哺乳类线粒体基因组

作为真核细胞的一种重要细胞器的线粒体含有独立的并自主复制和转录的DNA基因组。虽然线粒体蛋白质的大部分系核DNA编码,但有一小部分是线粒体DNA(mt DNA)编码,并由线粒体的蛋白质合成系统合成。线粒体蛋白质合成系统中的rRNA和tRNA也是mt DNA编码。mt DNA的复制、转录以及蛋白质合成系统均有其本身特点,既与非线粒体真核系统有所不同,又有别于原核细胞中者。因此,线粒体基因组的研究在生物学上有重要意义。此外,线粒体的起源和进化是许多生物学家所感兴趣的和长期争论的问题,而mt DNA的进化比较     

哺乳类线粒体基因组

作为真核细胞的一种重要细胞器的线粒体含有独立的并自主复制和转录的DNA基因组。虽然线粒体蛋白质的大部分系核DNA编码,但有一小部分是线粒体DNA(mt DNA)编码,并由线粒体的蛋白质合成系统合成。线粒体蛋白质合成系统中的rRNA和tRNA也是mt DNA编码。mt DNA的复制、转录以及蛋白质合成系统均有其本身特点,既与非线粒体真核系统有所不同,又有别于原核细胞中者。因此,线粒体基因组的研究在生物学上有重要意义。此外,线粒体的起源和进化是许多生物学家所感兴趣的和长期争论的问题,而mt DNA的进化比较     

蛹虫草线粒体DNA与细胞核DNA进化关系的比较

【目的】分析蛹虫草是否存在核内线粒体DNA片段,比较蛹虫草线粒体DNA与细胞核DNA的碱基变异程度及所反映的菌株间的系统发育关系。【方法】通过本地BLAST或LAST对蛹虫草线粒体基因组和核基因组进行序列相似性搜索;从10个已知线粒体基因组的蛹虫草菌株中分别扩增7个细胞核蛋白编码基因片段,并与其在14个线粒体蛋白编码基因上的碱基变异情况进行比较。【结果】蛹虫草核基因组中存在5处较短的核内线粒体DNA片段,总长只有278bp。蛹虫草核DNA的变异频率整体上高于线粒体DNA。核DNA和线粒体DNA所反映的蛹虫草菌株间的系统发育关系存在显著差异。【结论】蛹虫草线粒体DNA与核DNA间不存在长片段的基因交流,二者变异频率不同,所反映的蛹虫草菌株间的系统发育关系也有差异。本研究增加了对蛹虫草线粒体与细胞核DNA进化关系的认识。     

叶绿体的遗传进化

叶绿体是半自主性细胞器,其生长和增殖受核基因组和自身的基因组2套遗传系统的控制、关于叶绿体的起源有2种学说,近年来．大量叶绿体基因组全序列被测定,以及分子生物学的研究结果为内共生起源学说提供了更多证据。相对于线粒体,叶绿体DNA的结构更趋于保守一,叶绿体与核基因组所编码的蛋白质互相协调来维持叶绿体的正常功能。在进化过程中,基因可能从叶绿体大量转移到细胞核中。叶绿体基因组的信息常常表现出“母性遗传”特征．因而,使之更具生物反应器的优势。     

高等植物线粒体DNA的制备方法

自从六十年代发现线粒体DNA(mtDNA)以来,mtDNA在遗传上的功能引起了广泛的重视。由于线粒体具有自已的基因组,能够自我复制,又能编码一些酶,比如生物氧化链上的一部分酶的亚基就是由线粒体基因编码的,可以推测生物的某些性状的表达可能与mt-DNA有关;另外由于实现线粒体基因组的复制与表达所需的许多酶又是由核基因编码的(如DNA聚合酶,RNA聚合酶、DNA连接酶等),可以推测     

人线粒体DNA复制控制区与疾病和衰老的关系

线粒体DNA(mitochondrial DNA,mtDNA)复制控制区(又称D-环区)是线粒体非编码区中较为重要的区域,参与并调节线粒体DNA的复制与转录。然而,与核基因组不同的是,线粒体DNA的复制与转录并不是相互独立的,而是存在着密切的联系。从目前的研究看来,复制控制区的某些变化很可能会引起mtDNA复制、转录的变化,从而导致线粒体功能的变化,最终引起线粒体疾病或衰老的发生。     

与衰老相关的线粒体功能严重缺陷的胞质杂合细胞的分子特征

该研究组先前建立了含有不同年龄组小鼠神经细胞线粒体的胞质杂合细胞株(其细胞核基因组背景相同),发现老年组较年轻组的胞质杂合细胞线粒体总体功能显著降低。为了研究与衰老相关的线粒体功能严重缺陷的胞质杂合细胞的分子特征,该研究采用3个线粒体总体功能极其低下的老年组胞质杂合细胞株(1个正常功能年轻组胞质杂合细胞株作为对照)作为研究对象。首先,证实氧耗水平和ATP合成显著降低(P0.05或P0.01)。然后,对线粒体DNA(mitochondrial DNA,mt DNA)进行了高通量测序,特别是检出突变型比例低的mt DNA异质性突变,并进一步对呼吸链复合体依赖性氧耗进行了检测。测序结果显示,在老年组胞质杂合细胞中mt DNA点突变明显积累。这些突变包括非编码区的3个变异,据DNA保守性分析结果,其中2个(异质性m.15849GT、m.16289AT)可能为有害的;编码区的4个变异,据DNA和氨基酸保守性分析及蛋白质功能预测结果,其中2个(ND5基因的同质性m.12496TC、Cyt b基因的异质性m.15199AT)可能为有害的。进一步研究结果显示,同时具有复合体I亚基ND5(或复合体III亚基Cyt b)突变和2个调控区突变的胞质杂合细胞,其复合体I(或复合体III)依赖性呼吸显著降低(P0.05或P0.01)。综上,发生于老年组胞质杂合细胞的线粒体总体功能异常,其原因可能为,mt DNA调控区和编码区的异质性和同质性突变,以及多重mt DNA突变的累加引起线粒体呼吸链复合体功能的缺陷,进而导致线粒体总体功能异常,从而促进衰老。     

线粒体表观遗传学研究进展

作为细胞内的"动力工厂",线粒体是细胞内进行氧化磷酸化反应和形成ATP的主要场所。传统观点曾认为线粒体缺乏表观遗传机制,但线粒体DNA甲基化酶以及线粒体DNA中5-甲基胞嘧啶与5-羟甲基胞嘧啶的发现推翻了这一论断。在线粒体中,DNA甲基化酶、DNA甲基化模式及DNA羟甲基化模式与核基因组DNA相比均存在较大差异,而外界环境中不同因子的变化也会对线粒体DNA的甲基化状态造成影响。除此之外,线粒体DNA的表观遗传因素还包括线粒体长链非编码RNA、线粒体mi RNA和线粒体DNA结合蛋白。随着研究技术手段的不断完善,将线粒体DNA的甲基化状态作为生物标记的应用将日益广泛,其与基因组表观遗传调控的关联也将得到进一步的揭示。     

家马核基因组中线粒体核内插入序列分析

在各种真核生物核基因组中,存在一些由线粒体基因组转移进入核基因组中的DNA片段,这些被认为是分子化石的片段叫做线粒体核内插入序列(Numt)。由于Numt与真实的线粒体序列高度相似,因此它的存在必然会成为PCR扩增线粒体DNA的不利因素。利用已经公布的家马(Equus caballus)基因组序列(2007年9月公布,GenBank登录号为NC_009144-NC_009175)对家马Numt进行了深入分析,共发现200个可能的Numt,长度范围为29到3727bp,其中有10个的长度大于800bp。分析结果显示由于不存在线粒体控制区域的疑似Numt,因此对基于此区域的群体遗传学研究不会产生影响。本研究还发现在家马进化过程中,第1号和27号染色体更倾向于接受线粒体序列的转移。以上结果将为今后马科动物的研究提供重要的参考信息,有助于避免在线粒体DNA研究中由于Numt污染的存在而得出错误的实验结果。     

人类线粒体DNA异质性研究进展及其法医学应用

线粒体DNA(mitochondrial DNA mtDNA)的异质性自从被发现以来,一直被遗传学、进化学、发育遗传学以及法医遗传学、分子生物学领域所重视。由于线粒体异质性的存在,使得很多涉及疾病、进化、系统发育线粒体基因组与核基因组的相互作用关系、线粒体DNA复制机制以及法医学运用线粒体DNA进行实际案件评估的问题变得复杂化。此外线粒体DNA异质性的发生原因以及对线粒体异质性的检测方法标准化问题还没有一个统一的答案。针对线粒体DNA异质性带来的种种问题,近年来国内外取得了不少研究进展。     

Natural selection and the evolution of mtDNA-encoded peptides: evidence for intergenomic co-adaptation.

Mitochondrial DNA (mtDNA) variation is an important tool for the investigation of the population genetics of animal species. Recently, recognition of the role of mtDNA mutations in human disease has spurred increasing interest in the function and evolution of mtDNA and the 13 polypeptides it encodes. These proteins interact with a large number of peptides encoded in the nucleus to form the mitochondrial electron transport system (ETS). As the ETS is the primary energy generation system in aerobic metazoans, natural selection would be expected to favor mutations that enhance ETS function. Such mutations could occur in either the mitochondrial or nuclear genes encoding ETS proteins and would lead to positive intergenomic interactions, or co-adaptation. Direct evidence for intergenomic co-adaptation comes from functional studies of systems where nuclear-mitochondrial DNA combinations vary naturally or can be manipulated experimentally.     

Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys

Mitochondrial DNA (mtDNA) deletion mutations co-localize with electron transport system (ETS) abnormalities in rhesus monkey skeletal muscle fibers. Using laser capture microdissection in conjunction with PCR and DNA sequence analysis, mitochondrial genomes from single sections of ETS abnormal fibers were characterized. All ETS abnormal fibers contained mtDNA deletion mutations. Deletions were large, removing 20-78% of the genome, with some to nearly all of the functional genes lost. In one-third of the deleted genomes, the light strand origin was deleted, whereas the heavy strand origin of replication was conserved in all fibers. A majority (27/39) of the deletion mutations had direct repeat sequences at their breakpoints and most (36/39) had one breakpoint within or in close proximity to the cytochrome b gene. Several pieces of evidence support the clonality of the mtDNA deletion mutation within an ETS abnormal region of a fiber: (a) only single, smaller than wild-type, PCR products were obtained from each ETS abnormal region; (b) the amplification of mtDNA from two regions of the same ETS abnormal fiber identified identical deletion mutations, and (c) a polymorphism was observed at nucleotide position 16103 (A and G) in the wild-type mtDNA of one animal (sequence analysis of an ETS abnormal region revealed that mtDNA deletion mutations contained only A or G at this position). Species-specific differences in the regions of the genomes lost as well as the presence of direct repeat sequences at the breakpoints suggest mechanistic differences in deletion mutation formation between rodents and primates.     

Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 µm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4–9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.     

Mitochondrial DNA deletion mutations: a causal role in sarcopenia.

Mitochondrial DNA (mtDNA) deletion mutations accumulate with age in tissues of a variety of species. Although the relatively low calculated abundance of these deletion mutations in whole tissue homogenates led some investigators to suggest that these mutations do not have any physiological impact, their focal and segmental accumulation suggests that they can, and do, accumulate to levels sufficient to affect the metabolism of a tissue. This phenomenon is most clearly demonstrated in skeletal muscle, where the accumulation of mtDNA deletion mutations remove critical subunits that encode for the electron transport system (ETS). In this review, we detail and provide evidence for a molecular basis of muscle fiber loss with age. Our data suggest that the mtDNA deletion mutations, which are generated in tissues with age, cause muscle fiber loss. Within a fiber, the process begins with a mtDNA replication error, an error that results in a loss of 25-80% of the mitochondrial genome. This smaller genome is replicated and, through a process not well understood, eventually comprises the majority of mtDNA within the small affected region of the muscle fiber. The preponderance of the smaller genomes results in a dysfunctional ETS in the affected area. As a consequence of both the decline in energy production and the increase in oxidative damage in the region, the fiber is no longer capable of self-maintenance, resulting in the observed intrafiber atrophy and fiber breakage. We are therefore proposing that a process contained within a very small region of a muscle fiber can result in breakage and loss of muscle fiber from the tissue.     

Review: can diet influence the selective advantage of mitochondrial DNA haplotypes?

This review explores the potential for changes in dietary macronutrients to differentially influence mitochondrial bioenergetics and thereby the frequency of mtDNA haplotypes in natural populations. Such dietary modification may be seasonal or result from biogeographic or demographic shifts. Mechanistically, mtDNA haplotypes may influence the activity of the electron transport system (ETS), retrograde signalling to the nuclear genome and affect epigenetic modifications. Thus, differential provisioning by macronutrients may lead to selection through changes in the levels of ATP production, modulation of metabolites (including AMP, reactive oxygen species (ROS) and the NAD⁺/NADH ratio) and potentially complex epigenetic effects. The exquisite complexity of dietary influence on haplotype frequency is further illustrated by the fact that macronutrients may differentially influence the selective advantage of specific mutations in different life-history stages. In Drosophila, complex I mutations may affect larval growth because dietary nutrients are fed through this complex in immaturity. In contrast, the majority of electrons are provided to complex III in adult flies. We conclude the review with a case study that considers specific interactions between diet and complex I of the ETS. Complex I is the first enzyme of the mitochondrial ETS and co-ordinates in the oxidation of NADH and transfer of electrons to ubiquinone. Although the supposition that mtDNA variants may be selected upon by dietary macronutrients could be intuitively consistent to some and counter intuitive to others, it must face a multitude of scientific hurdles before it can be recognized.     

Mitochondrial genetics and human disease

Mitochondria contain a molecular genetic system to express the 13 protein components of the electron transport system encoded in the mitochondrial genome (mtDNA). Defects in the function of this system result in some diaseases, many of which are multisystem disorders, prominently involving highly aerobic, postmitotic tissues. These defects can be caused by large-scale rearrangements of mtDNA, by point mutations, or by nuclear gene mutations resulting in abnormalities in mtDNA. Although any of these mutations would be expected to produce a similar clinical phenotype by compromising oxidative phosphorylation, the surprising and puzzling result is that different clinical phenotypes are generally associated with specific mtDNA mutations. Moreover, the same mutation can produce a distinct clinical phenotype in different individuals or pedigrees. MtDNA rearrangements are also found in aged individuals, but at a subclinical level, suggesting that normal and pathological processes can differ by the effect of genetic or environmental factors on the error rate of mtDNA replication.     

Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers

Skeletal muscle-mass loss with age has severe health consequences, yet the molecular basis of the loss remains obscure. Although mitochondrial DNA (mtDNA)-deletion mutations have been shown to accumulate with age, for these aberrant genomes to be physiologically relevant, they must accumulate to high levels intracellularly and be present in a significant number of cells. We examined mtDNA-deletion mutations in vastus lateralis (VL) muscle of human subjects aged 49-93 years, using both histologic and polymerase-chain-reaction (PCR) analyses, to determine the physiological and genomic integrity of mitochondria in aging human muscle. The number of VL muscle fibers exhibiting mitochondrial electron-transport-system (ETS) abnormalities increased from an estimated 6% at age 49 years to 31% at age 92 years. We analyzed the mitochondrial genotype of 48 single ETS-abnormal, cytochrome c oxidase-negative/succinate dehydrogenase-hyperreactive (COX-/SDH++) fibers from normal aging human subjects and identified mtDNA-deletion mutations in all abnormal fibers. Deletion mutations were clonal within a fiber and concomitant to the COX-/SDH++ region. Quantitative PCR analysis of wild-type and deletion-containing mtDNA genomes within ETS-abnormal regions of single fibers demonstrated that these deletion mutations accumulate to detrimental levels (>90% of the total mtDNA).     

Evolution of interacting proteins in the mitochondrial electron transport system in a marine copepod

The extensive interaction between mitochondrial-encoded and nuclear-encoded subunits of electron transport system (ETS) enzymes in mitochondria is expected to lead to intergenomic coadaptation. Whether this coadaptation results from adaptation to the environment or from fixation of deleterious mtDNA mutations followed by compensatory nuclear gene evolution is unknown. The intertidal copepod Tigriopus californicus shows extreme divergence in mtDNA sequence and provides an excellent model system for study of intergenomic coadaptation. Here, we examine genes encoding subunits of complex III of the ETS, including the mtDNA-encoded cytochrome b (CYTB), the nuclear-encoded rieske iron-sulfur protein (RISP), and cytochrome c(1) (CYC1). We compare levels of polymorphism within populations and divergence between populations in these genes to begin to untangle the selective forces that have shaped evolution in these genes. CYTB displays dramatic divergence between populations, but sequence analysis shows no evidence for positive selection driving this divergence. CYC1 and RISP have lower levels of sequence divergence between populations than CYTB, but, again, sequence analysis gives no evidence for positive selection acting on them. However, an examination of variation at cytochrome c (CYC), a nuclear-encoded protein that transfers electrons between complex III and complex IV provides evidence for selective divergence. Hence, it appears that rapid evolution in mitochondrial-encoded subunits is not always associated with rapid divergence in interacting subunits (CYC1 and RISP), but can be in some cases (CYC). Finally, a comparison of nuclear-encoded and mitochondrial-encoded genes from T. californicus suggests that substitution rates in the mitochondrial-encoded genes are dramatically increased relative to nuclear genes.     

Disruption of mitochondrial function in interpopulation hybrids of Tigriopus californicus

Electron transport system (ETS) function in mitochondria is essential for the aerobic production of energy. Because ETS function requires extensive interactions between mitochondrial and nuclear gene products, coadaptation between mitochondrial and nuclear genomes may evolve within populations. Hybridization between allopatric populations may then expose functional incompatibilities between genomes that have not coevolved. The intertidal copepod Tigriopus californicus has high levels of nucleotide divergence among populations at mitochondrial loci and suffers F2 hybrid breakdown in interpopulation hybrids. We hypothesize that hybridization results in incompatibilities among subunits in ETS enzyme complexes and that these incompatibilities result in diminished mitochondrial function and fitness. To test this hypothesis, we measured fitness, mitochondrial function, and ETS enzyme activity in inbred recombinant hybrid lines of Tigriopus californicus. We found that (1) both fitness and mitochondrial function are reduced in hybrid lines, (2) only those ETS enzymes with both nuclear and mitochondrial subunits show a loss of activity in hybrid lines, and (3) positive relationships exist between ETS enzyme activity and mitochondrial function and between mitochondrial function and fitness. We also present evidence that hybrid lines harboring mitochondrial DNA (mtDNA) and mitochondrial RNA polymerase (mtRPOL) from the same parental source population have higher fitness than those with mtDNA and mtRPOL from different populations, suggesting that mitochondrial gene regulation may play a role in disruption of mitochondrial performance and fitness of hybrids. These results suggest that disruption of coadaptation between nuclear and mitochondrial genes contributes to the phenomenon of hybrid breakdown.     

Effect of mtDNA point mutations on cellular bioenergetics

This overview discusses the results of research on the effects of most frequent mtDNA point mutations on cellular bioenergetics. Thirteen proteins coded by mtDNA are crucial for oxidative phosphorylation, 11 of them constitute key components of the respiratory chain complexes I, III and IV and 2 of mitochondrial ATP synthase. Moreover, pathogenic point mutations in mitochondrial tRNAs and rRNAs generate abnormal synthesis of the mtDNA coded proteins. Thus, pathogenic point mutations in mtDNA usually disturb the level of key parameter of the oxidative phosphorylation, i.e. the electric potential on the inner mitochondrial membrane (Δψ), and in a consequence calcium signalling and mitochondrial dynamics in the cell. Mitochondrial generation of reactive oxygen species is also modified in the mutated cells. The results obtained with cultured cells and describing biochemical consequences of mtDNA point mutations are full of contradictions. Still they help elucidate the biochemical basis of pathologies and provide a valuable tool for finding remedies in the future. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).