Exploiting the complete yeast genome sequence

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.     

Experimental annotation of the human pathogen <Emphasis Type="Italic">Candida albicans</Emphasis> coding and noncoding transcribed regions using high-resolution tiling arrays

Background  

Compared to other model organisms and despite the clinical relevance of the pathogenic yeast Candida albicans, no comprehensive analysis has been done to provide experimental support of its in silico-based genome annotation.     

Mutation processes at the protein level: is Lamarck back?

The experimental evidence accumulated for the last half of the century clearly suggests that inherited variation is not restricted to the changes in genomic sequences. The prion model, originally based on unusual transmission of certain neurodegenerative diseases in mammals, provides a molecular mechanism for the template-like reproduction of alternative protein conformations. Recent data extend this model to protein-based genetic elements in yeast and other fungi. Reproduction and transmission of yeast protein-based genetic elements is controlled by the "prion replication" machinery of the cell, composed of the protein helpers responsible for the processes of assembly and disassembly of protein structures and multiprotein complexes. Among these, the stress-related chaperones of Hsp100 and Hsp70 groups play an important role. Alterations of levels or activity of these proteins result in "mutator" or "antimutator" affects in regard to protein-based genetic elements. "Protein mutagens" have also been identified that affect formation and/or propagation of the alternative protein conformations. Prion-forming abilities appear to be conserved in evolution, despite the divergence of the corresponding amino acid sequences. Moreover, a wide variety of proteins of different origins appear to possess the ability to form amyloid-like aggregates, that in certain conditions might potentially result in prion-like switches. This suggests a possible mechanism for the inheritance of acquired traits, postulated in the Lamarckian theory of evolution. The prion model also puts in doubt the notion that cloned animals are genetically identical to their genome donors, and suggests that genome sequence would not provide a complete information about the genetic makeup of an organism.     

101 Exploring the 3D spatial organization of the genome

Knowledge about the 3D organization of the genome will offer great insights into how cells retrieve and process the genetic information. Knowing the spatial probability distributions of individual genes will provide insights into gene regulatory and replication processes, and fill in the missing links between epigenomics, functional genomics, and structural biology. We will discuss an approach to determine 3D genome structures and structure–function maps of genomes by integrating divers types of data. To address the challenge of modeling highly variable genome structures, we discuss a population-based modeling approach, where we construct a large population of 3D genome structures that together are entirely consistent with all available experimental data including data from genome-wide chromosome conformation capture and imaging experiments. We interpret the result in terms of probabilities of a sample drawn from a population of heterogeneous structures. We will discuss results on the 3D spatial organization of genomes in human lymphoblastoid cells and budding yeast.     

Speciation through cytonuclear incompatibility: Insights from yeast and implications for higher eukaryotes

Several features of the yeast mitochondrial genome, including high mutation rate, dynamic genomic structure, small effective population size, and dispensability for cellular viability, make it a promising candidate for generating hybrid incompatibility and driving speciation. Cytonuclear incompatibility, a specific type of Dobzhansky‐Muller genetic incompatibility caused by improper interactions between mitochondrial and nuclear genomes, has previously been observed in a variety of organisms, yet its role in speciation remains obscure. Recent studies in Saccharomyces yeast species provide a new insight, with experimental evidence that cytonuclear incompatibility and DNA sequence divergence are both causes of the reproductive isolation of different yeast species. Interestingly, these two mechanisms seem to be perfectly complementary to each other in terms of their effects and evolutionary trajectories. Direct molecular analyses of the incompatible genes in yeasts have started to shed light on the evolutionary forces driving speciation. Editor's suggested further reading in BioEssays The cytoplasmic structure hypothesis for ribosome assembly, vertical inheritance, and phylogeny Abstract Mitochondrial bioenergetics as a major motive force of speciation Abstract     

Identification of Yeast Associated with the Planthopper, <Emphasis Type="Italic">Perkinsiella saccharicida</Emphasis>: Potential Applications for Fiji Leaf Gall Control

Yeasts associate with numerous insects, and they can assist the metabolic processes within their hosts. Two distinct yeasts were identified by PCR within the planthopper Perkinsiella saccharicida, the vector of Fiji disease virus to sugarcane. The utility of both microbes for potential paratransgenic approaches to control Fiji leaf gall (FLG) was assessed. Phylogenetic analysis showed one of the microbes is related to yeast-like symbionts from the planthoppers: Laodelphax striatellus, Nilaparvata lugens, and Sogetella furcifera. The second yeast was a member of the Candida genus, a group that has been identified in beetles and recently described in planthoppers. Microscopy revealed the presence of yeast in the fat body of P. saccharicida. The Candida yeast was cultured, and transformation was accomplished by electroporation of Candida albicans codon optimized plasmids, designed to integrate into the genome via homologous recombination. Transgenic lines conferred resistance to the antibiotic nourseothricin and expression of green fluorescent protein was observed in a proportion of the yeast cells. Stably transformed yeast lines could not be isolated as the integrative plasmids presumably replicated within the yeast without integration into the genome. If stable transformation can be achieved, then this yeast may be useful as an agent for a paratransgenic control of FLG.     

The syntaxins

Summary  

The SNARE hypothesis predicts that a family of SNAP receptors are localized to and function in diverse intracellular membrane compartments where membrane fusion processes take place. Syntaxins, the prototype family of SNARE proteins, have a carboxy-terminal tail-anchor and multiple coiled-coil domains. There are 15 members of the syntaxin family in the human genome and 7 syntaxin-like genes in the yeast Saccharomyces cerevisiae. In conjunction with other SNAREs and with the cytoplasmic NSF and SNAP proteins, syntaxins mediate vesicle fusion in diverse vesicular transport processes along the exocytic and the endocytic pathway. They are crucial components that both drive and provide specificity to the myriad vesicular fusion processes that characterize the eukaryotic cell.     

Identification of new cell size control genes in <Emphasis Type="Italic">S. cerevisiae</Emphasis>

Cell size homeostasis is a conserved attribute in many eukaryotic species involving a tight regulation between the processes of growth and proliferation. In budding yeast S. cerevisiae, growth to a “critical cell size” must be achieved before a cell can progress past START and commit to cell division. Numerous studies have shown that progression past START is actively regulated by cell size control genes, many of which have implications in cell cycle control and cancer. Two initial screens identified genes that strongly modulate cell size in yeast. Since a second generation yeast gene knockout collection has been generated, we screened an additional 779 yeast knockouts containing 435 new ORFs (~7% of the yeast genome) to supplement previous cell size screens. Upon completion, 10 new strong size mutants were identified: nine in log-phase cells and one in saturation-phase cells, and 97% of the yeast genome has now been screened for cell size mutations. The majority of the logarithmic phase size mutants have functions associated with translation further implicating the central role of growth control in the cell division process. Genetic analyses suggest ECM9 is directly associated with the START transition. Further, the small (whi) mutants mrpl49Δ and cbs1Δ are dependent on CLN3 for cell size effects. In depth analyses of new size mutants may facilitate a better understanding of the processes that govern cell size homeostasis.     

Recombinational Repair in Schizosaccharomyces pombe: A Role in Maintaining Genome Integrity

Recombinational DNA repair was first detected in budding yeast Saccharomyces cerevisiaeand was also studied in fission yeast Schizosaccharomyces pombeover the recent decade. The discovery of Sch. pombehomologs of the S. cerevisiae RAD52genes made it possible not only to identify and to clone their vertebrate counterparts, but also to study in detail the role of DNA recombination in certain cell processes. For instance, recombinational repair was shown to play a greater role in maintaining genome integrity in fission yeast and in vertebrates compared with S. cerevisiae. The present state of the problem of recombinational double-strand break repair in fission yeast is considered in this review with a focus on comparisons between Sch. pombeand higher eukaryotes. The role of double-strand break repair in maintaining genome stability is discussed.     

Budding Yeast for Budding Geneticists: A Primer on the Saccharomyces cerevisiae Model System

The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.     

Non-reciprocal chromosomal bridge-induced translocation (BIT) by targeted DNA integration in yeast

Several experimental in vivo systems exist that generate reciprocal translocations between engineered chromosomal loci of yeast or Drosophila, but not without previous genome modifications. Here we report the successful induction of chromosome translocations in unmodified yeast cells via targeted DNA integration of the KAN^R selectable marker flanked by sequences homologous to two chromosomal loci randomly chosen on the genome. Using this bridge-induced translocation system, 2% of the integrants showed targeted translocations between chromosomes V-VIII and VIII-XV in two wild-type Saccharomyces cerevisiae strains. All the translocation events studied were found to be non-reciprocal and the fate of their chromosomal fragments that were not included in the translocated chromosome was followed. The recovery of discrete-sized fragments suggested multiple pathway repair of their free DNA ends. We propose that centromere-distal chromosome fragments may be processed by a break-induced replication mechanism ensuing in partial trisomy. The experimental feasibility of inducing chromosomal translocations between any two desired genetic loci in a eukaryotic model system will be instrumental in elucidating the molecular mechanism underlying genome rearrangements generated by DNA integration and the gross chromosomal rearrangements characteristic of many types of cancer.Electronic Supplementary Material Supplementary material is available for this article at     

Microarray methods in Drosophila neurobiology

In the relatively short period since their development, DNA microarrays have been used increasingly in the study of genetic and cellular processes, thereby offering a genome-wide approach to gene expression studies. With the advent of genome sequencing programs for organisms from yeast to man, the number of organisms which now have ready-made commercial arrays continues to increase. Here, the principle of DNA microarrays is introduced, with particular attention being given to the role of this technology in studies of the nervous system of the fruitfly Drosophila melanogaster. The importance of experimental design and sample preparation, in line with minimum information about microarray experiment (MIAME) compliance, is emphasised. The technical platforms available to the Drosophila neurobiologist have been illustrated and a brief number of data analysis tools that are readily available reviewed.     

Open access to sequence: Browsing the Pichia pastoris genome

The first genome sequences of the important yeast protein production host Pichia pastoris have been released into the public domain this spring. In order to provide the scientific community easy and versatile access to the sequence, two web-sites have been installed as a resource for genomic sequence, gene and protein information for P. pastoris: A GBrowse based genome browser was set up at and a genome portal with gene annotation and browsing functionality at . Both websites are offering information on gene annotation and function, regulation and structure.     

Genes and genomes: Towards construction of an overlapping YAC library of the Arabidopsis thaliana genome

Arabidopsis thaliana (Thale cress, Arabidopsis) is an ideal model organism for the molecular genetic analysis of many plant processes. The availability of a complete physical map would greatly facilitate the gene cloning steps in these studies. The small genome size of Arabidopsis makes the construction of such a map a feasible goal. One of the approaches to construct an overlapping library of the Arabidopsis genome takes advantage of the many mapped markers and the availability of Arabidopsis yeast artificial chromosome (YAC) libraries. Mapped molecular markers are used to identify corresponding YAC clones and thereby place them on the genetic map. Subsequently, these YAC clones provide the framework for directed walking experiments aimed at closing the gaps between the YAC contigs. Adopting this strategy, YAC clones comprising about 10% of the genome have been assigned to the top halves of Arabidopsis chromosomes 4 and 5. Extensive walking experiments in a 10 cM interval of chromosome 4 have resulted in two contiguous regions in the megabase size range.     

Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae

The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast owing to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Because of problems associated with fusing genes encoding fluorescent proteins (FPs) to the native α‐tubulin (TUB1) gene, the FP‐Tub1 fusion is generally integrated into the genome such that the endogenous TUB1 locus is left intact. Although such modifications have no apparent consequences on cell viability, it is unknown if these genome‐integrated FP‐tubulin fusions negatively affect microtubule functions. Thus, a simple, economical and highly sensitive assay of microtubule function is required. Furthermore, the current plasmids available for generation of FP‐Tub1 fusions have not kept pace with the development of improved FPs. Here, we have developed a simple and sensitive assay of microtubule function that is sufficient to identify microtubule defects that were not apparent by fluorescence microscopy or cell growth assays. Using results obtained from this assay, we have engineered a new family of 30 FP‐Tub1 plasmids that use various improved FPs and numerous selectable markers that upon genome integration have no apparent defect on microtubule function.      

Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4

Autophagy is important for degradation and recycling of intracellular components. In a diversity of genera and species, orthologs and paralogs of the yeast Atg4 and Atg8 proteins are crucial in the biogenesis of double-membrane autophagosomes that carry the cellular cargoes to vacuoles and lysosomes. Although many plant genome sequences are available, the ATG4 and ATG8 sequence analysis is limited to some model plants. We identified 28 ATG4 and 116 ATG8 genes from the available 18 different plant genome sequences. Gene structures and protein domain sequences of ATG4 and ATG8 are conserved in plant lineages. Phylogenetic analyses classified ATG8s into 3 subgroups suggesting divergence from the common ancestor. The ATG8 expansion in plants might be attributed to whole genome duplication, segmental and dispersed duplication, and purifying selection. Our results revealed that the yeast Atg4 processes Arabidopsis ATG8 but not human LC3A (HsLC3A). In contrast, HsATG4B can process yeast and plant ATG8s in vitro but yeast and plant ATG4s cannot process HsLC3A. Interestingly, in Nicotiana benthamiana plants the yeast Atg8 is processed compared to HsLC3A. However, HsLC3A is processed when coexpressed with HsATG4B in plants. Molecular modeling indicates that lack of processing of HsLC3A by plant and yeast ATG4 is not due to lack of interaction with HsLC3A. Our in-depth analyses of ATG4 and ATG8 in the plant lineage combined with results of cross-kingdom ATG8 processing by ATG4 further support the evolutionarily conserved maturation of ATG8. Broad ATG8 processing by HsATG4B and lack of processing of HsLC3A by yeast and plant ATG4s suggest that the cross-kingdom ATG8 processing is determined by ATG8 sequence rather than ATG4.