沈哲鯤臺灣大學：分子醫學研究所林孟昭Lin, Meng-JauMeng-JauLin2007-11-262018-07-092007-11-262018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/51360DNA甲基化現象（DNA methylation）廣泛的存在於原核生物至真核生物，在基因調控上扮演極重要的角色。在哺乳動物中，已知DNA甲基化影響了包含基因表現、胚胎發育、X染色體不活化及癌症發生等生物反應。DNA甲基化是由DNA甲基化酵素（DNA (cytosine-5)-methyltransferase, Dnmt）進行。現在已經發現有多種DNA甲基化酵素存在於哺乳動物中，包含Dnmt1, Dnmt2, Dnmt3A與Dnmt3B。這些酵素的C-terminal都似有高度保留的motifs。其中Dnmt1是最早被發現的DNA甲基化酵素，已知它與DNA甲基化型式的保持有關，而Dnmt3A及Dnmt3B的作用主要是進行de novo methylation，即在新的位置產生甲基化DNA。另一個DNA甲基化酵素，Dnmt2，並未含有N-terminal的調節區（regulatory region），是唯一存在於果蠅中的DNA甲基化酵素。 雖然Dnmt2的甲基化活性已經被證實，不過其存在於生物體的功能尚未釐清。在此我們利用了基因轉殖等方式在果蠅中建立DNA甲基化的模式系統，以了解dDnmt2在果蠅中所扮演的角色。在研究過程中我們發現dDnmt2基因會影響果蠅的生命長短，當大量表現 dDnmt2時果蠅的存活時間有增長的現象，同時在果蠅dDnmt2的P-element mutant 中發現有存活天數降低的現象。而大量表現 dDnmt2的果蠅也具有較高的抗氧化性。 另外，在P element跳離(P element excision)的研究中我們得到一個dDnmt2基因的缺失突變(deletion mutant)。此突變的第三期幼蟲(third-instar larvae)無法正常的發育成蛹(pupa)，且其無法正常表現ecdysone signaling下游基因。由此我們推斷dDnmt2基因在果蠅的變態及ecdysone signaling pathway 扮演重要的角色。Methylation of DNA at the C5 position of cytosine residues is found from prokaryote to eukaryote. DNA methylation at the mammalian genomes participates in the control of several interlinked biological processes, e.g., gene expression, cell growth, genomic imprinting, X-chromosome inactivation, and embryogenesis, which in turn are mediated through chromatin remodeling. The mammalian DNA methyltransferases (Dnmt), including Dnmt1, Dnmt2, Dnmt3A, and Dnmt3B, are a family of proteins with highly conserved motifs in their carboxyl terminal regions. Of these, Dnmt1 is the maintenance enzyme. Dnmt3A and Dnmt3B carry out the de novo methylation reaction. Unlike the above three enzymes, Dnmt2 lacks the N-terminal regulatory region. Although recent studies have demonstrated residual DNA methylation activities of the mammalian Dnmt2 proteins and the Drosophila ortholog dDnmt2, the latter of which is the single DNA methyltransferase responsible for genome methylation of the fruit flies, the functional role of the Dnmt2 proteins remained unclear. We present evidence that intactness of the gene is required for maintenance of the normal lifespan of the fruit flies. In contrast, overexpression of dDnmt2 could extend Drosophila lifespan. The study links the Drosophila DNA methylation program with the longevity/aging, and has interesting implication on the eukaryotic DNA methylation programs in general. A dDnmt2 deletion mutant was obtained from P element excision. The mutant arrested at third-instar larvae stage and cannot develop to pupa stage. RT-PCR data shown the temporal profiles of nuclear receptor gene are abnormal. Suggest that dDnmt2 may contribute to the Drosophila metamorphosis and ecdysone-signaling pathway.Chinese abstract 1 Abstract 3 Introduction 5 DNA methylation 5 The cytosine methyltransferase families of eukaryotes 7 Methylated DNA binding proteins 11 DNA methylation in Drosophila 11 Longevity related genes in Drosophila 12 Material and Methods: 15 Drosophila stock and culture conditions 15 Isolation of fly genomic DNA 15 Inverse PCR 15 RNA isolation and RT-PCR for dDnmt2 gene 16 Life span analysis 17 Spetic injuries 18 Natural infection by Beauveria bassiana 18 RNA isolation and RT-PCR of immune challenged flies 18 Stress resistant 19 RT-PCR for longevity related genes 20 P- element excision 21 Screen if dDnmt2 P element excision mutants 22 Lethal Phase Determination 23 Developmental Staging 24 RT-PCR to determination of temporal profiles of ecdysone-inducible genes in dDnmt2 mutant 24 Results 26 I. Increasing dDnmt2 expression extends life span in Drosophila 26 II. dDnmt2 extend life span not via increase Drosophila immunity 27 III. Overexpression of dDnmt2 increase stress resistance 29 IV. dDnmt2 upregulate small heatshock proteins 31 V. Metamorphic development is blocked in dDnmt2 imprecise P element excision mutant 32 Discussion 35 Figure 40 Table 55 Reference 57 Appendix 66 Lin, M.-J., Tang, L.-Y., Reddy, M. N. and Shen, C.-K. ( 2005) DNA methyltransferase dDnmt2 and Drosophila longevity. J Biol Chem. 280, 861-864.3127465 bytesapplication/pdfen-US果蠅DNA甲基化酵素長壽DrosophilaDNA (cytosine-5)-methyltransferaselongevityotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51360/1/ntu-94-D89448003-1.pdf