2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/37514One of the applicant’s long-term research objectives has been to gain a better understanding of the structure and function of human chromosomes. Particularly, with the recent advances of molecular cytogenetic technologies, new approaches can now be undertaken to further explore the field of human chromosome biology and its implication to Bio-medicine. The following four related studies in this proposal were designed for achieving these goals. 1. Clinical cytogenetic studies Fluorescence in situ hybridization (FISH) using specific DNA probes to further delineate problematic constitutional cytogenetic cases, involving marker chromosomes, cryptic, translocations, duplication and microdeletions etc. FISH and multiple color-FISH will also be performed on interphase nuclei of paraffin-embedded brain tumor tissues and on metaphase chromosomes from glioma cell cultures to identify the specific chromosome aberrations associated with these brain tumors. Furthermore, the chromosomal origin of the double minute chromosomes and the homogeneously staining region of tumor cells will be determined by FISH studies. Results of those studies will provide more information on the nature of chromosome aberrations in relation to specific clinical manifestations. 2. Trinucleotide expansion site on chromosomes Attempts will be made to chromosomal localize trinucleotide repeat expansion which are associated with certain disorders as well as physically map all potential sites of expanded trinucleotide repeats in the human genome. New FISH techniques such as the ultrasensitive catalyzed reporter deposition amplification method will be used for these investigations. The sensitivity and reliability of FISH testing will be compared to routine molecular methods for detecting triplet expansions. These studies will proveide a better understanding on the extension, heterogeneity, polymorphism and disease association of trinucleotide expansions in man. 3. Study on human and other mammalian centromeric DN As Efforts will be made to isolate new subfamilies of human gamma satellite DNA and other mammalian centromeric satellite DNAs. DNA sequencing, chromosomal localization, genomic organization (by fiber-FISH) and protein binding studies will be conducted on each obtained centromeric DNA clone. These studies will undoubtedly lead to the discovery of new centromeric satellite DNAs and reveal aspects of its genomic organization and function. Positional cloning with the chromosome walking procedure will also be attempted across potential neocentromeric region of mitotic stable marker chromosomes which lack detectable amount of satellite DNAs. Such studies should provide new information on the nature of latent centromere DNAs and their centromeric function(s). 4. Mammalian cytogenetics and comparative gene mapping Systemic interspecific reciprocal painting studies will be carried out using chromosome painting probes generated from flow-sorted chromosomes of different species. Conserved chromosome regions between species will be determined and comparative cytogenetic maps can be established. Such comparative chromosome maps will greatly facilitate further mapping of cloned genes and DNA fragments from different mammalian species. The conserved region of synteny and their potential genomic function will also be better elucidated. .為了對人類染色體的構造與功能有更進一步的了解，特別是藉著分子細胞遺傳學技術的進展，現在已可以用新的方法來探究人類染色體生物學及其在生物醫學上的應用，為達此目的，本計劃設計四個相關研究主題： 1. 臨床細胞遺傳學之研究(clinical cytogenetic study) 利用特定DNA探針以螢光原位雜交法(Fluorescence in situ hybridization，FISH)以進一步了解可疑之組成性染色體(Problematic constitutional chromosome)個案，如標記染色體(maker chromosomes)，隱藏性轉位(cryptic translocation)，複製(duplication)和微缺失(microdeletion)等，FISH和多重顏色-FISH(multiple color-FISH)也可應用於paraffin包埋之腦部腫瘤組織的間期細胞核(interphase nuclei)或培養的神精膠質瘤細胞(glioma)之分裂中期染色體(metaphase chromosomes)。再者，發生於腫瘤細胞基因擴增之染色體如雙小染色體(double minute chromosomes)和均質染色區(homogeneously staining region, HSR)可利用FISH研究它們是來自一對染色體，這些研究結果將有助於更進一步的瞭解與臨床表徵有關之異常染色體的本質。 2. 染色體上三聯核甘酸擴增位置(trinucleotide expansion site on chromosome) 三聯核甘酸重複系列(trinucleotide repeated sequence)在人類基因組(genome)分佈很多，要定位三聯核甘酸擴增與某些疾病的關係以及分佈在那些染色體上，我們將採用新FISH的技術包括被催眠之reporter沉澱擴增法(catalyzed reporter deposition amplification methods)之超敏感FISH於此項研究，FISH檢定的敏感性和可靠度與例行偵測三聯核甘酸擴增所用之分子方法相比較並加以評估。此研究可提供對三聯核甘酸之擴增，異質性、多形性及其疾病關聯性的進一步了解。 3. 人類和其它哺乳類著絲點DNA的研究 我們將由具有大量repetitive DNA的特定染色體cosmid libraries分離出其他的人類γ-satellite DNA家族和新的人類著絲點satellite DNA。由所獲得的著絲點DNA純系(DNA clone)可以提供分析DNA序列，染色體定位，基因組結構(genomic organization)(利用fiber FISH)蛋白質結合之研究。這些研究將可能發現新著絲點satellite DNA和披露一些基因組結構和功能的訊息。我們也將利用染色體游走方法(chromosome walking procedure)的位置選殖(positional cloning)來含蓋小標記染色體之新著絲點區域(neocentromeric region)。標記染色體在有絲分裂時穩定但缺乏satellite DNA。研究標記染色體之著絲點DNA，將可提供潛在性著絲點DNA之本�閰M著絲點功能較新的訊息。 4. 哺乳動物細胞遺傳學及比較基因圖譜(Mammalian cytogenetics and comparative gene mapping) 我們將利用flow-sorted，不同品種染色體來做染色體繪圖(chromosome painting)的探針，並以此進行有系統的跨品種間相互繪圖(painting)的研究，在不同品種間保留的染色體區域將可被決定，從而建立比較染色體圖譜(comparative chromosome maps)。此比較圖譜對日後描繪不同哺乳動物純系基因及DNA片段的明細圖譜有非常大的幫助，而染色體上的保留區域及它們可能具有的基因組功能也會有較好的闡釋。螢光原位雜交法染色體著絲點三聯核甘酸臨床細胞遺傳人造人類染色體衛星DNA染色體顯微切割哺乳類核形之演化著絲點蛋白Fluorescence in situ hybridizationCentromereTrinucleotide repeatsClinical cytogeneticsArtifitial human chromosomeSatellite DNAChromosome microdissectionMammalian karyotypic evolutionCentromeric proteinProject