线粒体单体型与线粒体相关的人类疾病

线粒体是一种拥有自身遗传体系的半自主细胞器,它的遗传物质线粒体DNA(mitochondrial DNA,mt DNA)随着人类的迁移、隔离、进化而形成了广泛的线粒体基因组多态性,同一祖先所具有的一些相同mt DNA SNP位点的集合称为线粒体单体型.不同的线粒体单体型会在一定程度上影响线粒体功能,从而影响整个细胞的生长,并在某些情况下导致一些个体的病变,例如Leber遗传性视神经病变、母系遗传性耳聋、Ⅱ型糖尿病、帕金森以及各种癌症等复杂疾病.本文列举总结了几种线粒体相关疾病及其与线粒体单体型如A、B、D、F、G、H、J、K、M、N、T、U、Y及一些有特点的多态位点如G11778A、A1555G、T3394C、G10398A等的相关性.     

外源性线粒体DNA在细胞内的重建(简报)

近年来发现人类多种神经肌肉疾病存在线粒体电子传递链(electron transport chain,ETC)缺陷。由于线粒体在遗传上受核基因和线粒体基因双重控制,给确定ETC缺陷的来源造成困难。转线粒体DNA技术是线粒体同无线粒体DNA的细胞(ρ°cells)融合,形成转线粒体DNA细胞系(mtDNA-transferred cell line,也称cytoplasmic hybrids,简称cybrids),使病人的线粒体DNA(mito-     

线粒体DNA突变与相关人类疾病

在过去的20年里, 人们发现线粒体DNA(mitochondrial DNA, mtDNA)突变与多种人类疾病相关, 其致病范围从单器官组织损害到多系统受累。文章目的在于探讨mtDNA突变与人类疾病的关系。文章重点论述: (1)线粒体遗传学特征; (2) mtDNA突变与人类遗传性疾病; (3)体细胞mtDNA突变在衰老和肿瘤中的作用; (4)mtDNA疾病的诊断和治疗。     

靶向核酸酶在线粒体基因组编辑中的应用

线粒体基因组(mt DNA)的突变可导致多种人类疾病,其中绝大多数的mt DNA突变是异质性的:即在细胞中同时存在突变型和野生型的mt DNA,当突变型mt DNA的比例达到一定阈值时,就会引发疾病的发生。线粒体靶向的核酸内切酶可以诱导mt DNA异质性的改变,将突变型mt DNA的含量控制在发病阈值之下,从而达到疾病治疗的目的。本研究介绍了线粒体靶向的锌指核酸酶(ZFN)、类转录激活样效应因子核酸酶(TALEN)、规律成簇间隔短回文重复序列(CRISPR/Cas)以及常规的限制性核酸内切酶(restriction endonuclease,RE)在线粒体基因组编辑及疾病治疗中的应用。     

线粒体拟核结构及相关疾病研究进展

线粒体DNA(mitochondrial DNA,mtDNA)与一系列蛋白质相互作用形成核蛋白复合体,并包装折叠成类似原核生物拟核的结构,称为线粒体拟核(mitochondrial nucleoid)。参与线粒体拟核组成的相关蛋白包括线粒体转录因子、线粒体单链DNA结合蛋白以及多种参与线粒体中代谢途径的多功能蛋白。线粒体拟核结构的阐明对于进一步研究线粒体形态与功能以及mtDNA的遗传模式、基因表达调控具有重要意义。本文综述了线粒体拟核结构的最新研究进展,着重介绍组成拟核结构的重要蛋白,以及这些蛋白如何将mtDNA与柠檬酸循环等线粒体重要代谢途径相联系。同时,拟核相关蛋白(nucleoid-associated protein)的异常涉及多种人类疾病,这为研究线粒体相关疾病提供了新的思路。     

线粒体疾病小鼠模型的建立及病理生理学研究

线粒体疾病是机体ATP合成障碍、供能不足引起的多系统疾病。近十年来,随着线粒体疾病小鼠模型的不断建立和完善,发现核DNA（nuclear DNA,nDNA）或（和）线粒体DNA（mitochondrial DNA,mtDNA）突变造成线粒体氧化磷酸化功能缺陷是其发病的主要原因。将着重介绍线粒体氧化磷酸化功能缺陷导致线粒体疾病的小鼠模型的建立及其病理生理学特点。     

人类线粒体DNA的分子遗传特性

人类线粒体DNA(mitochodrialDNA,mtDNA)是存在于细胞核外唯一的遗传物质,具有独特的分子遗传特性,mtDNA突变可导致人类各种退行性疾病和与衰老相关的疾病发生。     

哺乳动物线粒体蛋白质翻译及其调控机制

线粒体是真核细胞内参与能量生成和物质代谢的重要细胞器。线粒体核糖体(mitochondrial ribosome, MR)作为细胞器中的翻译机器,用于表达线粒体DNA(mitochondrial DNA, mtDNA)编码的基因。近年来,随着研究的不断深入,人们对参与哺乳动物线粒体蛋白质翻译的蛋白质因子及其翻译的基本过程有了越来越清晰的认识,这对阐明线粒体蛋白质翻译的调控机制及研究人类线粒体疾病等方面具有重要的意义。线粒体蛋白质的翻译过程分为起始、延伸、终止和回收四个阶段。本文综述哺乳动物线粒体核糖体的结构与功能,以及线粒体蛋白质翻译因子的性质与功能,并进一步探讨翻译激活因子、微小RNA、线粒体COX翻译调控组装中间体(mt-translation regulation assembly intermediate of COX, MITRAC)以及核糖体的翻译后修饰对线粒体蛋白质翻译的调控及其机制,展望其对人类线粒体相关疾病研究的应用前景。     

线粒体DNA及其应用

线粒体DNA是在真核生物中普遍存在的一种核外遗传物质,具有分子小、结构简单、进化速度快、母性遗传、无组织特异性等特点。本综述了线粒体DNA在人类遗传疾病、动植物的亲缘关系、种群分化、群体多样性及植物细胞质雄性不育等研究领域中的应用情况。     

Mitochondrial DNA mutations regulate metastasis of human breast cancer cells

Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously expressed a highly metastatic potential, mitochondrial respiration defects, and ROS overproduction. Since mitochondrial respiratory function is controlled by both mtDNA and nuclear DNA, it is possible that nuclear DNA mutations contribute to the mitochondrial respiration defects and the highly metastatic potential found in MDA-MB-231 cells. To examine this possibility, we carried out mtDNA replacement of MDA-MB-231 cells by normal human mtDNA. For the complete mtDNA replacement, first we isolated mtDNA-less (ρ(0)) MDA-MB-231 cells, and then introduced normal human mtDNA into the ρ(0) MDA-MB-231 cells, and isolated trans-mitochondrial cells (cybrids) carrying nuclear DNA from MDA-MB-231 cells and mtDNA from a normal subject. The normal mtDNA transfer simultaneously induced restoration of mitochondrial respiratory function and suppression of the highly metastatic potential expressed in MDA-MB-231 cells, but did not suppress ROS overproduction. These observations suggest that mitochondrial respiration defects observed in MDA-MB-231 cells are caused by mutations in mtDNA but not in nuclear DNA, and are responsible for expression of the high metastatic potential without using ROS-mediated pathways. Thus, human tumor cells possess an mtDNA-mediated metastatic pathway that is required for expression of the highly metastatic potential in the absence of ROS production.     

A modified procedure for quantitative analysis of mtDNA, detecting mtDNA depletion

Quantitative analysis of mitochondrial DNA (mtDNA) is crucial for proper diagnosis of diseases that are caused by or associated with mtDNA depletion. However, such a quantitative characterization of mtDNA is not a simple procedure and requires several laboratory steps at which potential errors can accumulate. Here, we describe a modified procedure for quantitative human mtDNA analysis. The procedure is based on using two PCR-amplified, fluorescein-labeled DNA probes, complementary to mtDNA (detection probe) and chromosomal 18S rDNA (reference probe), both of similar length. Thus, equal amounts of these probes can be used and, contrary to previously published procedures, no mtDNA purification (apart from total DNA isolation) or 18S rDNA cloning is necessary for probe preparation. Two separate hybridizations (each with one probe) are suggested instead of one hybridization with both probes; this decreases background signals and enables adjustment of the strength of specific signals from both probes, which is useful in the subsequent densitometric analysis after superimposing of both pictures. Using different DNA amounts for reactions, we have proved that the procedure is quantitative in a broad range of sample DNA concentrations. Moreover, we were able to detect mtDNA depletion unambiguously in tissue samples from patients suffering from diseases caused by dysfunction of mtDNA.     

A novel mitochondrial DNA-like sequence in the human nuclear genome.

We describe here a nuclear mitochondrial DNA-like sequence (numtDNA) that is nearly identical in sequence to a continuous 5842 bp segment of human mitochondrial DNA (mtDNA) that spans nucleotide positions 3914 to 9755. On the basis of evolutionary divergence among modern primates, this numtDNA molecule appears to represent mtDNA from a hominid ancestor that has been translocated to the nuclear genome during the recent evolution of humans. This numtDNA sequence harbors synonymous and nonsynonymous nucleotide substitutions relative to the authentic human mtDNA sequence, including an array of substitutions that was previously found in the cytochrome c oxidase subunit 1 and 2 genes. These substitutions were previously reported to occur in human mtDNA, but subsequently contended to be present in a nuclear pseudogene sequence. We now demonstrate their exclusive association with this 5842-bp numtDNA, which we have characterized in its entirety. This numtDNA does not appear to be expressed as a mtDNA-encoded mRNA. It is present in nuclear DNA from human blood donors, in human SH-SY5Y and A431 cell lines, and in rho(0) SH-SY5Y and rho(0) A431 cell lines that were depleted of mtDNA. The existence of human numtDNA sequences with great similarities to human mtDNA renders the amplification of pure mtDNA from cellular DNA very difficult, thereby creating the potential for confounding studies of mitochondrial diseases and population genetics.     

Human mitochondrial DNA diseases and Drosophila models

Mutations that disrupt the mitochondrial genome cause a number of human diseases whose phenotypic presentation varies widely among tissues and individuals. This variability owes in part to the unconventional genetics of mitochondrial DNA(mtDNA), which includes polyploidy, maternal inheritance and dependence on nuclear-encoded factors. The recent development of genetic tools for manipulating mitochondrial genome in Drosophila melanogaster renders this powerful model organism an attractive alternative to mammalian systems for understanding mtDNA-related diseases. In this review, we summarize mtDNA genetics and human mtDNA-related diseases. We highlight existing Drosophila models of mtDNA mutations and discuss their potential use in advancing our knowledge of mitochondrial biology and in modeling human mitochondrial disorders. We also discuss the potential and present challenges of gene therapy for the future treatment of mtDNA diseases.     

Mitochondrial DNA mutations in disease and aging

The small mammalian mitochondrial DNA (mtDNA) is very gene dense and encodes factors critical for oxidative phosphorylation. Mutations of mtDNA cause a variety of human mitochondrial diseases and are also heavily implicated in age-associated disease and aging. There has been considerable progress in our understanding of the role for mtDNA mutations in human pathology during the last two decades, but important mechanisms in mitochondrial genetics remain to be explained at the molecular level. In addition, mounting evidence suggests that most mtDNA mutations may be generated by replication errors and not by accumulated damage.     

Production of mitochondrial DNA transgenic mice using zygotes

Several animal models of human disease, which have been developed by random or targeted modifications of genomic DNA sequences, have furthered our understanding of pathogenesis and the development of therapeutics. However, these models have not facilitated studies on mitochondrial diseases, since modifications to mitochondrial DNA (mtDNA) sequences are not possible using current recombination techniques. Consequently, information on human mitochondrial diseases is relatively sparse, and issues related to mitochondrial pathogenesis and inheritance remain unresolved. Recently, we reported the development of a new technique to generate mice carrying mutant mtDNA from a mouse cell line. In this report, we describe our techniques in detail, with emphasis on the preparation of donor cytoplasts and the micromanipulative procedures for electrofusion of cytoplasts and recipient zygotes. These steps are critically important for the successful introduction of exogenous mtDNA into embryos, and thereby into animals, so that the mutant mtDNA is efficiently propagated in subsequent generations.     

Relationship between mitochondrial DNA mutations and clinical characteristics in human lung cancer

Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations, some of which are related to various diseases, including cancers. However, roles of mutations and polymorphisms in some diseases are among heated debate, especially for cancer. To investigate the possible role of mtDNA mutations in lung cancer, we sequenced complete mtDNA of lung cancer tissues, corresponding normal (i.e., non-cancerous) lung tissues, and peripheral blood samples from 55 lung cancer patients and examined the relationship between mtDNA mutations or polymorphisms and clinical parameters. We identified 56 mutations in 33 (60%) of the 55 patients, including 48 point mutations, four single-nucleotide insertions, and four single-nucleotide deletions. Nineteen of these mutations resulted in amino acid substitution. These missense mtDNA mutations were distributed in 9 of 13 mitochondrial DNA coding genes. Three hundred eighty eight polymorphisms were identified among the 55 patients. Seventy-three polymorphisms resulted in amino acid substitution. There was no association of incidence of specific mtDNA mutation or polymorphism with patients' gender, age at diagnosis, smoking history, tumor type or tumor stage (P>0.05). This study revealed a variety of mtDNA mutations and mtDNA polymorphisms in human lung cancer, some of which might be involved in human lung carcinogenesis.     

The organization and inheritance of the mitochondrial genome

Mitochondrial DNA (mtDNA) encodes essential components of the cellular energy-producing apparatus, and lesions in mtDNA and mitochondrial dysfunction contribute to numerous human diseases. Understanding mtDNA organization and inheritance is therefore an important goal. Recent studies have revealed that mitochondria use diverse metabolic enzymes to organize and protect mtDNA, drive the segregation of the organellar genome, and couple the inheritance of mtDNA with cellular metabolism. In addition, components of a membrane-associated mtDNA segregation apparatus that might link mtDNA transmission to mitochondrial movements are beginning to be identified. These findings provide new insights into the mechanisms of mtDNA maintenance and inheritance.