Adi Beth Din1, Charles A. Specht2, Vered Zivl, Phillips W. Robbins2 and Oded Yarden1. 1 Department of Plant Pathology and Microbiology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel and 2Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
In Saccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by the CSD2/CAL1/DIT101/KTI2 gene. We have identified, isolated and structurally characterized a CSD2/CAL1/DIT101/KTI2 homologue in the filamentous ascomycete Neurospora crassa and have used a "reverse genetics" approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of a N. crassa gene (designated chs4) encoding a polypeptide belonging to the class of chitin syntheses, which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those of S. cerevisiae and Candida albicans. N. crassa strains in which chs-4 had been inactivated by the Repeat-Induced Point mutations (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in the chs-4RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme in N. crassa and that in contrast to yeasts, provide evidence for the presence of at least two complex-type chitin syntheses.
Vegetative Incompatibility in Podospora anserina Identification of Proteins Involved in Cell Death
Nathalie Bourges, Mathieu Paoletti, Corinne Clave and Joel Begueret. Laboratoire de Genetique des Champignons Filamenteux - UPR CNRS 9026 Institut de Biochimie et Genetique Cellulaires - 1, rue C. St Sadns, 33077 Bordeaux, France
In the filamentous fungus Podospora anserina, the coexistence in one cell of two allelic or non-allelic incompatible het genes leads to cell lysis. The avaibility of an incompatible thennosensitive strain, bearing two non-allelic genes (het-R and het-V), led to the display of transcriptional and/or translational regulation of genetic expression and an increase of the proteolytic activity in cells undergoing cell death.
Cloning genes under transcriptional regulation was initiated. A cDNA library was enriched in sequences preferentially expressed in cells undergoing lysis. Part of this library has been used to screen a P. anserina genomic library. Expression of the first two genes identified, lyt1 and lyt2, has been determined in other genetic backgrounds. No expression of lyt1 is observed when the lytic reaction is suppressed by mutation in specific genes (mod genes). Under the same conditions, lyt2 is expressed. The two genes might play different roles in the pathway leading to cell death. Expression of lyt1 is also observed when two other non-allelic genes (het-C and het-E) are responsible for incompatibility. Under the same conditions, lyt2 is not expressed. Some results obtained from the het-R/het- V incompatible reaction may be extrapolated to another non-allelic incompatible reaction. These different reactions exhibit common steps leading to cell death as it was suggested by the existence of common suppressor genes. lyt1 product is involved in one of these common steps. Consequences of inactivation of lyt genes and of their constitutive expression are under investigation.
Cloning P. anserina proteases involved in the incompatibilty reaction was also initiated. Previous results showed that aspartylproteases were implicated, the most active protease being protease C. Their activities are absent when incompatibility is suppressed by mutation in modA gene. Thus we initiated cloning of the corresponding genes. papA gene encoding a P. anserina aspartylprotease has been cloned by heterologous hybridization with the major aspartylprotease encoding gene of Cryphonectria parasitica. This gene has been inactivated. Comparative analysis of wild type strain and papa-inactivated strains by FPLC has allowed papa gene product identification. It doesn't correspond to protease C. No macroscopic modification of the incompatible reaction has been observed as a consequence of papA inactivation. Another aspartylprotease encoding gene has been isolated by low stringency hybridization with papA. Its sequence is in progress. Partial purification of protease C has been undertaken to establish a microsequence and to clone the corresponding gene.
Isolation and Microscopic Characterization of Oleate Non-utilizing Mutants in Aspergillus nidulans.
J.R. De Lucas, S. Valenciano, C. Amor & F. Laborda. Dept. Microbiologia & Parasitologia, Universidad Alcald de Henares, Madrid, Spain.
Despite the limited information on the role of microbodies in filamentous fungi, recent studies have stressed the importance of these organelles. The final enzyme of the penicillin biosynthesis pathway has been located in microbodies of Penicillium chrysogenum. More recently, peroxisomes have been shown to be required during karyogamy in Podospora anserina. Isolation of peroxisomes biogenesis mutants in A. nidulans, could help in further improvements in antibiotic production as well as in determine the role of these organelles in the metabolism of filamentous fungi.
In order to isolate peroxisomes mutants in A.nidulans, we used a new positive method of selection recently utilized in Saccharomyces cerevisiae. This procedure is based in the lethality of H2O2 during the -oxidation of fatty acids in the presence of the catalase inhibitor 3-aminotriazole. Cells that do not accumulate H,O, as a result of, either a non-functional -oxidation system or ill-assembled peroxisomes are able to grow.
Using UV-light mutagenesis, 400 stable mutants resistant to 3-AT were obtained. They were checked for their inability to grow in minimal oleic acid medium and their ability to grow in minimal medium plus glycerol or maltose. A total of 40 oleate non-utilizing (olu) mutants were isolated.
Complementation analyses through heterokaryons indicated that the olu mutants fall into 5 complementation groups. Preliminary characterization studies pointed out their inability to grow in acetate as sole carbon source, suggesting that the olu mutants do not seem to carry any lession in the -oxidation pathway. Since the selection method used in this work yielded peroxisome assembly mutants in S.cerevisiae, it is possible that lessions in our olu mutants are concerned with the biogenesis of peroxisomes. Electron microscopy studies in the wild-type and olu mutants are in progress and results will be presented.
Chemical Characterization of SC3 Hydrophobin
Onno M. H. de Vries, Han A.B. Wosten and Joseph G. H. Wessels. Groningen Biotechnology and Biomolecular Sciences Institute, Department of Plant Biology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
The Class I hydrophobin SC3 of Schizophyllum commune self-assembles at hydrophobic/hydrophilic interfaces into a 10 nm, highly insoluble amphipathic film, thereby changing the wettability of surfaces. To understand the mechanism underlying the process of self-assemblage and to explore various technical/medical applications a chemical and structural characterization of SC3 is required.
The secreted protein contains 100-101 a.a. with 8 cysteines located in a characteristic pattern. Amino acid composition analysis agreed well with that predicted. A Cys-count analysis using iodoacetate/iodoacetamide mixtures confirmed the presence of 8 Cys residues per molecule. Since SC3 was found not to contain any free SH groups it is likely that all Cys groups are involved in disulphide bridges.
SC3 is a glycoprotein binding ConA. TLC and GLC analysis after acid methanolysis showed the presence of 22-24 residues mannose per molecule, assuming a mass of 9.8 kD for the polypeptide. On the other hand, mass spectrometry showed an average mass of 14.2 kD, implicating 27 mannose residues assuming no other post-translational attachments. The mannose residues are likely linked to serine and threonine res. (25 in total) since no putative N-glycosylation site is present. They are mainly located at the hydrophilic side of the SC3 film after self-assembly as shown by XPS measurements.
Isoelectrofocusing revealed several isoforms: a major form with pI 5.4 (77 %), and minor forms with pl 4.9 (14 %) and pI 4.7 (1.5 %). The calculated pl of the unmodified protein is 6.8, indicating that SC3 contains acidic modifications.
The Promoter Region of the niiA-niaD Gene Cluster of Aspergillus nidulans contains a Meiosis-specific Recombination
Initiation Site
Hans Thijs, Henk van den Broek and Theo Goosen. Department of Genetics, Wageningen Agricultural University, Dreijenlaan 2, 6703HA Wageningen, The Netherlands
Genetic markers typically show a Mendelian segregation (2:2) in meiosis, but with a low frequency deviations are observed which, as a rule, affect only a single locus as adjacent loci usually segregate normally. The aberrant segregations are generally viewed as the consequence of gene conversion in which one DNA duplex (the acceptor) is altered using a non-sister duplex (the donor) as a template. This interaction probably reflects the chromosomal search for homology during meiotic prophase 1, prior to pairing and subsequent disjunction of the homologues. The mechanistic details of the detection of DNA homology between chromosomes are not thoroughly understood, but current models incorporate the formation of a heteroduplex tract between strands of nonsister chromatids that may include one or more mismatches and the physical linkage of the homologues by Holliday junctions. Aberrant segregations may originate in the repair of mismatches in the heteroduplex tract.
One characteristic of gene conversion is that the position of a marker within a locus is of strong influence on the frequency of its conversion. Typically a polarity of gene conversion is observed: markers on one side of the locus show a high gene conversion frequency in comparison with markers on the other side. This was observed genetically in many organisms, but practically all of the data correlating gene conversion frequency and physical position of a marker have been generated in the yeast Saccharomyces cerevisiae. Polarity of gene conversion is interpreted as a consequence of fixed initiation sites of heteroduplex formation and distance-dependent resolution. Markers located close to the initiation site have a higher probability to be included in the heteroduplex tract and thus to be converted. The initiation sites in the ARG4 and HIS4 genes of yeast (Petes et al, (1991) The Molecular and Cellular Biology of the Yeast Saccharomyces, Vol.1) and the niiA-niaD gene cluster of A. nidulans (Thijs et al. MGG (1995) 247 343-350) are located in the promoter regions of these genes.
We have taken advantage of the excellent possibilities for molecular manipulation in A. nidulans and introduced a series of silent point mutations in the niiA-niaD gene cluster by in vitro mutagenesis. These mutations alter restriction sites to allow detection by restriction enzyme analysis. Data from two-point crosses show that meiotic recombination events leading to functional restoration of the niiA and niaD genes are initiated in the promoter region of the cluster. The recombination tracts do not appear to cover the length of the genes and mutations included in a tract are always co-converted. The initiation site located in the promoter region is meiosis-specific; the analysis of mitotic recombination events indicate there is no specific initiation site. Implications for the mechanism of initiation and resolution of recombination intermediates will be discussed.
Mutants in Development and Differentiation of Coprinus cinereus
Ursula Kues, Jose Granado, Katerina Kertesz-Chaloupkova, Eline Polak and Markus Aebi. Institut fur Mikrobiologie, ETH Zurich, Schmelzbergstr. 7, CH-8092 Zurich
During life cycle, the Homobasidiomycete Coprinus cineretis switches from monokaryotic to dikaryotic cell growth in order to accomplish fruitbody development and sexual reproduction. Starting from a germinating haploid spore the monokaryotic mycelium is formed. It is uninucleate, has simple septa and produces asexual propagules, the so called oidiospores, on aerial hyphae. If two compatible monokaryons mate a dikaryon with two genetically different nuclei per cell and with clamp cells at each septum is formed. The sexual dikaryon represses oidiospore formation but may develop into fruitbodies where karyogamy and meiosis occur.
Asexual and sexual differentiation in Coprinus cinereus is controlled by the two mating type loci, A and B. Specific mutations in A and B can transform a monokaryon into a homokaryon that partly behaves like dikaryons. AmutBmut homokaryons are sexually fertile, form clamp cells and fruitbodies. Unlike dikaryons and similar to monokaryons, AmutBmut strains still produce oidiospores.
In our current project we use an AmutBmut strain of C. cinereus to study developmental processes in a homokaryotic background. Two different approaches were choosen to generate mutants in development. Oidiospores were either UV irradiated and or subjected to restriction enzyme-mediated DNA integration (REMI) by transformation. About 1200 UV treated colonies and 7500 REMI transformants were screened for both changes in oidiation and defects in fruitbody development. A range of specific mutations either in oidiation or in stages of fruitbody development were obtained.
Light Mediated Development in Aspergillus giganteus
T Hastings and A. J. Clutterbuck Molecular Genetics, University of Glasgow, Glasgow, GI I 6NJ, Scotland
Conidiation in the filamentous fungus Aspergillus giganieus, in common with many other fungi, is influenced by light. In this organism, two forms of conidiophore are produced in the light, short (<2mm) and long (2-20mm), while only short conidiophores are formed in the dark. In addition the long conidiophores developed in the light display positive phototropism.
To investigate the role of light on conidiophore development, conidia were mutated and resulting colonies screened by eye for altered conidiation phenotypes when grown in dark and light conditions. A number of mutant strains have been isolated, including white, yellow and pink conidial colour variants, and mutants similar in phenotype to the wetA and abaA developmental mutants of A. nidulans. In addition, four classes of mutants altered in their light response have been picked up;-
DARKGROWN LIGHTGROWNWe suggest that there is a balance between conidiophore elongation and conidial head formation, and that light promotes elongation and delays head formation. Mutants totally insensitive to light should have only short conidial heads and show no phototropism.short only very tall only
short+tall short+tall
short only short only
short only aconidial tall only
We have also isolated auxotrophic mutants suitable for use as transformation recipients and are using PCR and hybridisation approaches to identify the brlA gene.
Comparison of pheromone receptor genes in Schizophyllum commune.
Jorg Hegner, Jurgen Wendland and Erika Kothe. Philipps-University, Dept. Biology - Molecular Genetics, Karl-von-Frisch-Str., 35032 Marburg, F.R. Germany
The mating type locus B l of S. commune contains a pheromone receptor and putative pheromone genes. The pheromone receptor B l shows homology to other pheromone receptors such as Ste2 and Ste3 of Saccharomyces cerevisiae and pral and pra2 of Ustilago maydis. It also contains sites conserved among other G protein-linked receptors of the seven transmembrane domain family. The fact, that the mating system in S. commune provides nine different allelic specificities at the mating type locus B makes it a model system to investigate ligand interaction between different allelic specificities of pheromones and receptors.
To approach the mechanism of selective pheromone binding we constructed chimeric receptors of the two allelic genes bar1 and bar2 by exchanging the extracellular loops which are known to be involved in pheromone binding in other receptors.
Mutations Causing Chromosomal Aneuploidy in Aspergillus nidulans
Michelle A. Hughes and Susan J. Assinder. School of Biological Sciences, University of Wales, Bangor, Gwynedd, LL57 2UW, Wales, U.K.
When a eukaryotic cell divides, a complete set of chromosomes must be segregated faithfully to each of two daughter cells. Errors in segregation result in aneuploidy, which often causes cell death and is implicated in oncogenesis and human birth defects.
We are using Aspergillus nidulans as a model system to identify proteins that monitor chromosome stability and ensure accurate partitioning of the genetic material. Conditional-lethal, heat-sensitive (42 ) mutants were assayed at sub-restrictive temperature (37 ) for an inflated production of aneuploid colonies (1). Strains of A. nidulans mutant at one of several hfa (high frequency of aneuploidy) loci were found to be defective in the maintenance of ploidy during cell division, as shown by the generation of a high frequency of progeny exhibiting a spectrum of aneuploid phenotypes. A second group of mutations, designated sod (= stabilisation of disomy), produced progeny disomic for a specific chromosome (see accompanying poster, Whittaker et al.).
The hfa mutants have been characterised cytologically by fluorescence microscopy of DAPI- and anti-tubulin stained nuclei. For hfaB3 and hfaL1, aneuploid production can be attributed to defects in cell cycle control. The other mutants undergo apparently normal nuclear division at restrictive temperature and may represent defects in genes involved in spindle function or in monitoring cell ploidy as components of mitotic checkpoints. Data on the sensitivity of the mutants to DNA-damaging agents and spindle poisons will be presented and progress on cloning the genes will be discussed.
(I)Upshall & Mortimore (1984):Genetics 108, 107-21.
Genetics of Rhizctonia solani (Thanatephorus cucumeris)
María Julián and Jaap Keijer. Department of Virulence and Resistance, Research Institute for Plant Protection (IPO-DLO), PO Box 9060 GW Wageningen, The Netherlands
Rhizoctonia solani Kühn is the asexual form of the fungal species Thanatephorus cucumeris (Frank) Donk. R. solani is cosmopolitan in soils and is a destructive plant pathogen with a wide host range. The large variation between isolates of R. solani with respect to pathogenicity and growth characteristics and the concurrent lack of knowledge of the genetic basis of this variation make it very difficult to understand the population structure of this fungus. Isolates of R. solani are assigned to twelve anastomosis groups (AGs) based on the occurrence of hyphal fusion (anastomosis); isolates from the same AG anastomose, while in general isolates from different AGs are not able to anastomose.
Studies on the genetics of T. cucumeris have, been carried out in many of the AGs by studying the formation of heterokaryotic tufts arising in the area of contact between single-spore homokaryotic isolates. Until now only the genetics of AGs I and 4 is understood to a certain extent. It has been suggested that the genetics of AGs 2 and 3 may differ from that of AGs I and 4. We have studied the conditions for inducing the sexual stage in vitro of 76 isolates belonging to AGs 1, 2, 3 and 4. Homokaryons of AGs 1, 2 and 3 were obtained by growing sexual basidlospores into mycelium. To study the genetics of incompatibility, isolates were paired on cellophane placed over water agar and the area of contact between isolates was studied macro- and microscopically. Our first results reveal that:
- homokaryons from one AG anasiomose readily, but when homokaryons from different AGs are paired, no hyplial fusion takes place.
- heterokaryotic tufts form in the contact area when AG 1 homokaryons are paired in certain combinations, allowing to group the homokaryons in two mating types, but tufts are never observed when AG-2 or AG-3 homokaryons are paired.
- sexual and vegetative incompatibility are two different mechanisms that operate simultaneously in T. cucumeris AG- 1. Vegetative incompatibility does not prevent tuft formation.
Regulation of Asexual Spore Formation in the Homobasidiomycete Coprinus cinereus
Katerina Kertesz-Chaloupkova, Ursula Kues, Jose Granado and Markus Aebi. Institute of Microbiology, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland
The basidomycetous fungus Coprinus cinereus undergoes a life cycle with transitions between two types of mycelia. The asexual homokaryon has simple septa and forms vegetative spores (oidia) on aerial byphae. The sexual dikaryotic mycelium, by contrast, has clamp cells at each septum. It arises after fusion of two compatible monokaryons or a monokaryon with a compatible germinating oidium. The dikaryon differentiates under appropriate environmental conditions (light, temperature and nutrition) into a fruitbody in which the meiotic basidiospores are produced.
Differentiation processes in C. cinereus are controlled by two distinct mating type loci, A and B. To form a sexual dikaryon two nuclei with different A and different B specificity have to be present in the same cell. Specific activating mutations in both mating type loci lead from an asexual monokaryon to a homokaryon that shows growth characteristics of a dikaryon (formation of clamp cells) and produces fruitbodies in which caryogamy and meiosis occurs. These AmutBmut strains, however, still produce oidia typical of monokaryons. In dikaryons, oidia production is repressed by the A mating type locus.
In the work presented we have studied the effect of different environimental conditions on oidia formation in monokaryons, dikaryons and AmutBmut homokaryons. All investigated monokaryons produced oidia in the dark. The level of oldia produced was not significantly influenced by light. In contrast, spore production in AmutBmut strains was repressed in the dark, but oidia production could be induced by light at various temperatures. At 37 C, induction was at the same level as found in monokaryons. Light induction was seen already after 1 ininute of illumination. However, several hours of light treatment were required to reach the maximum of oidiation. A certain amount of light induction was also observed in all Amut B strain. This suggests that light induction must override the effect of A repression on oidiation over a range of temperatures, as well as that it induces fruitbody formation in dikaryoiis and AmutBmut strains at a temperature of 25 C.
The fact that oidiation is induced by light in AmutBmut homokaryons is not only interesting for developmental Studies but is also of practical value in preparing protoplasts for DNA transformations.
The A Mating Type Factor of Coprinus bilanatus Consists of Two Subloci Encoding Homeodomain Proteins
Ursula Kues1 and Mike P. Challen 2. 1lnstitut fur Mikrobiologie, ETH Zurich, Schmelzbergstr. 7, CH-8092 Zurich
and 2Hortictulture Research International, Wellesbourne, Warwickshire CV35 9EF, UK
Coprinus bilanatus is a two-spored secondarily homothallic basidiomycete with a multiallelic bifactorial breeding system. For dikaryon formation nuclei with different A and B mating type factor specificities have to come together either by mating or by inclusion of two distinct nuclei in a single basidiospore. A and B regulate different steps in the development of a dikaryon. A is responsible for synchronised nuclear division and clamp cell formation, B for nuclear migration and clamp cell fusion. To date seven distinct A and seven distinct B mating type specificities have been identified in four different collections of the species.
The A1 factor of C. bilanatus was identified by homology to the mep gene of C. cinereus which flanks its well characterised A locus. Molecular analysis of the C. bilanatus A1 factor revealed structural similarities to the A factors of C cinereus and S. commune. There are two closely linked subloci encoding two types of proteins with homeodomain motifs (HDI and HD2). Homology to other A factors of the same species is low. Localisation of the mep gene defines the -sublocus. The pab1 gene which was found by chromosome walking is about 40 kb apart from mep and the sublocus.
Reciprocal heterologous expression of the C bilanatus A1 factor and the C cinereus A42 factor have been demonstrated using transformation. However, not all individual genes elicit sexual development in their respective heterologous host. Active interactions of A factor products are formed between compatible HDI and HD2 proteins. Heterologous expression of individual mating type genes provide evidence for interactions between HDI products of C bilanatus and HD2 products of C. cinereus. Attempts to heterologously express the C. bilanatus HD2 and C cinereus HDI genes have not proved successful; this may be due to inactivity of individual genes in the respective heterologous host or, alternatively, there miglit be no recognition between products of HD2 genes of C.bilanatus and HD I genes of C cinereus.
Hydrophobin rodlet layers in Agaricus bisporus and Schizophyllum commune fruit bodies
Luis G. Lugones and Joseph G. H. Wessels Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Department of Plant Biology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
The hydrophobins ABHI and SC4 typically occur in the fruit bodies of Agaricus bisporus and Schizophyllum commune respectively. Both hydrophobins assemble into an SDS insoluble amphipathic membrane at hydrophilic-hydrophobic interfaces. When this occurs at a water-air interface the hydrophobic side of the membrane exhibits the typical ultrastructure of rodlet fascicles.
In S. commune SC4 is immunologically detected inside the fruit bodies, lining air channels present in the plectenchyma. Rodlets can be seen on freeze fractured surfaces from the plectenchyma. In A. bisporus, ABHI was immunologically localized primarily at the outer surface of the fruit body, conferring a high hydrophobicity to this surface (contact angles up to 120 deg) but was present also throughout the fruit-body plectenchyma. Since rodlets were seen both at the outer surface and at the surface of freeze-fractured fruit bodies, we surmise that in A. bisporus too, air channels are lined with a hydrophobin membrane exposing its hydrophobic side towards the air. This would prevent these air cavities and channels to collapse and become flooded with water. In addition, we propose that these hydrophobins may play a role in early aggregation of hyphae during fructification.
The Putative RNA-helicase Encoded by the Pah-4 Gene Is Essential for Viability of the Ascomycete Podospora anserina
A. Luking, U. Schmidt & U. Stahl. Department of Microbiology and Genetics, University of Technology Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
RNA-helicases are known to be involved in various cellular processes such as translation, ribosomal biogenesis, cell growth and development. They belong to the so-called DEAD-box family of proteins, which are widely distributed among a variety of organisms ranging from bacteria to human (Linder et al., 1989).
We have identified four genes from the ascomycete Podospora anserina encoding typical DEAD box proteins by PCR screening using degenerated primers derived from two highly conserved regions of the protein family. One of these genes, pah-4, was characterized in detail. The putative PAH-4 protein shows high homology to the RNA-helicases DED1 from Saccharomyces cerevisiae and PL10 from mouse. The yeast gene was originally isolated as a nuclear suppressor of a nuclear pre-mRNA splicing defect (Jamieson et al., 1991) and seems to play a role in initiation of translation (Chang et al.,1995). The PL10 protein of mouse was found to be involved in spermatogenesis (Leroy et al., 1989).
In order to examine the biological role of the putative Podospora RNA-helicase, we have disrupted the gene by homologous recombination. However, viable homokaryotic spores isolated from back-crosses of pah-4 mutants to the wildtype strain were exclusively found to carry the wildtype gene. Moreover, under conditions selecting for the inactivated gene, heterokaryotic mycelia show an altered phenotype and sexual development is impaired. These results indicate that the PAH-4 protein is essential for the life cycle of the fungus.
We are now planning to restore the phenotype of the heterozygote by complementation with a helicase gene displaying a high homology to the Podospora gene.
Linder, P. et al., Nature 337, 121- 122 (1989)
Jamieson, D. J., Rahe, B., Pringle, J. & Beggs, J. D., Nature 349, 715- 717 (1991)
Chang, T. H., Chuang, R. Y. & Weaver, P., Poster presented at the RNA Processing Meeting, CSH, May 17- 21, 1995
Disruption of Rco-3, a Gene Involved in Glucose Sensing in Neurospora Abolished the Glucose Repression Control and Altered Conidia Development
Lea Madi1, Sheila A. McBride and Daniel J. Ebbole2 1present Address: Department of Plant Pathology and Microbiology,
Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel. 2Department of Plant Pathology and Microbiology,
Texas A &M University, College Station TX 77843, USA.
Conidiophore development in Neurospora is regulated by rco-3, a gene that shares homology with hexose transporters. Disruption of rco-3 gene in the wildtype strain by gene replacement, resulted in phenotype similar to the rco-3 strain. Growth in media containing non-glucose carbon sources did not suppress conidiation in submerged culture. However, a rich medium containing peptone did prevent development. Glucose transport studies suggested alterations in the kinetics of glucose uptake of the repressed rco-3 cells. Moreover, qa-2, a glucose repressible gene is consititively expressed in rco-3. The properties of the mutant suggest that rco-3 is primarily involved in glucose sensing. Mutation in rco-3 resulted in signaling for starvation in nonlimited glucose levels, loss of glucose repression and altered conidia development. Our results indicating that conidiation in Neurospora is under glucose repression.
Cloning, Characterization and in Vivo Inactivation of the Phenoloxidase Genes of Podospora anserina. What Role Do They Play in Morphogenesis, Pigmentation and Alternative Respiration?
Karin Marbach, Juan Fernandez-Larrea & Ulf Stahl. FG Microbiology and Genetics, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
Podospora anserina is a filamentous fungus that produces four different phenol oxidase enzymes: laccases 1, II and III and one tyrosinase (MOLITORIS & ESSER, 1971). There are several developmental mutants of P. anserina which show abnormalities in their phenol oxidase expression, raising the question whether these enzymes play a role in morphogenesis. Additionally, most of these mutants are incapable of using the alternative respiratory pathway, which suggests a link between alternative respiration and phenol oxidases (FRESE & STAHL, 1992). Indeed, most of these nuclear mutants were originally isolated as phenol oxidase-negative mutants (PRILLINGER, 1976). Although these mutations cause similar defects, they are located on different chromosomes, indicating that the primary defect is not in the phenol oxidase genes but is caused by more general defects. For example, ihe mutant grisea has a defective copper metabolism, resulting in reduced female fertility and no detectable phenol oxidase activity, phenol oxidases being copper dependent enzymes (MARBACH & STAHL, 1994). With these regulatory mutants it is difficult to clarify the biological function of phenol oxidases in P. anserina.
We used a "reverse genetic approach" to obtain some insight into the function of the above laccases and tyrosinase in P. anserina. The laccase II gene and the tyrosinase gene have both been cloned and characterized on the molecular level (FERNANDEZ-LARREA, 1993). These genes were then inactivated in vivo and the transformants analysed with regard to differentiation, alternative respiration and pigmentation. The biological function of laccase II and tyrosinase will be discussed in respect to these mutants.
FERNANDEZ-LARREA (1993), Thesis, University of Technology
FRESE & STAHL (1992), Mech Ageing & Dev. 65: 277
MARBACH & STAHL (1994), Curr Genet. 26: 184
MOLITORIS & ESSER (1971), Arch Mikrobiol, 77: 99
PRILLINGER (1976); Bibliotheca Mycologica, 51
Transformation and Karyotype Analysis of a Developmental Mutant from Sordaria macrospora
S. Masloff, M Nowrousian, S. Jacobsen, S. Poggeler and U Kuck. Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat, D-44780 Bochum, Germany
Sordaria macrospora is a filamentous ascomycete, which is closely related to Neurospora crassa and Podospora anserina. In contrast to the latter species S. macrospora is homothallic and shows no incompatibility. After apandrous fruit body development, asci with eight linearly arranged ascospores are formed, which can be used as ordered tetrads for a genetic analysis (1). Several developmental mutants impaired in perithecial or ascospore formation were isolated using UV mutagenesis. In this contribution genetic analysis of a mutant, showing a defect in fruit body development, is provided. This mutant was named prol (protoperithecia) and was further characterized by electrophoretic karyotyping. Using CHEF gel electrophoresis we succeeded in separation of seven chromosomal bands, with a total size of 38 Mb. In addition, nuclear genes (pro1-, rDNA-, tubA-, ura3-, ura5- and mating-type-genes) were mapped on chromosomal bands using homologous gene probes. Using a recently established transfomation system (2) molecular tagging was carried out for ectopic integration of vector pRP81-1 (3) into genomic DNA. Chromosomal mapping of the integrated vector molecules was performed by Southern hybridization of chromosomal DNA separated by pulsed-field gel electrophoresis. Genetic analysis shows close linkage of a chromosomal marker with mutation prol. In a further approach mutation pro1 was complemented to fertility by transformation with a wild-type cosmid library. We defined the complementing region of the cosmid clone to a 2,55 kb DNA-fragment.The molecular characterization of mutant prol allows identification of gene products involved in fruit body morphogenesis in ascomycetes.
[1] Esser K, Straub J (1958) Z Vererbungslehre 89:729-746 (2)Walz M, Kuck U (1995) Curr Genet (in press) (3)Ridder R, Osiewacz HD (1992) Curr Genet 21:207-213
Differential Expression of the Vegetative and Spore-bound Hydrophobins of Trichoderma reesei
Tiina Nakari-Setala1, Nina Aro1, Maria Ilmen1, Gaston Munoz1,2, Nisse Kalkkinen3 and Merja Penttila1. 1VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Finland. 2Dept. of Chemical and Bioprocess Engineering, School of Engineering, Universidad Catolica de Chile, P.O. Box 306-22, Santiago, Chile. 3 Institute of Biotechnology, P.O. Box 45, FIN-00014 University of Helsinki, Finland
We have characterized hydrophobins from the cellulolytic filamentous fungus T. reesei and previously reported the isolation of the hfb1 gene. The gene is expressed in vegetative glucose-containing cultures and the protein is secreted into the culture medium. It is also found in aggregated form in fungal cell wall wherefrom it can be extracted with trifluoroacetic acid- acetonitrile solution. As a typical hydrophobin, HFBI also aggregates on air-liquid interfaces and by freezing of HFBI containing solutions. A second hydrophobin gene, hfb2, was isolated by heterologous hybridization using the hfb1 gene as a probe. The HFBII protein encodes a protein of 71 amino acids (mature protein) that has a high amino acid similarity towards HFBI. The HFBII proteins was isolated from the fungal spores and it was also present in the culture medium of vegetative lactosegrown cultures. Expression of the hfb1 and hfb2 genes is divergent. hfb1 expression was only observed in vegetative cultures on glucose- and sorbitol-containing media. It was not found on media containing complex plant polysaccharides, cellulose, xylan, cellobiose or lactose, whereas hfb2 was highly expressed in vegetative cultures on these media. Expression of hfb2 was also strongly induced by N- and C-starvation, by light and in conidiating cultures.
Mating-type Genes in the Homothallic Ascomycete Sordaria Macrospora
Stefanie Poggeler, Siegfried Risch, Heinz Dieter Osiewacz* Ulrich Kuck. Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat, 44780 Bochum, Germany; * Biozentrum Niederursel, AK Entwicklungsbiologie Johann-WolfgangGoethe- Universitat, Marie-Curie-Str. 9, 60439 Frankfurt, Germany
In the homothallic ascomycete Sordaria macrospora (Pyrenomycetidae, Sordariaceae) a single ascospore gives rise to hyphae that are able to enter the sexual reproductive pathway and to produce fruiting bodies (perithecia) that enclose the meiotic ascospore progeny. Sexual reproduction in heterothallic ascomycetes is similar to that of S. macrospora but it differs in that a haploid ascospore is not capable of completing the sexual cycle. Heterothallic species of the Sordariaceae like Neurospora crassa and Podospora anserina are composed of two mating type populations A and a or mat- and mat+, respectively and mating occurs only between sexual structures of opposite mating-type. The sequences conferring the mating-type behaviour in N. crassa and P. anserina have been cloned and characterized. They consist of dissimilar DNA sequences (idiomorphs), which are present at a single locus in haploid strains (1,2,3). Total DNA of S. macrospora was probed with A and a mating-type sequences of N. crassa and as was already shown (4), S. macrospora contains single copy sequences from both A and a idiomorphs.
To better understand the molecular basis of homothallism and to elucidate the role of mating-products during fruting body development, we cloned and sequenced the mating-type locus of Sordaria macrospora. For further investigation of the functional conservation of the Sordaria mating-type genes we transformed the mating-type DNA into mat- and mat+ strains of the closeley related fungus P. anserina. The mating-type genes of Sordaria were partially able to complement fruiting body formation in both mat- and mat+ strains of P. anserina.
(1) Glass NL, Grotelueschen J, Metzenberg RL (1990). Proc Natl Acad Sci USA 87:4912-1916
(2) Staben C, Yanofsky C (1990) Proc Nati Acad Sci USA 87: 4917-1921
(3) Picard M, Debuchy R, Coppin E (1991) Genetics 128: 539-547
(4) Glass NL, Metzenberg RL, Raju NB (1990) Exp Mycol 14: 274-289
Gene Isolation from Sordaria macrospora Using an Indexed Genomic Cosmid Library
Stefanie Poggeler, Minou Nowrousian, Sabine Jacobsen, Ulrich Kuck. Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat, 44780 Bochum, Germany
Numerous mutants with defects in sexual morphogenesis have been isolated from the homothallic ascomycete Sordaria macrospora (Pyrenomycetidae, Sordariaceae) (1).
So far, genes responsible for these mutations have not been cloned and sequenced. To isolate these fungal genes, either by complementation or by chromosome walking we constructed a cosmid library of the S. macrospora genomic DNA using the double-cos-site cosmid vector pANsCosl (2). The average insert size of recombinant cosmid clones carrying fungal genomic DNA is about 38 kb. To improve the efficiency with which genes complementing a particular mutation can be isolated, we have established an indexed cosmid library of 4224 individual clones contained in the seperate wells of 44 microliter plates. Rapid screening methods with cosmid DNAs pooled from individual microliter dishes have been applied successfully to isolate the S. macrospora ura5 gene encoding the orotate phosphoribosyl transferase and the tubA gene encoding -tubulin. For further analysis, the ura5 gene was used for genomic complementation of a S. macrospora mutant bearing an inactive ura5 gene.
(1) Esser K, Straub J (1958) Z Vererbungslehre 89: 729-746
(2) Osiewacz HD (I 994). Curr Genet 26: 87-90
Reverse Transcriptase Activity of Group II Introns in Mitochondria of Senescing Mycelia of the Ascomycete Podospora anserina
Ralf Sagebarth, Udo Schmidt and Ulf Stahl Department of Microbiology and Genetics, University of Technology Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
The mitochondrial genome of the filamentous fungus Podospora anserina race sl contains two group IIA introns: intron cox1-I1, which is the first intron of the cytochrome-c-oxidase subunit I gene (coxl), and intron ND5-14, which is the fourth intron of the NADH-dehydrogenase subunit 5 gene. The cox1 gene of race A contains an additional group IIA intron (coxl-14). In senescing cultures of P. anserina the complete coxl-I1 accumulates as a circular plasmid (p1DNA or -senDNA). The introns complete cox1-I1 and coxl-14 but not ND5-I4 integrate at their homologous position of the corresponding intron-containing gene leading to tandem repeats of the introns (Sainsard-Clianet et al. 1994, R. Sdgebarth, unpublished results).
As discussed for the integration of group II introns into intronless alleles during crosses (intron homing) in yeast (Kennell et al., 1993), this mobility very likely depends on the reverse trancriptase activitiy of the intron encoded protein.
Since p1DNA and the spliced intron lariat coxl-Il accumulates during the course of aging, we are planning to investigate, whether this accumulation also results in an increase of RT activity.
Therefore, mitochondrial ribonucleoprotein (RNP) particles from young, middle-aged and old mycelia of race A and s were prepared and the RT activity was assayed by digesting the endogenous RNA template with RNase A and using the artificial template-primer substrate poly(A)-dT18.
Sainsard-Chanet, A., Begel, O., and Belcour, L. (1994). J.Mol.Biol. 242, 630-643.
Kennell, J.C., Morati, J.V., Perlman, P.S., Butow, R.A., and Lambowitz, A.M. (1993). Cell 73, 133-146.
Hyphae of the thn Mutant of Schizophyllum commune Have a Different Cell-wall Biosynthesis than Wild-type Hyphae
Frank H.J Schuren Microbiological Institute, Swiss Federal Institute of Technology, 8092 Zurich, Switzerland
Development of aerial structures in the basidiomycete Schizophyllum commune is regulated by the THN I gene.
A frequently occurring spontaneous mutation in this gene prevents formation of aerial mycelium in a monokaryon and, if present in both nuclei, formation of aerial hyphae and fruit-bodies in a dikaryon, Genes specifically expressed during formation of aerial structures (e.g. hydrophobin genes) are not expressed in mutant colonies. These colonies are further characterized by very characteristic wavy (sometimes corkscrew-like) hyphae having a larger diameter than normal hyphae, by a faster radial growth rate than wild-type colonies (although the biomass of a thn colony is only about half of that of a wild-type colony) and by producing a pungent smell. It was observed that the tips of thn hyphae are easily damaged by mechanical stress not harmful to wild-type hyphae. In agar-grown cultures thn hyphae quite frequently showed balloon-like vesicles out of which sometimes hyphae can start growing again. These balloons were not observed in liquid cultures and only very rarely in wild-type colonies. Addition of Congo red (binding to non-cross-linked B-1,4linked sugars) to the medium increased the number of thn hyphae ending in these balloons dramatically whereas this was not seen in wildtype hyphae. Calcofluor white (binding non-cross-linked B-1,4-linked sugars) and aniline blue (reacting with B-1,3 glucan) stained a far larger part of the apical region of thn hyphae than of wild-type hypliae and also showed dispersed staining in more subapical parts of thn hyphae. All these observations indicate a difference in the cell-wall biosyntliesis of thn hyphae as compared to wild-type hyphae. Further studies have to show whether this is caused e.g. by a problem with cross-linking of secreted non-polymerized cell-wall components in cell-walls of thn hypliae or by altered secretion of certain cell-wall components at the hyphal tips of thn hyphae.
apsA and apsB, Two Genes for Nuclear Positioning in Aspergillus nidulans
Nicole Sievers, Danuta Galetzka and Reinhard Fischer . Philipps-Universitat Marburg, Laboratorium fur Mikrobiologle, Karl-von-Frisch-Str., D-35043 Marburg, Germany
Nuclear migration is very important in many eukaryotic cells and absolutely crucial for apical extension of fungal hyphae. Microtubuies and the microtubule dependent motor protein dynein are essential components for the translocation process. Nothing is yet known about the regulation of the basic machinery and its coordination to other cellular functions, e.g. cell cycle, cell differentiation and morphogenesis. However, in Aspergillus nidulans two mutants have been isolated in which nuclear positioning is affected, suggesting an effect on the regulation of the process. In these mutants nuclei are clustered in hyphae in contrast to evenly distributed nuclei in the wild type. The nuclear positioning defect has a dramatic effect on asexual development. Metulaeremain anucleate and thus do not proliferate. Occasionally, nuclei enter metulae, development proceeds and thus single chains of conidia are generated.
The two corresponding genes, apsA and apsB (anucleate primary sterigmata) were cloned and sequenced. apsA encodes a 180 kD coiled coil protein with similarity to the yeast nuclear migration protein NUM1. Amino acid sequence motifs suggest a role in signal transduction or an association to the cytoskeleton. The ApsA protein and a hemeagglutinine epitope tagged version of the protein were detected in Aspergillus protein extracts with polyclonal or monoclonal antibodies respectively. apsB will be further analyzed at a molecular level. Expression studies of apsA and apsB on the transcript and the protein level are under way.
PCR Cloning of Chitin Synthase Genes from the Cultivated Mushroom Agaricus bisporus
S. Sreenivasaprasad, K.S. Burton and D.A. Wood. Department of Microbial Biotechnology, Horticulture Research
International, Wellesbourne, Warwick CV35 9EF, U.K.
Mushroom fruit bodies exhibit considerable expansion during morphogenesis. Cell wall biosynthesis is linked to chitin synthase activity. PCR of Agaricus bisporus genomic DNA with degenerate primers based on fungal chitin synthase gene sequences ainplified two fragments of 960 and 680bp. Southern hybridisation analysis of these putative chitin synthase gene fragments with a chitin synthase 2 clone from Candida albicans yielded positive signals. The chitin synthase gene fragments from A. bisporus were individually gel purified and cloned into vector pCRII using the TA cloning kit. Nucleotide sequences of these fragments were determined and analysis of the sequence data indicated homology between the A. bisporus 680bp fragment and the chitin synthase 2 gene from other fungi. Preliminary screening of a genomic library yielded positive signals; results of further screening and expression analysis will be presented.
Meiosis in Aspergillus nidulans, Cytology of Wild Type and Mutants
Klaas Swart and Edu Holub. Department of Genetics, Wageningen Agricultural University, Dreijenlaan 2, NL-6703 HA Wageningen, The Netherlands
The cytology of meiosis in Aspergillus nidulans was studied by haematoxylin staining and by acriflavin fluorescence. This study was undertaken to describe normal meiosis in this fungus and to characterise mutants blocked in meiosis. Such mutants are of' interest to identify and to clone genes that have a particular role in the progress of meiosis, either as differentiation genes or as genes involved in the process of recombination.
The meiotic cells of A.nidulans are small compared to other well studied fungi like e.g. Neurospora crassa and Sordaria macrospora. The sequence of events in meiosis could clearly be established and appeared not to differ significantly from that described for other fungi.
A uvsC mutant, defective in mitotic and meiotic recombination, was studied and was shown to be blocked at meiotic prophase 1. (Characterization of the uvsC gene revealed the homology to the yeast Rad5l gene, see poster by D. van Heemst et al). Specific meiotic mutants were isolated on basis of absence of mature ascospores in well developed cleistothecia (fruiting bodies). A meiA1 Mutant appeared to be blocked at anaphase 1. The cytology of wild type and mutant meiosis will be shown and discussed.
Isolation of meiosis specific genes from Aspergillus nidulans.
Diana van Heemst, Edu Holub, Klaas Swart, Henk van den Broek, Christa Heijting and Theo Goosen. Department of Genetics, Wageningen Agricultural University, Dreijenlaan 2, 6703HA Wageningen, The Netherlands
A first step towards a molecular analysis of meiosis in any organism, is the cloning of meiosis specific genes from that organism. In order to clone genes involved in meiosis in the filamentous fungus A. nidulans several different strategies can be followed. The strategy that will be discussed here, is transformation complementation of mutants defective in meiosis. In A. nidulans two classes of such mutants exist.
T'he first class consists of mutants that are both defective in the repair of DNA damage as well as in meiosis. Mutants in the A. nidulans uvsC gene have such a phenotype. We will describe the cloning of the uvsC gene of A. nidulans by transformation complementation of an A. nidulans uvsCII4 mutant. Sequence analysis of the smallest fragment still giving full complementation, revealed strong homology of the predicted protein sequence with all known RAD51 homologs.
T'he second class are meiosis specific mutants. Since these were not already available in A. nidulans, we have set up a screening to isolate such mei mutants. One of them, meiA1, has been cytologically characterized and seems to be blocked at one of the later stages of the first meiotic division. We will report the cloning and characterization of the corresponding wild type meiA gene, which we are currently undertaking.
Targeted Mutation of the Sc3 Gene of Schizophyllum commune Suppresses Formation of Aerial Hyphae
Marianne A. van Wetter, Theo A. Schuurs, Frank H.J. Schuren and Joseph G.H. Wessels. Department of Plant Biology, University of Groningen, 9750 AA Haren, The Netherlands
Correlative evidence suggests that in Schizophyllum commune activation of the SC3 hydrophobin gene is necessary for formation of aerial mycelium. Here we report disruption of the SC3 gene by homologous integration of an SC3 genomic fragment interrupted by a phleomycin resistance cassette. Two strains with a targeted mutation in the SC3 gene were obtained in a sample of 163 transformants by screening for the absence of SC3 secretion followed by Southern analysis. The phenotype of the mutants is particularly clear in sealed plates in which no aerial hyphae are formed at all. In open plates the mutants do form aerial hyphae which are however hydrophilic and not hydrophobic as in wild-type strains. In a dikaryon which is homozygous for the SC3 mutation normal fniit bodies are produced but aerial hyphae formed by this dikaryon are hydrophilic. Dikaryon-specific hydrophobins (SC1, SC4, SC6) are apparently unable to substitute for the SC3 hydrophobin. Complementation of a disniptant strain with an SC3 genomic clone restores formation of hydrophobic aerial hyphae in sealed plates. Currently we are working on the disruption of the dikaryon-expressed hydrophobin genes SCI, SC4 and SC6 by using a double selection system as is also used in mammalian systems and has been described for Neurospora crassa.
Reversible Dimorphism and Growth Behaviour of the thermoresistant yeast Arxula adeninivorans Ls3
T Wartmann, A. Kruger, K. Adler, I. Kunze and G, Kunze. Institut fur Pflanzengenetik und Kulturpflanzenforschung Gatersicben, Corrensstr. 3, D-06466 Gatersleben, Germany.
Arxula adeninivorans Ls3 is an anamorphic, ascomycetous, arthroconidial and xerotolerant yeast, ,which was selected from wood hydrolysates in Siberia. This yeast is able to grow at temperatures as high as 48 C in minimal salts medium or yeast-extract-peptone-medium with glucose or maltose as carbon source. A morphological change from yeast to mycelia can be induced by temperature shifts and is accompanied by an altered gene expression programme This dimorphism is reversible and the mycelia can be induced at a cultivation temperature of 43 C or higher. Depending on the morphology of the strain Ls3 (yeast phase or mycelia) the secretion behaviour as well as the spectrum of polypeptides accumulated in the culture medium is altered. Besides higher concentrations of secretary proteins, the activities of the accumulated extracellular enzymes glucoamylase and invertase, were 2 to 3 times higher in cultures grown as yeast cells than in those grown as mycelia.
Role of the sodVIC Gene in Aspergillus nidulans
Susan L. Whittaker, Susan J. Assinder and John H. Doonan1 School of Biological Sciences, University of Wales, Bangor,
Gwynedd, Wales, LL5 7 2LTW, U.K.; 1John Innes Research Centre, Norwich, NR4 7UH, U.K.
Strains of Aspergillus nidulans carrying the sodVIC1 (=stabilisation of disomy) mutation are unable to grow at the restrictive temperature of 42 (1). However, the mutation is only conditionally lethal, allowing growth at the sub-restrictive temperature of 37 . Upon transfer from permissive to sub-restrictive temperature, sodVIC1 strains produce stable disomic sectors carrying an extra copy of chromosome VI. Subsequent downshift to permissive temperature causes immediate reversion to the haploid state. Study of the sodVIC1 mutation, and others affecting the maintenance of ploidy such as sodVIAl (1), sodVI BI (1) and the hfa family ((2), see accompanying poster, Hughes & Assinder), will allow further insight into cell cycle controls ensuring high fidelity of chromosome segregation during mitosis.
The sodVIC1 mutation has been characterised cytologically by fluorescence microscopy of DAPI-stained nuclei. Asexual conidia arrest at restrictive temperature with 1-2 nuclei and fail to produce a germ tube. Temperature shift experiments indicate that the germination block is reversible up to 4-5 hours but that more prolonged incubation at restrictive temperature is lethal.
The sodVIC1 gene has been cloned by complementation using a chromosome VI-specific cosmid library (3). DNA sequence data for genomic and cDNA clones will be presented.
(1)Upshall et al. (1979) In: Sebek (ed): Procs. 3rd Int. Symp. Genetics Indust. Micro. pp 197-204.
(2)Upshall & Mortimore (1984):Genetics 108, 107-2 1. (3)Brody et al. (1991): Nucl. Acids Res. 19, 3105-9
Membranous Vesicles (Chitosomes) Are Involved in Chitin Synthase
Compartmentalization and Trafficking in Neurospora crassa
J. Hans Sietsma1, Adi Beth Din2, Vered Ziv2, Klaas A. Sjollemal and Oded Yarden2
1Department of Plant Biology and Groningen Biomolecular Sciences and Biotechnology Institute, University of
Groningen, Biological Center, Nl 9715 NN Haren, The Netherlands;
and 2Department of Plant Pathology and Microbiology and The Otto Warburg Center for
Agricultural Biotechnology, The Faculty of Agriculture, The Hebrew University
of Jerusalem, Rehovot, 76100 Israel
Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa. A single band of approximately I 10 kDa was observed in Western blots of total protein extracts of N. crassa, probed with these antibodies. However, several additional bands were labeled when membrane fraction proteins (microsomes) were probed. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are highly abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labeling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips.
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