Studies on the tandem duplications in the D-loop region of human mitochondrial DNA

• Main Authors: You, Ban-Jau, 游邦照
• Other Authors: Wei, Yau-Huei
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/29879589008706396150

碩士 === 國立陽明大學 === 生物化學研究所 === 83 === 1993年Brockington在粒線體肌病和腦肌病患者的粒線體DNA(mtDNA)之D－loop區域發現了第一型序列重複突變，Poulton等，認為在所發現得序列重複突變可能是先產生類似在病所看到的序列重複突變種，的過程所產生的。最近又有第二型的序列重複突變被證實存在於正常老年人的肌肉組織中，而且可能與人類老化的過程有關。第一型序列重複突變亦可於老年人的組織中發現。因此，我們乃進一步研究在正常老化組織中mtDNA的D－loop區域所發生的序列重複突變，探討其發生的分子機轉。 在本研究中，我們在mtDNA的D－loop區域發現了另外九種序列重複突變，依序列重複突變發生的位置，接合位置的核甘酸序列以及在各組織之出現的頻率，我們將其歸為二類。第一類從近一個重股DNA合成的起始點開始產生重複，接合位置為polyC序列或12bp的正向重複序列(direct repeat)，在不同組織中各型序列重複突變出現的頻率不同，有相當高的組織特異性。而且在較老的組織中出現的頻率亦較高，可能與老化有關，其中第五型重複突變在較耗能的組織中出現的頻率較高，我們認為這一類序列重複突變可能是在重股DNA合成接近完成時，藉由DNA末端的poly C序列或正向重複序列與模板上其他位置的相同序列產生錯誤配對(mis－pairing)，再經由DNA polymerase重複合成該段DNA所造成。 第二類序列重複突變由核甘酸序列第16257或16191位置附近開始產生重複，這個位置與第一類一至四型開始產生重複的位置之序列相似，而開始被重複的位置，也和第一類的第一、二、三型一樣，為第567、493及455位置附近。因此，第455、493、567位置區域是序列重複突變的好發生部位;所以第二類與第一類序列重複突變的產生必有一些共同的因素，有可能是因為未合成完全的(incompletely replicated)7S DNA與模板DNA經由錯誤配對而產生；但因其接合位置之核甘酸序列與在各組織中出現頻率之趨勢與第一類相差甚多，所以也有可能經由其他約方式產生，因為其產生在特定的核甘酸位置，所以可能與DNA結構或與某些特定的蛋白質結合有關係；這一類mtDNA序列重複突變在年輕人的組織、血液、以及幾對的母子血液檢體均可測得，所以有可能會經由母系遺傳的方式傳給子代。 A heteroplasmic tandem duplication (type) with a size of about 260 bp in the D-loop of human mitochondial DNA (mtDNA) has recently been identified in the muscle of several patients with mitochondrial myopathy. An aging-associated type Ⅱ tandem duplicated mtDNA was also observed in the mtDNA of aging human muscle. In the study, we have tried to search for the other tandem duplications in the D-loop region of human mtDNA and analyzed the nucleotide sequences flanking the insertion sites with an aim to answer how the tandem duplications are generated. By use of several back-to-back primers, we have identified with PCR techniques another nine types of tandem duplications in the D-loop region of human mtDNA. These tandem duplications are classified into two groups according to the duplicated regions in the mtDNA, the nucleotide sequences in the junction sites and the frequency of occurrence in different tissues. Group I tandem duplications bear some important characteristics. First, the duplications start from the CSB Ⅱ and CSB Ⅲ region near the start site of the heavy-strand DNA synthesis. Second, the junction sites are localized in the poly C sequence or a 12 bp direct repeat sequence in the D-loop region. Third, they are tissue-specific and aging-associated. In addition, typeⅤ tandem duplication occurs much more frequently in the tissues of high-energy demand. On the basis of these observations, we propose that group I tandem duplications are caused by mis-pairing of directly repeated poly C runs or similar ones at the end of replication of the heavy strand mtDNA. In the group Ⅱ tandem duplications, the nucleotide sequence in the start sites of the duplicated regions and the positions of the start sites are similar to those of group Ⅰ tandem duplications. Thus, there are some common factors involved in the generation of groupⅠand group Ⅱ tandem duplications. Group Ⅱ tandem duplications are most likely caused by the mis-pairing of the incompletely replicated 7S DNA with template DNA. Moreover, there are some characteristics that differentiate group Ⅰ from group Ⅱ tandem duplications. Most notably, the nucleotide sequences flanking the junction sites and frequency of occurrence in different tissues were found to be highly variable. It may be related to a unique mtDNA structure or DNA-protein binding as judged by the junction sites of these duplications. Thus, the group Ⅱ tandem duplications are caused by a different mechanism different from that of group Ⅰ tandem duplications. Interestingly, the group Ⅱ tandem duplications were detectable in the tissues from very young subjects and the blood cells of several mothers and their children. This indicates that the group Ⅱ tandem duplications may be transmitted through maternal lineage.