Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA

The manifestation of mitochondrial DNA (mtDNA) diseases depends on the frequency of heteroplasmy (the presence of several alleles in an individual), yet its transmission across generations cannot be readily predicted owing to a lack of data on the size of the mtDNA bottleneck during oogenesis. For deleterious heteroplasmies, a severe bottleneck may abruptly transform a benign (low) frequency in a mother into a disease-causing (high) frequency in her child. Here we present a high-resolution study of heteroplasmy transmission conducted on blood and buccal mtDNA of 39 healthy mother–child pairs of European ancestry (a total of 156 samples, each sequenced at ∼20,000× per site). On average, each individual carried one heteroplasmy, and one in eight individuals carried a disease-associated heteroplasmy, with minor allele frequency ≥1%. We observed frequent drastic heteroplasmy frequency shifts between generations and estimated the effective size of the germ-line mtDNA bottleneck at only ∼30–35 (interquartile range from 9 to 141). Accounting for heteroplasmies, we estimated the mtDNA germ-line mutation rate at 1.3 × 10−8 (interquartile range from 4.2 × 10−9 to 4.1 × 10−8) mutations per site per year, an order of magnitude higher than for nuclear DNA. Notably, we found a positive association between the number of heteroplasmies in a child and maternal age at fertilization, likely attributable to oocyte aging. This study also took advantage of droplet digital PCR (ddPCR) to validate heteroplasmies and confirm a de novo mutation. Our results can be used to predict the transmission of disease-causing mtDNA variants and illuminate evolutionary dynamics of the mitochondrial genome

AST: An Automated Sequence-Sampling Method for Improving the Taxonomic Diversity of Gene Phylogenetic Trees

A challenge in phylogenetic inference of gene trees is how to properly sample a large pool of homologous sequences to derive a good representative subset of sequences. Such a need arises in various applications, e.g. when (1) accuracy-oriented phylogenetic reconstruction methods may not be able to deal with a large pool of sequences due to their high demand in computing resources; (2) applications analyzing a collection of gene trees may prefer to use trees with fewer operational taxonomic units (OTUs), for instance for the detection of horizontal gene transfer events by identifying phylogenetic conflicts; and (3) the pool of available sequences is biased towards extensively studied species. In the past, the creation of subsamples often relied on manual selection. Here we present an Automated sequence-Sampling method for improving the Taxonomic diversity of gene phylogenetic trees, AST, to obtain representative sequences that maximize the taxonomic diversity of the sampled sequences. To demonstrate the effectiveness of AST, we have tested it to solve four problems, namely, inference of the evolutionary histories of the small ribosomal subunit protein S5 of E. coli, 16 S ribosomal RNAs and glycosyl-transferase gene family 8, and a study of ancient horizontal gene transfers from bacteria to plants. Our results show that the resolution of our computational results is almost as good as that of manual inference by domain experts, hence making the tool generally useful to phylogenetic studies by non-phylogeny specialists. The program is available at http://csbl.bmb.uga.edu/~zhouchan/AST.php

Heterogeneous patterns of tissue injury in NARP syndrome

Point mutations at m.8993T>C and m.8993T>G of the mtDNA ATPase 6 gene cause the neurogenic weakness, ataxia and retinitis pigmentosa (NARP) syndrome, a mitochondrial disorder characterized by retinal, central and peripheral neurodegeneration. We performed detailed neurological, neuropsychological and ophthalmological phenotyping of a mother and four daughters with NARP syndrome from the mtDNA m.8993T>C ATPase 6 mutation, including 3-T brain MRI, spectral domain optical coherence tomography (SD-OCT), adaptive optics scanning laser ophthalmoscopy (AOSLO), electromyography and nerve conduction studies (EMG-NCS) and formal neuropsychological testing. The degree of mutant heteroplasmy for the m.8993T>C mutation was evaluated by real-time allele refractory mutation system quantitative PCR of mtDNA from hair bulbs (ectoderm) and blood leukocytes (mesoderm). There were marked phenotypic differences between family members, even between individuals with the greatest degrees of ectodermal and mesodermal heteroplasmy. 3-T MRI revealed cerebellar atrophy and cystic and cavitary T2 hyperintensities in the basal ganglia. SD-OCT demonstrated similarly heterogeneous areas of neuronal and axonal loss in inner and outer retinal layers. AOSLO showed increased cone spacing due to photoreceptor loss. EMG-NCS revealed varying degrees of length-dependent sensorimotor axonal polyneuropathy. On formal neuropsychological testing, there were varying deficits in processing speed, visual–spatial functioning and verbal fluency and high rates of severe depression. Many of these cognitive deficits likely localize to cerebellar and/or basal ganglia dysfunction. High-resolution retinal and brain imaging in NARP syndrome revealed analogous patterns of tissue injury characterized by heterogeneous areas of neuronal loss

ViralORFeome: an integrated database to generate a versatile collection of viral ORFs

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such ‘ORFeome’ resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway® system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins

In Silico Insights into the Symbiotic Nitrogen Fixation in Sinorhizobium meliloti via Metabolic Reconstruction

BACKGROUND: Sinorhizobium meliloti is a soil bacterium, known for its capability to establish symbiotic nitrogen fixation (SNF) with leguminous plants such as alfalfa. S. meliloti 1021 is the most extensively studied strain to understand the mechanism of SNF and further to study the legume-microbe interaction. In order to provide insight into the metabolic characteristics underlying the SNF mechanism of S. meliloti 1021, there is an increasing demand to reconstruct a metabolic network for the stage of SNF in S. meliloti 1021. RESULTS: Through an iterative reconstruction process, a metabolic network during the stage of SNF in S. meliloti 1021 was presented, named as iHZ565, which accounts for 565 genes, 503 internal reactions, and 522 metabolites. Subjected to a novelly defined objective function, the in silico predicted flux distribution was highly consistent with the in vivo evidences reported previously, which proves the robustness of the model. Based on the model, refinement of genome annotation of S. meliloti 1021 was performed and 15 genes were re-annotated properly. There were 19.8% (112) of the 565 metabolic genes included in iHZ565 predicted to be essential for efficient SNF in bacteroids under the in silico microaerobic and nutrient sharing condition. CONCLUSIONS: As the first metabolic network during the stage of SNF in S. meliloti 1021, the manually curated model iHZ565 provides an overview of the major metabolic properties of the SNF bioprocess in S. meliloti 1021. The predicted SNF-required essential genes will facilitate understanding of the key functions in SNF and help identify key genes and design experiments for further validation. The model iHZ565 can be used as a knowledge-based framework for better understanding the symbiotic relationship between rhizobia and legumes, ultimately, uncovering the mechanism of nitrogen fixation in bacteroids and providing new strategies to efficiently improve biological nitrogen fixation

Mitochondrial ATP synthase: architecture, function and pathology

Human mitochondrial (mt) ATP synthase, or complex V consists of two functional domains: F1, situated in the mitochondrial matrix, and Fo, located in the inner mitochondrial membrane. Complex V uses the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP. This review covers the architecture, function and assembly of complex V. The role of complex V di-and oligomerization and its relation with mitochondrial morphology is discussed. Finally, pathology related to complex V deficiency and current therapeutic strategies are highlighted. Despite the huge progress in this research field over the past decades, questions remain to be answered regarding the structure of subunits, the function of the rotary nanomotor at a molecular level, and the human complex V assembly process. The elucidation of more nuclear genetic defects will guide physio(patho)logical studies, paving the way for future therapeutic interventions