A novel analysis of gene array data: yeast cell cycle

Many gene array studies of the yeast cell cycle have been performed (Cho RJ, Campbell MJ, Winzeler EA et al. A genome-wide transcriptional analysis of the mitotic cell cycle. Mol Cell 1998;2:65–73; Orlando DA, Lin CY, Bernard A et al. Global control of cell-cycle transcription by coupled CDK and network oscillators. Nature 2008;453:944–7; Pramila T, Wu W, Miles S et al. The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle. Genes Dev 2006;20:2266–78; Spellman PT, Sherlock G, Zhang MQ et al. Comprehensive identification of cell cycle–regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. MBoC 1998;9:3273–97). Largely, these studies contain elements drawn from laboratory experiments. The present investigation determines cell division cycle (CDC) genes solely from the data (Orlando DA, Lin CY, Bernard A et al. Global control of cell-cycle transcription by coupled CDK and network oscillators. Nature 2008;453:944–7). It is shown by simple reasoning that the dynamics of the approximately 6000 yeast genes are described by an approximately six-dimensional space. This leads a precisely determined cell-cycle period, along with the quality and timing of the identified CDC genes. Convincing evidence for the role of the identified genes is obtained. While these show good agreement with standard CDC gene representatives (Orlando DA, Lin CY, Bernard A et al. Global control of cell-cycle transcription by coupled CDK and network oscillators. Nature 2008;453:944–7; Spellman PT, Sherlock G, Zhang MQ et al. Comprehensive identification of cell cycle–regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. MBoC 1998;9:3273–97; de Lichtenberg U, Jensen LJ, Fausbøll A et al. Comparison of computational methods for the identification of cell cycle-regulated genes. Bioinformatics 2005;21:1164–71) several hundred newly revealed CDC genes appear, which merit attention. The present approach employs an adaptation of a method introduced to study turbulent flows (Schmid PJ. Dynamic mode decomposition of numerical and experimental data. J Fluid Mech 2010;656:5–28), “dynamic mode decomposition” (DMD). From this, one can infer that singular value decomposition, analysis of the data entangles the underlying (gene) dynamics implicit in the data; and that DMD produces the disentangling transformation. It is the assertion of this study that a new tool now exists for the analysis of the gene array signals, and in particular for investigating the yeast cell cycle.

Genomic features and evolution of the conditionally dispensable chromosome in the tangerine pathotype of Alternaria alternata

The tangerine pathotype of the ascomycete fungus Alternaria alternata is the causal agent of citrus brown spot, which can result in significant losses of both yield and marketability for tangerines and tangerine hybrids worldwide. A conditionally dispensable chromosome (CDC), which harbors the host-selective ACT toxin gene cluster, is required for tangerine pathogenicity of A. alternata. To understand the genetic makeup and evolution of the tangerine pathotype CDC, we analyzed the function and evolution of the CDC genes present in the A. alternata Z7 strain. The 1.84Mb long CDC contains 512 predicted protein-coding genes, which are enriched in functional categories associated with ‘metabolic process’ (132 genes, p-value = 0.00192) including ‘oxidation-reduction process’ (48 genes, p-value = 0.00021) and ‘lipid metabolic process’ (11 genes, p-value = 0.04591). Relatively few of the CDC genes can be classified as CAZymes (13), kinases (3) and transporters (20). Differential transcriptome analysis of H2O2 treatment and control conditions revealed that 29 CDC genes were significantly up-regulated and 14 were significantly down-regulated, suggesting that CDC genes may play a role in coping with oxidative stress. Evolutionary analysis of the 512 CDC proteins showed that their evolutionary conservation tends to be restricted within the genus Alternaria and that the CDC genes evolve faster than genes in the essential chromosomes. Interestingly, phylogenetic analysis suggested that the genes of 13 enzymes and one sugar transporter residing in the CDC were likely horizontally transferred from distantly related species. Among these, one carboxylesterase gene was transferred from bacteria but functionally knocking out this gene revealed no obvious biological role. Another 4 genes might have been transferred from Colletotrichum (Sordariomycetes) and 5 were likely transferred as a physically linked cluster of genes from Cryptococcus (Basidiomycota) or Penicillium (Eurotiomycetes). Functionally knocking out the 5-gene cluster resulted in an 80% decrease in asexual spore production in the deletion mutant. These results provide new insights into the function and evolution of CDC genes in Alternaria.Author SummaryMany fungal phytopathogens harbor conditionally dispensable chromosomes (CDCs). CDCs are variable in size, contain many genes involved in virulence, but their evolution remains obscure. In this study, we investigated the origin of the CDC present in the tangerine pathotype of Alternaria alternata Z7 strain. We found that most of the Z7 CDC proteins are highly conserved within the genus Alternaria but poorly conserved outside the genus. We also discovered that a small number of genes originated via horizontal gene transfer (HGT) from distantly related fungi and bacteria. These horizontally transferred genes include a carboxylesterase gene that was likely acquired from bacteria, a cluster of 4 physically linked genes likely transferred from Colletotrichum, and a cluster of 5 physically linked genes likely transferred from Cryptococcus (Basidiomycota) or Penicillium (Eurotiomycetes). To gain insight into the functions of these transferred genes, we knocked out the bacterial carboxylesterase and the 5-gene cluster. Whereas the carboxylesterase deletion mutant showed no obvious phenotype, the 5-gene cluster mutant showed a dramatically reduced production of asexual spores (conidia). The results of our study suggest that Alternaria CDCs are largely comprised from rapidly evolving native genes; although only a few genes were acquired via horizontal gene transfer, some of them appear to be involved in functions critical to the phytopathogenic lifestyle.

Forty-five years of cell-cycle genetics

In the early 1970s, studies in Leland Hartwell’s laboratory at the University of Washington launched the genetic analysis of the eukaryotic cell cycle and set the path that has led to our modern understanding of this centrally important process. This 45th-anniversary Retrospective reviews the steps by which the project took shape, the atmosphere in which this happened, and the possible morals for modern times. It also provides an up-to-date look at the 35 original CDC genes and their human homologues.

Horizontal Chromosome Transfer, a Mechanism for the Evolution and Differentiation of a Plant-Pathogenic Fungus

ABSTRACT The tomato pathotype of Alternaria alternata produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene, ALT1, which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of ALT1 and another PKS gene, both of which reside on the CDC in the tomato pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five A. alternata tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the A. alternata strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among A. alternata strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.

p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains.

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

MAPPING CDC MUTATIONS IN THE YEAST S. Cerevisiae BY RAD52-MEDIATED CHROMOSOME LOSS

ABSTRACT Using the chromosome loss-mapping method of Schild and Mortimer, I have mapped several new temperature-sensitive mutations that define five CDC genes. Modified procedures were used to facilitate mapping temperature-sensitive mutations in general, and these modifications are discussed. The mutations were assigned to specific chromosomes by chromosome loss procedures, and linkage relationships were determined subsequently by standard tetrad analysis. Four of the mutations define new loci. The fifth mutation, cdc63-1, is shown to be allelic to previously known mutations in the PRT1 gene.

Septum pattern in ts mutants of Schizosaccharomyces pombe defective in genes cdc3, cdc4, cdc8 and cdc12

Septum-defective mutants of Schizosaccharomyces pombe impaired in cdc genes 3, 4, 8 and 12 were compared by fluorescence microscopy, freeze-etching and ultrathin sectioning. This approach made it possible to recognize the internal organization of defective phenotypes under restrictive conditions. Of special interest in this study was the pattern of unusual septum malformations found to be regular features of the terminal phenotypes of the mutants. Their overall topology was visualized at the cellular level by primulin fluorescence. The subcellular location of septum defects was found to be identical in origin to the compartment where normal septum was assembled in the wild type. Delocalized septation involved both microfibrillar and matrix components, which participated in the final assembly of malformations. Unique contour views of delocalized septa were exposed by freeze-fracturing. Cytoplasmic microtubules and microfilaments were detected in ultrathin sections of the cytoplasm of mutant cells. The internal organization of malformation-accumulating phenotypes suggested a disruption of the directional mechanism that steers septum material to the periplasm at the cell equator.