Transposable elements in yeasts

With the development of new sequencing technologies in the past decade, yeast genomes have been extensively sequenced and their structures investigated. Transposable elements (TEs) are ubiquitous in eukaryotes and constitute a limited part of yeast genomes. However, due to their ability to move in genomes and generate dispersed repeated sequences, they contribute to modeling yeast genomes and thereby induce plasticity. This review assesses the TE contents of yeast genomes investigated so far. Their diversity and abundance at the inter- and intraspecific levels are presented, and their effects on gene expression and genome stability is considered. Recent results concerning TE-host interactions are also analyzed.     

Mitochondrial mutagenesis in yeast: mutagenic specificity of EMS and the effects of RAD9 and REV3 gene products

EMS is capable of inducing point mutations in mitochondrial genomes of yeast. It induces efficiently the mitochondrial suppressor mutation of the mitochondrial ochre mutation oxi 1-V25. The base changes leading to the suppression effect have not been identified. AT----GC base substitutions in mitochondrial genomes are inefficiently induced by EMS. The RAD9 and REV3 gene products participate in EMS mutagenesis in nuclear, as well as mitochondrial genomes of yeast.     

Identification and characterization of a novel testicular germ cell-specific gene Ggnbp1

A novel gene Ggnbp1 was identified during yeast two-hybrid screening of gametogenetin protein 1 (GGN1)-interacting proteins. Ggnbp1 gene was found in mouse, rat, and human genomes but not in sequenced yeast, worms, fly, or fish genomes. Northern blotting analysis revealed that the gene was specifically expressed in the testis but not expressed in the other tissues. In situ hybridization showed that it was testicular germ cell-specific and was specifically expressed in later primary spermatocytes, meiotic cells, and early round spermatids. Western blotting analysis detected a protein of expected size in and only in the testis. By making membrane and cytosolic fractions of germ cells, we were able to show that GGNBP1 associated with the membrane. The identification and characterization of a novel germ cell-specific gene Ggnbp1 is the first step toward the defining of the functions of Ggnbp1 in spermatogenesis.     

A genome phylogeny for mitochondria among alpha-proteobacteria and a predominantly eubacterial ancestry of yeast nuclear genes

Analyses of 55 individual and 31 concatenated protein data sets encoded in Reclinomonas americana and Marchantia polymorpha mitochondrial genomes revealed that current methods for constructing phylogenetic trees are insufficiently sensitive (or artifact-insensitive) to ascertain the sister of mitochondria among the current sample of eight alpha-proteobacterial genomes using mitochondrially-encoded proteins. However, Rhodospirillum rubrum came as close to mitochondria as any alpha-proteobacterium investigated. This prompted a search for methods to directly compare eukaryotic genomes to their prokaryotic counterparts to investigate the origin of the mitochondrion and its host from the standpoint of nuclear genes. We examined pairwise amino acid sequence identity in comparisons of 6,214 nuclear protein-coding genes from Saccharomyces cerevisiae to 177,117 proteins encoded in sequenced genomes from 45 eubacteria and 15 archaebacteria. The results reveal that approximately 75% of yeast genes having homologues among the present prokaryotic sample share greater amino acid sequence identity to eubacterial than to archaebacterial homologues. At high stringency comparisons, only the eubacterial component of the yeast genome is detectable. Our findings indicate that at the levels of overall amino acid sequence identity and gene content, yeast shares a sister-group relationship with eubacteria, not with archaebacteria, in contrast to the current phylogenetic paradigm based on ribosomal RNA. Among eubacteria and archaebacteria, proteobacterial and methanogen genomes, respectively, shared more similarity with the yeast genome than other prokaryotic genomes surveyed.     

Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene.

The ATF1 gene, which encodes alcohol acetyltransferase (AATase), was cloned from Saccharomyces cerevisiae and brewery lager yeast (Saccharomyces uvarum). The nucleotide sequence of the ATF1 gene isolated from S. cerevisiae was determined. The structural gene consists of a 1,575-bp open reading frame that encodes 525 amino acids with a calculated molecular weight of 61,059. Although the yeast AATase is considered a membrane-bound enzyme, the results of a hydrophobicity analysis suggested that this gene product does not have a membrane-spanning region that is significantly hydrophobic. A Southern analysis of the yeast genomes in which the ATF1 gene was used as a probe revealed that S. cerevisiae has one ATF1 gene, while brewery lager yeast has one ATF1 gene and another, homologous gene (Lg-ATF1). Transformants carrying multiple copies of the ATF1 gene or the Lg-ATF1 gene exhibited high AATase activity in static cultures and produced greater concentrations of acetate esters than the control.     

Bacteriophage Mu integration in yeast and mammalian genomes

Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed ‘transpososomes’. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.     

TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution

The structural and functional analysis of mammalian genomes would benefit from the ability to isolate from multiple DNA samples any targeted chromosomal segment that is the size of an average human gene. A cloning technique that is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae satisfies this need. It is a unique tool to selectively recover chromosome segments that are up to 250 kb in length from complex genomes. In addition, TAR cloning can be used to characterize gene function and genome variation, including polymorphic structural rearrangements, mutations and the evolution of gene families, and for long-range haplotyping.     

Detection of gene duplications and block duplications in eukaryotic genomes

Several eukaryotic genomes have been completely sequenced and this provides an opportunity to investigate the extent and characteristics (e.g., single gene duplication, block duplication, etc.) of gene duplication in a genome. Detecting duplicate genes in a genome, however, is not a simple problem because of several complications such as domain shuffling, the existence of isoforms derived from alternative splicing, and annotational errors in the databases. We describe a method for overcoming these difficulties and the extents of gene duplication in the genomes of Drosophila melanogaster, Caenorhabditis elegans, and yeast inferred from this method. We also describe a method for detecting block duplications in a genome. Application of this method showed that block duplication is a common phenomenon in both yeast and nematode. The patterns of block duplication in the two species are, however, markedly different. Yeast shows much more extensive block duplication than nematode, with some chromosomes having more than 40% of the duplications derived from block duplications. Moreover, in yeast the majority of block duplications occurred between chromosomes, while in nematode most block duplications occurred within chromosomes.     

Genomic exploration of the hemiascomycetous yeasts: 20. Evolution of gene redundancy compared to Saccharomyces cerevisiae

We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others.     

Yeast genome sequencing: the power of comparative genomics

For decades, unicellular yeasts have been general models to help understand the eukaryotic cell and also our own biology. Recently, over a dozen yeast genomes have been sequenced, providing the basis to resolve several complex biological questions. Analysis of the novel sequence data has shown that the minimum number of genes from each species that need to be compared to produce a reliable phylogeny is about 20. Yeast has also become an attractive model to study speciation in eukaryotes, especially to understand molecular mechanisms behind the establishment of reproductive isolation. Comparison of closely related species helps in gene annotation and to answer how many genes there really are within the genomes. Analysis of non-coding regions among closely related species has provided an example of how to determine novel gene regulatory sequences, which were previously difficult to analyse because they are short and degenerate and occupy different positions. Comparative genomics helps to understand the origin of yeasts and points out crucial molecular events in yeast evolutionary history, such as whole-genome duplication and horizontal gene transfer(s). In addition, the accumulating sequence data provide the background to use more yeast species in model studies, to combat pathogens and for efficient manipulation of industrial strains.     

Fungal Smn and Spf30 homologues are mainly present in filamentous fungi and genomes with many introns: implications for spinal muscular atrophy

Spinal muscular atrophy is an important rare genetic disease characterized by the loss of motor neurons, where the main gene responsible is smn1. Orthologous genes have only been characterized in a single fungal genome: Schizosaccharomyces pombe. We have searched for putative SMN orthologues in publically available fungal genomes, finding that they are predominately present in filamentous fungi. SMN binding partners and the SPF30 SMN paralogue, which are all involved in mRNA splicing, were found to be present in a similar but non-identical subset of fungal genomes. The Saccharomycces cerevisiae yeast genome contains neither smn1 orthologues nor paralogues and it has been suggested that this might be related to the low number of introns in this yeast. Here we have tested this hypothesis by looking at other fungal genomes. Significantly, we find that fungal genomes with high numbers of introns also possess an SMN orthologue or at least its paralogue, SPF30.     

Microbial genescapes: a prokaryotic view of the yeast genome

We examine the translated open reading frames (ORFs) of the yeast Saccharomyces cerevisiae, focusing on those that have FASTA matches in phyletically defined sets of completely sequenced genomes. On this basis, we identify archaeal yeast, bacterial yeast, universal yeast, and yeast ORFs that do not have a match in any of nine prokaryote genomes. Similarly, we examine the yeast mitochondrial genome and the subset of the yeast nuclear ORFs identified as being involved in mitochondrial biogenesis. For the yeast ORFs that match one or more ORFs in these prokaryote genomes, we examine the phyletic and functional distributions of these matches as a function of match strength. These results provide genome level insights into the origin of the eukaryotic cell and the origin of mitochondria. More generally, they exemplify how the growing database of prokaryote genome sequences can help us understand eukaryote genomes.     

Identification of cryptic lox sites in the yeast genome by selection for Cre-mediated chromosome translocations that confer multiple drug resistance.

The Cre recombinase efficiently causes site-specific DNA recombination at loxP sites placed into the eukaryotic genome. Since the loxP site of phage P1 is 34 base-pairs in size, the natural occurrence of this exact sequence is unlikely in any eukaryotic genome. However, related sequences may exist in eukaryotic genomes that could recombine at low efficiency with an authentic loxP site. This work identifies such cryptic lox sites in the yeast genome using a positive selection procedure that allows the detection of events occurring at a frequency of less than 1 x 10(-7). The selection is based on the disruption/reconstruction of the yeast gene YGL022. Disruption of YGL022 confers multiple drug sensitivity. Recombination events at a loxP site 5' to the structural gene restore expression of YGL022 and result in a multiple drug resistant phenotype. These drug resistant mutants all display chromosomal rearrangements resulting from low-frequency Cre-mediated recombination with an endogenous cryptic lox site. Ten such sites have been found and they have been mapped physically to a number of different yeast chromosomes. Although the efficiency of Cre-mediated recombination between loxP and such endogenous sites is quite low, it may be possible to redesign recombination substrates to improve recombination efficiency. Because of the greater complexity of the human and mouse genomes compared with yeast, an analogous situation is likely to exist in these organisms. The availability of such sites would be quite useful in the development of alternative strategies for gene therapy and in the generation of transgenic animals.     

酿酒酵母基因组学研究进展

酿酒酵母是第一个完成基因组测序的真核生物,在基因组序列信息研究上取得了重大的进展,并且成为后基因组研究的主要模式材料,在基因功能、转录组、蛋白质组等方面获得了许多重要的成果,为高等生物,以及人基因组的研究提供了很好的借鉴,并为深入认识酵母以及生命的进化提供了基本的信息。     

Study of animal viruses in yeast

Yeast is often considered to be a model eukaryotic organism, in a manner analogous to E. coli as a model prokaryotic organism. Yeast has been extensively characterized and the genomes completely sequenced. Despite the small genome size, yeast displays most of features of higher eukaryotes. The facts that most of cellular machinery is conserved among different eukaryotes and that the powerful technologies of genetics and molecular biology are available have made yeast model eukaryotic cells in biological and biomedical sciences including virology. Cumulative data indicate that yeast can be a host for animal viruses. I briefly describe yeast gene expression and review viral replication in yeast. Great discovery include complete replication of animal viruses and production of virus-like particle vaccines in yeast. Current studies on yeast focus on identification of host factors and machinery used for viral replication. The studies are based on traditional yeast genetics and genome-wide identification using a complete set of yeast deletion strains.     

Mitochondrial fusion and inheritance of the mitochondrial genome

Although maternal or uniparental inheritance of mitochondrial genomes is a general rule, biparental inheritance is sometimes observed in protists and fungi, including yeasts. In yeast, recombination occurs between the mitochondrial genomes inherited from both parents. Mitochondrial fusion observed in yeast zygotes is thought to set up a space for DNA recombination. In the last decade, a universal mitochondrial fusion mechanism has been uncovered, using yeast as a model. On the other hand, an alternative mitochondrial fusion mechanism has been identified in the true slime mold Physarum polycephalum. A specific mitochondrial plasmid, mF, has been detected as the genetic material that causes mitochondrial fusion in P. polycephalum. Without mF, fusion of the mitochondria is not observed throughout the life cycle, suggesting that Physarum has no constitutive mitochondrial fusion mechanism. Conversely, mitochondria fuse in zygotes and during sporulation with mF. The complete mF sequence suggests that one gene, ORF640, encodes a fusogen for Physarum mitochondria. Although in general, mitochondria are inherited uniparentally, biparental inheritance occurs with specific sexual crossing in P. polycephalum. An analysis of the transmission of mitochondrial genomes has shown that recombinations between two parental mitochondrial genomes require mitochondrial fusion, mediated by mF. Physarum is a unique organism for studying mitochondrial fusion.