. 1997. Adjacent GATA and κB-like motifs regulate the expression of a Drosophila immune gene. Nucl. Acids Res. 25(6): 1233-1239.
Kanno H, H Fujii, S Miwa. 1992. Structural analysis of human pyruvate kinase L-gene and identification of the promoter activity in erythroid cells. Biochem. Biophys. Res.commun. 188: 516-523.
Kaufman TC, SE Tasaka, OT Suzuki. 1973. The interaction of two complex loci zeste and bithorax in Drosophila melanogaster. Genetics. 75: 299-321.
Ko LJ, JD Engel. 1993. DNA-binding specificities of the GATA transcription factor family. Mol. Cell Biol. 13(7): 4011-4022.
Lee H-S, KW Kraus, MF Wolfner, JT Lis. 1992. DNA sequence requirements for generating paused polymerase at the start of hsp 70. Genes Dev. 6: 284-295.
Lee YM, T Park, RA Schulz, Y Kim. 1997. Twist-mediated activation of the NK-4 homeobox gene in the visceral mesoderm of Drosophila requires two distinct clusters of E-box regulatory element. J.Biol. Chem. 272(28):17531-17541.
Lone YC, MP Simon, A Kahn, J Marie. 1986. Complete nucleotide and deduced amino acid sequences of rat L-type pyruvate kinase. FEBS 195(1,2): 97-100
Mariani C, B Pirrotta, E Manet. 1985. Isolation and characterization of the zeste locus of Drosophila. EMBO J. 4: 2045-2052.
McKnight SL, R Kingsbury. 1982. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 217(23): 316-324.
Mclean C, A Bucheton, DJ Finnegan. 1993. The 5’-untranslated region of the I factor, a long interspersed nuclear element-like retrotransposon of Drosophila melanogaster, contains an internal promoter and sequences that regulate expression. Mol. Cell Biol. 13: 1042-1050.
Merika M, SH Orkin. 1993. DNA-binding specificity of GATA family transcription factors. Mol. Cell Biol. 13(7): 3999-4010.
Minchiotti G, PP Di Noncera. 1991. Convergent transcription initiates from oppositely oriented promoters within the 5’end regions of Drosophila melanogaster F elements. Mol. Cell Biol. 11: 5171-5180.
Minchiotti G, C Contursi, PP Di Nocera. 1997. Multiple downstream promoter modules regulate the transcription of the Drosophila melanogaster I. Doc and F elements. J. Mol. Biol. 267: 37-46.
Noguchi T, H Inoue, Y Nakamura, HL Chen, Ki Matsubara, T
Tanaka.1984. Molecular cloning of cDNA sequences for rat M2-type pyruvate kinase and regulation of its mRNA. J. Biol. Chem. 259(4): 2651-2655
Noguchi T, H Inoue, T Tanaka. 1986. The M1 and M2-type isozymes of rat pyuuvate kinase are produced from the same gene by alternative RNA splicing. J. Biol. Chem. 261(29): 13807-13812.
Noguchi T, K Yamada, H Inoue, T Matsuda, T Tanaka. 1987. The L- and R- type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. J. Biol. Chem. 262(29): 14366-14371.
Noguchi T, K Yamada, K Yamagata, M Takenaka, H Nakajima, E Imai, Z Wang, T Tanaka. 1991. Expression of liver type pyruvate kinase in insulinoma cells: involvement of LF-B1 (HNF1). Biochem. Biophys. Res. Commun. 181(1): 259-264.
O’Connell P, M Rosbash. 1984. Sequence, structure and codon preference of the Droslphila ribosomal protein 49 gene. Nucl. Acids Res. 12(13): 5495-5513
O’Donnell KH, PC Wensink. 1994. GAGA factor and TBF1 bind DNA elements that direct ubiquitous transcription of the Drosophila α1-tubulin gene. Nucleic Acids Res. 22: 4712-4718.
Olson EN. 1990. MyoD family: a paradigm for development. Genes Dev. 4: 1454-1461.
Orkin SH. 1992. GATA-binding transcription factors in hematopoietic cells. Blood. 80(3): 575-581.
Perkins KK, GM Dailey, R Tjian. 1988. In vitro analysis of the Antennapedia P2 promoter: identification of a new Drosophila transcription factor. Genes Dev. 2: 1615-1626.
Pilar P, U Cristina, C Manuel, G Rafael. 1995. Analysis of the mitochondrial ATP synthase β—subunit gene in Drosophidae: structure, transcriptional regulatory features and developmental pattern of expression in Drosophila melsnogaster. Biochem. J. 312: 887-897.
Pirrotta V, E Manet, E Hardon, SE Bickel, M Benson. 1987. Structure and sequence of the Drosophila zeste gene. EMBO J. 6: 791-799.
Rasmussen EB, JT Lis. 1993. In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. Proc. Natl. Acad. Sci. USA. 90: 7923-7927.
Roberts DB. 1986. Drosophila: a practical approach. IRL Press Limited. pp.25-27
Rust KJ, GE Collier. 1985. Localization of a dosage sensitive region for pyruvate kinase in Drosophila melanogaster. J. Heredity. 76:39-44.
Sambrook J, EE Fritsch, T Maniatis. 1989. Molecular cloning: A laboratory manual, 2nd ed., Cold Spring Haubor Laboratory Press.
Segravaves WA, DS Hogness. 1990. The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes.Dev. 4: 204-219.
Soeller W, SJ Poole, T Kornberg. 1988. In vitro transcription of the Drosophila engrailed gene. Genes Dev. 2: 68-81.
Soller WC, CE Oh, TB Kornberg. 1993. Isolation of cDNAs encoding the Drosophila GAGA transcription factor. Mol. Cell. Biol. 113: 7961-7970.
Southern EM. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503
Strickberger MW.1962. Experiments in genetics with Drosophila, John Wiley and Sons, Inc., New York pp.8-12.
Takenaka M, T Noguchi, H Inoue, K Yamada, T Matsuda, T Tanaka. 1989. Rat pyruvate kinase M gene. Its complete structure and characterization of the 5’-flanking region.J. Biol. Chem. 264(4): 2363-2367 .
Takenaka M, T Noguchi, S Sadahiro, H Hirai, K Yamada, T Matsuda, E Imai, T Tanaka. 1991. Isolation and characterization of the human pyruvate kinase M gene. Eur. J. Biochem. 198: 101-106..
Takenaka M, K Yamada, T Lu, R Kang, T Tanaka, T Noguchi. 1996. Alternative splicing of the pyruvate kinase M gene in a minigene system. FEBS. 235: 366- 371.
Tanaka T, YH Harano, R Mori. 1965. Evidence for the presence of two type of pyruvate kinase in rat liver liver. Biochim. Biophys. Res. Commun. 21: 55-60.
Tanaka T, YH Harano, R Mori. 1967. Crystallization, characterization and metabolic regulation of two type of pyruvate kinase isolated from rat tissues. J. Biochem. (Tokyo) 62: 71.
Tremp GL, D Boquet, MA Ripoche, M Cognet, YC Lonet, J Jami, A Kahn, D Daegelen. 1989. Expression of the rat L-type pyruvate kinase gene from its dual erythrois- and liver-specific promoter in transgenic mice. J. Biol. Chem. 264(33): 19904-19910.
Tsukiyama T, P Becker, C Wu. 1994. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor . Nature. 367: 525-532.
Wasylyk B, SL Hahn, A Giovane. 1993. The Ets family of transcription factors. FEBS. 211: 7-18
Wikins RC, JT Lis. 1998. GAGA factor binding to DNA via a single trinucleotide sequence element. Nucleic Acids Res. 26(11): 2672-26