Whole-genome sequencing of a Plasmodium vivax isolate from the China-Myanmar border area

Currently, there is a trend of an increasing number of Plasmodiumvivaxmalaria cases in China that are imported across its Southeast Asiaborder, especially in the China-Myanmar border area (CMB). To date, little is knownabout the genetic diversity of P. vivax in this region. In thispaper, we report the first genome sequencing of a P. vivaxisolate(CMB-1) from a vivax malaria patient in CMB. The sequencing data were aligned onto96.43% of the P. vivax Salvador I reference strain (Sal I) genomewith 7.84-fold coverage as well as onto 98.32% of 14 Sal I chromosomes. Usingthe de novo assembly approach, we generated 8,541 scaffolds andassembled a total of 27.1 Mb of sequence into CMB-1 scaffolds. Furthermore, weidentified all 295 known virgenes, which is the largest subtelomericmultigene family in malaria parasites. These results provide an important foundationfor further research onP. vivax population genetics.     

The putative mitochondrial genome of Plasmodium falciparum

Intraerythrocytic stages of mammalian malarial parasites employ glycolysis for energy production but some aspects of mitochondrial function appear crucial to their survival since inhibitors of mitochondrial protein synthesis and electron transport have antimalarial effects. Investigations of the putative mitochondrial genome of Plasmodium falciparum have detected organellar rRNAs and tRNAs encoded by a 35 kb circular DNA. Some features of the organization and sequence of the rRNA genes are reminiscent of chloroplast DNAs. The 35 kb DNA also encodes open reading frames for proteins normally found in chloroplast but not mitochondrial genomes. An apparently unrelated 6 kb tandemly repeated element which encodes two mitochondrial protein coding genes and fragments of rRNA genes is also found in malarial parasites. The malarial mitochondrial genome thus appears quite unusual. Further investigations are expected to provide insights into the possible functional relationships between these molecules and perhaps their evolutionary history.     

The origins of replication of the mitochondrial genome of yeast

Recent investigations have provided information on the origin of replication of the mitochondrial genome of yeast and an explanation for the phenomenon of the suppressivity.     

Mitochondrial genome sequences support ancient population expansion in Plasmodium vivax

Examination of nucleotide diversity in 106 mitochondrial genomes of the most geographically widespread human malaria parasite, Plasmodium vivax, revealed a level of diversity similar to, but slightly higher than, that seen in the virulent human malaria parasite Plasmodium falciparum. The pairwise distribution of nucleotide differences among mitochondrial genome sequences supported the hypothesis that both these parasites underwent ancient population expansions. We estimated the age of the most recent common ancestor (MRCA) of the mitochondrial genomes of both P. vivax and P. falciparum at around 200,000-300,000 years ago. This is close to the previous estimates of the time of the human mitochondrial MRCA and the origin of modern Homo sapiens, consistent with the hypothesis that both these Plasmodium species were parasites of the hominid lineage before the origin of modern H. sapiens and that their population expansion coincided with the population expansion of their host.     

The complete mitochondrial genome of Fusarium oxysporum: insights into fungal mitochondrial evolution

The complete mitochondrial DNA (mtDNA) sequence was determined for the phytopathogenic fungus Fusarium oxysporum. It is 34,477 bp long, maps circularly, and encodes for 14 protein-coding, 25 tRNA and 2 rRNA genes. The nucleotide and amino acid data sets from its 14 concatenated protein-coding mitochondrial (mt) genes were used along with gene order comparisons for an extensive phylogenetic study of the Subphylum Pezizomycotina. Our results are in agreement with current taxonomic treatments and additionally provide better statistical support for all relationships within Pezizomycotina when compared to analyses based on single or few gene data sets. The gene order of F. oxysporum was consistent with that established in the order Hypocreales (Class: Sordariomycetes) and enhanced previous suppositions on the ancestral state of Sordariomycetes. In comparison with mt genomes of the other orders it added further insights to the evolution of Pezizomycotina.     

线粒体基因组测序策略和方法

本文综述了线粒体基因组测序策略和方法,在传统测序方法中介绍了基于物理分离线粒体DNA的克隆文库测序方法和基于PCR扩增产物的直接测序方法,后者重点介绍了基于长PCR扩增产物的引物步移法和基于总DNA的引物步移法;应用新一代高通量测序方法有基于总DNA样品的方法,包括需要预扩增mtDNA的多物种平行高通量和无需预扩增mtDNA的高通量方法,基于总RNA样品的转录组测序方法等。在实际工作中,选择哪种方法取决于研究规模、样品大小和保存状态、经费情况等。总的来说,基于长PCR扩增产物的引物步移法尤其适合小规模昆虫线粒体基因组研究,而对于大规模线粒体基因组研究来说,NGS技术无疑是省时省力的最佳选择。     

The paroxysm of Plasmodium vivax malaria

The paroxysms of Plasmodium vivax malaria are antiparasite responses that, although distressing to the human host, almost never impart serious acute pathology. Using plasma and blood cells from P. vivax patients, the cellular and noncellular mediators of these events have been studied ex vivo. The host response during a P. vivax paroxysm was found to involve T cells, monocytes and neutrophils, and the activity, among others, of the pyrogenic cytokines tumor necrosis factor alpha and interleukin 2 in addition to granulocyte macrophage-colony stimulating factor. However, interferon gamma activity, associated with serious acute pathogenesis in other studies on malaria, was absent. Induction of the cytokines active during a P. vivax paroxysm depends upon the presence of parasite products, which are released into the plasma before the paroxysm. Chemical identification of these natural parasite products will be important for our understanding of pathogenesis and protection in malaria.     

The Plasmodium vivax rhoptry-associated protein 1

Rhoptries are cellular organelles localized at the apical pole of apicomplexan parasites. Their content is rich in lipids and proteins that are released during target cell invasion. Plasmodium falciparum rhoptry-associated protein 1 (RAP1) has been the most widely studied among this parasite species' rhoptry proteins and is considered to be a good anti-malarial vaccine candidate since it displays little polymorphism and induces antibodies in infected humans. Monoclonal antibodies directed against RAP1 are also able to inhibit target cell invasion in vitro and protection against P. falciparum experimental challenge is induced when non-human primates are immunized with this protein expressed in its recombinant form. This study describes identifying and characterizing RAP1 in Plasmodium vivax, the most widespread parasite species causing malaria in humans, producing more than 80 million infections yearly, mainly in Asia and Latin America. This new protein is encoded by a two-exon gene, is proteolytically processed in a similar manner to its falciparum homologue and, as observed by microscopy, the immunofluorescence pattern displayed is suggestive of its rhoptry localization. Further studies evaluating P. vivax RAP1 protective efficacy in non-human primates should be carried out taking into account the relevance that its P. falciparum homologue has as an anti-malarial vaccine candidate.     

The malaria genome sequencing project: complete sequence of Plasmodium falciparum chromosome 2

An international consortium has been formed to sequence the entire genome of the human malaria parasite Plasmodium falciparum. We sequenced chromosome 2 of clone 3D7 using a shotgun sequencing strategy. Chromosome 2 is 947 kb in length, has a base composition of 80.2% A + T, and contains 210 predicted genes. In comparison to the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, a greater proportion of genes containing introns, and nearly twice as many proteins containing predicted non-globular domains. A group of putative surface proteins was identified, rifins, which are encoded by a gene family comprising up to 7% of the protein-encoding gene in the genome. The rifins exhibit considerable sequence diversity and may play an important role in antigenic variation. Sixteen genes encoded on chromosome 2 showed signs of a plastid or mitochondrial origin, including several genes involved in fatty acid biosynthesis. Completion of the chromosome 2 sequence demonstrated that the A + T-rich genome of P. falciparum can be sequenced by the shotgun approach. Within 2-3 years, the sequence of almost all P. falciparum genes will have been determined, paving the way for genetic, biochemical, and immunological research aimed at developing new drugs and vaccines against malaria.     

Cultivation of Plasmodium vivax

Establishment of a continuous line of Plasmodium vivax parasite is crucial to understand the parasite's biology; however, this has not yet been achieved. Beginning in the 19th century, there were several efforts to cultivate this malaria parasite but without much success until the late 1980s. In addition, to date, only minor modifications of the methodology have been investigated, which has resulted in extending the cultivation period to around four weeks by supplying reticulocytes obtained from normal blood or rare hemochromatotic blood. However, the use of laboratory-produced erythroblasts to cultivate P. vivax enables maintenance of a continuous line of the parasite stably in the laboratory. Here, we summarize and compare the available methodologies and conditions for the in vitro cultivation of P. vivax.     

The origin and age of Plasmodium vivax

The evolutionary history of Plasmodium vivax has recently been addressed in terms of its origin as a parasite of humans and the age of extant populations. The consensus is that P. vivax originated as a result of a host switch from a non-human primate to hominids and that the extant populations did not originate as recently as previously proposed. Here, we show that, in a comparison of parasite isolates from across the world, Asian populations of P. vivax are the oldest. We discuss how this result, together with the phylogenetic evidence that P. vivax derived from Plasmodium found in Southeast Asian macaques, is most simply explained by assuming an Asian origin of this parasite. Nevertheless, the available data show only the tip of the iceberg. We discuss how sampling might affect time estimates to the most recent common ancestor for P. vivax populations and suggest that spatially explicit estimates are needed to understand the demographic history of this parasite better.     

Origins of human malaria: rare genomic changes and full mitochondrial genomes confirm the relationship of Plasmodium falciparum to other mammalian parasites but complicate the origins of Plasmodium vivax

Despite substantial work, the phylogeny of malaria parasites remains debated. The matter is complicated by concerns about patterns of evolution in potentially strongly selected genes as well as the extreme AT bias of some Plasmodium genomes. Particularly contentious has been the position of the most virulent human parasite Plasmodium falciparum, whether grouped with avian parasites or within a larger clade of mammalian parasites. Here, we study 3 classes of rare genomic changes, as well as the sequences of mitochondrial ribosomal RNA (rRNA) genes. We report 3 lines of support for a clade of mammalian parasites: 1) we find no instances of spliceosomal intron loss in a hypothetical ancestor of P. falciparum and the avian parasite Plasmodium gallinaceum, suggesting against a close relationship between those species; 2) we find 4 genomic mitochondrial indels supporting a mammalian clade, but none grouping P. falciparum with avian parasites; and 3) slowly evolving mitochondrial rRNA sequences support a mammalian parasite clade with 100% posterior probability. We further report a large deletion in the mitochondrial large subunit rRNA gene, which suggests a subclade including both African and Asian parasites within the clade of closely related primate malarias. This contrasts with previous studies that provided strong support for separate Asian and African clades, and reduces certainty about the historical and geographic origins of Plasmodium vivax. Finally, we find a lack of synapomorphic gene losses, suggesting a low rate of ancestral gene loss in Plasmodium.     

Differentiating between monozygotic twins through next-generation mitochondrial genome sequencing

Monozygotic (MZ) twins, considered to be genetically identical, cannot be distinguished from one another by standard forensic DNA testing. A recent study employed whole genome sequencing to identify extremely rare mutations and reported that mutation analysis could be used to differentiate between MZ twins. Compared with nuclear DNA, mitochondrial DNA (mtDNA) has higher mutation rates; therefore, minor differences theoretically exist in MZ twins' mitochondrial genome (mtGenome). However, conventional Sanger-type sequencing (STS) is neither amenable to, nor feasible for, the detection of low-level sequence variants. The recent introduction of massively parallel sequencing (MPS) has the capability to sequence many targeted regions of multiple samples simultaneously with desirable depth of coverage. Thus, the aim of this study was to assess whether full mtGenome sequencing analysis can be used to differentiate between MZ twins. Ten sets of MZ twins provided blood samples that underwent extraction, quantification, mtDNA enrichment, library preparation, and ultra-deep sequencing. Point heteroplasmies were observed in eight sets of MZ twins, and a single nucleotide variant (nt15301) was detected in five sets of MZ twins. Thus, this study demonstrates that ultra-deep mtGenome sequencing could be used to differentiate between MZ twins.     

Tracing the dawn of Plasmodium falciparum with mitochondrial genome sequences

Until recently, little light had been shed on the murky origins of human malaria. Did Plasmodium falciparum, the most virulent malaria parasite, emerge as a common pathogen only in the past few thousand years, as suggested by some analyses of its nucleotide sequence diversity? Or, was it an ancient scourge of early humans >100 000 years ago, as suggested by others? A recent study, using complete mitochondrial DNA sequence polymorphism data and new analytical methods, points to an intermediate date of origin and expansion out of Africa. Subsequent population growth in each continent is less well resolved.     

Genetics of psychosis; insights from views across the genome

The major psychotic illnesses, schizophrenia and bipolar disorder (BD), are among the most heritable common disorders, but finding specific susceptibility genes for them has not been straightforward. The reasons are widely assumed to include lack of valid phenotypic definition, absence of good theories of pathophysiology for candidate gene studies, and the involvement of many genes, each making small contributions to population risk. Within the last year or so, a number of genome wide association (GWAS) of schizophrenia and BD have been published. These have produced stronger evidence for association to specific risk loci than have earlier studies, specifically for the zinc finger binding protein 804A (ZNF804A) locus in schizophrenia and for the calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C) and ankyrin 3, node of Ranvier (ANK3) loci in bipolar disorder. The ZNF804A and CACNA1C loci appear to influence risk for both disorders, a finding that supports the hypothesis that schizophrenia and BD are not aetiologically distinct. In the case of schizophrenia, a number of rare copy number variants have also been detected that have fairly large effect sizes on disease risk, and that additionally influence risk of autism, mental retardation, and other neurodevelopmental disorders. The existing findings point to some likely pathophysiological mechanisms but also challenge current concepts of disease classification. They also provide grounds for optimism that larger studies will reveal more about the origins of these disorders, although currently, very little of the genetic risk of either disorder is explained.     

Long PCR amplification of the entire mitochondrial genome from individual helminths for direct sequencing

Exploring mitochondrial (mt) genomes has significant implications for various fundamental research areas, including mt biochemistry and physiology, and, importantly, such genomes provide a rich source of markers for population genetics and systematic studies. Although some progress has been made, there is a paucity of information on mt genomes for many metazoan organisms, particularly invertebrates such as parasitic helminths, which relates mainly to the technical limitations associated with sequencing from tiny amounts of material. In this article, we describe a practical long PCR approach for the amplification and subsequent sequencing of the entire mt genome from individual helminths, which overcomes these limitations. The protocol includes the isolation of genomic DNA, long PCR amplification, electrophoresis and sequencing, and takes approximately 1-3 weeks to carry out. The present user-friendly, cost-effective approach has demonstrated utility to the study of a range of parasites, and has the potential to be applied to a wide range of organisms.     

Neglect of Plasmodium vivax malaria

Plasmodium vivax infects 130-435 million of the 2.6 billion people living at risk of infection. Recent studies suggest that vivax malaria can become lethal in a similar way to severe falciparum malaria. First-line therapies remain unchanged after 50 years. Despite evidence of failing chloroquine efficacy, little work has assessed the problem or explored alternative therapies. Primaquine treatment, the only therapeutic option against relapse, might also be failing. No licensed primary chemoprophylactic agent protects travelers from relapse. Misdiagnosis of species now affects clinical decisions resulting in inadequate therapy for P. falciparum and P. vivax. All of these factors demonstrate the lack of research on P. vivax.     

Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins

Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading-deficient mtDNA polymerase (mtDNA mutator mice) have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.