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Reinvestigation of the Saccharomyces cerevisiae genome annotation by comparison to the genome of a related fungus: Ashbya gossypii.

BACKGROUND: The recently sequenced genome of the filamentous fungus Ashbya gossypii revealed remarkable similarities to that of the budding yeast Saccharomyces cerevisiae both at the level of homology and synteny (conservation of gene order). Thus, it became possible to reinvestigate the S. cerevisiae genome in the syntenic regions leading to an improved annotation. RESULTS: We have identified 23 novel S. cerevisiae open reading frames (ORFs) as syntenic homologs of A. gossypii genes; for all but one, homologs are present in other eukaryotes including humans. Other comparisons identified 13 overlooked introns and suggested 69 potential sequence corrections resulting in ORF extensions or ORF fusions with improved homology to the syntenic A. gossypii homologs. Of the proposed corrections, 25 were tested and confirmed by resequencing. In addition, homologs of nearly 1,000 S. cerevisiae ORFs, presently annotated as hypothetical, were found in A. gossypii at syntenic positions and can therefore be considered as authentic genes. Finally, we suggest that over 400 S. cerevisiae ORFs that overlap other ORFs in S. cerevisiae and for which no homolog can be detected in A. gossypii should be regarded as spurious. CONCLUSIONS: Although, the S. cerevisiae genome is rightly considered as one of the most accurately sequenced and annotated eukaryotic genomes, we have shown that it still benefits substantially from comparison to the completed sequence and syntenic gene map of A. gossypii, an evolutionarily related fungus. This type of approach will strongly support the annotation of more complex genomes such as the human and murine genomes.

Amino Acid Sequence↗

The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081.

The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B) and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common themes in the genome evolution of other human enteropathogens.

Evolution, Molecular↗

Genome projects and the functional-genomic era.

The problems we face today in public health as a result of the -- fortunately -- increasing age of people and the requirements of developing countries create an urgent need for new and innovative approaches in medicine and in agronomics. Genomic and functional genomic approaches have a great potential to at least partially solve these problems in the future. Important progress has been made by procedures to decode genomic information of humans, but also of other key organisms. The basic comprehension of genomic information (and its transfer) should now give us the possibility to pursue the next important step in life science eventually leading to a basic understanding of biological information flow; the elucidation of the function of all genes and correlative products encoded in the genome, as well as the discovery of their interactions in a molecular context and the response to environmental factors. As a result of the sequencing projects, we are now able to ask important questions about sequence variation and can start to comprehensively study the function of expressed genes on different levels such as RNA, protein or the cell in a systematic context including underlying networks. In this article we review and comment on current trends in large-scale systematic biological research. A particular emphasis is put on technology developments that can provide means to accomplish the tasks of future lines of functional genomics.

Animals↗

One-dimensional genome scanning: identification of the basis of a mouse mutation and identification of genomic changes in ovarian carcinoma.

We have developed a simple one-dimensional electrophoretic method, genome scanning, that can be used to identify large-scale genomic differences between two or more DNA samples. Genome scanning is especially useful in the detection of genetic amplifications, deletions, and rearrangements. The assay is essentially a high-resolution Southern analysis, comparing equivalent amounts of genomic DNA samples that are variant for a given trait. The Southern blots are hybridized to a probe sequence derived from a medium copy number repetitive element (1000-2000 copies per haploid genome) naturally dispersed throughout the genome. The hybridization pattern that results is complex and consists of hundreds of bands. If the DNA samples are otherwise equivalent, a net difference in hybridization intensity between homologous bands of different samples indicates a genetic change. In this report, we discuss the origin of the method, its premise, and review its application to mouse mutational analysis and to human cancer research (a more detailed discussion of the theory is presented elsewhere in this issue; Y. Gondo and M. H. Brilliant, Electrophoresis 1995, 16, 174-178).

Animals↗

Genomic mismatch scanning identifies human genomic DNA shared identical by descent.

Genomic mismatch scanning (GMS) is a high-throughput, high-resolution identity by descent mapping technique that enriches for genomic DNA fragments that are shared between related individuals. In GMS, DNA heteroduplexes are formed from restriction-digested genomic DNA fragments from two relatives. Mismatch-free DNA heteroduplexes, likely representing DNA shared identical by descent between the two individuals, are relatively purified by depleting the mismatch-containing heteroduplexes using the Escherichia coli mismatch repair proteins and exonuclease. Here, we demonstrate using quantitative microsatellite genotyping that, despite the complexity of the human genome, GMS can enrich the majority of restriction fragments that are identical by descent between two related humans. As the entire genome is selected in GMS, an extraordinarily dense set of markers (up to 200,000 markers) may be screened in parallel. The demonstration of the molecular enrichment of identical DNA fragments in the context of the whole human genome establishes conditions for the application of GMS to human genetics. This forms a frame-work for the further development of GMS as a hybridization-based mapping technique that utilizes DNA microarray technology to map the selected identical by descent DNA fragments.

DNA↗

Comparative analysis of human genome assemblies reveals genome-level differences.

Previous comparative analysis has revealed a significant disparity between the predicted gene sets produced by the International Human Genome Sequencing Consortium (HGSC) and Celera Genomics. To determine whether the source of this discrepancy was due to underlying differences in the genomic sequences or different gene prediction methodologies, we analyzed both genome assemblies in parallel. Using the GENSCAN gene prediction algorithm, we generated predicted transcriptomes that could be directly compared. BLAST-based comparisons revealed a 20-30% difference between the transcriptomes. Further differences between the two genomes were revealed with protein domain PFAM analyses. These results suggest that fundamental differences between the two genome assemblies are likely responsible for a significant portion of the discrepancy between the transcript sets predicted by the two groups.

Chromosome Mapping↗

Tomato spotted wilt Tospovirus genome reassortment and genome segment-specific adaptation.

A system to associate specific genome segments with viral phenotypes and to study factors influencing genome reassortment was developed for tomato spotted wilt Tospovirus (TSWV). Reassortant isolates were generated by co-inoculating a TSWV isolate, TSWV-D, with TSWV-10 or TSWV-MD. The parental origin of each genome segment in putative reassortant isolates was determined by segment-specific restriction fragment length polymorphisms. The TSWV isolates readily exchanged genome segments in a nonrandom fashion. The S RNA from TSWV-D was dominant over the S RNA from TSWV-10. The intergenic region (IGR) of the S RNA was correlated with competitiveness of this genome segment in reassortant isolates. The less competitive S RNA contained a net increase of 62 nt, including a 33-nt duplication in the IGR. This duplicate sequence was highly conserved among isolates from the southeastern United States and an isolate from Bulgaria. The evidence supports the hypothesis that the IGR of the S RNA with an ambisense coding strategy serves a regulatory function which influences the occurrence of this segment in the viral population. In addition, it was demonstrated that stable parental phenotypes can be mapped to specific genome segments as well as generating novel phenotypes not associated with either parent.

Adaptation, Biological↗

The complete genome sequence of Perina nuda picorna-like virus, an insect-infecting RNA virus with a genome organization similar to that of the mammalian picornaviruses.

Perina nuda picorna-like virus (PnPV) is an insect-infecting RNA virus with morphological and physicochemical characters similar to the Picornaviridae. In this article, we determine the complete genome sequence and analyze the gene organization of PnPV. The genome of PnPV consists of 9476 nucleotides (nts) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 8958 nts (2986 codons) flanked by 473 and 45 nt noncoding regions on the 5' and 3' ends, respectively. Northern blotting did not detect the presence of any subgenomic RNA. The PnPV genome codes for four structural proteins (CP1-4), and determination of their N-terminal sequences by Edman degradation, showed that all four are located in the 5' region of the genome. The 3' part of the PnPV genome contains the consensus sequence motifs for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) in that order from the 5' to the 3' end. In all of these characters, the genome organization of PnPV resembles the mammalian picornaviruses and two other insect picorna-like viruses, infectious flacherie virus (IFV) of the silkworm and Sacbrood virus (SBV) of the honeybee. In a phylogenetic tree based on the eight conserved domains in the RdRp sequence, PnPV formed a separate cluster with IFV and SBV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses.

Amino Acid Sequence↗

Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: module evolution of the filamentous phage genome.

The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E. coli phages Ff(M13, fl, or fd) and IKe. The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff. Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff. The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe. Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff. No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes. Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism.

Amino Acid Sequence↗

An infectious cDNA copy of the genome of a non-cardiovirulent coxsackievirus B3 strain: its complete sequence analysis and comparison to the genomes of cardiovirulent coxsackieviruses.

The genome of the non-cardiovirulent coxsackievirus B3 (CVB3) strain CVB3/0 was cloned and sequenced to aid in the elucidation of the viral genetic basis for the CVB3 cardiovirulent phenotype. Reverse-transcribed sub-genomic complementary DNA (cDNA) fragments were enzymatically amplified using generic oligonucleotide primers and were assembled as a complete infectious genomic copy (pCVB3-0) downstream of the T7 RNA polymerase promoter. Positive-strand viral RNA transcribed from pCVB3-0 using T7 RNA polymerase and transfected into HeLa cells produced infectious virus (CVB3/0c). No differences in phenotype were observed comparing growth of CVB3/0c to the parental CVB3/0 in HeLa single-step growth curves, virus yields, or plaque size. When inoculated into C3H/HeJ mice, CVB3/0c achieved cardiac titers equivalent to the parental CVB3/0 and like the parental virus, demonstrated a non-cardiovirulent phenotype. The nucleotide sequence of the cloned CVB3/0 genome was determined and compared to the genomes of infectious cDNA clones of cardiovirulent CVB3 strains. Two consistent differences among nucleotides in non-translated regions and 8 amino acid differences relative to two well-characterized infectious cDNA copies of genomes from cardiovirulent CVB3 strains were identified.

Animals↗

[Public Health Genomics. The integration of genome-based knowledge into public health research, policies and health services].

Which consequences can be drawn from genome-based knowledge and how can it be responsibly and timely translated into policies and practice? What are recent developments in genetics and molecular biology, what are the challenges, what are the risks of these developments? Which policies can provide an acceptable balance between providing strong protection of individuals'interests and needs while enabling society to benefit from the genomic advances and empowering individuals? How can molecular medicine contribute to more effective and efficient health care services, and what infrastructures and policies can already now be implemented to assure a benefit for population health? Thus, Public Health Genomics (PHG) tries to answer these challenging questions. This integration of genomics into the aims of public health is called Public Health Genomics (PHG) and is defined as "the responsible and effective translation of genome-based knowledge and technologies into public policy and health services for the benefit of population health".

Delivery of Health Care, Integrated↗

Complete chloroplast genome sequences of Solanum bulbocastanum, Solanum lycopersicum and comparative analyses with other Solanaceae genomes.

Despite the agricultural importance of both potato and tomato, very little is known about their chloroplast genomes. Analysis of the complete sequences of tomato, potato, tobacco, and Atropa chloroplast genomes reveals significant insertions and deletions within certain coding regions or regulatory sequences (e.g., deletion of repeated sequences within 16S rRNA, ycf2 or ribosomal binding sites in ycf2). RNA, photosynthesis, and atp synthase genes are the least divergent and the most divergent genes are clpP, cemA, ccsA, and matK. Repeat analyses identified 33-45 direct and inverted repeats >or=30 bp with a sequence identity of at least 90%; all but five of the repeats shared by all four Solanaceae genomes are located in the same genes or intergenic regions, suggesting a functional role. A comprehensive genome-wide analysis of all coding sequences and intergenic spacer regions was done for the first time in chloroplast genomes. Only four spacer regions are fully conserved (100% sequence identity) among all genomes; deletions or insertions within some intergenic spacer regions result in less than 25% sequence identity, underscoring the importance of choosing appropriate intergenic spacers for plastid transformation and providing valuable new information for phylogenetic utility of the chloroplast intergenic spacer regions. Comparison of coding sequences with expressed sequence tags showed considerable amount of variation, resulting in amino acid changes; none of the C-to-U conversions observed in potato and tomato were conserved in tobacco and Atropa. It is possible that there has been a loss of conserved editing sites in potato and tomato.

Base Sequence↗

Heteroplasmy suggests paternal co-transmission of multiple genomes and pervasive reversion of maternally into paternally transmitted genomes of mussel (Mytilus) mitochondrial DNA.

Marine mussels of the genus Mytilus have two types of mitochondrial DNA with separate paternal and maternal inheritance. Females are homoplasmic for an F genome that is transmitted to all offspring, whereas males are heteroplasmic for this F genome and for a highly diverged (> 20%) M genome that is transmitted only to sons. Here we provide phylogenetic evidence based on lrRNA sequence data that most of the paternal genomes in European M. trossulus have an introgressive female M. edulis origin and are nearly indistinguishable in sequence from F types of M. trossulus. This observation is best explained by the hypothesis that introgressed F type molecules have recently invaded the paternal route and have assumed the role of M molecules, then resetting to zero the time of sequence divergence between M and F lineages. European M. trossulus shows a high prevalence of males heteroplasmic for three different mitochondrial DNA types all having the same two paternal types and the same maternal type, consistent with paternal co-transmission of multiple genomes. Co-transmission of the same genomes must apparently operate uninterruptedly for several generations in spite of the very different evolutionary origin of the specific molecules that are transmitted paternally and maternally in European M. trossulus.

Animals↗

"Word" preference in the genomic text and genome evolution: different modes of n-tuplet usage in coding and noncoding sequences.

Extensive work on n-tuplet occurrence in genomic sequences has revealed the correlation of their usage with sequence origin. Parallel to that, there exist different restrictions in the nucleotide composition of coding and noncoding sequences that may result in distinct modes of usage of n-tuplets. The relatively simple approaches described herein focus on such differences. They are based on simple summation measures of n-tuplet frequencies, computed after filtering the background nucleotide composition. Among the main targets of this work is to draw some conclusions on the qualitative differences in the composition of genomic sequences depending on their functionality. Moreover, an evolutionary model is formulated, including simple forms of ubiquitous events of genome dynamics: genomic fusions, genome shuffling due to transpositions, replication slippage, and point mutations. This model is shown to be able to reproduce all the statistical features of genomic sequences discussed herein.

Base Sequence↗

From genetical genomics to systems genetics: potential applications in quantitative genomics and animal breeding.

This article reviews methods of integration of transcriptomics (and equally proteomics and metabolomics), genetics, and genomics in the form of systems genetics into existing genome analyses and their potential use in animal breeding and quantitative genomic modeling of complex traits. Genetical genomics or the expression quantitative trait loci (eQTL) mapping method and key findings in this research are reviewed. Various procedures and potential uses of eQTL mapping, global linkage clustering, and systems genetics are illustrated using actual analysis on recombinant inbred lines of mice with data on gene expression (for diabetes- and obesity-related genes), pathway, and single nucleotide polymorphism (SNP) linkage maps. Experimental and bioinformatics difficulties and possible solutions are discussed. The main uses of this systems genetics approach in quantitative genomics were shown to be in refinement of the identified QTL, candidate gene and SNP discovery, understanding gene-environment and gene-gene interactions, detection of candidate regulator genes/eQTL, discriminating multiple QTL/eQTL, and detection of pleiotropic QTL/eQTL, in addition to its use in reconstructing regulatory networks. The potential uses in animal breeding are direct selection on heritable gene expression measures, termed "expression assisted selection," and genetical genomic selection of both QTL and eQTL based on breeding values of the respective genes, termed "expression-assisted evaluation."

Animals↗

Genomic characterization of Rim2/Hipa elements reveals a CACTA-like transposon superfamily with unique features in the rice genome.

The availability of huge amounts of rice genome sequence now permits large-scale analysis of the structure and molecular characteristics of the previously identified transposase-encoding Rim2 (also called Hipa) element, which is transcriptionally activated by infection with the fungal pathogen Magnaporthe grisea and by treatment with the corresponding fungal elicitor. Based on genomic cloning and data mining from 230 Mb of rice genome sequence, 347 Rim2 elements, with an average size of 5.8 kb, were identified. This indicates that an estimated total of 600-700 Rim2 elements are present in the whole genome. Rim2 insertions occur non-randomly on the chromosomes, as visualized by fluorescence in situ hybridization. The elements harbor 16-bp terminal inverted repeats with the core sequence CACTG, 16-bp sub-terminal repeats, internal variable regions, 3-bp target sequence duplications in the flanking regions, and genes coding for Rim2 proteins (the putative transposase) and hydroxyproline-rich glycoproteins. High levels of insertion into genic regions are observed for members of this family, and the transposition history of the family can be deduced from the high level of shared sequences and analysis of repeat target sites of the elements. Phylogenetic analysis indicates that the putative RIM2 proteins fall into a subgroup distinct from the TNP2-like subgroup of transposases. Southern hybridization with genomic DNA from monocotyledonous and dicotyledonous plants demonstrates that the RIM2-coding sequence is unique to the Oryza genome. Our results demonstrate that the Rim2 elements from rice belong to a distinct superfamily of CACTA-like elements with evolutionary diversity.

Amino Acid Sequence↗

Non-methylated Genomic Sites Coincidence Cloning (NGSCC): an approach to large scale analysis of hypomethylated CpG patterns at predetermined genomic loci.

We have developed a new approach to the analysis of hypomethylated CpG patterns within predetermined, megabase long, genome regions. The approach, which we term Non-methylated Genomic Sites Coincidence Cloning (NGSCC), includes three main steps. First, total genomic DNA is digested with a methylation sensitive restriction endonuclease, such as HpaII or HhaI. Then the fragments corresponding to the genomic area of interest are selected. To this end the fragmented genome DNA is hybridized with a mixture of clones (BACs, cosmids etc.) representing a given region and digested with the same restriction enzyme(s). A special version of the coincidence cloning procedure was developed to make this hybridization selection highly efficient and specific. Finally, fragments of the locus under study are mapped and sequenced. The technique proved to be efficient and specific. As a test, it was applied to the analysis of hypomethylated CpG patterns along the 1-Mb D19S208-COX7A1 (Chr 19q13.12) locus, on human chromosome 19, in normal testis and in seminoma tissues. Some differences in the distribution of hypomethylated CpGs between the two tissues were demonstrated. The methylation profiles in both tissues revealed a clear trend to clustering of non-methylated sites. We also analyzed the expression of genes located within hypomethylated clusters in both tissues. It was shown that, whereas the expression of some of the genes investigated was correlated with hypomethylation of the region, other genes were expressed regardless of their methylation status. NGSCC thus promises to be a useful approach for the analysis of the role of dynamic epigenetic factors in genome function.

Base Sequence↗

Gradual genome stabilisation by progressive reduction of the Saccharomyces uvarum genome in an interspecific hybrid with Saccharomyces cerevisiae.

Considerable amounts of molecular and genetic data indicate that interspecific hybridisation may not be rare among natural strains of Saccharomyces sensu stricto. Although a post-zygotic barrier operating during meiosis usually prevents the production of viable spores, stable hybrids can arise which can even evolve into distinct species. This study was aimed to analyse the genome of a fertile Saccharomyces cerevisiae x S. uvarum hybrid and monitor its changes over four filial generations of viable spores. The molecular genetic analysis demonstrated that the two species did not contribute equally to the formation and stabilisation of the hybrid genome. S. cerevisiae provided the mitochondrial DNA and the more stable part of the nuclear genome. The S. uvarum part of the hybrid nuclear genome became progressively smaller by loosing complete chromosomes and genetic markers in the course of successive meiotic divisions. Certain S. uvarum chromosomes were eliminated and/or underwent rearrangements in interactions with S. cerevisiae chromosomes. Numerous S. uvarum chromosomes acquired S. cerevisiae telomere sequences. The gradual elimination of large parts of the S. uvarum genome was associated with a progressive increase of sporulation efficiency. We hypothesise that this sort of genomic alterations may contribute to speciation in Saccharomyces sensu stricto.

Chromosomes, Fungal↗