Rearranged mitochondrial genes in the yeast nuclear genome

We have found a contiguous DNA sequence in the yeast nuclear genome with extensive homology to non-contiguous yeast mitochondrial DNA sequences. Closely linked to this nuclear sequence in some, but not all, yeast strains is a tandem pair of transposable (Ty) elements. Certain features of the content and organization of this nuclear DNA sequence suggest that it may have originated from petite mitochondrial DNA which integrated into the nuclear genome.     

Expression of a bacterial modification methylase gene in yeast

Methylation of specific cytosines in the DNA is generally believed to play some role in the regulation of gene expression in eukaryotes. However, some eukaryotes, such as Drosophila and yeast (S. Hattman, personal communication) seem not to contain 5-methylcytosine in their DNA. It would be interesting to test, how gene expression in such organisms would respond to the methylation of specific cytosines in the genome. As a first step towards this goal, we have introduced the gene encoding the Bacillus sphaericus R modification methylase, which methylates the internal cytosine within the recognition sequence 5'-GGCC, into yeast cells. Southern-type hybridization to DNAs isolated from the transformed yeast clones revealed that the yeast plasmid carrying the prokaryotic methylase gene, as well as the two chromosomal genes tested (his3 and leu2) were methylated, whereas the bulk of the yeast DNA remained largely unmethylated. This indicates that the Bacillus sphaericus modification methylase was expressed in yeast but it modified only certain parts of the yeast DNA.     

Genome sequencing and analysis of Aspergillus oryzae

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.     

Mitochondrial DNA repairs double-strand breaks in yeast chromosomes

The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.     

线粒体假基因--核基因组中的分子化石

核基因组中线粒体DNA片段是线粒体基因向核基因组转移造成的．这些线粒体来源的核DNA片段都是不能表达的假基因，这种序列容易被通用引物从总DNA模板中优先扩增出来，它的存在必然给mtDNA的应用带来负面影响．总结了脊椎动物线粒体假基因的特点，结合笔在GenBank上发现的登录为mtDNA而实际为假基因的序列提出假基因存在的普遍性，分析这种序列对mtDNA在人类线粒体病理学、脊椎动物系统进化研究等方面应用的负面影响．对假基因在线粒体和细胞核两基因组进化研究以及物种系统发育学研究中的应用前景进行展望．     

Construction of a bacterial artificial chromosome library of S-type CMS maize mitochondria

In order to isolate mitochondrial genes easily, we have developed a new method to construct S-type CMS maize mitochondrial gene library by means of embedding mitochondria and enzymatic digesting mitochondriain situ, preparing mtDNA by electrophoresis, digesting LMP agarose with β-agarase, using BAC vector and electroporation. About 2 500 white clones of Mo17 CMS-J mitochondrial gene library were obtained with the average size of 18.24 kb, ranging from 5 to 40 kb, 63.6% inserts came from mitochondrial genome and represented 48 × mitochondrial genome equivalents. All the probes had detected the positive clones in the gene library. It is helpful to elucidating the maize mitochondrial genome structure and mechanism of S-type CMS, and may give some valuable reference to the construction of other plant mitochondrial genome library.     

The genome sequence of Schizosaccharomyces pombe

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.     

Widespread horizontal transfer of mitochondrial genes in flowering plants

Horizontal gene transfer--the exchange of genes across mating barriers--is recognized as a major force in bacterial evolution. However, in eukaryotes it is prevalent only in certain phagotrophic protists and limited largely to the ancient acquisition of bacterial genes. Although the human genome was initially reported to contain over 100 genes acquired during vertebrate evolution from bacteria, this claim was immediately and repeatedly rebutted. Moreover, horizontal transfer is unknown within the evolution of animals, plants and fungi except in the special context of mobile genetic elements. Here we show, however, that standard mitochondrial genes, encoding ribosomal and respiratory proteins, are subject to evolutionarily frequent horizontal transfer between distantly related flowering plants. These transfers have created a variety of genomic outcomes, including gene duplication, recapture of genes lost through transfer to the nucleus, and chimaeric, half-monocot, half-dicot genes. These results imply the existence of mechanisms for the delivery of DNA between unrelated plants, indicate that horizontal transfer is also a force in plant nuclear genomes, and are discussed in the contexts of plant molecular phylogeny and genetically modified plants.     

Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene

The structure equivalent to higher eukaryotic centrosomes in fission yeast, the nuclear membrane-bound spindle pole body, is inactive during interphase. On transition from G2 to M phase of the cell cycle, the spindle pole body duplicates; the daughter pole bodies seed microtubules which interdigitate to form a short spindle that elongates to span the nucleus at metaphase. We have identified two loci which, when mutated, block spindle formation. The predicted product of one of these genes, cut7+, contains an amino-terminal domain similar to the kinesin heavy chain head domain, indicating that the cut7+ product could be a spindle motor. The cut7+ gene resembles the Aspergillus nidulans putative spindle motor gene bimC, both in terms of its organization with a homologous amino-terminal head and no obvious heptad repeats and in the morphology of the mutant phenotype. But we find no similarity between the carboxy termini of these genes, suggested that either the cut7+ gene represents a new class of kinesin genes and that fission yeast may in addition contain a bimC homologue, or that the carboxy termini of these mitotic kinesins are not evolutionarily conserved and that the cut7+ gene belongs to a subgroup of bimC-related kinesins.     

The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein

Ubiquitin, a 76-residue protein, occurs in cells either free or covalently joined to a variety of protein species, from chromosomal histones to cytoplasmic proteins. Conjugation of ubiquitin to proteolytic substrates is essential for the selective degradation of intracellular proteins in higher eukaryotes. We show here that a protein homologous to human ubiquitin exists in the yeast Saccharomyces cerevisiae, and that yeast extracts conjugate human ubiquitin to a variety of endogenous proteins in an ATP-dependent reaction. We have isolated the S. cerevisiae ubiquitin gene and found it to contain six consecutive ubiquitin-coding repeats in a found it to contain six consecutive ubiquitin-coding repeats in a head-to-tail arrangement. This apparently unique gene organization suggests that yeast ubiquitin is generated by processing of a precursor protein in which several exact repeats of the ubiquitin amino acid sequence are joined directly via Gly-Met peptide bonds between the last and first residues of mature ubiquitin, respectively. Ubiquitin-coding yeast DNA repeats are restricted to a single genomic locus; although the sequenced repeats differ in up to 27 of 228 bases per repeat, they encode identical amino acid sequences. As this predicted amino acid sequence differs in only 3 of 76 residues from that of ubiquitin in higher eukaryotes, ubiquitin is apparently the most conserved of known proteins.     

中国猛禽类线粒体DNA遗传多态性研究进展

鸟类线粒体ＤＮＡ的研究在种群生物学和进化生物学研究方面越来越显示出重要的作用,特别是在遗传多态性和基因流研究方面更具特殊意义。简要回顾了鸟类线粒体ＤＮＡ的研究历史,并分析了中国猛离线粒体ＤＮＡ遗传多态性的研究现状及进展,要点：１．猛禽类线粒体基因组大小存在遗传多态性,２：猛禽类线粒体ＤＮＡ的进化速率与哺乳类相同,３;种间或种内存在丰富的遗传变异;４．不同地理种群存在ｍｔＤＮＡ克隆群的连续性。     

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi.

Microsporidia are obligate intracellular parasites infesting many animal groups. Lacking mitochondria and peroxysomes, these unicellular eukaryotes were first considered a deeply branching protist lineage that diverged before the endosymbiotic event that led to mitochondria. The discovery of a gene for a mitochondrial-type chaperone combined with molecular phylogenetic data later implied that microsporidia are atypical fungi that lost mitochondria during evolution. Here we report the DNA sequences of the 11 chromosomes of the approximately 2.9-megabase (Mb) genome of Encephalitozoon cuniculi (1,997 potential protein-coding genes). Genome compaction is reflected by reduced intergenic spacers and by the shortness of most putative proteins relative to their eukaryote orthologues. The strong host dependence is illustrated by the lack of genes for some biosynthetic pathways and for the tricarboxylic acid cycle. Phylogenetic analysis lends substantial credit to the fungal affiliation of microsporidia. Because the E. cuniculi genome contains genes related to some mitochondrial functions (for example, Fe-S cluster assembly), we hypothesize that microsporidia have retained a mitochondrion-derived organelle.     

Large-scale analysis of the yeast genome by transposon tagging and gene disruption

Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.     

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.     

Analysis of the genome sequence of the flowering plant Arabidopsis thaliana

The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans--the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement.     

Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum

The origin of all mitochondria can be traced to the symbiotic arrangement that resulted in the emergence of eukaryotes in a world that was exclusively populated by prokaryotes. This arrangement, however, has been in continuous genetic flux: the varying degrees of gene loss and transfer from the mitochondrial genome in different eukaryotic lineages seem to signify an ongoing 'conflict' between the host and the symbiont. Eukaryotic parasites belonging to the phylum Apicomplexa provide an excellent example to support this view. These organisms contain the smallest mitochondrial genomes known, with an organization that differs among various genera; one genus, Cryptosporidium, seems to have lost the entire mitochondrial genome. Here we show that erythrocytic stages of the human malaria parasite Plasmodium falciparum seem to maintain an active mitochondrial electron transport chain to serve just one metabolic function: regeneration of ubiquinone required as the electron acceptor for dihydroorotate dehydrogenase, an essential enzyme for pyrimidine biosynthesis. Transgenic P. falciparum parasites expressing Saccharomyces cerevisiae dihydroorotate dehydrogenase, which does not require ubiquinone as an electron acceptor, were completely resistant to inhibitors of mitochondrial electron transport. Maintenance of mitochondrial membrane potential, however, was essential in these parasites, as indicated by their hypersensitivity to proguanil, a drug that collapsed the membrane potential in the presence of electron transport inhibitors. Thus, acquisition of just one enzyme can render mitochondrial electron transport nonessential in erythrocytic stages of P. falciparum.     

Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies

In vitro studies of muscle mitochondrial metabolism in patients with mitochondrial myopathy have identified a variety of functional defects of the mitochondrial respiratory chain, predominantly affecting complex I (NADH-CoQ reductase) or complex III (ubiquinol-cytochrome c reductase) in adult cases. These two enzymes consist of approximately 36 subunits, eight of which are encoded by mitochondrial DNA (mtDNA). The increased incidence of maternal, as opposed to paternal, transmission in familial mitochondrial myopathy suggests that these disorders may be caused by mutations of mtDNA. Multiple restriction endonuclease analysis of leukocyte mtDNA from patients with the disease, and their relatives, showed no differences in cleavage patterns between affected and unaffected individuals in any single maternal line. When muscle mtDNA was studied, nine of 25 patients were found to have two populations of muscle mtDNA, one of which had deletions of up to 7 kilobases in length. These observations demonstrate that mtDNA heteroplasmy can occur in man and that human disease may be associated with defects of the mitochondrial genome.     

Frequent mtDNA mutations and its role in gastric carcinogenesis

In order to disclose the relationship between mutations of mitochondrial DNA (mtDNA) and gastric carcinogenesis, we screened the entire mtDNA sequence in 30 cases of human gastric cancer and matched normal tissues by using denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. Our data showed that high frequency (66.7%, 20/30) of mitochondrial genome mutation occurred in gastric cancer. Among these variants, 17 cases (56.7%, 17/30) were identified to be somatic mutation. High level mutant frequency was found in ND4, ND5 coding genes and D-loop control region, which was 36.7%, 26.7% and 30% respectively. Comparing with complexes Ⅲ, Ⅳ and Ⅴof the electron transport chain, we found that variants appeared to be more frequent in the subunit genes of complexⅠ. Most of mutations were base substitutions (85.4%, 41/48). Our results suggested that mutations of subunit genes encoding complexⅠ, especially ND3, ND4 and ND5 genes, might contribute to human gastric carcinogenesis.     

An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region

Deletions of muscle mitochondrial DNA (mtDNA) have recently been found in patients with mitochondrial myopathy. However, as most of the described cases were sporadic, and individual deletions involved different portions of mtDNA, the mechanism(s) producing the molecular lesions, as well as their mode of transmission, remain unclear. By studying families with mtDNA heteroplasmy, valuable information can be obtained about the role of inheritable factors in the pathogenesis of these disorders. We have studied four members of a family with autosomal dominant mitochondrial myopathy. Multiple deletions, involving the same portion of muscle mtDNA, were identified in all patients. Sequence analysis of the mutant mtDNAs, performed after DNA amplification by the polymerase-chain reaction showed that all the deletions start within a 12-nucleotide stretch at the 5' end of the D-loop region, a site of active communication between the nucleus and the mtDNA. The data indicate that a mutation of a nuclear-coded protein can destroy the integrity of the mitochondrial genome in a specific, heritable way.