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題 名 | 第一型黏多醣儲積症:突變及多型性分析 |
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作 者 | 李桂楨; 程幼青; 賴秋雁; 劉美能; 胡務亮; 王作仁; | 書刊名 | 師大生物學報 |
卷 期 | 30:1 1995.10[民84.10] |
頁 次 | 頁1-10 |
分類號 | 362.61 |
關鍵詞 | 第一型黏多醣儲積症; 突變; 多型性; MPS I; IDUA; Mutation; Polymorphism; |
語 文 | 中文(Chinese) |
中文摘要 | 本實驗在以分子生物的技術,進行第一型黏多醣儲積症(MPSI)的基因突變及多型性分析。首先由正常人及患者的皮層纖維母細胞萃取出poly(A)+RNA作為模板,用特殊序列的寡核甘酸引子及鼠類白血病毒(Mo-MLV)反轉錄酵素,合成和mRNA序列互補的第一鏈cDNA,再用聚合酵素鏈反應(PCR),將α-Liduronidase (IDUA)基因,以部份重疊的二片段選殖出來。置入pGEM-T載體後,將篩選出的純系(clo□e)進行DNA序列分析,將比較正常人及患者的基因序列,來了解患者的分子病因。思者W3為異型合子□在對偶基因1上,轉錄起始密碼ATG之G突變為A,而且第2表現子的47個胺基駿片段,發生在同一解讀架構的缺失。南方吸潰分析顯示患者基因體DNA上,一個包含第二表現子的4.7Kb片段發生了缺失;在對偶基因2上。 第193個胺基酸呈現多型性的變化,由蛋胺酸(Met)轉變為異白胺酸(Ile),而且第8表現子5端的19個胺基酸片段,也發生在同一解讀架構的缺失。南方吸潰分析則顯示患者的IDUA基因,並無相對應的缺失。相反的,患者W2的IDUA cDNA,則含有數種改變胺基酸的突變和多型性的變化,包括同型合子的R105Q、A361T、T364M、V454I和異型合子的H240L、F495L、G549E。北方吸潰分析顯示,患者的纖維母細胞中,可偵測到少量的2.3Kb及2.0 Kb mRNA及大量的6.4Kb和3.9Kb processed pre-mRNA。應用PCR及限制片段長度多型性(RFLP)或不同數目重覆單位(VNTR)的方法,我們對正常人及第一型黏多醣儲積症患者,進行KpnI及VNTR多型性的對偽基因(allele)及單套型(haplotype)頻率的分析。此多型性的研究,或可作為臨床上MPSI檢視的參考。 |
英文摘要 | Molecular lesions of two Chinese patients with type I mucopolysaccharidosis (MPSI) are currently being investigated. Poly(A) + RNA was prepared from established skin biopsy fibroblast of MPSI patients and a normal person. First strand α-L-iduronidase (IDUA) cDNA was synthesized from poly(A)+ RNA using sequence-specific oligonucleotide primers and Moloney murine leukemia virus reverse transcriptase. The complete coding region was amplified as two overlapping fragments by polymerase chain reaction (PCR) and sequenced to determine the molecular lesions. Patient W3 is heteroallelic. One IDUA allele has a G-to-A transition in the initiation codon ATG and a 47 amino acid fragment in-frame deletion in exon II. Southern analysis of genomic DNA indicated deletion of a 4.7 Kb fragment containing exon II. Whereas the other IDUA allele has a polymorphism M193I and a 19 amino acid fragment in-frame deletion in exon VIII. No corresponding deletion in genomic DNA was detected by Southern analysis. In patient W2, a number of mutated and polymorphic changes which result in amino acid alteration are observed, including homoallelic R105Q, A36IT, T364M, V4541 and heteroallelic H240L, F495L, G549E. Northern analysis of fibroblast poly(A)+ RNA indicated a low level of 2.3 Kb and 2.0 Kb mRNAs and larger amount of 6.4 Kb and 3.9 Kb processed pre-mRNAs present in patients. By combining restriction fragment length polymorphism (RFLP) or variable number of tandem repeats (VNTR) with PCR, we examined an exonⅢKpnI polymorphism and an intron II VNTR polymorphism for normal and MPSI individuals to generate the allele and haplotype frequencies. Polymorphisms can be used as a reference for patient diagnosis. |
本系統中英文摘要資訊取自各篇刊載內容。