Plenary Abstracts






Genome reconstruction and gene expression for the rice blast fungus, Magnaporthe grisea. Ralph A. Dean. Fungal Genomics Laboratory, NC State University, Raleigh NC 27695

Rice blast disease, caused by Magnaporthe grisea, is one of the most devastating threats to food security worldwide. The fungus is amenable to classical and molecular genetic manipulation and is a compelling experimental system for elucidating numerous aspects of pathogenesis, including infection-related morphogenesis, host species and cultivar specificity, and signaling pathways. In 1998, an international consortium (IRBGP) was established to sequence the rice blast genome. For this initiative, we used a 25X large insert (130 kb) HindIII BAC library to construct a physical map of the genome. BAC clones were fingerprinted and assembled into 188 contigs. These were aligned into a physical map by anchoring to mapped RFLP markers. Chromosome 7 (4.2 Mb) has been studied in the greatest detail and a set of 42 BAC clones representing a minimum tiling path covering >95% of the chromosome has been deduced. The sequence of one BAC clone (6J18) has been completed. The entire BAC library was end sequenced providing sequence tag connectors (STC) every 3-4 kb across the genome. A federated database integrating physical, genetic and expression data from relational and object-oriented databases is being developed. We have initiated a draft sequence (~5X coverage) of chromosome 7 using the "BAC by BAC" approach coupled with information from our STC/fingerprint databases. A comprehensive EST program has been launched. 30,000 ESTs will be derived from 8 cDNA libraries prepared from different stages of growth and development as well as cells subjected to various stress conditions. A set of ~5,000 ESTs representing unique genes will be further sequenced. The current status of the genome project and database development will be presented.

Partial reconstruction of the metabolic capacity of Pneumocystis carinii through genomics and identification of potential therapeutic targets. Melanie T. Cushion1, Bradley E. Slaven1, Jonathan Arnold2, John Wunderlich2, Chuck Staben3, Tom Sesterhenn1 and A. George Smulian1.

1University of Cincinnati College of Medicine, Department of Internal Medicine, Division of Infectious Diseases, Cincinnati, OH 45267-0560 and the Cincinnati VAMC, Cincinnati, OH 45220, 2Department of Genetics, University of Georgia, Athens, GA 30602, 3University of Kentucky, T.H. Morgan School of Biological Sciences, Lexington, KY 40506

Pneumonia caused by Pneumocystis carinii (PcP) remains the leading opportunistic infection associated with AIDS patients, even in the era of Highly Active Anti-Retroviral Therapy (HAART). Despite concerted efforts to identify additional therapeutic agents, trimethoprim-sulfamethoxazole (TMP-SMX) continues to be the standard prophylactic and therapeutic modality in use today, as it was over 20 years ago. Recently, treatment failures of TMP-SMX were linked to mutations in the target genes in P. carinii making the search for new targets a priority. Unlike any extant fungus, P. carinii possesses a single copy of the nuclear ribosomal RNA locus and has little to no ergosterol. It is refractory to standard anti-fungals, including amphotericin B and standard azoles such as fluconazole. These data suggest there are distinct differences in some metabolic pathways of other fungi and Pc.

Genetic data resulting from the Pneumocystis Genome Project offers promise for development of new drug targets. Analysis of sequences from a partial Expressed Sequence Tag (EST)-, cosmid end sequence-, and cosmid library databases by homology searches revealed the presence of several metabolic pathways with drug targeting potential, including sterol and heme biosynthesis. P. carinii appears to possesses most of the genes in the sterol biosynthetic pathway, including ERG4, the final enzyme that converts ergosta-5, 7, 22, 24, (28)-tetraen-3, B-ol to ergosterol, and squalene synthase (erg9), squalene epoxidase (ERG1), lanosterol cyclase (ERG7) and SAM:SMT (ERG6). Assessment of the efficacy of compounds targeting the enzymatic steps along the sterol biosynthetic pathway using a short term drug screening assay based on measurement of ATP by bioluminescence correlated with the presence or absence of the gene in the sequence databases. These data will be used to formulate combination therapies that will be assessed in pre-clinical trials using rodent models of infection.

Phytophthora genomics. Brett M Tyler1, Felipe R. Arredondo1, Howard S. Judelson2, Ralph A. Dean3, Peter Hraber4, Mark E.Waugh4, Bruno W. Sobral5, Callum J. Bell4, Dinah Qutob6 , Mark Gijzen6. 1Department of Plant Pathology, University of California, Davis, CA95616; 2Department of Plant Pathology, University of California, Riverside CA 92521; 3Fungal Genomics Laboratory, NC State University, Raleigh, NC 27695-7251; 4National Center for Genome Resources, Santa Fe, New Mexico 87505; 5Virginia Bioinformatics Institute, Virginia Tech University, Blacksburg, VA24061; 6Agriculture Canada, London Research Centre, London, Ontario N5V 4T3, Canada.

The more than 40 species of the oomycete Phytophthora cause serious diseases of a huge range of crop and ornamental plants. To facilitate isolation of infection-related genes from the soybean pathogen Phytophthora sojae, we are constructing a BAC contig of the entire genome, using a hybridization fingerprinting strategy with random probes, most of them repetitive, and with ESTs. Computer software has been developed to collect and analyze the data. At present, 19% of the BACs have received the minimum number of hybridization hits needed to establish contigs. Of these BACs, 34% have been placed into contigs. We have confirmed the authenticity of three of the largest contigs by HindIII digestion. With the long term goal of sequencing the entire genome of P. sojae and selected sequences from other Phytophthora species, such as P. infestans we have established the Phytophthora genome initiative (PGI). We have begun preliminary sequencing of a 200 kb BAC contig spanning two avirulence genes from P. sojae. Sequencing of the first 60 kb BAC reveals that the gene density is extremely high. Both the sequencing data and the BAC hybridization data suggest that the P. sojae genome is composed of gene-rich regions separated by regions rich in repetitive sequences. Sequencing of 55,000 new ESTS from P. sojae and P. infestans, and development of a synteny map of the two species, has recently begun, funded by USDA-IFAFS.

Chromosomes II and V of Neurospora crassa. Ulrich Schulte, Institute of Biochemistry, Heinrich-Heine-University Dusseldorf, Germany

As part of the German Neurospora Genome Project the chromosomes (LG) II and V with a total of 14 Mbp were sequenced. Cooperating partners in the project are Jöörg Hoheisel (DKFZ, Heidelberg), Gerald Nyakatura (MWG Biotech, Ebersberg) and H. Werner Mewes (MIPS, Martinsried). The web accessible MIPS Neurospora crassa data base (MNCDB) provides the assembled sequences as well as the identified and annotated open reading frames. More than 3000 genes have been identified sofar. At least 13.000 genes are expected for the entire genome. The deduced proteins are classified according to matches in sequence data bases and attributed to functional categories based on the identified relatives. Concurrently the protein sequences are analysed using the PEDANT system employing a wide spectrum of sequence analysis and structure prediction tools such as attribution of proteins to protein-superfamilies, secondary structure, transmembrane regions, coiled-coil regions etc. Sequencing and annotation data are provided in a comprehensive and multidimensional way. Tools to explore and query the information have been implemented and will be extend further. Generous funding of the project by the Deutsche Forschungsgemeinschaft is greatfully acknowledged.

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The TOX2 locus of Cochliobolus carbonum for cyclic peptide biosynthesis: genomic organization and the basis of auto-resistance. Jonathan D. Walton1, Dipnath Baidyaroy1, Gerald Brosch2, and Stefan Graessle2 1DOE-Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824 2Department of Microbiology, University of Innsbruck Medical School, Austria

The high virulence of the filamentous fungal pathogen C. carbonum on maize of genotype hm/hm is due to production of a cyclic tetrapeptide, HC-toxin (cyclo[D-Pro-L-Ala-D-Ala-L-2-amino-9,10-epoxi-8-oxodecanoic acid]). Cyclic tetrapeptides containing Aeo are also known to be made by five other unrelated filamentous fungi. In crosses, production of HC-toxin is controlled by a single locus, TOX2. At the molecular level TOX2 spans at least 560 kb and contains multiple copies of multiple genes. The central gene of TOX2, HTS1, contains a 16-kb ORF that encodes a tetrapartite 570-kDa non-ribosomal peptide synthetase. Other characterized genes of TOX2 include TOXA, encoding a putative HC-toxin efflux carrier; TOXE, encoding a unique pathway-specific transcription factor, and TOXG, encoding an alanine racemase. The site of action of HC-toxin is histone deacetylase (HDAC), a key enzyme linking transcriptional regulation (i.e., repression) and chromatin structure in all eukaryotes. We are investigating the mechanism by which C. carbonum protects its HDACs from inhibition by its own toxin. The bulk HDAC activity of strains of C. carbonum that make HC-toxin (Tox2+) is resistant to HC-toxin and to other HDAC inhibitors, whereas the HDAC activity of other fungi, including N. crassa, A. nidulans, and yeast, are sensitive. We have obtained evidence for two mechanisms of self-protection in C. carbonum. One is an apparent extrinsic protective factor, present only in Tox2+ isolates, that can protect the toxin-sensitive HDAC activity extracted from toxin non-producing (Tox2-) isolates. A second mechanism is an apparent post-translational modification of the major sensitive HDAC activity (ccRpd3) of C. carbonum. Whereas yeast has at least five genes that encode NAD+-independent HDACs (RPD3, HDA1, HOS1, HOS2, and HOS3), to date only three have been found in filamentous fungi including A. nidulans and C. carbonum. ccRPD3 in C. carbonum is apparently an essential gene, but strains mutated in ccHOS2 and ccHDA1 are viable. The cchos2 mutant has altered spore morphology, reduced growth on alternate carbohydrate and polysaccharide carbon sources, reduced expression of genes encoding extracellular cell wall degrading enzymes, and reduced virulence on maize. These results indicate that although HDACs normally function as co-repressors of gene transcription, HDACs also have a role in gene activation.

Basidiomycete pathogens: sex, signaling and morphogenesis. J. Kronstad, N. Lee, K. Wake, D. Laidlaw, M. Moniz de Sa, K. Tangen, B. Steen, K. MacDonald, T. Lian, G. Jiang, M. Marra* and S. Jones*. University of British Columbia, Biotechnology Laboratory, Vancouver, B.C., Canada; *B.C. Genome Sequence Centre, B.C. Cancer Agency, Vancouver, B.C., Canada

Experiments with basidiomycete pathogens such as Ustilago maydis and Ustilago hordei (the smut fungi), and the human pathogen Cryptococcus neoformans, have provided considerable insight into the role of mating and signaling in fungal virulence. In the smut species, the a and b mating-type genes control cell fusion as well as subsequent establishment and maintenance of the filamentous, infectious dikaryon. Our comparison of mating-type regions from U. maydis and U. hordei led to the discovery that the MAT region in U. hordei is over 500 kb in size. We have recently constructed a physical map of the genome by BAC clone fingerprinting; this map will serve as a platform for additional comparisons of mating-type regions from the smut fungi and for sequencing the MAT region. In the smut fungi, MAPK and cAMP signaling pathways are interconnected with mating-type regulation and play critical roles in virulence. We have characterized downstream targets of cAMP signaling in U. maydis and this work identified the product of the hgl1 gene as a potential regulatory factor that controls dimorphism, pigmentation and sporulation. We also recently identified a gene whose transcription is directly or indirectly regulated by the hgl1 product. This gene plays a role in pigmentation and morphogenesis. Investigation of the cAMP pathway also revealed a role for a ras gene in the transition between budding and filamentous growth. Interestingly, mating and cAMP signaling are also involved in virulence and morphogenesis in C. neoformans. To gain a deeper understanding of these aspects of virulence, there is a need for genomic resources for C. neoformans (and for the smut pathogens). Therefore, we have constructed BAC fingerprint maps of the JEC21 (serotype D) and H99 (serotype A) strains of C. neoformans. These maps will contribute to the efforts of an international consortium to obtain the genomic sequence of these strains of C. neoformans. As part of this genomic effort with C. neoformans, we have also defined the transcriptome of both strains using serial analysis of gene expression (SAGE).

Signal transduction cascades regulating fungal development and virulence. Joseph Heitman, Department of Genetics, Duke University Medical Center

Our studies address how cells sense and respond to their environments. We focus on the opportunistic human fungal pathogen Cryptococcus neoformans. This organism is a basidiomycete with a defined sexual cycle involving mating between haploid MAT and MATa cells. Interestingly, the MAT mating type has been linked to prevalence, virulence, and ability to undergo monokaryotic fruiting. Our studies have identified components of conserved signaling pathways that regulate differentiation and virulence. These pathways include a G-protein-cAMP pathway that senses nutrients and a pheromone responsive MAP kinase cascade in which several components are encoded by the mating type loci. Related signaling pathways operate during differentiation and mating in the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and during infection of both humans and plants by fungi including Candida albicans and Ustilago maydis. These findings reveal how common pathways have adapted as organisms diverged to exploit different environments, and illustrate the power of comparative studies to uncover conserved and unique facets of signal transduction.

The arbuscular mycorrhizal fungi and their symbiotic association with plants. Maria Harrison. The Noble Foundation, Plant Biology, Ardmore, OK

Arbuscular mycorrhizas are symbiotic associations formed between plant roots and zygomycetes from the order, Glomales. These fungi, commonly termed arbuscular mycorrhizal (AM) fungi, are obligate symbionts and form associations with plant roots in order to obtain carbon from their plant partner. The symbiosis also benefits the plant and in many cases the AM fungi assist the plant with the acquisition of phosphate from the soil. AM fungi exist in the soil as large multi-nucleate, asexual spores, which are capable of germination and limited hyphal growth in the absence of a plant root. Following entry into a root, the fungus rapidly invades the intercellular spaces of the cortex and also differentiates within the cortical cells to form highly branched hyphae termed arbuscules. Nutrient exchange (phosphate and carbon) between the symbionts is assumed to occur over the arbuscule /cortical cell interface. Once intra-radical hyphae have developed, the fungus also proliferates in the surrounding soil. AM fungal hyphae are coenocytic and the hyphae in the interior of the root form a single continuum with those in the soil. The internal and external parts of this hyphal system however, exist in highly different environments and show differentiation at the metabolic and functional levels. A model legume, M. truncatula, and AM fungi, Glomus versiforme and G. intraradices, have been selected for studies of the molecular mechanisms underlying development and functioning of the AM symbiosis. High affinity phosphate transporters have been cloned from these AM fungi and studies of the regulation of phosphate transporter gene expression are in progress. EST sequencing and arrays are being used to analyze and profile gene expression in these arbuscular mycorrhizas.

Molecular background to the interaction between parasitic fungi and nematodes

Anders Tunlid, Dag Ahrén, Eva Friman, Tomas Johansson, Maja Lindeblad and Johan Åhman. Department of Microbial Ecology, Lund University, Lund, Sweden.

The nematode-trapping fungi comprise a rather large group of soil living fungi that can infect nematodes by forming special morphological structures (traps) like mycelial networks, adhesive knobs or constricting rings. Following the development of the traps, the fungi infect the nematodes through a sequence of events: attachment to the host surface, penetration, followed by invasion and digestion of the host tissue. We are examining the molecular background to the development of traps by insertional mutagenesis of the fungus Arthrobotrys oligospora. The trap of this fungus consists of a three-dimensional network of loops of specialized, differentiated cells. Among ca 5000 mutants, we have isolated a mutant in which the trap development stops after two cell divisions, thus it cannot form a loop and a three-dimensional net. These mutants are presently examined in more detail at genetical and cellular levels. To identify genes and metabolic patways that are uniquely expressed in the trap cells, we have started an EST sequencing project in the fungus Monacrosporium haptotylum which captures nematodes with the aid of adhesive knobs. In this fungus, functionally intact trap cells can be isolated from the mycelium. Three different cDNA libraries are analyzed, representing transcripts of mycelium, knobs and knobs infecting C. elegans. Finally, we are interested in the role of extracellular proteases in the penetration and digestion of infected nematodes. Using a mutational approach, we have analyzed the function of a cuticle degrading serine protease (PII) in A. oligospora. PII deletion mutants had significantly lower levels of protease activity compared to the wild-type. In bioassays with nematodes, these mutants developed significantly lower number of infection structures (traps) and had a slightly lower ability to capture nematodes compared to the wildtype. However, the mutants did not appear to be affected in the penetration of the cuticle. The above data, and experiments with "over-expressing" mutants, demonstrate that the activity of PII is important for the digestion of the infected nematodes, thus making nutrients available that the fungus can utilize for the development of new infection structures or, alternatively, for vegetative growth.

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The cytoskeleton and tip growth. I. Brent Heath, Biology Department, York University, Toronto, Canada.

Continuous tip growth and the initiation of new tips (branches) are the dominant forms of fungal growth and morphogenesis, yet we lack even a basic understanding of the essential features of both processes. The multi-component, highly dynamic and localized features of tip growth contribute to the difficulties of analysis. Nevertheless, specific patterns of F-actin and tip-high gradients of cytoplasmic calcium ions seem to be universal components of both maintenance and initiation of hyphen tip growth. Among the oomycetes, the dynamic organization of actin filaments and the response of tips to actin disruption with latrunculin B indicate that these filaments function to regulate the extensibility of the tips, localize calcium ion channels and localize exocytosis of apical wall vesicles. The formation of filament arrays characteristic of growing tips early in branch formation indicates their function as one of the early initiation intermediates. In contrast, in eufungi, the apical actin patterns are very different from those of the oomycetes. However, previous observations showing the intimate relationship between the Spitzenkörper and tip growth and the enrichment of actin in the Spitzenkörper demonstrate the importance of actin in tip growth of the eufungi too. The changes in actin patterns and the growth response of eufungal hyphae in latrunculin B indicate that among these hyphae the apical actin may be less important in controlling tip extensibility and more related to exocytosis localization. A spectrin-like protein and or cell wall properties may be more important in extensibility regulation. Interestingly, while latrunculin B clearly affects its predicted target, it does not depolymerize all forms of F-actin and slow polarized tip growth can transiently reform in its presence without formation of a detectable Spitzenkörper. Irrespective of the role of apical actin, tip growth, and thus presumably apical actin, appears to be regulated by a tip-high gradient of cytoplasmic calcium ions which can apparently be generated and maintained by both localized channel-mediated influx from exogenous sources and internal recycling mediated by the apical wall vesicles.

Motile and stationary vacuole compartments in filamentous fungi. Anne E. Ashford. University of New South Wales, School Biological Science, Sydney, Nsw, Australia

Motile vacuole systems have two distinct components: spherical vacuoles that are linked to the plasma membrane and relatively fixed in position, and tubules that extend from these and interconnect them. A number of fluorescent probes that accumulate in the vacuole lumen allow changes in vacuole dynamics in living cells to be recorded by video microscopy. Vacuolar accumulation of probes such as carboxyfluorescein is inhibited by probenecid, indicating that the probe is cleaved from the acetate in the cytoplasm and subsequently taken up across the tonoplast via non-specific anion transporters. Evidence that microtubules and not microfilaments are essential in maintenance and motility of tubular vacuole networks will be reviewed. Tubular vacuole networks are reversibly converted into a series of spherical vacuoles by addition of 40 micromolar oryzalin, while latrunculin B at 25 micromolar has no obvious effect on vacuolar tubules but removes immunocytochemcal staining of the actin caps in the hyphal tips. Under these conditions growth is inhibited while the vacuole system continues to move forward, resulting in an accumulation of tubules in the tip region. This apparent uncoupling of tip growth and vacuole motility is seen with other growth- inhibiting treaments. The effect of drugs known to perturb processes regulated by GTP-binding proteins on tubular vacuole systems indicates that tubule formation by vacuoles is regulated by GTP-binding proteins and also that different types may be involved in the apical and basal regions of the same hyphal tip cell. The responses are consistent with involvement of a dynamin-like protein in regulation of tubule formation.

Bioimaging of the Aspergillus nidulans secretory system. Susan J. Assinder1, Kelly J. Milward1, Nicholas D. Read2 and John H. Doonan3. 1School of Biological Sciences, University of Wales, Bangor, Gwynedd, Wales UK. 2 Institute of Cell and Molecular Biology, University of Edinburgh, Scotland UK, 3 Department of Cell Biology, John Innes Centre, Norwich, UK.

Filamentous fungi are exploited commercially for the production of a variety of secreted proteins. Despite the increasing industrial importance of these organisms, our knowledge of the secretory process is limited and the endomembrane system is poorly characterised. We have used green fluorescent protein (GFP) fusion constructs as vital reporters to study the dynamics of protein secretion in vivo in Aspergillus nidulans. The A. nidulans sodVIC gene encodes a protein homologous to the -COP subunit of the coatomer complex involved in protein secretion in yeast and higher eukaryotes. The gene is essential and strains carrying the temperature-sensitive sodVIC1 mutation are defective at restrictive temperature in both hyphal extension and nuclear division. The wild-type gene has been cloned by complementation of sodVIC1 from a chromosome VI-specific cosmid library. A chimeric protein containing a plant-adapted GFP fused in-frame to the C-terminus of SodVIC also complemented the temperature-sensitivity of sodVIC1 and was therefore deduced to be functional in vivo. This, and other GFP-tagged constructs, have been used visualise components of the A. nidulans secretory pathway using confocal microscopy. GFP fused to a plant ER-retention signal targeted to the endoplasmic-reticulum (ER), whereas the sodVIC:GFP fusion protein showed localisation to putative Golgi bodies concentrated at the hyphal tip. The secretory blocker Brefeldin A led to an accumulation of GFP in the ER, and decreased hyphal tip localisation by the sodVIC:GFP fusion. The data support the view that secretion in filamentous fungi occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip.

Struture and function of the Woronin body. Gregory Jedd and Nam-Hai Chua. Laboratory of Plant Molecular Biology, The Rockefeller University

Filamentous fungi have adopted a syncytial mode of cellular organization that allows the movement of cytoplasm and subcellular organelles between cells. This type of organization is likely to provide an advantage based on cooperation between cells, but also carries risks: when hyphae lyse the septal pore must be sealed to maintain cellular integrity. This function is executed by the Woronin body; a dense-core vesicle that rapidly occludes the septal pore following cell lysis. To determine both the origin and precise function of the Woronin body we purified it from Neurospora crassa and isolated a peroxisome-targeting signal-1 (PTS1) containing protein that we called HEX-1. HEX-1 is localized to the matrix of the Woronin body by immunoelectron microscopy and a GFP-HEX-1 fusion protein is targeted to the yeast peroxisome in a PTS1 and peroxin dependent manner, suggesting that the Woronin body originates in the peroxisome. In addition, the expression of hex-1 in yeast results in the formation of intra-peroxisomal Woronin-body-like structures, suggesting a HEX-1 encoded mechanism of Woronin body-core formation. Deletion of hex-1 in N. crassa eliminates Woronin bodies from the cytoplasm and results in hyphae that exhibit a cytoplasmic bleeding phenotype following cell lysis. Together, these results show that HEX-1 is necessary and sufficient for the formation of the Woronin body-core. In addition, we conclude that the Woronin body is required for the maintenance of cellular integrity following cell lysis. We are currently comparing Woronin body structure and composition in several model fungi. Preliminary results suggesting a mechanism of Woronin body formation will be presented.

COT1 kinase - a regulator of hyphal elongation - can we place it on the MAPK? Oded Yarden, Rena Gorovits, Oshrat Propheta, Saar Cohen and Zipora Resheat-Eini. Department of Plant Pathology and Microbiology , Faculty of Agricultural Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Neurospora crassa grows by forming spreading colonies. cot-1 belongs to a class of N. crassa colonial temperature-sensitive (cot) mutants, and encodes a Ser/Thr protein kinase, which is highly similar to the mammalian myotonic dystrophy kinase (DMPK). Two COT1 isoforms are produced and can be detected in cytoplasmic, nuclear and membrane fractions of the fungal cell. When grown at restrictive temperature, alterations in cell shape, changes in plasma membrane structure and a reduction in the abundance of the lower MW COT1 isoform were found to occur in a cot-1 background. The lower MW COT1 isoform was also less abundant in two additional unlinked mutants exhibiting defects in polar growth - cot-3 and cot-5 (determined to encode translation elongation factor 2 and an alpha 1,3 mannosyltransferase, respectively), when grown at the restrictive temperatures. A reduction in membrane-associated COT1 was detected at elevated growth temperatures. This was accompanied by a significant increase in proton eflux and a concomitant 2.5-fold decrease in intracellular sodium content. As amending the growth medium with inhibitors of ion pumps, sodium or sorbitol partially suppressed the cot-1 phenotype, we concluded that cellular osmoticum is altered in cot-1. The osmoeffect of sodium and sorbitol and the 3-fold levels of glycerol measured in cot-1 indicated a possible linkage between COT1 and OS2 (a component of the hyperosmotic stress response MAP kinase pathway). However, as a functional OS2 is not essential for conferring the cot-1 phenotype and glycerol accumulation (as determined by analysis of a cot-1;os-2 mutant), we suggest that an alternative cascade may be involved.

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Fungal chromosome rearrangements in the new era. David D. Perkins. Department of Biological Sciences, Stanford University, Stanford, CA

Rearrangements have long served as basic tools for the genetics of eukaryotes, providing information on genome organization and on the behavior of chromosomes in meiosis. They have been used to determine gene sequence and to relate genetic and physical maps, to reveal position effects and transvection, to examine dosage effects and gene-silencing in segmental aneuploids, to determine relationships between species, and to construct evolutionary trees. Animals and plants have led the way in these studies. The fungi were late starters in the study of rearrangements. Advantageous features of fungi such as haploidy and the survival of all four meiotic products have compensated for the disadvantage of small chromosome size. Knowledge of fungal rearrangements is still fragmentary, however, and their potentialities as experimental tools and as tags in tracing phylogenies are still largely untapped. -- Far from making the use of chromosome rearrangements obsolete, new developments in molecular genetics and genomics can be expected to increase their relevance and usefulness. DNA sequencing is certain to reveal rearrangements that are invisible to classical cytogenetics, as was shown dramatically when the completely sequenced genomes of Saccharomyces and Candida were compared. Existing examples from fungi and other organisms illustrate what may be expected from future research with fungal rearrangements. Rearrangements are already being used in new ways, such as for studying the RIP machinery, intracellular trafficking in heterokaryons, and the restoration of fertility to barren duplications. Rearrangemetns are potentially useful for determining whether segmental domains exist that are subject to epigenetic control. Junction-sequences will provide information on the origin of rearrangements, showing whether recombination occurs between relic transposable elements. Species comparisons will enable conserved, displaced segments to be identified in the genomes of related taxa, while ancient rearrangements can provide unique markers for establishing phylogenetic relationships among distant taxa..

Break-dancing chromosomes: meiosis and DNA repair in Coprinus. Mimi Zolan, Sonia Acharya, Martina Celerin, Jason Cummings, Erin Gerecke, Alex Many, Daniel Maillet, Sandra Merino, Elizabeth Sierra, and Kevin Young Department of Biology, Indiana University, Bloomington, IN 47405 USA

We study genes necessary for DNA repair and meiosis in Coprinus cinereus, a basidiomycete in which the meiotic process is naturally synchronous. Using screens for mutants sensitive to ionizing radiation, we identified four genes (rad3, rad9, rad11 and rad12) necessary for both surviving irradiation and the completion of meiosis. The genes rad11 and rad12 encode the C. cinereus orthologs of Mre11 and Rad50, respectively; these proteins form a complex necessary for pathways of DNA double strand break repair. The rad9 gene encodes a large protein whose homologs have been shown to function in mitotic sister chromatid cohesion. To examine meiotic sister chromatid cohesion, we used B mating type locus-specific probes to differentially label homologs by fluorescence in situ hybridization. We found that Rad9 is necessary for sister chromatid cohesion during meiosis, and that about half the homolog pairing defects in the rad9-1 mutant are due to defects in meiotic sister chromatid cohesion. Using direct screens for meiotic mutants, we isolated the C. cinereus spo11-1 mutant, which is completely defective in meiotic chromosome synapsis and yet fairly proficient at homolog pairing. The spo11 gene encodes a topoisomerase-like protein shown in other systems to be required for the initiation of meiotic recombination. We found that the specific inhibition of premeiotic DNA replication (by use of the C. cinereus spo22 mutation) suppresses synapsis defects for both spo11-1 and for a rad50 mutant, rad50-4. Our results show that Spo11-induced recombination is not absolutely required for synapsis in C. cinereus, and that the early meiotic role of both Spo11 and Rad50 in synapsis partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.

Post-transcriptional gene silencing: a defense mechanism conserved in eukaryotes. Carlo Cogoni, Caterina Catalanotto, Gianluca Azzalin, Giuseppe Macino. Universita' di Roma, Genetica Molecolare, Rome, Italy

Post-transcriptional gene silencing (PTGS) as a consequence of the introduction of either DNA or RNA molecules, has been found to occur in a number of species. Trangenes can induce sequence-specific mRNA degradation in plants in a phenomenon termed co-suppression, in which the expression of both the introduced transgenes and the homologous endogenous genes were co-ordinately suppressed. A similar transgene-induced PTGS mechanism called quelling occurs in the fungus Neurospora crassa. Recently, it has been found that not only transgenes but also double-stranded RNA (dsRNA) molecules when injected into C. elegans specifically interfere with the expression of homologous resident genes. Analogous examples of dsRNA interference (RNAi) were subsequently documented in a number of invertebrate and vertebrate species. Although quelling, co-suppression and RNAi show consistent differences in the events that trigger PTGS, evident similarities have lead to the idea that the underlying molecular mechanisms could be related. Genetic dissection of the quelling phenomenon in Neurospora crassa, with the identification of quelling-deficient (qde) mutants and the isolation of the corresponding genes has paved the way for the identification cellular components of the PTGS machinery. Similar genetic approaches used in different systems such as C. elegans and A. thaliana have indeed demonstrated the existence of a common genetic base for PTGS. In the last year, these genetic approaches together with biochemical studies have produced a spectacular progress in the comprehension of PTGS mechanisms and a unified model for PTGS phenomena is now emerging.

Respiration controls mitochondrial DNA stability and longevity in Podospora anserina. Annie Sainsard-Chanet, Eric Dufour and Séverine Lorin. Centre de Génétique Moléculaire du CNRS, 91118 Gif sur Yvette, France.

In Podospora anserina, vegetative growth is systematically limited and the senescence process is always correlated with mitochondrial DNA instability. To date, the control of this process is poorly understood. We recently demonstrated that respiration plays a key role in this control. Inactivation of the nuclear COX5 gene encoding subunitV of the cytochrome c oxidase complex and in consequence, the loss of the cytochromic pathway and the exclusive use of the alternative one, leads to a striking increase of longevity ( > 30-fold for some subcultures) and to stabilization of the mitochondrial chromosome. We also showed that this respiratory modification leads to a decrease in ROS and energy production. In order to determine the respiratory parameters involved in the control of longevity, we decided to directly test whether the level of expression of the alternate oxidase, assumed to limit the production of ROS in plant mitochondria, affects lifespan in P. anserina. For this, we isolated and characterized the AOX1 gene encoding the alternative oxidase. We demonstrated that both overexpression and disruption of this gene have no effect on mitochondrial DNA stability or longevity. We are now testing the effects of the overexpression of the alternative oxidase in long-lived cytochrome c oxidase deficient mutants. Our preliminary results support the hypothesis that longevity is controlled by the global respiration efficiency and not by the expression level of the alternative oxidase.

Interdependency of chromosomal and cell cycle processes. Denise Zickler1, Diana van Heemst1,2, Etta Kaffer3, Sophie Tessé1, Gwenaël Ruprich-Robert1, Marguerite Picard1. 1 Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France
2 Dept of Genetics, Agricultural University, Wageningen, The Netherlands. 3 Dept of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., Canada

SPO76/BIMD genes of Sordaria macrospora (1) and Aspergillus nidulans (2) encode a conserved protein which provides a molecular link between mitotic and meiotic chromosome morphogenesis: it localizes on chromosomes in analogous periods and both mutants exhibit closely related defects in chromosome morphogenesis in both divisions. In the absence of the protein, cohesion and compaction are coordinately affected (1). Moreover, such a transition occurs additionally at Sordaria meiotic midprophase, temporally correlated in wild-type cells with strong localization of Spo76p along the chromosome axes, synaptonemal complex (SC) formation and the loss of early recombination nodules (1). Spo76-GFPp allows direct visualization of chromosome behavior during synapsis in both wild-type and in mutants of the six genes, screened as suppressors of the sister cohesion defect of spo76-1.
Interestingly, while Spo76-GFP forms strong lines of continuous supra-axial staining during meiotic prophase in Sordaria (1), BIMD-GFP does not localize preferentially to the chromosome axes during the meiotic prophase of Aspergillus. This difference may be related to the absence of synaptonemal complex and crossover interference in A. nidulans (3, 4).
Spo76p and BIMD are functional homologues. Both mutants are sensitive to DNA-damaging agents and bimD6 displays mitotic recombination defects. It reduces the level of mitotic interhomolog recombination but does not change the ratio between crossover and noncrossover outcomes and also is normal for intrachromosomal "gene conversion". BIMD also influences cell cycle progression: overexpression causes an arrest in G1-S (2) and, when compared to wild type, the rate of progression through G1/S/G2/M is increased in the bimD6 mutant. Moreover, BIMD function is required during both G1/S and S/M phases.
Mutations in the Podospora anserina mitochondrial citrate synthase gene (cit1) reveal that the meiotic diffuse stage is likely a metabolic checkpoint for meiotic completion. This gene was identified as a suppressor of the metabolic defects of the peroxysomal assembly car1/pex2 mutants (6). All mutants are viable, show wild-type respiration rates but higher catalase activity. All cit1 mutations impair meiotic progression and most (including the null allele) block meiosis after synapsis, at the same diffuse stage when oocytes commonly stop for a long period of time.
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