| Abstract: | 著絲點在有絲分裂及減數分裂的過程中扮演了相當重要的角色，它負責將姐妹染色分體平均分配至兩個子代細胞中有關。而功能不正常或失去功能的著絲點通常會有染色體不穩定的現象發生，而造成所謂的Aneuploid。在細胞遺傳學上的研究報告指出aneuploidy 和自發性的流產，先天性的染色體疾病，如唐氏症和透納氏症，甚至與某些癌症的發生有關。近年來，有許多實驗室為了改良基因治療載體，於是致力於研究人類人造染色體的建構上。但想要了解Aneuploid 形成的機制及成功地建構人類人造染色體為基因治療的載體，就必須先透澈著絲點的功能及構造。著絲點在染色體上是以一種收縮的結構型態顯現，而其分子結構的基本元素包括鹼基序列及特殊的蛋白質結構名Kinetochore(是以一種三層板狀的結構所構成，最外層是和紡錘絲連結，最內層是相連於著絲點DNA，中間是一層狹窄的透明區)。就目前所知，在高等的真核生物內，存在著高度複雜性的著絲點DNA 族群，其中已確定的著絲點DNA 主要是由重複性的衛星DNA 所組成，舉例來說，阿爾發衛星DNA 是最主要著絲點DNA 且存在每個人類染色體著絲點上，而且阿爾發衛星DNA 的某些單元體內具有17 個鹼基對的CENP-B 鍵結序列。因此曾認為是支配人類染色體著絲點功能的重要且必須的衛星DNA。可惜的是，後來在某些異常染色體(marker chromosomes)上發現具有功能的新生著絲點上並沒有阿爾發衛星DNA 的存在，而且近來研發的人造染色體構築中，若只是以重複的阿爾發衛星DNA作為著絲點DNA 的基本架構元素，通常無法穩定存在人造染色體的轉殖細胞中。從低等到高等的生物體，著絲點的衛星DNA 往往差異相當大，且在不同種族甚至同一種不同個體間也都不同；然而，著絲點的蛋白質的功能及構造卻保留相當大的一致性。正因如此，使得著絲點的構造及功能更加複雜且不易了解。由於著絲點衛星DNA 在各種族中差異相當大，因而推算著絲點衛星DNA 在演化過程中變化極為快速，因此著絲點衛星DNA 的差異度可作為種族間血緣遠近的依據。在尋找具有著絲點功能的著絲點DNA 的同時，也可研究著絲點衛星DNA 的演化過程。鹿科的染色體變化差異極大，而且在先前的研究中，發現衛星DNA 在山羌的核型演化中扮演極重要的角色。在此次的計劃中，我們更進一步研究原先已選殖到的三種印度山羌衛星DNA (分別為I、II 和IV)在著絲點上的分布結構：在原位螢光雜交及3-D影像建構的結果中，顯示衛星II DNA及衛星IV DNA 是以相同的螺旋方式圍繞在著絲點上，圍繞二圈而在X+3 複合著絲點上則圍繞四圈，每一圈約由六個原位螢光訊號所形成；而Kinetochore 仍是以兩條平行對稱的方式排列在衛星DNA 的外側，每條約有四個免疫螢光訊號在X+3 的複合著絲點上。此一結果說明了衛星DNA 的螺旋結構可能是為了將著絲點染色質絲表現在染色體的外側，使其能夠支配kinetochore 的蛋白質的包裝，且和紡錘絲作用。然而這並不足以說明這兩種衛星DNA可和kinetochore 上的蛋白質直接鍵結。因此我們利用染色質絲免疫沉澱法直接找到kinetochore 蛋白質的鍵結DNA 片段，再以此一DNA 當作探針搜尋BAC 基因庫以找到完整的kinetochore 鍵結序列。首先我們已成功取得kinetochore 上建構蛋白質的鍵結DNA 片段，至於基因庫的部分，我們已得到涵蓋2 個基因組的BAC 菌株，目前仍繼續建構中。在找尋功能性著絲點DNA 的過程中，我們從BAC 菌珠中找到了Y 特異性的衛星DNA，由於它只存在亞洲系的鹿類中，因此更加確認了印度山羌核型是由2n=70 類似中國水鹿的核型演化而來。另外我們也利用一致性相當高的著絲點衛星IV DNA 和衛星I、II DNA 加以鑑定中國山羌及台灣山羌的血緣關係。為了更加確認印度山羌的核型演化和中國水鹿的核型的關係，我們更進一步分析了三種衛星DNA（I、II、III）在中國水鹿染色體中的分布情形。在此次計劃執行中，共有四篇已發表的論文，一篇論文已送出，一篇正在撰寫的論文，已有涵蓋2個印度山羌基因組的BAC 菌株及許多由染色質絲免疫沉澱法得到的DNA 菌株，目前正在分析這些DNA 在染色體上的分布情形及和CENP 鍵結的可能性。
Centromere plays a pivotal role during mitosis and meiosis. Malfunctional centromere would result in prematured centromere division (PCD) that causes aneuploidy, an aberrant chromosome number in the complement. Epidemically reported that aneuploidy is associated with disorders such as spontaneous abortion, birth defect (e.g. Down syndrome, Klinfelter syndrome, Tuner syndrome, etc.) and some neoplasia. Recently, several laboratories have endowed in the construction of human artificial chromosomes with centromeric DNA for gene therapy intervention. In order to unveil the enigma of aneuploidy and to construct successfully artificial chromosomes for gene therapy intervention, it is an important and necessary to understanding the molecular architecture and composition of centromere. Several detailed analyses have demonstrated that the centromere contains various centromeric DNA and an exquisite and dramatic proteinaceous structure, the kinetochore, which in turn interacts with the spindle microtubules. Satellite DNA is a predominant and ubiquitous centromeric DNA in mammals. Alphoid satellite DNA, the major centromeric satellite DNA of human chromosomes, was considered a sufficient component for a functional centromere in the construction of human artificial minichromosomes. It was not questioned until alphoid satellite DNA was not found in some mitotically stable markers with functional centromere. Moreover, taken the available data from yeast to man, the centromere sequence and sequence organization have diverged significantly, even amongst different chromosomes of a single organism; however, overall centromere and kinetochore components might be significantly more conserved than thought previously. The centromeric DNAs found so far are quite complex so that it becomes even less clear about the structure and function of the mammalian centromeres. In order to understand completely the centromere structure and function in mammals, it will require detailed sequence analysis of centromeric DNA. The diversity of the centromeric satellite DNA among species resulted from the quick evolution. Therefore, satellite DNA can be traced during genome evolution and speciation. Several studies had used satellite DNA a marker to identify the karyotype evolution and phylogenetic relationship. In the mean time searching a functional centromeric DNA, there should be a lot of novel satellite DNAs identified. Those novel satellite DNAs can be further characterized to use in karyotype evolution and speciation. The chromosomes of the Indian muntjac (Muntiacus muntjak vaginalis) is unique among mammals due to their low diploid number (2N=6♀,7♂), giant size, and unusual large centromeres. Particularly, the centromere of X+3 chromosome appears having a compound kinetochore. Moreover, the karyotype of Indian muntjac resulted from the centric fusion. Therefore, we are interested in the functional and molecular structure study of centromeric DNA of Indian munjtac. In this project, we not only endeavor to find a functional centromeric DNA, but also characterize the role of the found novel satellite DNA in muntjac’s karyotype evolution. We further characterized the 3D structure of three previously isolated cervid centromeric satellite DNA elements (satellite I, II and IV) of Indian muntjac. In the simultaneous 3D-FISH and
immunofluorescence study, the CENP immunofluorescence signals parallels along with centromere as well as both satellite II and IV signals are organized into a spiral structure. The spiral structure may be to present centromeric chromatin to the exterior of the chromosome, where it can mediate kinetochore assembly and interactions with the spindle. However, it is no enough to prove that the satellite II or IV can directly bind with kinetochore protein. Therefore, we would uncover the whole DNAs that exactly associate with kinetochore protein. First step, we have successfully use chromatin immunoprecipitation to isolate the DNA fragments that associate with kineotchore protein. Subsequently, we would use the isolated DNA fragments as a probe to screen the Indian muntjac’s BAC library.We have constructed 2 coverages of library. The BAC library is still under construction to achieve 6 coverages. During finding a functional satellite DNA, there is a novel Y-specific satellite DNA found. This satellite DNA existed only in Asian deer species. This found Y-specific satellite DNA further interpreted that the karyotype of Indian muntjac derived from the Chinese water deer-like karyotype. For understanding the karyotype relationship between Indian muntjac and Chinese water deer more clearly, we identified the chromosomal distribution of satellite I, II, and III DNA in Chinese water deer.We also confirmed the phylogenetic relationship between Formosan muntjac and Chinese muntjac using satellite I, II,
III as a tracing marker. Totally, we have four published papers, one submitted paper, and one preparing paper in this project. |
