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1.
Nucleic Acids Res ; 32(Database issue): D560-7, 2004 Jan 01.
Article in English | MEDLINE | ID: mdl-14681481

ABSTRACT

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.


Subject(s)
Cell Differentiation/genetics , Databases, Genetic , Gene Expression Profiling , Germ Cells/cytology , Germ Cells/metabolism , Animals , Computational Biology , Genomics , Humans , Information Storage and Retrieval , Internet , Meiosis/genetics , Mitosis/genetics , Oligonucleotide Array Sequence Analysis , Proteins/metabolism , Proteome , Proteomics , Rats
2.
J Cell Biol ; 155(6): 979-90, 2001 Dec 10.
Article in English | MEDLINE | ID: mdl-11733545

ABSTRACT

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Delta mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Delta cells suggest the existence of various levels of control involved in the partitioning of peroxisomes.


Subject(s)
Actins/metabolism , Carrier Proteins/metabolism , Cytoskeletal Proteins , GTP-Binding Proteins , Myosin Heavy Chains/metabolism , Myosin Type V/metabolism , Peroxisomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Actin Cytoskeleton/metabolism , Bacterial Proteins/genetics , Base Sequence , Carrier Proteins/genetics , Gene Deletion , Green Fluorescent Proteins , Indicators and Reagents/metabolism , Luminescent Proteins/genetics , Membrane Proteins/genetics , Microtubule-Associated Proteins/genetics , Microtubules , Molecular Sequence Data , Mutagenesis/physiology , Oligonucleotide Probes , Peroxisome-Targeting Signal 1 Receptor , Polymers/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Saccharomyces cerevisiae/genetics , Vesicular Transport Proteins
3.
Mol Microbiol ; 42(1): 121-32, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11679072

ABSTRACT

Conventional kinesin is a microtubule-dependent motor protein believed to be involved in a variety of intracellular transport processes. In filamentous fungi, conventional kinesin has been implicated in different processes, such as vesicle migration, polarized growth, nuclear distribution, mitochondrial movement and vacuole formation. To gain further insights into the functions of this kinesin motor, we identified and characterized the conventional kinesin gene, kinA, of the established model organism Aspergillus nidulans. Disruption of the gene leads to a reduced growth rate and a nuclear positioning defect, resulting in nuclear cluster formation. These clusters are mobile and display a dynamic behaviour. The mutant phenotypes are pronounced at 37 degrees C, but rescued at 25 degrees C. The hyphal growth rate at 25 degrees C was even higher than that of the wild type at the same temperature. In addition, kinesin-deficient strains were less sensitive to the microtubule destabilizing drug benomyl, and disruption of conventional kinesin suppressed the cold sensitivity of an alpha-tubulin mutation (tubA4). These results suggest that conventional kinesin of A. nidulans plays a role in cytoskeletal dynamics, by destabilizing microtubules. This new role of conventional kinesin in microtubule stability could explain the various phenotypes observed in different fungi.


Subject(s)
Aspergillus nidulans/metabolism , Fungal Proteins/genetics , Kinesins/genetics , Microtubules/metabolism , Amino Acid Sequence , Aspergillus nidulans/cytology , Cell Nucleus/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Genes, Reporter , Kinesins/chemistry , Kinesins/metabolism , Microscopy, Fluorescence , Molecular Motor Proteins/genetics , Molecular Motor Proteins/metabolism , Molecular Sequence Data , Phenotype , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Temperature
4.
Mol Biol Cell ; 12(8): 2519-33, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11514632

ABSTRACT

In Saccharomyces cerevisiae, the spindle pole body (SPB) is the functional homolog of the mammalian centrosome, responsible for the organization of the tubulin cytoskeleton. Cytoplasmic (astral) microtubules essential for the proper segregation of the nucleus into the daughter cell are attached at the outer plaque on the SPB cytoplasmic face. Previously, it has been shown that Cnm67p is an integral component of this structure; cells deleted for CNM67 are lacking the SPB outer plaque and thus experience severe nuclear migration defects. With the use of partial deletion mutants of CNM67, we show that the N- and C-terminal domains of the protein are important for nuclear migration. The C terminus, not the N terminus, is essential for Cnm67p localization to the SPB. On the other hand, only the N terminus is subject to protein phosphorylation of a yet unknown function. Electron microscopy of SPB serial thin sections reveals that deletion of the N- or C-terminal domains disturbs outer plaque formation, whereas mutations in the central coiled-coil domain of Cnm67p change the distance between the SPB core and the outer plaque. We conclude that Cnm67p is the protein that connects the outer plaque to the central plaque embedded in the nuclear envelope, adjusting the space between them by the length of its coiled-coil.


Subject(s)
Cell Nucleus/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , Saccharomyces cerevisiae/physiology , Active Transport, Cell Nucleus/physiology , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Genes, Reporter , Mutagenesis , Nuclear Envelope/metabolism , Phosphorylation , Protein Sorting Signals/genetics , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
5.
EMBO J ; 20(14): 3695-704, 2001 Jul 16.
Article in English | MEDLINE | ID: mdl-11447111

ABSTRACT

Ribosomal precursor particles are assembled in the nucleolus before export into the cytoplasm. Using a visual assay for nuclear accumulation of 60S subunits, we have isolated several conditional-lethal strains with defects in ribosomal export (rix mutants). Here we report the characterization of a mutation in an essential gene, RIX7, which encodes a novel member of the AAA ATPase superfamily. The rix7-1 temperature-sensitive allele carries a point mutation that causes defects in pre-rRNA processing, biogenesis of 60S ribosomal subunits, and their subsequent export into the cytoplasm. Rix7p, which associates with 60S ribosomal precursor particles, localizes throughout the nucleus in exponentially growing cells, but concentrates in the nucleolus in stationary phase cells. When cells resume growth upon shift to fresh medium, Rix7p-green fluorescent protein exhibits a transient perinuclear location. We propose that a nuclear AAA ATPase is required for restructuring nucleoplasmic 60S pre-ribosomal particles to make them competent for nuclear export.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Nucleus/enzymology , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins , Adenosine Triphosphatases/genetics , Base Sequence , Biological Transport , Cell Nucleolus/metabolism , Cytoplasm/metabolism , DNA Primers , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Mutation , Nuclear Proteins , RNA Processing, Post-Transcriptional/genetics , RNA, Ribosomal/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
6.
Genetics ; 157(2): 601-10, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11156982

ABSTRACT

Polarized cell growth requires a polarized organization of the actin cytoskeleton. Small GTP-binding proteins of the Rho-family have been shown to be involved in the regulation of actin polarization as well as other processes. Hyphal growth in filamentous fungi represents an ideal model to investigate mechanisms involved in generating cell polarity and establishing polarized cell growth. Since a potential role of Rho-proteins has not been studied so far in filamentous fungi we isolated and characterized the Ashbya gossypii homologs of the Saccharomyces cerevisiae CDC42, CDC24, RHO1, and RHO3 genes. The AgCDC42 and AgCDC24 genes can both complement conditional mutations in the S. cerevisiae CDC42 and CDC24 genes and both proteins are required for the establishment of actin polarization in A. gossypii germ cells. Agrho1 mutants show a cell lysis phenotype. Null mutant strains of Agrho3 show periodic swelling of hyphal tips that is overcome by repolarization and polar hyphal growth in a manner resembling the germination pattern of spores. Thus different Rho-protein modules are required for distinct steps during polarized hyphal growth of A. gossypii.


Subject(s)
Cell Division/genetics , Guanine Nucleotide Exchange Factors , Saccharomyces cerevisiae Proteins , Saccharomycetales/genetics , rho GTP-Binding Proteins/physiology , Actins/genetics , Actins/metabolism , Amino Acid Sequence , Cell Cycle Proteins/genetics , Cytoskeleton/genetics , Cytoskeleton/metabolism , DNA/metabolism , DNA Primers/metabolism , Gene Deletion , Genetic Complementation Test , Models, Genetic , Molecular Sequence Data , Mutagenesis , Mutation , Phenotype , Proto-Oncogene Proteins/genetics , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/physiology , rho GTP-Binding Proteins/genetics
7.
J Cell Sci ; 114(Pt 5): 975-86, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11181180

ABSTRACT

We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network.


Subject(s)
Ascomycota/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Dyneins/metabolism , Actins/ultrastructure , Ascomycota/cytology , Ascomycota/ultrastructure , Base Sequence , Benomyl/pharmacology , DNA Primers , Dyneins/genetics , Microscopy, Electron , Microtubules/drug effects , Microtubules/ultrastructure , Mitosis , Mutagenesis
8.
J Cell Sci ; 113 Pt 24: 4563-75, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11082049

ABSTRACT

Filamentous fungi grow by hyphal extension, which is an extreme example of polarized growth. In contrast to yeast species, where polarized growth of the tip of an emerging bud is temporally limited, filamentous fungi exhibit constitutive polarized growth of the hyphal tip. In many fungi, including Ashbya gossypii, polarized growth is reinforced by a process called hyphal maturation. Hyphal maturation refers to the developmental switch from slow-growing hyphae of young mycelium to fast-growing hyphae of mature mycelium. This process is essential for efficient expansion of mycelium. We report for the first time on the identification and characterization of a fungal gene important for hyphal maturation. This novel A. gossypii gene encodes a presumptive PAK (p21-activated kinase)-like kinase. Its closest homolog is the S. cerevisiae Cla4 protein kinase; the A. gossypii protein is therefore called AgCla4p. Agcla4 deletion strains are no longer able to perform the developmental switch from young to mature hyphae, and GFP (green fluorescent protein)-tagged AgCla4p localizes with much higher frequency in mature hyphal tips than in young hyphal tips. Both results support the importance of AgCla4p in hyphal maturation. AgCla4p is also required for septation, indicated by the inability of Agcla4 deletion strains to properly form actin rings and chitin rings. Despite the requirement of AgCla4p for the development of fast-growing hyphae, AgCla4p is not necessary for actin polarization per se, because tips enriched in cortical patches and hyphae with a fully developed network of actin cables can be seen in Agcla4 deletion strains. The possibility that AgCla4p may be involved in regulatory mechanisms that control the dynamics of the actin patches and/or actin cables is discussed.


Subject(s)
Fungal Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomycetales/enzymology , Saccharomycetales/growth & development , Actins/metabolism , Base Sequence , DNA, Fungal , Fungal Proteins/genetics , Fungal Proteins/physiology , Genes, Fungal , Molecular Sequence Data , Phenotype , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins , Subcellular Fractions , p21-Activated Kinases
9.
Mol Biol Cell ; 11(4): 1197-211, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10749924

ABSTRACT

Saccharomyces cerevisiae cnm67Delta cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein-labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the gamma-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Delta cells Spc72-gamma-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Delta cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.


Subject(s)
Cell Nucleus/genetics , Saccharomyces cerevisiae/genetics , Spindle Apparatus/physiology , Cell Division , Cell Nucleus/physiology , Cell Polarity , Centrosome/physiology , Diploidy , Green Fluorescent Proteins , Luminescent Proteins , Microscopy, Video , Microtubules/physiology , Mutation , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/physiology , Spindle Apparatus/genetics
10.
J Cell Sci ; 113 ( Pt 9): 1611-21, 2000 May.
Article in English | MEDLINE | ID: mdl-10751152

ABSTRACT

In the filamentous ascomycete Ashbya gossypii, like in other filamentous fungi onset of growth in dormant spores occurs as an isotropic growth phase generating spherical germ cells. Thereafter, a switch to polarized growth results in the formation of the first hyphal tip. The initial steps of hyphal tip formation in filamentous fungi, therefore, resemble processes taking place prior to and during bud emergence of unicellular yeast-like fungi. We investigated whether phenotypic similarities between these distinct events extended to the molecular level. To this end we isolated and characterized the A. gossypii homolog of the Saccharomyces cerevisiae BEM2 gene which is part of a network of rho-GTPases and their regulators required for bud emergence and bud growth in yeast. Here we show that the AgBem2 protein contains a GAP- (GTPase activating protein) domain for rho-like GTPases at its carboxy terminus, and that this part of AgBem2p is required for complementation of an Agbem2 null strain. Germination of spores resulted in enlarged Agbem2 germ cells that were unable to generate the bipolar branching pattern found in wild-type germ cells. In addition, mutant hyphae were swollen due to defects in polarized cell growth indicated by the delocalized distribution of chitin and cortical actin patches. Surprisingly, the complete loss of cell polarity which lead to spherical hyphal tips was overcome by the establishment of new cell polarities and the formation of multiple new hyphal tips. In conclusion these results and other findings demonstrate that establishment of cell polarity, maintenance of cell polarity, and polarized hyphal growth in filamentous fungi require members of &rgr;-GTPase modules.


Subject(s)
Ascomycota/cytology , GTPase-Activating Proteins , Saccharomyces cerevisiae Proteins , Spores, Fungal , rho GTP-Binding Proteins/physiology , Amino Acid Sequence , Ascomycota/genetics , Ascomycota/growth & development , Base Sequence , DNA Primers , Fungal Proteins/chemistry , Fungal Proteins/genetics , Molecular Sequence Data , Sequence Deletion , Sequence Homology, Amino Acid , rho GTP-Binding Proteins/chemistry
11.
Gene ; 242(1-2): 381-91, 2000 Jan 25.
Article in English | MEDLINE | ID: mdl-10721732

ABSTRACT

We have investigated a PCR-based approach for one-step gene targeting in the filamentous fungus Ashbya gossypii. Short guide sequences with 40-46 bp of homology to two sequences of a targeted gene, provided by PCR, were sufficient to mediate homologous recombination. The PCR products used for transformation were generated from the newly constructed chimeric selection marker GEN3. This consists of the open reading frame of the Escherichia coli kanR gene under the control of promoter and terminator sequences of the Saccharomyces cerevisiae TEF2 gene and allows selection of G418/geneticin-resistant transformants. Verification of gene targeting was performed either by PCR or by DNA hybridization analyses, and in all 18 cases tested, correct targeting was confirmed. This approach was used for the complete deletion of the open reading frame of the A. gossypii RHO4 gene for which a double-strand sequence was available as information source for the design of PCR primers. We also demonstrated successful partial deletion of four other ORFs using single-read sequences (SRS) as sole information for the design of targeting primers. A gossypii is the first filamentous fungus in which a PCR-based gene disruption technique has been established. Since short target guide sequences are sufficient to direct homologous integration into the A. gossypii genome it is not necessary to obtain and sequence large DNA fragments from a target locus to provide the long flanking homology regions usually required for efficient targeting of cloned disruption cassettes in filamentous fungi. Thus functional analysis of A. gossypii genes is already possible, based on single-pass sequence information.


Subject(s)
Ascomycota/genetics , GTP-Binding Proteins , Gene Targeting/methods , Saccharomyces cerevisiae Proteins , Base Sequence , DNA, Fungal/chemistry , DNA, Fungal/genetics , Fungal Proteins/genetics , Gene Deletion , Genetic Markers , Molecular Sequence Data , Open Reading Frames/genetics , Polymerase Chain Reaction , Sequence Analysis, DNA , Transformation, Genetic , rho GTP-Binding Proteins
12.
Yeast ; 16(3): 241-53, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10649453

ABSTRACT

As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c-GFP) and the nucleus (Ynl075w-GFP) as the sites of action of two proteins. Ynl080c-GFP and Ynl081c-GFP fluorescence was visible in dot-shaped and elongated structures, whereas the Ynl022c-GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C-terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5'-GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP-Ynl020c and GFP-Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases.


Subject(s)
Gene Deletion , Genes, Fungal , Luminescent Proteins/metabolism , Open Reading Frames/genetics , Saccharomyces cerevisiae/genetics , Green Fluorescent Proteins , Luminescent Proteins/genetics , Phenotype , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/classification , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/physiology , Spores, Fungal
13.
Curr Genet ; 35(6): 618-25, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10467006

ABSTRACT

The rDNA cluster in the phytopathogenic fungus Ashbya gossypii consists of approximately 50 tandem repeat units of 8197 bp. Each unit carries a gene for the 35S pre-rRNA, processed into 18S, 5.8S and 25S rRNA, and a divergently transcribed gene for 5S rRNA. The well-characterized rDNA of the yeast Saccharomyces cerevisiae is the only other example of a completely sequenced rDNA unit (9137 bp) carrying both a 35S pre-rRNA and a 5S rRNA gene. The coding regions for the 5S, 5.8S, 18S and 25S rRNAs are 95-100% identical whereas transcribed and non-transcribed spacers show 43-66% sequence identity. Functionally characterized rDNA and rRNA elements of S. cerevisiae can be unambiguously recognized in the A. gossypii sequence, including the RNA polymerase-I transcription start site, two Reb1p enhancer binding sites and numerous recognition sequences for rRNA modification and processing. In addition to these functionally characterized sequences eight highly conserved elements from 10 to 71 bp were detected in the over 600-bp transcribed region upstream of the 18S rRNA gene which most likely play as yet uncharacterized functions at the DNA or RNA level. In addition to this work we started to identify A. gossypii homologs of S. cerevisiae nucleolar proteins involved in rDNA maturation.


Subject(s)
Ascomycota/genetics , Genes, rRNA/genetics , Amino Acid Sequence , Ascomycota/chemistry , Base Sequence , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal/genetics , Genes, Fungal/genetics , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 18S/genetics , Repetitive Sequences, Nucleic Acid , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
14.
Science ; 285(5429): 901-6, 1999 Aug 06.
Article in English | MEDLINE | ID: mdl-10436161

ABSTRACT

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.


Subject(s)
Gene Deletion , Genes, Essential , Genome, Fungal , Open Reading Frames , Saccharomyces cerevisiae/genetics , Culture Media , Gene Expression Regulation, Fungal , Gene Targeting , Genes, Fungal , Phenotype , Polymerase Chain Reaction , Recombination, Genetic , Saccharomyces cerevisiae/growth & development
15.
Yeast ; 14(10): 943-51, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9717240

ABSTRACT

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.


Subject(s)
Gene Targeting/methods , Polymerase Chain Reaction , Schizosaccharomyces/genetics , Drug Resistance, Microbial/genetics , Gene Deletion , Gene Expression , Genetic Markers , Gentamicins/pharmacology , Plasmids , Recombination, Genetic , Transformation, Genetic
16.
Yeast ; 14(10): 953-61, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9717241

ABSTRACT

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.


Subject(s)
Molecular Biology/methods , Plasmids , Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , DNA Primers , Gene Deletion , Gene Expression , Genetic Vectors , Green Fluorescent Proteins , Luminescent Proteins , Recombinant Fusion Proteins , Reproducibility of Results , Transformation, Genetic
17.
Mol Biol Cell ; 9(5): 977-91, 1998 May.
Article in English | MEDLINE | ID: mdl-9571234

ABSTRACT

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Delta1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Delta1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.


Subject(s)
Cell Nucleus/physiology , Microtubules/physiology , Saccharomyces cerevisiae/physiology , Spindle Apparatus/physiology , Benomyl/pharmacology , Cytoskeleton , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungal Proteins/physiology , Fungicides, Industrial/pharmacology , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/physiology , Microtubules/metabolism , Microtubules/ultrastructure , Mutagenesis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Spindle Apparatus/metabolism , Spindle Apparatus/ultrastructure , Spores, Fungal
18.
Yeast ; 13(11): 1065-75, 1997 Sep 15.
Article in English | MEDLINE | ID: mdl-9290211

ABSTRACT

We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosac-charomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1.4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0.9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2.4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter.


Subject(s)
Cell Cycle Proteins/genetics , Luminescent Proteins/genetics , Polymerase Chain Reaction/methods , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Transcription Factors/genetics , Acyltransferases/genetics , Chromosome Mapping , Cloning, Molecular , Genes, Reporter , Genetic Complementation Test , Gentamicins , Green Fluorescent Proteins , Open Reading Frames , Peptide Elongation Factor 1 , Peptide Elongation Factors/genetics , Plasmids/genetics , Promoter Regions, Genetic , Recombination, Genetic , Saccharomyces/genetics , Schizosaccharomyces/genetics
19.
Nature ; 387(6632 Suppl): 93-8, 1997 May 29.
Article in English | MEDLINE | ID: mdl-9169873

ABSTRACT

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.


Subject(s)
Chromosomes, Fungal , Evolution, Molecular , Saccharomyces cerevisiae/genetics , Base Sequence , Molecular Sequence Data , Multigene Family , Open Reading Frames , Restriction Mapping
20.
Science ; 274(5287): 546, 563-7, 1996 Oct 25.
Article in English | MEDLINE | ID: mdl-8849441

ABSTRACT

The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.


Subject(s)
Chromosome Mapping , Genes, Fungal , Genome, Fungal , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Chromosomes, Fungal/genetics , Computer Communication Networks , DNA, Fungal/genetics , Evolution, Molecular , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/physiology , Gene Library , International Cooperation , Multigene Family , Open Reading Frames , RNA, Fungal/genetics , Sequence Analysis, DNA
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