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研究生:王中茹
研究生(外文):Chung-Ju Rachel Wang
論文名稱:利用螢光原位雜交定位玉米重複性與單拷貝序列於粗絲期染色體
論文名稱(外文):Localization of repetitive and single-copy sequences on maize pachytene chromosomes by fluorescence in situ hybridization
指導教授:陳其昌陳其昌引用關係
指導教授(外文):Chi-Chang Chen
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:植物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:英文
論文頁數:97
中文關鍵詞:實質定位細胞遺傳圖輿玉米螢光原位雜交粗絲期染色體異染色質結
外文關鍵詞:physical mappingcytogenetic mapmaizefluorescence in situ hybridization (FISH)pachytene chromosomeknob
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180-bp和TR-1是聚集在玉米異染色質結 (heterochromatic knob) 的兩種銜接排列的重複性序列 (tandemly repeated sequences)。在本論文中,從玉米及近緣種teosinte中分離出59個TR-1選殖體,由南方氏雜交和DNA序列分析發現TR-1由三種次序列所組成: A (67 bp)、 B (184 bp) 或 B' (184 bp),2/3 B (115 bp) 或 2/3 B' (115 bp) 和 C (108 bp),並且排列組合成以180 bp為重複單位的複雜構造。根據序列的結構,我們提出TR-1可能的演化機制。DNA序列比對顯示180-bp和TR-1這兩種異染色質結序列與B染色體中節序列 (B repeat) 具有同源性 (homology),因此我們推測B repeat是由A染色體上的異染色質結序列演化而來。利用螢光原位雜交技術,B repeat被定位於B染色體的中節,而180-bp和TR-1被定位於B染色體中節旁的異染色質結上。
以一種中節特有的重複性序列CentC,與KYS及A344兩種玉米自交系的粗絲期染色體做原位雜交,由訊號強度的差異可將中節區分為兩類,並發現與利用傳統aceto-carmine染色法所觀察到的兩種中節相互吻合。此外,以端粒序列(5'-TTTAGGG-3')n為探針,與KYS和A344的粗絲期染色體雜交,根據訊號強弱推測A344的端粒序列重複次數較KYS多。從本論文及之前的研究結果顯示:由CentC雜交訊號的強弱,染色體長度,長短臂的比例,和異染色質結序列的分佈,玉米的十條粗絲期染色體可以被清楚鑑定。我們進一步利用所建立的螢光原位雜交技術,將選殖於載體中長度為5-7 kb的單拷貝DNA序列定位在玉米粗絲期染色體上。

Two families of tandem repeats, 180-bp and TR-1, have been found in the knobs of maize. In this thesis, we isolated 59 clones belonging to the TR-1 family from maize and teosinte. Southern hybridization and sequence analysis revealed that members of this family are composed of three basic sequences, A (67 bp), B (184 bp) or its variants B' (184 bp), 2/3 B (115 bp), 2/3 B' (115 bp), and C (108 bp), which are arranged in various combinations to produce repeat units which are multiples of approximately 180 bp. Based on the structure, we proposed a hypothesis for the origin of TR-1 elements. Sequence comparisons showed partial homologies between knob repeats and the centromeric sequence (B repeat) of the B chromosome, suggesting that the B repeat is derived from knob repeats in A chromosomes. Fluorescence in situ hybridization (FISH) localized the B repeat to the B centromere and the 180-bp and TR-1 repeats to the proximal heterochromatin knob on the B chromosome.
The strength of hybridization signals from a centromere specific repeat, CentC, on pachytene chromosomes of inbred lines KYS and A344 could be classified into two types corresponding to the appearance of centromeres stained by aceto-carmine. The telomeres of A344 are longer than those of KYS as revealed by pachytene FISH with telomeric repeat 5'-TTTAGGG-3'. This and previous studies demonstrate that based on the intensity of CentC signals, chromosome length, arm ratio and distribution of knob sequences, ten pachytene chromosomes of maize can be identified unambiguously. We further show that with the technique developed by us, single-copy sequences as small as 5-7 kb inserted in plasmid vectors can be mapped on maize pachytene chromosomes.

Chapter 1 Molecular characterization of a family of tandemly
repeated DNA sequences, TR-1, in heterochromatic
knobs of maize and its relatives----------------20
Chapter 2 Localization of centromere and telomere sequences
on maize pachytene chromosomes by fluorescence
in situ hybridization --------------------------53
Chapter 3 Localization of single-copy DNA sequences on maize
pachytene chromosomes by fluorescence in situ
hybridization ----------------------------------70
Conclusion-----------------------------------------------93

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