Mitochondrial purine and pyrimidine metabolism and beyond

ABSTRACT

Carefully balanced deoxynucleoside triphosphate (dNTP) pools are essential for both nuclear and mitochondrial genome replication and repair. Two synthetic pathways operate in cells to produce dNTPs, e.g., the de novo and the salvage pathways. The key regulatory enzymes for de novo synthesis are ribonucleotide reductase (RNR) and thymidylate synthase (TS), and this process is considered to be cytosolic. The salvage pathway operates both in the cytosol (TK1 and dCK) and the mitochondria (TK2 and dGK). Mitochondrial dNTP pools are separated from the cytosolic ones owing to the double membrane structure of the mitochondria, and are formed by the salvage enzymes TK2 and dGK together with NMPKs and NDPK in postmitotic tissues, while in proliferating cells the mitochondrial dNTPs are mainly imported from the cytosol produced by the cytosolic pathways. Imbalanced mitochondrial dNTP pools lead to mtDNA depletion and/or deletions resulting in serious mitochondrial diseases. The mtDNA depletion syndrome is caused by deficiencies not only in enzymes in dNTP synthesis (TK2, dGK, p53R2, and TP) and mtDNA replication (mtDNA polymerase and twinkle helicase), but also in enzymes in other metabolic pathways such as SUCLA2 and SUCLG1, ABAT and MPV17. Basic questions are why defects in these enzymes affect dNTP synthesis and how important is mitochondrial nucleotide synthesis in the whole cell/organism perspective? This review will focus on recent studies on purine and pyrimidine metabolism, which have revealed several important links that connect mitochondrial nucleotide metabolism with amino acids, glucose, and fatty acid metabolism.     

金鱼草中央细胞中线粒体DNA拷贝数的定量

为探究金鱼草(Antirrhinum majus)中央细胞的线粒体DNA拷贝数,采用竞争型定量PCR技术进行了测定。结果表明,金鱼草中央细胞的体积为(61570±732)μm~3,平均携带(783±25)个拷贝的线粒体DNA,并且中央细胞与卵细胞具有相似的体积/线粒体DNA拷贝数比值。推测金鱼草中央细胞包含如此高丰度线粒体DNA可能是为早期胚乳细胞的发育而储备的。     

芥菜型油菜脂肪酸含量的变异、相关性分析及芥酸调控基因FAE1特异引物设计

芥菜型油菜是我国芸苔属的三大油料作物之一,具有耐旱、抗病虫等优良特性;而我国是芥菜型油菜的重要起源中心,具有丰富的种质资源.该研究从全国各地搜集了34份芥菜型油菜,在贵阳环境条件下种植,其脂肪酸含量(芥酸、油酸、硬脂酸、亚麻酸和亚油酸)表现出丰富的变异,并呈正态分布.结果表明:这些芥菜型油菜种质资源的不同脂肪酸含量间的相关性发现,芥酸和油酸间呈极显著负相关,亚麻酸和硬脂酸呈极显著的正相关,亚麻酸和亚油酸呈现负相关.利用这些材料的脂肪酸含量进行主成分分析,发现绝大部分材料(30份,占88.2%)集中在二维图的特定区域,只有少数其它材料散落在图中其它区域,他们分别是SL63、棱角油菜、T6342和长阳黄芥,这些变异较大的材料在芥菜型油菜的育种中可以发挥特殊作用.此外,运用来自甘蓝型油菜和甘蓝的芥酸调控基因FAE1的已知序列,并设计了FAE1特异引物,而引物在全部34份芥菜型油菜种质资源中均表现出了较好的扩增效果.因此证实芥菜型油菜中至少含有一个FAE1拷贝.该研究结果对于芥菜型油菜育种在我国的开展及其未来的分子育种具有重要的指导意义.     

白芨SSR引物筛选及群体遗传多样性研究

白芨(Bletilla striata Rchb.)为中国珍稀濒危植物,重要的药用植物。该研究基于Illumina测序技术构建白芨基因组文库和微卫星文库,设计白芨微卫星引物,用白芨4个野生种群80个个体对引物进行多态性检测,应用4个白芨近缘种中进行引物的通用性检测,并在此基础上分析了白芨的遗传多样性和遗传分化,以探讨白芨的遗传结构和进化潜能。结果表明:(1)白芨基因组中微卫星片断丰富,共检测出17 841条微卫星片断。基于白芨微卫星库对100个位点设计了引物对,经PCR扩增和检测筛选出能够稳定扩增的多态性位点20个,每个位点的复等位基因数(Na)在2~6之间,平均为3.85;20对引物的大部分能够在4个白芨近缘种中成功扩增。(2)白芨在物种水平均有较高的遗传多样性(Na=3.85,I=1.07,H=0.614 7),白芨种群遗传分化强烈(Gst=0.43),居群间的基因流较弱(Nm=0.867 6),居群聚类分析结果均表明地理距离较近的居群具有较近的遗传关系。     

Fragile X‐associated tremor/ataxia syndrome: Regional decrease of mitochondrial DNA copy number relates to clinical manifestations

Fragile X‐associated tremor/ataxia syndrome (FXTAS) is a late‐onset neurodegenerative disorder that appears in at least one‐third of adult carriers of a premutation (55‐200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. Several studies have shown that mitochondrial dysfunction may play a central role in aging and also in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease as well as in FXTAS. It has been recently proposed that mtDNA copy number, measured by the number of mitochondrial genomes per nuclear genome (diploid), could be a useful biomarker of mitochondrial dysfunction. In order to elucidate the role of mtDNA variation in the pathogenesis of FXTAS, mtDNA copy number was quantified by digital droplet Polymerase chain reaction. In human brain samples, mtDNA levels were measured in the cerebellar vermis, dentate nucleus, parietal and temporal cortex, thalamus, caudate nucleus and hippocampus from a female FXTAS patient, a FMR1 premutation male carrier without FXTAS and from three male controls. The mtDNA copy number was further analyzed using this technology in dermal fibroblasts primary cultures derived from three FXTAS patients and three controls as well as in cortex and cerebellum of a CGG knock in FXTAS mice model. Finally, qPCR was carried out in human blood samples. Results indicate reduced mtDNA copy number in the specific brain region associated with disease progression in FXTAS patients, providing new insights into the role of mitochondrial dysfunction in the pathogenesis of FXTAS.     

Voluntary exercise normalizes the proteomic landscape in muscle and brain and improves the phenotype of progeroid mice

The accumulation of mitochondrial DNA (mtDNA) mutations is a suspected driver of aging and age‐related diseases, but forestalling these changes has been a major challenge. One of the best‐studied models is the prematurely aging mtDNA mutator mouse, which carries a homozygous knock‐in of a proofreading deficient version of the catalytic subunit of mtDNA polymerase‐γ (PolgA). We investigated how voluntary exercise affects the progression of aging phenotypes in this mouse, focusing on mitochondrial and protein homeostasis in both brain and peripheral tissues. Voluntary exercise significantly ameliorated several aspects of the premature aging phenotype, including decreased locomotor activity, alopecia, and kyphosis, but did not have major effects on the decreased lifespan of mtDNA mutator mice. Exercise also decreased the mtDNA mutation load. In‐depth tissue proteomics revealed that exercise normalized the levels of about half the proteins, with the majority involved in mitochondrial function and nuclear–mitochondrial crosstalk. There was also a specific increase in the nuclear‐encoded proteins needed for the tricarboxylic acid cycle and complex II, but not in mitochondrial‐encoded oxidative phosphorylation proteins, as well as normalization of enzymes involved in coenzyme Q biosynthesis. Furthermore, we found tissue‐specific alterations, with brain coping better as compared to muscle and with motor cortex being better protected than striatum, in response to mitochondrial dysfunction. We conclude that voluntary exercise counteracts aging in mtDNA mutator mice by counteracting protein dysregulation in muscle and brain, decreasing the mtDNA mutation burden in muscle, and delaying overt aging phenotypes.     

New mitochondrial primers for metabarcoding of insects,designed and evaluated using in silico methods

Insect metabarcoding has been mainly based on PCR amplification of short fragments within the “barcoding region” of the gene cytochrome oxidase I (COI). However, because of the variability of this gene, it has been difficult to design good universal PCR primers. Most primers used today are associated with gaps in the taxonomic coverage or amplification biases that make the results less reliable and impede the detection of species that are present in the sample. We identify new primers for insect metabarcoding using computational approaches (ecoprimers and degeprime ) applied to the most comprehensive reference databases of mitochondrial genomes of Hexapoda assembled to date. New primers are evaluated in silico against previously published primers in terms of taxonomic coverage and resolution of the corresponding amplicons. For the latter criterion, we propose a new index, exclusive taxonomic resolution, which is a more biologically meaningful measure than the standard index used today. Our results show that the best markers are found in the ribosomal RNA genes (12S and 16S); they resolve about 90% of the genetically distinct species in the reference database. Some markers in protein‐coding genes provide similar performance but only at much higher levels of primer degeneracy. Combining two of the best individual markers improves the effective taxonomic resolution with up to 10%. The resolution is strongly dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S primers detect 12% of Collembola. Our results indicate that amplicon‐based metabarcoding of insect samples can be improved by choosing other primers than those commonly used today.     

Genetic differentiation of Pseudoregma bambucicola population based on mtDNA COII gene

mtDNA COII gene sequences were identified and analyzed using different types of software, namely, MEGA5.0, DNAMAN, and DnaSP5.0 in four Chinese provinces, namely, Sichuan, Zhejiang, Guizhou and Shanghai. Analysis of molecular genetic variation and its genetic structure and differentiation, combined with NJ tree, MP tree analysis and analysis of molecular variance (AMOVA), at Fst = 0.0582 conclude that the genetic differentiation is low, gene flow is Nm = 8.0911, and gene exchange is sufficient. However, for the geographic populations of Pseudoregma bambucicola in the four provinces, their gene exchange is relatively weak at Nm = 0.8284, whereas the genetic differentiation is high at Fst = 0.3764. Based on the data, total nucleotide diversity between the populations is 0.00158 ± 0.00021. The results showed that the total population of Tajima’s D and Fu’s Fs results are D = ?0.885 and Fs = 0.226, respectively. The experimental numerical results showed that this total population is not significant (P > 0.10), indicating that nine different geographic populations are short-term. No expansion occurred in the internal population. This study provided a theoretical and practical basis for the comprehensive prevention and control of P. bambucicola.