臺灣博碩士論文加值系統

綠豆子葉粒線體DNA結構、型態和複雜性在綠豆種子發芽過程中呈現高度的動態變化，本研究目的在藉此了解植物粒線體DNA複雜性和複製過程的相關性。研究結果顯示，在早期子葉發育過程中，粒線體中短的DNA片段轉換為長的線性DNA以及花瓣狀結構的DNA。隨著DNA在子葉發育過程中複製活力越高，伴隨著越來越複雜的花瓣狀結構出現。在DNA複製過程中，複製叉會出現在這種花瓣結構核心周圍部分。以低溫處理種子或低溫儲存已純化之子葉粒線體DNA，會誘導DNA轉成長線狀結構，花瓣結構的核心部分消失。這種長線結構上可明顯觀察到D環 (D-loop)、霍利迪交叉 (Holliday junction)等複製結構。長線結構型態的DNA總長度可能超過花瓣結構型態的長度30倍以上。本研究推測這種花瓣核心結構包含大量聚集的DNA，是依賴重組複製模式(Recombination dependent replication, RDR)中DNA複製的主要場所以及複製起點位置。在花瓣結構上出現的衛星核心結構代表另一個重新開始的複製起點，進而形成巨大且複雜、具有大量複製中間體的粒線體DNA分子結構。此研究結果可證實花瓣狀DNA結構是植物粒線體複製之中間產物，並且確定粒線體DNA是依循依賴重組複製模式進行複製。

The highly dynamic changes in mitochondrial DNA (mtDNA) conformation, structure and complexity correspond to the activity of mtDNA recombination dependent replication (RDR). This study aimed to elucidate the interrelationship between mtDNA replication and genesis of the multi-genomic, highly complex structure of plant mtDNA in mung bean cotyledons. The results suggest that short DNA fragments convert to longer linear and rosette structures early in the development of cotyledon mitochondria. Consequently, a large number of rosette structures appear, with simultaneous elevation of mtDNA synthesis. A fork-like structure near the rosette core appears during mtDNA replication. With in vivo prolonged-cold incubation or in vitro freeze-thaw treatment, the rosette structure is converted to a much longer linear DNA structure and the rosette core disappears. This large linear DNA with displacement loops, Holliday junctions and other structures attached may increase in size more than 30 times as compared to the original rosette entity. The rosette core may consist of condensed mtDNA and play an initial and central role in RDR. The satellite cores in the rosette structure may represent the re-initiation sites of mtDNA RDR in the same parental molecule. Consequently, highly complex and giant mitochondrial DNA molecules, representing RDR intermediates, are formed in vivo. The core-and-rosette structures represent replicating DNA and almost certainly use RDR.

List of Tables iv
List of Figures v
中文摘要 vii
Abstract viii
Abbreviation ix
I. Introduction 1
1. Mitochondrial genome 1
2. Mitochondrial nucleoid 2
3. Plant mitochondrial genome 3
4. Mitochondrial genome replication 5
5. The aim of the study 7
II. Materials and Methods 8
1. Materials 8
2. Mitochondria isolation from mung bean cotyledons 8
3. MtDNA purification of mung bean cotyledons 9
4. Flow cytometric of isolated mitochondria 10
5. Pulsed-field gel electrophoresis 10
6. Analytical ultracentrifugation analysis 11
7. EM examination of mtDNA 11
8. PCR analysis 12
9. In vivo mtDNA synthesis 12
10. LC-MS/MS 13
11. Mung bean nuclease and RNaseH treatment for mtDNA 13
12. DNA purification from PFGE gels 14
13. Real-time PCR analysis 14
14. Immunoblot Analysis 14
III. Results 16
1. Altered quantity of mtDNA at different stages of cotyledon development 16
2. Physical characters of cotyledon mtDNA during seed germination 17
3. Fluctuation of cotyledon mtDNA synthesis in organello during seed germination 18
4. The difference between wb and fm mtDNA in terms of buoyant density, ultra-structure and restriction enzyme patterns 20
5. MtDNA ultrastructural dynamics during cotyledon development 20
6. Structural characteristics of giant mtDNA rosette complex 22
7. Unraveling the mtDNA rosette structure by freeze-thaw treatment in vitro 23
8. RDR recombination assessed by real-time PCR 26
9. Mitochondrial nucleoproteins present in cotyledon mitochondria with cotyledon aging during mung bean seed germination 26
IV. Discussion 28
V. Conclusions 38
VI. References 39

Tables 45
Figures 50

Alverson AJ, Rice DW, Dickinson S, Barry K, Palmer JD. 2011a. Origins and recombination of the bacterial-sized multichromosomal mitochondrial genome of cucumber. Plant Cell 23(7): 2499-2513.
Alverson AJ, Zhuo S, Rice DW, Sloan DB, Palmer JD. 2011b. The mitochondrial genome of the legume Vigna radiata and the analysis of recombination across short mitochondrial repeats. PLoS One 6(1): e16404.
Andre C, Levy A, Walbot V. 1992. Small repeated sequences and the structure of plant mitochondrial genomes. Trends Genet 8(4): 128-132.
Arrieta-Montiel MP, Shedge V, Davila J, Christensen AC, Mackenzie SA. 2009. Diversity of the Arabidopsis mitochondrial genome occurs via nuclear-controlled recombination activity. Genetics 183(4): 1261-1268.
Backert S, Borner T. 2000. Phage T4-like intermediates of DNA replication and recombination in the mitochondria of the higher plant Chenopodium album (L.). Curr Genet 37(5): 304-314.
Backert S, Dorfel P, Lurz R, Borner T. 1996. Rolling-circle replication of mitochondrial DNA in the higher plant Chenopodium album (L.). Mol Cell Biol 16(11): 6285-6294.
Backert S, Lurz R, Oyarzabal OA, Borner T. 1997a. High content, size and distribution of single-stranded DNA in the mitochondria of Chenopodium album (L.). Plant Mol Biol 33(6): 1037-1050.
Backert S, Nielsen BL, Borner T. 1997b. The mystery of the rings: structure and replication of mitochondrial genomes from higher plants. Trends in Plant Science 2(12): 477–483.
Bendich AJ. 1993. Reaching for the ring: the study of mitochondrial genome structure. Curr Genet 24(4): 279-290.
Bendich AJ. 1996. Structural analysis of mitochondrial DNA molecules from fungi and plants using moving pictures and pulsed-field gel electrophoresis. J Mol Biol 255(4): 564-588.
Bendich AJ. 2004. Circular chloroplast chromosomes: the grand illusion. Plant Cell 16(7): 1661-1666.
Bendich AJ. 2010. The end of the circle for yeast mitochondrial DNA. Mol Cell 39(6): 831-832.
Bogenhagen DF. 2010. Does mtDNA nucleoid organization impact aging? Exp Gerontol 45(7-8): 473-477.
Bogenhagen DF. 2012. Mitochondrial DNA nucleoid structure. Biochim Biophys Acta 1819(9-10): 914-920.
Bogenhagen DF, Rousseau D, Burke S. 2008. The layered structure of human mitochondrial DNA nucleoids. J Biol Chem 283(6): 3665-3675.
Bogenhagen DF, Wang Y, Shen EL, Kobayashi R. 2003. Protein components of mitochondrial DNA nucleoids in higher eukaryotes. Mol Cell Proteomics 2(11): 1205-1216.
Bowmaker M, Yang MY, Yasukawa T, Reyes A, Jacobs HT, Huberman JA, Holt IJ. 2003. Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone. J Biol Chem 278(51): 50961-50969.
Cappadocia L, Marechal A, Parent JS, Lepage E, Sygusch J, Brisson N. 2010. Crystal structures of DNA-Whirly complexes and their role in Arabidopsis organelle genome repair. Plant Cell 22(6): 1849-1867.
Chang S, Wang Y, Lu J, Gai J, Li J, Chu P, Guan R, Zhao T. 2013. The mitochondrial genome of soybean reveals complex genome structures and gene evolution at intercellular and phylogenetic levels. PLoS One 8(2): e56502.
Chen XJ, Butow RA. 2005. The organization and inheritance of the mitochondrial genome. Nat Rev Genet 6(11): 815-825.
Cupp JD, Nielsen BL. 2014. Minireview: DNA replication in plant mitochondria. Mitochondrion 19 Pt B: 231-237.
D''Aurelio M, Gajewski CD, Lin MT, Mauck WM, Shao LZ, Lenaz G, Moraes CT, Manfredi G. 2004. Heterologous mitochondrial DNA recombination in human cells. Hum Mol Genet 13(24): 3171-3179.
Dai H, Lo YS, Litvinchuk A, Wang YT, Jane WN, Hsiao LJ, Chiang KS. 2005. Structural and functional characterizations of mung bean mitochondrial nucleoids. Nucleic Acids Res 33(15): 4725-4739.
Dai H, Lo YS, Wu CY, Tsou CL, Hsu GS, Chern CG, Ruddat M, Chiang KS. 1991. Protein Synthesis in Isolated Mitochondria of Rice (Oryza sativa L.) Seedlings. Plant Physiol 96(1): 319-323.
Dudareva NA, Kiseleva EV, Boyarintseva AE, Maystrenko AG, Khristolyubova NB, Salganik RI. 1988. Structure of the mitochondrial genome of Beta vulgaris L. Theor Appl Genet 76(5): 753-759.
Edmondson AC, Song D, Alvarez LA, Wall MK, Almond D, McClellan DA, Maxwell A, Nielsen BL. 2005. Characterization of a mitochondrially targeted single-stranded DNA-binding protein in Arabidopsis thaliana. Mol Genet Genomics 273(2): 115-122.
Gerhold JM, Aun A, Sedman T, Joers P, Sedman J. 2010. Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication. Mol Cell 39(6): 851-861.
Gualberto JM, Kuhn K. 2014. DNA-binding proteins in plant mitochondria: implications for transcription. Mitochondrion 19 Pt B: 323-328.
Gualberto JM, Mileshina D, Wallet C, Niazi AK, Weber-Lotfi F, Dietrich A. 2014. The plant mitochondrial genome: dynamics and maintenance. Biochimie 100: 107-120.
He J, Mao CC, Reyes A, Sembongi H, Di Re M, Granycome C, Clippingdale AB, Fearnley IM, Harbour M, Robinson AJ, et al. 2007. The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization. J Cell Biol 176(2): 141-146.
Heinhorst S, Cannon GC. 1993. DNA replication in chloroplasts. Journal of Cell Science 104: 1-9.
Ifft JB, Voet DH, Vinograd J. 1961. The determination of density distributions and density gradients in binary solutions at equilibrium in the ultracentrifuge. J. Phys. Chem. 65: 1138-1145.
Junier I, Martin O, Kepes F. 2010. Spatial and topological organization of DNA chains induced by gene co-localization. PLoS Comput Biol 6(2): e1000678.
Kang D, Kim SH, Hamasaki N. 2007. Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions. Mitochondrion 7(1-2): 39-44.
Kasiviswanathan R, Collins TR, Copeland WC. 2012. The interface of transcription and DNA replication in the mitochondria. Biochim Biophys Acta 1819(9-10): 970-978.
Kaufman BA, Durisic N, Mativetsky JM, Costantino S, Hancock MA, Grutter P, Shoubridge EA. 2007. The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures. Mol Biol Cell 18(9): 3225-3236.
Kaufman BA, Kolesar JE, Perlman PS, Butow RA. 2003. A function for the mitochondrial chaperonin Hsp60 in the structure and transmission of mitochondrial DNA nucleoids in Saccharomyces cerevisiae. J Cell Biol 163(3): 457-461.
Kazak L, Reyes A, Holt IJ. 2012. Minimizing the damage: repair pathways keep mitochondrial DNA intact. Nat Rev Mol Cell Biol 13(10): 659-671.
Kolodner R, Tewari KK. 1972. Molecular size and conformation of chloroplast deoxyribonucleic acid from pea leaves. J Biol Chem 247(19): 6355-6364.
Kucej M, Butow RA. 2007. Evolutionary tinkering with mitochondrial nucleoids. Trends Cell Biol 17(12): 586-592.
Kucej M, Kucejova B, Subramanian R, Chen XJ, Butow RA. 2008. Mitochondrial nucleoids undergo remodeling in response to metabolic cues. J Cell Sci 121(11): 1861-1868.
Kumar RA, Oldenburg DJ, Bendich AJ. 2014. Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development. J Exp Bot 65(22): 6425-6439.
Kumar RA, Oldenburg DJ, Bendich AJ. 2015. Molecular integrity of chloroplast DNA and mitochondrial DNA in mesophyll and bundle sheath cells of maize. Planta 241(5): 1221-1230.
Li JF, Li L, Sheen J. 2010. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology. Plant Methods 6(1): 1.
Lo YS, Cheng N, Hsiao LJ, Annamalai A, Jauh GY, Wen TN, Dai H, Chiang KS. 2011. Actin in mung bean mitochondria and implications for its function. Plant Cell 23(10): 3727-3744.
Lonsdale DM. 1984. A review of the structure and organization of the mitochondrial genome of higher plants. Plant Mol Biol 3(4): 201-206.
Losada L, Pakala SB, Fedorova ND, Joardar V, Shabalina SA, Hostetler J, Pakala SM, Zafar N, Thomas E, Rodriguez-Carres M, et al. 2014. Mobile elements and mitochondrial genome expansion in the soil fungus and potato pathogen Rhizoctonia solani AG-3. FEMS Microbiol Lett 352(2): 165-173.
Maleszka R, Skelly PJ, Clark-Walker GD. 1991. Rolling circle replication of DNA in yeast mitochondria. EMBO J 10(12): 3923-3929.
Marechal A, Brisson N. 2010. Recombination and the maintenance of plant organelle genome stability. New Phytol 186(2): 299-317.
Naito K, Kaga A, Tomooka N, Kawase M. 2013. De novo assembly of the complete organelle genome sequences of azuki bean (Vigna angularis) using next-generation sequencers. Breed Sci 63(2): 176-182.
Oldenburg DJ, Bendich AJ. 1996. Size and Structure of Replicating Mitochondrial DNA in Cultured Tobacco Cells. Plant Cell 8(3): 447-461.
Oldenburg DJ, Bendich AJ. 2001. Mitochondrial DNA from the liverwort Marchantia polymorpha: circularly permuted linear molecules, head-to-tail concatemers, and a 5'' protein. J Mol Biol 310(3): 549-562.
Oldenburg DJ, Bendich AJ. 2004. Changes in the structure of DNA molecules and the amount of DNA per plastid during chloroplast development in maize. J Mol Biol 344(5): 1311-1330.
Oldenburg DJ, Bendich AJ. 2015. DNA maintenance in plastids and mitochondria of plants. Front Plant Sci 6: 883.
Palmer JD, Herbon LA. 1986. Tricircular mitochondrial genomes of Brassica and Raphanus: reversal of repeat configurations by inversion. Nucleic Acids Res 14(24): 9755-9764.
Petersen G, Cuenca A, Moller IM, Seberg O. 2015. Massive gene loss in mistletoe (Viscum, Viscaceae) mitochondria. Sci Rep 5: 17588.
Pohjoismaki JL, Holmes JB, Wood SR, Yang MY, Yasukawa T, Reyes A, Bailey LJ, Cluett TJ, Goffart S, Willcox S, et al. 2010. Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. J Mol Biol 397(5): 1144-1155.
Rebelo AP, Dillon LM, Moraes CT. 2011. Mitochondrial DNA transcription regulation and nucleoid organization. J Inherit Metab Dis 34(4): 941-951.
Reyes A, He J, Mao CC, Bailey LJ, Di Re M, Sembongi H, Kazak L, Dzionek K, Holmes JB, Cluett TJ, et al. 2011. Actin and myosin contribute to mammalian mitochondrial DNA maintenance. Nucleic Acids Res 39(12): 5098-5108.
Reyes A, Kazak L, Wood SR, Yasukawa T, Jacobs HT, Holt IJ. 2013. Mitochondrial DNA replication proceeds via a ''bootlace'' mechanism involving the incorporation of processed transcripts. Nucleic Acids Res 41(11): 5837-5850.
Sakai A, Takano H, Kuroiwa T. 2004. Organelle nuclei in higher plants: structure, composition, function, and evolution. Int Rev Cytol 238: 59-118.
Sala F, Parisi B, Burroni D, Amileni AR, Pedrali-Noy G, Spadari S. 1980. Specific and reversible inhibition by aphidicolin in the alpha-like DNA polymerase of plant cells. FEBS Lett 117(1): 93-98.
Sambrook J, Fritsch E, Maniatis T 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 382 - 389.
Sambrook J, Russell D. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Shedge V, Arrieta-Montiel M, Christensen AC, Mackenzie SA. 2007. Plant mitochondrial recombination surveillance requires unusual RecA and MutS homologs. Plant Cell 19(4): 1251-1264.
Sloan DB. 2013. One ring to rule them all? Genome sequencing provides new insights into the ''master circle'' model of plant mitochondrial DNA structure. New Phytol 200(4): 978-985.
Takanashi H, Arimura S, Sakamoto W, Tsutsumi N. 2006. Different amounts of DNA in each mitochondrion in rice root. Genes Genet Syst 81(3): 215-218.
Thresher R, Griffith J. 1992. Electron microscopic visualization of DNA and DNA-protein complexes as adjunct to biochemical studies. Methods Enzymol 211: 481-490.
Valach M, Farkas Z, Fricova D, Kovac J, Brejova B, Vinar T, Pfeiffer I, Kucsera J, Tomaska L, Lang BF, et al. 2011. Evolution of linear chromosomes and multipartite genomes in yeast mitochondria. Nucleic Acids Res 39(10): 4202-4219.
Wells R, Ingle J. 1970. The constancy of the buoyant density of chloroplast and mitochondrial deoxyribonucleic acids in a range of higher plants. Plant Physiol 46(1): 178-179.
Williamson D. 2002. The curious history of yeast mitochondrial DNA. Nat Rev Genet 3(6): 475-481.
Woloszynska M. 2010. Heteroplasmy and stoichiometric complexity of plant mitochondrial genomes--though this be madness, yet there''s method in''t. J Exp Bot 61(3): 657-671.
Woloszynska M, Gola EM, Piechota J. 2012. Changes in accumulation of heteroplasmic mitochondrial DNA and frequency of recombination via short repeats during plant lifetime in Phaseolus vulgaris. Acta Biochim Pol 59(4): 703-709.
Woloszynska M, Trojanowski D. 2009. Counting mtDNA molecules in Phaseolus vulgaris: sublimons are constantly produced by recombination via short repeats and undergo rigorous selection during substoichiometric shifting. Plant Mol Biol 70(5): 511-521.
Zhang L, Chan SS, Wolff DJ. 2011. Mitochondrial disorders of DNA polymerase gamma dysfunction: from anatomic to molecular pathology diagnosis. Arch Pathol Lab Med 135(7): 925-934.