哺乳類動物的著絲點異染色質絲--鹿類的衛星DNA家族之研究

• Main Authors: 李月君, Yueh-chun Li
• Other Authors: 許宗雄 博士, 林齊強 博士, Dr. Tzong-Hsiung Hseu, Dr. Chyi-Chyang Lin
• Language: Chinese; English
• Published: 2008
• Subjects: 著絲點; 異染色質絲; 衛星DNA; 鹿類; centromere; heterochromatin; satellite DNA; cervidae; 39; Indian; The ''Y''; Rangifer tarandus
• Online Access: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/5565

博士 國立清華大學 生命科學系 我的博士論文主要是以鹿科的著絲點衛星DNA家族研究鹿科的染色體核形的演化情形及這些著絲點衛星DNA家族在著絲點的排列結構。目前只有衛星I DNA已經被選殖且詳細鑑定的鹿科著絲點衛星DNA家族，而衛星II 和III DNA分別只在白尾鹿及小鹿中被選殖出但並未詳細研究。而我將針對從其它鹿類選殖出來的三種衛星DNA家族及新選殖出的1kb 衛星DNA家族作更詳盡之探討。 首先利用白尾鹿的衛星II DNA序列設計一對引子，然後以聚合鋂鏈鎖反應擴大加拿大麋鹿、中國水鹿、哥倫比亞黑尾鹿、印度山羌及中國山羌內的衛星II DNA家族。在這些鹿種內皆可得到主要長度約0.7 kb的PCR產物，而除了中國水鹿外其餘的PCR產物中有一長度約1kb。分別將此兩種PCR產物和pGEMT載體結合，然後加以選殖並定序之。其中0.7 kb的PCR產物和白尾鹿的衛星II DNA有70%以上的相似性，然而1 kb的PCR產物和白尾鹿的衛星II DNA甚至其它的衛星DNA家族並沒有任何相似性。另外也利用小鹿的衛星III DNA序列設計一對引子，同樣以聚合鋂鏈鎖反應擴大加拿大麋鹿、中國水鹿、 哥倫比亞黑尾鹿、印度山羌及中國山羌內的衛星III DNA家族。除了中國水鹿有一長度約2.2 kb 的PCR產物外，其餘皆無法得到此一衛星III DNA的PCR產物。然後將此2.2 kb 的PCR產物和pGEMT載體結合，轉送到大腸桿菌XL1-blue中然後加以選殖並定序之。此一中國水鹿的衛星III DNA和小鹿的衛星III DNA有85%以上的相似性。這些選殖出的衛星II、衛星III及衛星1 kb的DNA clones皆分別以南方吸漬法分析這些衛星DNA家族的單元體大小及限制鋂的圖譜、以染色體及染色質絲的原位螢光雜交法觀察在染色體上的分布情形及染色質絲上的排列方式。在這些衛星DNA clones中，我選定來自加拿大麋鹿及印度山羌的衛星II DNA clones加以分析，分別以Rt-0.7及Mmv-0.7命名之。Rt-0.7的GC含量佔了63 %，且佔了加拿大麋鹿的基因組含量的3.9 %。在南方吸漬法的分析中以Rt-0.7做探針顯示衛星II DNA是以0.7 kb的單元體前後排列方式形成一個高層次結構。在染色體的原位螢光雜交結果中，得知衛星II DNA分布在每一加拿大麋鹿的染色體著絲點上，且和衛星I DNA有相同的位置。因此以染色質絲的原位螢光雜交法觀察到這兩種衛星DNA是以前後排列且每種衛星DNA中之單元體都是以高度重複的方式連續排列。其中衛星II DNA高度重複連續約200微米相當於2x103 kb。此部分的結果已發表在Cytogenetic Cell Genetics 89:192-198, 2000。另一Mmv-0.7 clone，其GC含量佔了62.1 %，且佔了印度山羌的基因組含量的2.1 %。在南方吸漬法的分析中以Mmv-0.7做探針顯示衛星II DNA也是以0.7 kb的單元體前後排列方式形成一個高層次結構。我利用此一衛星II DNA clone (Mmv-0.7)及先前實驗室已選殖到並已鑑定的中國大陸山羌的衛星I DNA clone （C5）當作探針以原位螢光雜交到印度山羌的染色體上，發現衛星II DNA及衛星I DNA除了在著絲點有較強的訊號外，在染色體的兩臂上同樣也有些許較微弱的訊號。這些染色體臂上的微弱訊號共有27個，合併其他實驗室發現的染色體臂上衛星I DNA的訊號共有29個，和理論上由70個雙套染色體核形演化成6個雙套染色體核形所須的29個訊號是非常吻合的。因此推論印度山羌的染色體核形並非直接由中國大陸山羌的染色體核形演化而來的，而是可能由擁有70個雙套染色體的某一鹿科祖先演化而來的。此一結果已發表於Chromosome research 8:363-373, 2000。 中國水鹿中的三種衛星DNA（衛星I DNA、衛星II DNA、衛星III DNA）在染色體上的分布情形，分別用先前選殖出的衛星I DNA、衛星II DNA、衛星III DNA clones 當作探針以原位螢光雜交到中國水鹿的染色體上觀察。結果顯示每個染色體上皆有衛星I DNA及衛星II DNA的訊號，其中衛星II DNA的訊號以緊密方式集中在著絲點兩側上，但衛星I DNA的訊號則以大量且鬆散如火花狀分布在著絲點旁的異染色質絲區域上，而衛星III DNA只分布在某些染色體著絲點旁的異染色質絲區域上，而這三種衛星DNA在某些染色體上的分布順序，從短臂的末端往長臂方向依序是衛星III DNA－衛星II DNA－衛星I DNA。一般而言，異染色質絲上的衛星DNA不論在何種細胞週期都是以緊密的方式呈現出，但在中國水鹿的間期細胞中發現衛星I DNA卻是以較鬆散的方式表現，而衛星II DNA仍是緊密地纏繞成點狀呈現。這種呈現鬆散的衛星I DNA可能由於染色質絲纏繞較鬆散所造成或此種衛星I DNA中有某種散佈的DNA序列分佈其中所構成的。另外，衛星II DNA的原位螢光雜交訊號在著絲點上的位置和CREST的免疫螢光測定之kinetochore的位置是相同的，因此推論衛星II DNA緊密的結構可能是著絲點蛋白鍵結在著絲點DNA上的必要條件之一。此部份結果已展示在第五十屆的美國人類遺傳學會國際會議的壁報中。 至於其它鹿種的衛星DNA clones 也在我的論文研究中，其結果將陸續彙整發表。 This thesis describes the characterization of several cervid centromeric satellite DNA families and their use in elucidating the karyotypic evolution within the Asian muntjac species. Cervid satellite I DNA has been well characterized previously in a number of deer species. Cervid satellite II and III DNA are relatively novel and have not been study in detail. The first part of thesis research involves the direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA, satellites I and II. Two cervid satellite II DNA clones of the Canadian woodland caribou (Rangifer tarandus caribou) were generated by PCR using primer sequences derived from the white tailed deer satellite II clone OvDII (Qureshi and Blake, 1995). These two clones were designated as Rt-0.5 and Rt-0.7, respectively, and found to share 96% sequence similarity between each other. The caribou satellite II clones are 63% GC-rich, and comprises some 3.9% of the caribou genome. Dual-color fluorescence in situ hybridisation (FISH) studies were performed with caribou satellite I DNA (Rt-Pst3) (Lee et al., 1994) and caribou satellite II DNA (Rt-0.7) probes to caribou metaphase chromosomes and extended chromatin fibers. Direct visualization of the genomic organization of these two satellite DNA families revealed the following: (a) Sat. I and sat.II co-localized at the centromeres of acrocentric chromosomes, whereas the centromeres of bi-armed chromosomes revealed only sat. II signals. The centromere of the Y chromosome appeared to be devoid of either satellite DNA repeat. (b) Rt-0.5 and Rt-0.7 repeats could represent specific subsets of caribou satellite II DNA that have a differential chromosomal distribution in addition to higher-order organization. (c) FISH studies on highly extended chromatin fibers demonstrated that satellite I and satellite II arrays were juxtaposed the length of a given satellite II array usually reached 200 mm, corresponding to 2 x 103 kb of DNA at a given centromere. Results of this study have been published (Li et al., 2000a). In the second part of this thesis, a cervid satellite II DNA clone was generated from PCR amplification of Indian muntjac genomic DNA using primer sequences derived from OvDII. The Indian Muntjac satellite II clone (Mmv-0.7) was characterized by a tandem repetition of 0.7-kb monomers, 62.1% GC-rich, and comprised approximately 2.1% of the Indian muntjac genome. Dual colored FISH studies were performed with the Indian muntjac satellite I DNA (C5 clone) (Lin et al., 1991) and this satellite II DNA (Mmv-0.7 clone) as probes to Indian muntjac metaphase chromosomes. The results obtained enabled us to more precisely define the chromosome breakage and fusion sites that are likely associated with the formation of the present-day Indian muntjac karyotype (2n=6/7). Furthermore, the study showed a total of 27 distinct interstitial hybridization sites, in addition to pericentromeric signals. This is remarkably close to the theoretical maximum number of 29 interstitial sites expected from chromosome fusions involving a deer species with 70 acrocentric chromosomes. This new finding further hints at the possibility that the Indian muntjac karyotype may have evolved directly from a ancestral deer species with a 2n=70 karyotype rather than from an intermediate Chinese muntjac-like species with a 2n=46 karyotype (Lin et al., 1991). Results from this part of study have also now been published (Li et al., 2000b). The third part of this thesis research deals with genomic organization of several cervid satellite DNA families in the Chinese water deer (Hydropotes inermis), the muntjac species (Muntiacus muntjak vaginalis and Muntiacus reevesi) and Columbian black tailed deer (Odocoileus hemionus hemionus). The water deer appeared to have two types of centromeric heterochromatin which are resolvable by FISH analysis using cervid satellite I, II and III DNAs (water deer satellite III clone was derived from PCR products using primer sequences of the roe deer satellite III DNA (Buntjer et al., 1998) probes to metaphase chromosomes or resting nuclear preparations. The large cluster of hybridization signal in the centromeric and pericentromeric region produced by satellite I and satellite III DNA probes appeared as a group of small fluorescent spots. This unique hybridization signal pattern was also observed in the resting nuclei. These findings suggested that the satellite I and III DNA chromatin is more diffuse with chromatin fiber extended out over a large area. Whereas, the hybridization signal with satellite II DNA appeared as pairs of distinct fluorescent spots located at the primary constrictions. These pairs of satellite DNA II signals also co-localized with the immunofluorescent signals produced by the human CREST anti-sera. These observations suggested that satellite II chromatin is more condensely packaged. Such a manner of chromatin packing may be a prerequisite for CENPs binding. During the PCR cloning experiments of cervid satellite II DNAs, another satellite DNA organized as 1-kb monomer repeat was also obtained. This 1-kb satellite DNAs has little significant homology to satellites I, II and III and is thought to potentially be a new cervid satellite DNA family which exists in the pericentric regions of the majority of the Munticus chromosomes and in the centromeric regions of certain chromosomes of the mule deer as well. Part of the results from the above study have been presented in the 50th Annual Meeting of the American Society of Human Genetics, Philadelphia, Oct. 3-7, 2000 （American Journal of Human Genetics 67:A155, 2000）