Other

557. Molecular cloning of chitin synthase gene from Rhizopus oryzae. Ayumi Abe, *Yuji Oda, Teruo Sone, Kozo Asano . (Grad. Sch. Agr., Hokkaido Univ., Sapporo JAPAN *Natl. Agr. Res. Center Hokkaido, Memuro JAPAN)

Rhizopus oryzae and Amylomyces rouxii are well-known zygomycetes for fermentation foods in Asian countries They have the same rDNA ITS sequence and are a closely related to genome level. The remarkable difference between these two species is spore formation: R. oryzae makes abundant sporangiospore but less chlamydospore whereas A. rouxii rarely makes sporangiospore but makes abundant chlamydospore. In order to clarify this difference at molecular level, it is necessary to investigate the genes related to spore formation. In the recent research, the gene of class IV chitin synthase in R. oligosporus is revealed to be related to sporangiospore formation. Thus we analyzed chitin synthase genes in R. oryzae.

We tried to amplify chitin synthase genes (chs) from R. oryzae CBS 112.07 by using primer pairs for class I-III and class IV-V conserved amino acid sequences. Class I-III chs were divided into 6 clusters and all clusters indicated homology to class II conserved sequenceDClass IV-V chs were divided into 3 clusters and two clusters indicated homology to class IV and the other was class V. Southern hybridization analysis showed that R. oryzae had multiple chs genes in classes II and IV. Expression analysis revealed that one clsss II chs gene, named chs3, was expressed at spore formation and one class II chs gene, named chs2, was suppressed at spore formation. The chs2 is 2726 bp, encoding 856 amino acids and contain 3 putative introns. The chs3 is 2674 bp, encoding 852 amino acids and contains 2 putative introns.

558. Genetic analysis of cytoplasmic dynein structure and function in Neurospora crassa. Michael Plamann, David Madole, Dipti Gandhe, and Sonali Joshi. University of Missouri-Kansas City, School of Biological Sciences, Division of Cell Biology and Biophysics, Kansas City, MO 64110

Cytoplasmic dynein is a large, microtubule-associated motor complex that facilitates minus-end-directed transport of various cargoes. Dynein heavy chain (DHC) is >4000 residues in length, with the last two-thirds of the heavy chain forming the motor head. Six domains within the dynein motor exhibit varying degrees of homology to the AAA+ superfamily of ATPases. These domains are followed by a distinct C-terminal domain and together form a ring-like structure from which a microtubule-binding domain protrudes. Using a genetic assay in the filamentous fungus Neurospora crassa, we have isolated over 50 DHC mutants that produce full-length protein, but are defective in function. We have identified DHC point mutations in various areas including 1) AAA#1, the AAA module known to bind and hydrolyze ATP; 2) AAA#3, an AAA domain thought to regulate microtubule binding; 3) AAA#6, a domain with degenerate AAA homology lacking the conserved Walker boxes; and 4) AAA”#0”, a globular domain with feint AAA homology which is N-terminal to AAA#1. In filamentous fungi such as N. crassa, cytoplasmic dynein is required for nuclear migration and retrograde vesicle transport of organelles. While all DHC point mutations examined so far are deficient in vesicle transport, some DHC point mutants exhibit apparently normal nuclear distribution phenotypes.

559. Reactive oxygen species regulate fungal cell differentiation. Karen Alvarez-Delfín, Nallely Cano, Teresa Lara-Ortíz, David Hewitt*, Wilhelm Hansberg and Jesús Aguirre. IFC, Universidad Nacional Autónoma de México and *Farlow Herbarium, Harvard University.

Reactive oxygen species (ROS) have been regarded as inevitable harmful by-products of aerobic metabolism. Growing evidence, however, suggests that ROS play important physiological roles. The phagocyte NADPH oxidase was the first example of an enzyme dedicated to ROS production. The catalytic component of this oxidase (NOX2) utilizes NADPH and O2 to generate O2•-. Novel members of the NOX family have been described in plants and animal non-phagocytic cells. In fungi, a phylogenetic analysis shows the presence of three new subfamilies (NOXA, B and C). The number of nox genes in fungi ranges from none in yeasts and dimorphic U. maydis and C. neoformans to one, two and three, revealing an interesting correlation between the presence of nox genes and the capability to develop multicellular fruiting bodies. In A. nidulans, ROS are generated during differentiation of cleistothecia. Inactivation of the NOX NoxA markedly reduces ROS production and blocks cleistothecia development at an early stage , while noxA derepression correlates with premature and abundant development of cleistothecia. Likewise, in N. crassa the inactivation of nox-1 impairs fruiting body development, whereas the inactivation of nox-2 prevents ascospore germination. Our results show that manipulation of reactive species, as strategy to regulate cell differentiation, is ubiquitous in eukaryotes.

560. Molecular karyotypes of the phytopathogenic fungus Ascochyta rabiei and related legume-infecting Ascochyta spp. Hajime Akamatsu and Tobin L. Peever. Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, U.S.A.

The ascomycete fungus Ascochyta rabiei (Pass.) Labr. is the causal agent of Ascochyta blight of chickpea. Although it is possible to perform in vitro crosses with this fungus, electrophoretic karyotyping using pulsed-field gel electrophoresis (PFGE) has not been reported. PFGE was performed on a worldwide collection of A. rabiei and karyotypes were compared to other Ascochyta spp. including A. fabae, A. lentis, A. pisi and A. viciae-villosae from faba bean, lentil, pea, and hairy vetch, respectively. PFGE profiles of 45 isolates of A. rabiei from 19 countries revealed 10-14 chromosomes between 0.9 and 3.9 Mb and an estimated genome size of 20.5 to 30.5 Mb. Chromosome-length polymorphisms among chromosomes larger than 2.2 Mb were detected. PFGE of other Ascochyta spp. revealed a high level of interspecific variation in karyotype and all were highly dissimilar to A. rabiei. Numbers of chromosomes ranged from 13-17, 11-13 and 11-12 in A. fabae, A. lentis and A. pisi with sizes of 0.6 to 6.0, 0.6 to 4.6 and 0.4 to 3.8 Mb, respectively. The genome sizes of these species were estimated to be 30.4-39.5, 21.0-25.3 and 21.0-25.5 Mb, respectively. Assignment of nuclear ribosomal RNA and other genetic markers to chromosomes is being carried out by hybridizing cloned A. rabiei sequences to PFGE blots and linkage analyses. This data, combined with data from directed in vitro crosses, mating system analyses, host inoculations and phylogenetic analyses is being used to study the host specificity, genome evolution, and speciation of these fungi.

561. Human Bcl-2 genes affect vegetative growth, stress adaptation and apoptosis in the plant pathogen Colletotrichum gloeoesporioides. Sima Barhoom and Amir Sharon. Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel

Members of the evolutionarily conserved Bcl-2 family of proteins are central regulators of programmed cell death. They include apoptosis promoting (e.g. Bax, Bak) and inhibiting (e.g. Bcl-2, Bcl-xL) proteins, which regulate the mitochondria-mediated apoptosis pathway. In the present study we tested the ability of human Bcl-2 proteins to affect growth and survival in the plant pathogenic fungus Colletotrichum gloeosporioides f. sp. Aeschynomene (C.g.a.). Transgenic fungal isolates expressing the apoptosis-inhibiting Bcl-2 gene had various phenotypes including elevated levels of stress resistance, longevity, and modified spore and mycelium production. As a result, liquid cultures of the transgenic isolates had an extended logarithmic phase leading to over production of mycelium, enhanced conidia production in solid cultures, and higher tolerance to stresses. Transcriptional analysis revealed that the Bcl-2 isolates had elevated levels of stress resistance genes under non stress conditions. Furthermore, vegetative growth genes were found to retain high levels of expression under stress conditions. These and other results suggest that fungal vegetative growth and stress adaptation are tightly coordinated, and are regulated through a common mechanism which involves apoptosis-related proteins.

562. Adaptation of the TAP tag method to decipher protein complexes in filamentous fungi. Özgür Bayram, Kerstin Helmstaedt, Sven Krappmann and Gerhard H. Braus. Institute of Microbiology & Genetics, Georg-August-University Goettingen, Grisebachstr.8, 37077 Goettingen, Germany

Protein purification methods employing tandem affinity purification (TAP) tags have become an increasingly useful tool to gain information about the composition of cellular protein complexes and interactions among proteins. Generally, TAP tag constructs consist of two tandemly repeated Staphylococcus aureus protein A domains, one TEV protease cleavage site and a small peptide comprising a calmoduline binding domain. Originally designed for yeast expression, when expressed in the filamentous fungus Aspergillus nidulans detectable levels of TAP tag fusion proteins are rather low owing to the species' codon usage.

By application of site directed mutagenesis, we have altered all the rarely used codons in the commonly employed TAP tag construct to achieve higher rates of translation in the endogenous host. Both versions of the tag suited for N-terminal and C-terminal fusions were modified. Expression levels of these modified TAP tags were tested by construction of fusions to the green flourescent protein (GFP), the expression of which was driven by the inducible alcA promoter. After functionality could be validated, chimeric constructs with one regulator of A. nidulans fruit body formation were expressed and a purification protocol for complex enrichment could be established.

563. Quantitative trait locus (QTL) analysis in Neurospora crassa for discovery of new circadian clock genes. Cornelia Boesl¹, Elizabeth Turner², Elisabetta Trevellin³, Martha Merrow⁴, John Taylor² and Till Roenneberg¹. ¹Ludwig-Maximilians-Uiversity, Munich, Germany; ²University of California, Berkeley, USA; ³University of Padua, Padua, Italy; ⁴University of Groningen, Haren, The Netherlands.

We hypothesize that the Neurospora circadian clock is a multi-oscillator network that involves numerous genes and we are using a QTL approach to challenge our hypothesis. We measured free-running period (FRP) in constant darkness and the phase of entrainment (PoE) in a 12h/12h light-dark-cycle in each member of a QTL mapping population comprising 200 offspring of a cross between two wild type strains of N. crassa. Despite the fact that the parental strains have a similar FRP and phase, we found a wide distribution amongst the progeny for both FRP and PoE, consistent with the involvement of multiple genes in these traits. Contrary to circadian theory and experimental evidence, but similar to findings in Arabidopsis, we found no straightforward correlations between phase and FRP. The statistically strongest QTL that we identified was linked to the known clock genes, frequency and white collar-1, offering proof of principle.

564. Differentiation of onion neck rot Botrytis spp. in onion seed crops in Washington State and development of a real-time PCR assay for detection in onion seed. M.I. Chilvers¹, L.J. du Toit² and T.L. Peever¹. ¹Washington State University, Department of Plant Pathology, PO Box 646430, Pullman, WA 99164-6430, ²Washington State University - NWREC, 16650 State Route 536, Mount Vernon, WA 98273-4768.

Botrytis allii and B. aclada are the predominant species causing neck rot of onion, but are indistinguishable morphologically, with similar growth patterns on media and overlapping spore sizes. Botrytis byssoidea also causes neck rot but is far less prevalent and can be distinguished morphologically from B. allii and B. aclada. A collection of isolates of Botrytis spp. from the Columbia Basin of Washington State was examined using a PCR-RFLP technique developed by Nielsen et al (2002) for neck rot Botrytis spp. The primers specific to Botrytis spp. reported on onion were used to amplify the anonymous region of DNA of 469 isolates identified morphologically as B. allii or B. aclada, and digested with ApoI restriction endonuclease. The digested product was separated on agarose gel and stained with ethidium bromide. Sixty and forty percent of the isolates were identified as B. aclada and B. allii, respectively. The results demonstrate that both species are prevalent in onion seed crops in this semi-arid region of central Washington.

A real-time fluorescent PCR assay was developed using SYBR green chemistry to quantify the amount of neck rot Botrytis spp. (B. allii, B. aclada, and B. byssoidea) present in onion seed. The nuclear ribosomal intergenic spacer (IGS) region of target and non-target Botrytis spp. was sequenced, aligned, and used to design primers specific to B. allii, B. aclada, and B. byssoidea. Primers and amplification parameters were optimized to avoid amplifying the related species B. cinerea, B. porri, B. squamosa, and Sclerotinia sclerotiorum, as well as 15 other fungal species commonly found on or in onion seed. The assay was capable of detecting 10 fg genomic DNA extracted from pure cultures of B. allii and B. aclada. Preliminary analyses demonstrate that the real-time PCR assay can be performed without inhibition of the PCR reactions by the seed extract.

565. Molecular and cellular mycology: using fungal models to illustrate advanced biological concepts to students.. Angus L. Dawe, Biology Department and Molecular Biology Program, New Mexico State University, Las Cruces, NM 88003

Traditional mycology courses have likely formed a part of the education of most of the attendees at the 23rd FGC. Such curricula provide essential insights into basic biology and phylogeny and often incorporate laboratory exercises that teach students the characteristics of different groups as they relate to fungal identification. However, in diverse departments that handle students in a wide variety of biological disciplines, basic fungal biology may not provide the challenges that more advanced students are seeking. Many courses that straddle the divide between upper-division undergraduates and graduate students explore advanced biological topics such as the detailed analyses of intracellular or extracellular communication, for instance, or comprehensive studies of pathways critical for development and differentiation. These examples are topics that are usually described in the context of high-profile model systems, and yet fungal molecular biologists are well placed to offer similar material using examples from fungal model systems. A new course recently developed in the Biology Department at New Mexico State University seeks to exploit the vast resources available from the fungal community in a syllabus that covers advanced topics in molecular and cellular biology, genetics and genomics. The syllabus content will be discussed, together with the issue of providing mycology-centered courses that will attract students from other biology disciplines.

566. An essential role for the Neurospora frequency gene in circadian entrainment to temperature cycles. Antonio M. Pregueiro¹, Nathan Price-Lloyd², Deborah Bell-Pedersen³, Christian Heintzen², Jennifer J. Loros¹, Jay C. Dunlap¹.¹Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, ²School of Biological Sciences, The University of Manchester, Manchester, M13 9PT, UK, ³Department of Biology, Texas A&M University, College Station TX 77843

Circadian systems include slave oscillators and central pacemakers, and the cores of eukaryotic circadian clocks described to date are composed of transcription and translation feedback loops (TTFLs). In the model system Neurospora, normal circadian rhythmicity requires a TTFL in which a White Collar Complex (WCC) activates expression of the frequency (frq) gene, and the FRQ protein feeds back to attenuate that activation (reviewed in Dunlap and Loros, J. Biol. Rhythms 19, 414-424, 2004). To further test the centrality of this TTFL to the circadian mechanism in Neurospora, we exposed wild-type and frq-null strains to low-amplitude temperature cycles. We followed the peak in conidiation (a standard phase reference point) as well as the trough, and also novel (for analysis of Neurospora rhythms) phase reference points of "onset" and "offset" corresponding to the points where the daily cycle crosses the line represented by the running average level of conidiation. We found that the temperature cycle altered the waveform of the rhythm, especially the rate of rise to the peak, so that the "onset" reference point could not provide reliable estimates of phase among groups exposed to different period length temperature cycles. Based on all reliable reference markers, however, wild-type cultures were entrained to all the temperature cycles. Unlike normal strains, however, frq-null mutants did not truly entrain to the same cycles. Their peaks and troughs always occurred in the cold and warm periods respectively, strongly suggesting that the rhythm in Neurospora lacking frq function is driven by the temperature cycles. Altogether, these data as well as results of additional tests indicate that the FLO, at best, reflects the output of a weak oscillator that is a slave to the TTFL which underlies circadian rhythm generation in Neurospora.

567. A simplified method for collecting secreted proteins from Botrytis cinerea. H. El Mubarek, G.K. Podila and M.R. Davis. University of Alabama in Huntsville, Huntsville, AL.

Botrytis cinerea is an economically important plant pathogenic fungus with a broad plant host range. Conidia of B. cinerea loosely attach to the substrata by hydrophobic interactions that are easily interrupted. Upon germination, the previous interactions are superseded by the secretion of an extracellular matrix (ECM). The ECM is extremely resistant to removal from various strata and suggested to be important for the infection process. In liquid culture, the ECM has also been found to trap many of the organism's secreted proteins and masks their activities. We describe a method that anchors the fungus to cellophane or nylon membranes that are also permeable to the secreted protein. This anchoring of the fungus to a membrane reduces the deposition of the ECM, which may be excessively released as the organism searches for a substratum in a liquid culture. The pool of enzymes secreted from B. cinerea is measurably increased and can be used for subsequent purification and protein characterization. Similarly, this method allows for isolation of ECM from B. cinerea, away from secreted proteins, for further characterization.

568. NpkA, a cdc2-related kinase from Aspergillus nidulans, interacts with the UvsB^ATR kinase. Fagundes, Mrvzk¹, Savoldi, M¹, Lima, JF¹, Malavazi, I¹, Larson, RE², Goldman, MH³; Goldman, GH¹. ¹Faculdade de Ciências Farmacêuticas de Ribeirão Preto; ²Faculdade de Medicina de Ribeirão Preto; ³ Faculdade de Filosofia Ciências e Letras de Ribeirão Preto,USP, Brazil.

The DNA damage response is a protective mechanism that ensures the maintenance of genomic integrity. We have been using Aspergillus nidulans as a model system to characterize the DNA damage response caused by the anti-topoisomerase I drug, camptothecin (CPT). We report the molecular characterization of a p34Cdc2-related gene, npkA, that is transcriptionally induced by CPT and other DNA-damaging agents, and its induction in the presence of CPT is dependent on the uvsB^ATR gene. The npkA deleted strain can partially suppress HU-sensitivity caused by the uvsB^ATR and uvsD153^ATRIP checkpoint mutations. We demonstrated that uvsB^ATR gene is involved in DNA replication and the intra-S-phase checkpoints, and that the npkA deleted strain can suppress its intra-S-phase checkpoint deficiency. There is a defect in both the intra-S-phase and DNA replication checkpoints due to the npkA inactivation when DNA replication is slowed at 6 mM HU. Our results suggest that npkA gene plays a role in cell cycle progression during S phase as well as in a DNA-damage signal transduction pathway in A. nidulans.

Financial Support: FAPESP e CNPq

569. Amplification of fungal genomes by multiple displacement amplification. Simon J Foster and Brendon J Monahan. Centre for Functional Genomics, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.

The availability of genomic DNA (gDNA) of sufficient quality and quantity is fundamental to molecular genetic analysis. With the increasing amount of genome sequence data available for filamentous fungi, high-throughput methods for gDNA isolation are required, predominantly for rapid screening of deletion mutants. Many fungi are slow growing or even unculturable (e.g. obligate pathogens of plant or animals) and current DNA isolation methods for these fungi are often unsatisfactory, giving low yields and poor quality DNA. We present here the use of a whole genome amplification method for the rapid production of fungal genomic DNA.

Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes for two fungal species, Penicillium paxilli and the slow growing endophyte of grasses Epichloë festucae. Up to 10 micrograms of high molecular weight DNA was routinely amplified from less than 10 ng of crude template DNA. Rapid methods used to obtain template DNA for MDA reactions were optimized and differed for the two fungi. For P. paxilli, which sporulates profusely, mechanical disruption of spores using glass beads was sufficient; for the non-sporulating endophyte, alkaline lysis of mycelium was required.

The use of MDA-derived gDNA for downstream applications was assessed. PCR of target sequences up to 10 kb was possible and the DNA could be digested using restriction enzymes. Southern blot analysis was successful, although results depended on the size of hybridizing bands. We also show, by hybridisation to a cosmid library, that MDA-amplified DNA is representative of the genome.

570. Agrobacterium tumefaciens -mediated genetic transformation of Aspergillus carbonarius. Maria Helena P. Fungaro, Luiz Rodrigo Ito, Rubens Tadeu Duarte, Márcia Cristina Furlaneto. Centro de Ciências Biológicas, Universidade Estadual de Londrina, CP, 6001, CEP 86051-990, Londrina-Paraná. Brazil. *Corresponding author

Aspergillus carbonarius is a potent ochratoxin A (OTA) producer that has been found in cereal and food commodities. OTA has nephrotoxic effect and it has been classified as a possible carcinogenic substance for humans. Genetic studies of Aspergillus carbonarius are poorly developed and here we described a genetic transformation system for this filamentous fungus, providing an important step towards the genetic manipulation of this ochratoxigenic species. The fungus was transformed to hygromycin B resistance using the AGL-1 strain of Agrobacterium tumefaciens harboring the binary vector pPK2. Transformation frequencies ranged from 23 to 101 transformants per 10⁵ conidia. Highly resistant transformants were obtained which showed capacity of growing on increased concentrations of hygromycin B (up to 1200 ug ml^-1).High mitotic stability of the transformants (94%) was demonstrated after ten successive transfers on non-selective media.Considering the efficiency and flexibility of A. tumefaciens-mediated transformation, this method appears to be an efficient alternative to other insertional mutagenesis techniques in characterizing genes related to ochratoxin biosynthesis.

Acknowledgements CAPES/CNPq/Fundação Araucária/CPG-UEL

571. Agrobacterium tumefaciens-mediated transformation and protease mutants isolation in the entomopathogenic fungus Metarhizium anisopliae var acridum. Marcia Cristina Furlaneto¹, Rubens Tadeu Delgado Duarte¹, Ariane Coelho Donatti¹, Celso Vataru Nakamura², Augusto Schrank³, and Maria Helena Pelegrinelli Fungaro¹. ¹Universidade Estadual de Londrina, Microbiologia, Londrina, PR. ²Universidade Estadual de Maringá, Análises Clínicas, Maringá-PR. ³Universidade Federal do Rio Grande do Sul, Centro de Biotecnologia, Porto Alegre-RS, Brazil.

Metarhizium anisopliae var. acridum is regarded as one of the most promising species in the development of practical insect biological control agents of grasshoppers In Brazil, a biocontrol program has been assembled for the purpose of developing a native strain (CG423) of M. anisopliae var. acridum as a bioinsecticide against the grasshopper Rhammatocerus schistocercoides which causes severe agricultural losses. We report for the first time that Agrobacterium tumefaciens strain AGL-1 attaches to and genetically transforms M. anisopliae var. acridum strain CG423. The Agrobacterium-mediated transformation was applied using two distinct binary vectors carrying a benomyl resistance (b-tubulin) gene as a selection marker. The efficiency of transformation was up to 53 transformants per 10⁵ target conidia. High mitotic stability of the transformants (89-95%) was demonstrated after five successive transfers on non-selective media. Highly resistant transformants were obtained which showed capacity of growing on increased concentrations of benomyl (up to 100 ug ml^-1). We obtained three putative T-DNA-tagged mutants with altered protease production. Thus, the described protocol could provide a useful tool to tag genes that may be important for pathogenesis and virulence of this fungus.

Acknowledgements: CNPq/CAPES/CPG-UEL

572. Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus using T-DNA from Agrobacterium tumefaciens . Marcia Cristina Furlaneto, Ivan Gláucio Paulino Lima, Rubens Tadeu Delgado Duarte, Luciana Furlaneto, and Maria Helena Pelegrinelli Fungaro. Universidade de Londrina, Microbiologia, Londrina, Parana – Brazil.

Paecilomyces fumosoroseus causes diseases in the whitefly Bemisia tabaci which is responsable for damage to a variety of high value crops worldwide. Agrobacterium tumefaciens-mediated transformation was successfully applied to P. fumosoroseus. Conidia of P. fumosoroseus were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous gpd promoter and trpC terminator from Aspergillus nidulans. The efficiency of transformation was up to 74, 127 and 247 transformants per 10⁵, 10⁶ and 10⁷ target conidia, respectively. Following 5 serial passages of transformants on non-selective medium, 100% of the transformants were found to be mitotically stable by a conidial germination test. A.tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B (up to 900 ug/ml).The presence of hph gene was confirmed by PCR. Southern analysis revealed that the P. fumosoroseus transformants contained a randomly integrated single ou multiple T-DNA copies in the genome. Inverse PCR will be used to recover the P. fumosoroseus DNA flanking the T-DNA. Considering the efficiency and flexibility of A. tumefaciens-mediated transformation, this method appears to be an efficient alternative to other insertional mutagenesis techniques in characterizing genes that are important for the pathogenicity of P. fumosoroseus.

Acknowledgements: CNPq/CAPES/CPG-UEL

573. Analysis of MAT ideomorphs in Colletotrichum lindemuthianum. Garcia-Serrano, Monica¹, Rodriguez-Guerra, Raul² and Simpson June¹. 1. Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Unidad Irapuato, Irapuato, Gto., México. 2. INIFAP, Unidad Celaya, Celaya, Gto., México.

Colletotrichum lindemuthianum causes anthracnose on common bean (P. vulgaris) and Under favorable conditions can cause up to 100% losses in bean production. Although classified as a Deuteromycete, sporadic reports have shown that under laboratory conditions Colletotrichum lindemuthianum can be made to undergo a sexual reproductive cycle.

We have isolated two Mexican strains of C. lindemuthianum capable of reproducing sexually and our interest is to study mating in this species and compare this process with a closely related species G. gloesporoides (G. cingulata) which freely undergoes sexual reproduction in nature.

Degenerate oligonucleotide primers specific for the MAT 1-2 locus of ascomycetes produce fragments in both parental strains as is the case for other plant pathogens such as C. Graminicola and B. sacchari .

We are currently characterizing the MAT 1-2 ideomorphs of the parental C. lindemuthianum strains by TAIL-PCR and the construction of mini-genomic libraries. This characterization will be presented and discussed in relation to other ascomycetes.

574. The role of faculty-graduate collaborative course design in teaching scientific reasoning to undergraduates. Caleb A. Hodson ¹ and Patricia J. Pukkila ², ¹Graduate Program in Cell and Molecular Physiology and ² Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Effective undergraduate science education must include opportunities for students to engage in original scientific inquiry. We have found that a faculty-graduate student team is ideally suited to design and implement a sophomore-level seminar ("Introduction to Research in Biology") in which students learn to interpret scientific conclusions as the products of research, to generate and evaluate original ideas, and to develop proposals for their own work. Throughout the course, the instructors focus both on the elements of scientific reasoning (particularly the evaluation of scientific claims) and also on the attitudes and values of practicing scientists (especially the need to convince one's peers of the validity of one's arguments). These two elements allow the undergraduates to apply new habits of mind to questions of interest to them during the seminar and to other courses in both scientific and non-scientific areas both during the semester and after. The collaborative design and implementation of the course enhances its effectiveness and contributes positively to graduate professional development. We conclude that our model is adaptable to a wide range of disciplines and allows faculty, graduate and undergraduate students to share in the stimulating intellectual environment offered by a research university.

575. Recombination is enhanced in regions flanking the Cryptococcus mating type locus: implications for the evolution of a fungal sex chromosome. Yen-Ping Hsueh¹, Alex Idnurm¹, and Joseph Heitman¹. ¹Department of Molecular Genetics and Microbiology, Duke University, Durham, NC

Meiotic recombination is unevenly distributed throughout the genome. As a consequence, genetic and physical map distances do not have a simple linear relationship. Recombination hot spots and cold spots have been described in many fungi and recombination rates may in part reflect global features of chromosome structure. Here, we report that recombination is elevated adjacent to the mating type locus (MAT) in the pathogenic basidiomycete Cryptococcus neoformans. C. neoformans has an unusually large MAT locus, and recombination is suppressed between the alleles of this sexually dimorphic region of the genome. By introducing genetic markers at defined physical distances on both sides of MAT, we scored the meiotic recombination frequency on the MAT chromosome. The frequency of meiotic recombination is ~20% between the MAT locus and a flanking marker at 5, 10, 50 or 100 kb from the right or left border of MAT. This is approximately ten times higher than the recombination frequency between the markers at 10 and 100 kb from the MAT right border. Moreover, we found that recombination frequently occurs on both the left and the right sides of MAT. This result suggests that during meiosis, C. neoformans may exchange the MAT locus onto different genetic backgrounds and meanwhile, restrict the MAT locus from expanding to prevent capture of the entire chromosome. MAT linked recombinational enhancers may also have facilitated capture of genes into MAT by enabling rapid assimilation into both mating type alleles. We are currently investigating whether recombinational enhancers flank MAT or reside in the a or alpha MAT allele, and testing if MAT heterozygosity is required to stimulate recombination.

576. Growth defect and mutator phenotypes of RecQ-deficient Neurospora crassa mutants. Akihiro Kato and Hirokazu Inoue (Saitama University, Japan).

RecQ helicases function in the maintenance of genome stability in many organisms. The filamentous fungus Neurospora crassa has two RecQ homologues, QDE3 and RECQ2. We found that the qde-3 recQ2 double mutant showed a severe growth defect. In addition, sequences introduced by transfection integrated homologously much more frequently in the qde-3 recQ2 double mutant than in the wild type, in which homologous integration is rare. The growth defect was alleviated and the increase in homologous integration suppressed by mutation in mei-3, the homologue of yeast RAD51, which is required for homologous recombination (HR). These results suggest that HR is responsible for both phenotypes.

577. Infecting an introductory bioinformatics course with your favorite fungus. Steve James. Biology Department, Gettysburg College, Gettysburg, PA

Bioinformatic tools are indispensable in the daily life and work of molecular biologists. Putting these powerful tools into the hands of aspiring undergraduates provides a rigorous intellectual challenge that fosters development of original thinking and problem-solving. Fungal genomes furnish an ideal training ground for in silico gene discovery and analysis, owing to their high gene density, the relative diminution of introns, and the increasing availability and variety of fungal genomes. In a new bioinformatics course at Gettysburg College, students undertake a comprehensive project to annotate 50,000 bp of a recently released, pre-annotation fungal sequence. In the inaugural course offering, during fall of 2004, students studied the Coccidioides immitis genome. Using NCBI tools such as ORFinder, tblastn, and blastp, students identified C. immitis genes, learned to judge the quality of hits, merge exons, recognize conserved domains, and predict orthologs. The students identified paralogs within C. immitis, and constructed phylogenies among paralogs and orthologs using ClustalW. In addition, students examined synteny between the closely related C. immitis and A. nidulans genomes. Students also employed the Saccharomyces Genome Database (SGD) to investigate expression profiles and protein-protein interactions of the yeast homologs of C. immitis genes. An example of a web-based final project will be presented to show the breadth and quality of learning that can be achieved by this type of project-based approach.

578. Molecular mechanisms of stress response in the arbuscular mycorrhizal fungus Gigaspora margarita. Luisa Lanfranco and Paola Bonfante. Dipartimento Biologia Vegetale, Università di Torino and Istituto Protezione Piante, C.N.R., Torino, Italy.

Arbuscular mycorrhizal (AM) fungi form a highly compatible root symbiosis. In natural and agricultural environments, they significantly contribute to plant growth by improving mineral nutrition and by protecting plants against a variety of biotic and abiotic stresses. Little information is currently available on the molecular mechanisms of stress response operating in these organisms and whether they contribute to the dialogue with the host plant.

An EST collection from germinated spores of the AM fungus Gigaspora margarita (BEG 34) turned out to be a valuable source of genes related to defence responses. A gene encoding a metallothionein was characterised (Lanfranco et al., 2002, Plant Physiol). By screening a genomic library the promoter region was isolated and its transcriptional activity is currently under investigation. Another clone showed high similarity to CuZn superoxide dismutases (SODs). This gene was differentially expressed during the fungal life cycle. In two different host plants the highest transcript levels were found in the intraradical fungal structures. These structures were also positive to DAB reaction, used to detect H₂O₂ accumulation. These results suggest that fungal ROS scavenging systems may be an unexpected component of the plant/fungus dialogue, which allows a functional compatibility between the partners. Investigations on whether the gene is modulated upon exposure to different stresses are under way.

579. The alignment between physical and genetic maps of Gibberella zeae J.Lee(1), J.E. Jurgenson(2). J.F. Leslie(1) and R.L. Bowden(3). (1) Kansas State University, Manhattan,KS; (2) University of Northern Iowa, Cedar Falls, IA; (3) USDA-ARS Plant Science and Entomology Research Unit, Manhattan, KS.

 Jurgenson et al. (2002) previously published a genetic map of Gibberella zeae (Fusarium graminearum) based on a cross between Kansas strain Z-3639 (lineage 7) and Japanese strain R-5470 (lineage 6). The genetic map was based on 1048 AFLP markers and consisted of nine linkage groups. We aligned the genetic map with the first assembly of the genomic sequence of strain PH-1 (lineage 7) that was released by The Broad Institute (Cambridge, MA). We used 7 sequenced structural genes and 129 sequenced AFLP markers from all nine linkage groups (LG) of the genetic map. One hundred and fourteen markers were associated with nine supercontigs (SC) of the genomic sequence. LG1, LG7, LG8 and LG9 aligned with SC2 and SC5; LG2 aligned with SC3, SC8 and SC9; LG 3 aligned with SC4 and SC6; and LG4, LG5 and LG6 aligned with SC1 and SC7. Approximately 99% of the sequence was anchored to the genetic map, indicating the high quality of the sequence assembly and the relative completeness and validity of the genetic map. The alignments grouped the linkage groups and supercontigs into four sets, suggesting that there are four chromosomes in this fungus.

580. Interspecific Interactions between Ustilago maydis and fungal endophytes of maize. Keunsub Lee¹, Jean Pan², Georgiana May^1,2. University of Minnesota ¹Plant Biological Sciences Graduate Program. ²Department of Ecology, Evolution & Behavior, Minnesota, United States

Ustilago maydis, a basidiomycete fungus, can infect any above ground tissues causing smut disease in maize and teosinte. We isolated and identified a number of maize endophytes. These are asymptomatic in terms of disease symptoms but endophytes are known to have other effects against other pathogens and pests. However, the interactions between endophytes and Ustilago maydis are unknown. As a first step to investigate interspecific interactions between Ustilago maydis and endophytes, we identified Fusarium species - the most common fungal endophytes in maize - using the TEF-1 alpha sequence. Among 82 isolates, we identified 22 F. sporotrichioides, 20 F. verticillioides, 15 F. graminearum, 15 F. subglutinans, 6 F.proliferatum, 1 F.cerealis, 1 F. poae, and 1 F. oxysporum. We examined their competitive interactions in vitro.

581. The Coprinus cinereus Genome Project in the Teaching Laboratory. Walt Lilly and Allen Gathman, Southeast MO State Univ., Cape Girardeau, MO

To be well prepared for careers in research, students need significant, inquiry-based laboratory activities. For several years we have offered an inquiry-based molecular biology laboratory course, Investigative Molecular Biology and Biotechnology (IMBB), which focuses on individual student investigations. We have recently integrated materials and objectives from the C. cinereus Genome Project into IMBB. Students isolate random clones from cDNA libraries, sequence them, determine insert size, and assess the presence of their clones in the genomes of C. cinereus and related basidiomycetes using Southern hybridization. Students also perform bioinformatics analyses on the isolated gene sequences and their predicted products, including mapping them to the genomic sequence using GBrowse. Ultimately, each student prepares a publication-style manuscript. The challenges of this approach include designing research around pre-arranged lab time, keeping students focused on the semester-long objective, the cost per student, and the reality that experiments don't always proceed as expected. Technical problems of failed PCR reactions, poor quality genomic DNA isolations, and poor student lab skills present a failure risk. Students also face the reality of our limited knowledge of basidiomycete genes. In fall 2004, students selected 18 clones from a cDNA library. Nine of 14 successful sequences failed to match any known genes using Blastx. Despite the challenges, we find that student understanding of and interest in research are improved by this teaching model.

582. A mitochondrial short-chain acyl-CoA dehydrogenase from Aspergillus nidulans. Lori A. Maggio-Hall and Nancy P. Keller. Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin, USA

We have recently described a mitochondrial fatty acid beta-oxidation pathway in Aspergillus nidulans (Maggio-Hall & Keller. 2004. Mol. Microbiol. 54: 1173-85). Here we describe the disruption and characterization of another gene in the pathway, that encoding the acyl-CoA dehydrogenase (scdA). The phenotypes of the scdA deletion strain further confirmed this pathway's role in degrading short-chain fatty acids (C4-C6) as well as the amino acids isoleucine and valine. Unlike the previously described disruption of the enoyl-CoA hydratase enzyme in the pathway, the scdA mutant showed no defect during growth on long- and very long-chain fatty acids. Furthermore, the scdA echA double mutant also grew just as well as wild type on these fatty acids, indicating that the echA deletion phenotype—extremely restricted growth on long- and very long-chain fatty acids—was due entirely to toxicity of accumulated intermediates and not a failure to metabolize the fatty acids. Biochemical analysis of the scdA (dehydrogenase) mutant 12 h after transfer to hexanoate-containing medium showed that whole-cell enoyl-CoA hydratase activity was significantly diminished, suggesting a build-up of short-chain fatty acids inhibits other beta-oxidation pathways in the cell. This inhibition is not at the level of transcription, based on Northern analysis of identified enoyl-CoA hydratase-encoding genes (echA and foxA).

583. Aspergillus nidulans rad50 gene inactivation leads to hypersensitivity to DNA damaging agents, and sexual and cell cycle checkpoint defects. Iran Malavazi¹, Joel Fernandes Lima¹, Márcia Regina von Zeska Kress Fagundes¹, Vladimir P. Efimov², Maria Helena de Souza Goldman³ and Gustavo Henrique Goldman¹. ¹Faculdade de Ciências Farmacêuticas de Ribeirão Preto and ³Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil. ²Department of Pharmacology, University of Medicine and Dentistry of New Jersey, USA.

The hMRE11-hRAD50-NBS1 protein complex has emerged as a central player in the human cellular DNA damage response, and recent observations suggest that these proteins are at least partially responsible for the linking of DNA-damage detection to DNA-repair and cell cycle-checkpoint functions. We have identified A. nidulans rad50 mutant in a screen for dynein synthetic lethals. The rad50 gene was cloned by complementation of the sporulation deficiency phenotype of this mutant. A transversion G-C at the position 2509 (Ala-692-Pro amino acid change) in the rad50 mutant causes sensitivity to several DNA damaging agents. This mutation is novel and occurs at the coiled-coil region of the Rad50. We have deleted part of the coiled-coil region and few aminoacids of the Rad50-Mre11 interaction region and assessed several phenotypic traits in this deletion strain. Besides sensitivity to several DNA damaging agents, this deletion strain is also impaired in the intra-S-phase and DNA replication checkpoints. There is a genetic interaction between rad50 and bimE^APC1 at both S-phase checkpoints. Interestingly, we have also seen epistasis between rad50 and uvsB^ATR when germinating conidia of the corresponding double mutant are exposed to ultraviolet light. In addition, the rad50 gene is also important for ascospore viability.

Financial support: FAPESP and CNPq, Brazil

584. Diversity of fungal-like protists cultured from marine invertebrate guts. Wyth L. Marshall and Mary L. Berbee. University of British Columbia.

Zoosporic fungi, an assemblage of poorly understood aquatic eukaryotes, are both effective parasites and primary decomposers in aquatic ecosystems. Some species are also known to proliferate inside the guts of marine invertebrates with apparently benign, possibly mutualistic, effects. There are three lineages of these micro-eukaryotes: the Labyrinthulomycota and Oomycota (Stramenopiles), the Chytridiomycota (basal fungi), and the Mesomycetozoans (a newly discovered clade near the animal-fungal divergence). Despite their differences in origin, these groups are strikingly similar in many aspects of their reproductive, colonization, and nutritional strategies -- in effect occupying very similar ecological niches. Traditional fungal culturing techniques are being used to investigate the diversity of these fungal or fungal-like microbes within marine invertebrate digestive tracts. Host species have included echinoderms, bivalves, sipunculids, tunicates and polychaetes. As expected, one or more distinct thraustochytrid species (based on sequence identity) can be cultured from each animal, however, Mesomycetozoans are also present in many of these cultures. Sequence data from the 18S rDNA from the Mesomycetozoan isolates show 95-98% similarity to Pseudoperkinsus tapetis and Spaeroforma actica. Within this class less than ten genera have been described, all have been isolated from animals, and most are intractable to culture. The discovery of these new isolates suggests that the biodiversity and prevalence of this class is much higher.

585. Raft domains in AM fungi? Aurora Ocon and Natalia Requena. University of Tuebingen, Physiological Ecology, Tuebingen, Germany

Glomus intraradices GiGIN1 gene is the orthologue of a novel gene, GmGIN1, found in Glomus mosseae, only expressed during early developmental stages of the life cycle of the arbuscular mycorrhizal fungus. The predicted GiGin1 protein, have a two-domain structure with a putative self-splicing activity. The N-terminal domain shares sequence similarity with a novel family of GTP binging proteins while the C-terminus has a striking homology to the C-terminal part of hedgehog protein family from metazoa. The C-terminal part of hedgehog proteins is known to participate in the covalent modification of the N-terminus by cholesterol, and in the self-splicing activity which renders the active form of the protein with signalling function. Raft domains are microdomains at the plasma membrane enriched in the cholesterol and sphingolipids. They serve as platform for proteins to interact with each other during signalling events. Hh-Np, the mature signalling form of hedgehog proteins, has been shown to partition into the rafts due to its cholesterol moiety. We speculate that the active N-terminal domain from Gin1 also localizes to the plasma membrane, most likely at the raft domains, where presumably participates in the signalling events prior to symbiosis formation. To prove this hypothesis, we have first investigated the presence of lipid rafts in G. intraradices. They have been purified from spores and hyphae using Triton X-100. The presence of Gin1 in lipid rafts is being analyzed by western blot using specific antibodies. The plasma membrane H+ATPase, a raft domain marker, is being used as a positive control. The protein content of the rafts will be analyzed by 2D-gel electrophoresis and mass spectrometry.

586. Rapid RNA expression profiling of the fungus Trichoderma reesei in production conditions. Jari Rautio,^1,2 Kari Kataja,¹ Reetta Satokari,¹ Merja Penttilä,¹ Hans Söderlund,¹ and Markku Saloheimo¹. ¹VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Finland; ²Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, Finland

Microorganisms used for various types of bioprocesses are subjected to constantly changing environmental conditions to which they adapt by adjusting their cellular physiology. Changes in the genomic expression program are the first signs of adaptation to chancing conditions or to potential process disturbances. However, tools suitable for high-throughput expression monitoring of process-relevant genes are scarce. Moving to postgenomic era with a growing number of organisms has increased the interest in functional genomics, and the need for fast and reliable transcriptional profiling methods is thus growing. We have developed a rapid method for transcriptional profiling of microbial cultivations based on a novel technique called TRAC “transcriptional profiling with the aid of affinity capture”. This method allows fast gene expression analysis for sets of mRNAs by solution hybridisation with a pool of target-specific oligonucleotide probes of distinct sizes that are identified and quantified by capillary electrophoresis. The assay procedure has been semi-automated for simultaneous treatment of 96 samples using magnetic bead particle processor. To further enhance the robustness of the method it was set up to work with crude cell lysates. TRAC has been shown to produce results highly consistent with mRNA quantification by Northern hybridisation. Computational methods have been applied for design of target-specific oligonucleotide probes and to assign them into minimal number of pools. The whole assay procedure can be performed in three hours, implying its usefulness in bioprocess monitoring and control.

The developed TRAC method application has been used for monitoring the levels of a set of mRNAs in the filamentous fungus Trichoderma reesei in fermentation conditions. Chosen gene markers for bioprocess monitoring are involved in various cellular pathways including unfolded protein response, protection against various stress conditions, oxygen and nutrient limitation responses, protein synthesis and growth. Data collected from different types of fermentations, batch, fed-batch and continuous cultures, shows the potential of the method for use in optimisation of production processes and provides novel information about regulation of various genes during different phases of long cultivations. The TRAC method has also been used successfully for assessment of the steady states in chemostat cultures.

587. Agrobacterium tumefaciens-mediated transformation of the arbuscular mycorrhizal fungus Glomus intraradices. Margret Ecke, Reinhard Fischer and Natalia Requena. University of Tubingen, Plant Ecophysiology, Tubingen, Germany

Genetic manipulation of arbuscular mycorrhizal (AM) fungi has been hindered by their obligate biotrophism that narrows the cultivation under axenic conditions. In order to establish a stable transformation methodology for these fungi, we took advantage of the Agrobacterium tumefaciens mediated transformation and of an in vitro mycorrhizal system where large amounts of extraradical mycelium can be produced under axenic conditions. We carried out sensitivity screenings to different fungicides (hygromycin, benomyl and phosphinothricin) to be used as positive selection markers in this system. We found that hygromycin and benomyl were very effective at low concentrations (25 and 0.01 ?g/ml respectively) against both spore germination and hyphal growth of G. intraradices in contrast to the herbicide phosphinotricin. A binary vector was constructed containing the GFP reporter gene and hygromycin as selection marker, both under the control of the heterologous gpd promoter from Aspergillus nidulans. Extraradical hyphae of G. intraradices were transformed with this construct by co-cultivation with A. tumefaciens. Fluorescence microscopy showed a high number of transformed AM hyphae expressing GFP fluorescence. This is the first report of a mycorrhizal fungus where GFP fluorenscence has been observed and it shows that the Agrobacterium-mediated transformation could be further developed for functional analysis of fungal genes in the AM symbiosis.

588. Biochemical chacterization of the self-splicing protein GIN1 from the arbuscular mycorrhizal fungus Glomus mosseae. Natalia Requena and Esther Serrano. University of Tubingen, Plant Ecophysiology, Tubingen, Germany

GmGin1 is a mycorrhizal two-domain protein expressed during early developmental stages in G. mosseae . The C-terminus has a striking homology to the C-terminal part of the hedgehog protein family from metazoa. Hedgehog proteins are able to undergo self-splicing thanks to the Hint domain located in their C-terminus. As a result the mature N-terminal part is released covalently modified by a cholesterol moiety at its carboxy end. This lipid modification determines the proper localization of the protein at the cell membrane. We hypothesized that GIN1 would be also able to undergo self-splicing due to the conservation of the Hint domain. To assess this hypothesis the C-terminal domain was His-tagged purified. Gin1-C was shown to undergo splicing by addition of small nucleophiles such as DTT. To determine which molecule provokes the splicing in vivo, splicing assays with cholesterol and lipid extracts from spores, external mycelium and mycorrhized roots of G. intraradices were performed. The N-terminus of GIN1 shares similarity with the GTP binding protein family (IAN) evolutionary conserved from plants to humans. They are related to the control of local host defense against pathogens. We have expressed a His-tag Gin1-C protein in order to perform affinity and hydrolysis assays with radiolabelled nucleotides using cdc42 from Ustilago maydis as a positive control. Our hypothesis is that GmGIN1-N covalently bound to cholesterol will localize at a cell membrane and have GTPase signaling activity.

589. Truncation of the C-terminal domain of the ammonium transporter Mep2 abolishes transport but spares ligand sensing during Saccharomyces cerevisiae pseudohyphal growth. Julian Rutherford, John Rohde, Sandra Vergara and Joe Heitman. Molecular Genetics and Microbiology, Duke University, Research Drive, Box 3546, Durham, NC 27710 USA.

Diploid cells of the yeast Saccharomyces cerevisiae undergo pseudohyphal growth when both a fermentable carbon source is present and nitrogen is limiting. Previous studies indicate that this developmental process is dependent on the ammonium transporter, Mep2, which functions to both transport and sense ammonium ions. Our aim is to understand how Mep2 is able to sense ammonium levels that are low but sufficient to support a dimorphic switch. We have identified a novel MEP2 allele, MEP2-T, which encodes a truncated Mep2 that lacks the C-terminal cytoplasmic domain. Expression of the MEP2-T allele results in increased levels of pseudohyphal growth in cells that contain the wild-type MEP2 allele. However, the Mep2-T mutant fails to restore filamentous growth in a mep2/mep2 mutant. Together, these data establish that the C-terminus of Mep2 has a regulatory function and that Mep2 and the Mep2-T mutant function co-ordinately to signal. In addition, the Mep2-T mutant is unable to transport ammonium, indicating that the transport and sensing functions of Mep2 are separate. Interactions of the C-terminal domain with other cytoplasmic domains of Mep2 and other proteins is under investigation.

590. The distribution of chronotypes in Neurospora crassa. Shahana Sultana, Till Roenneberg, David Jacobson§ and Martha Merrow. Institute for Medical Psychology, University of Munich, Munich, Germany; §Stanford University, CA, USA; *Biologisch Centrum, University of Groningen, Haren, The Netherlands.

Circadian clocks in all phyla control functions from activity and wakefulness to gene expression. Individual clocks synchronize (entrain) differently to the 24-hour day, creating different 'chronotypes'. A chronotype is determined by the interaction of the circadian clock with environmental factors, such as light. Chronotype has been used as a quantitative tool to determine the complex genetic basis of the circadian clock. It can be quantified in Neurospora by measuring the phase of entrainment (PoE).

We measured period in constant darkness and PoE in 12 h light/12 h dark cycles for 98 N. crassa strains collected from between 0̊ and 47̊ latitude. The sam-ples were thus subjected to a large range of day lengths in their natural environment. We used long race tubes with a novel media formulation to ensure visualization of the circadian phenotype in wild type strains. For the Neurospora isolates, there is no significant correlation of phase with free running period in constant darkness, con-sistent with other recent results but overturning decades of basic research in mam-mals. Also, there is not a significant correlation between chronotype and latitude. The chronotype distribution of N. crassa is similar to that for our database of 30,000 Europeans showing a close to normal distribution, with few individuals (colloquially known as larks and owls) at the two extreme ends. These studies establish this sim-ple, fungal, genetic model system as a substrate for investigation of the clock as a complex trait.

591. Visualization of mitotic chromosomes and fluorescence in situ hybridization in Phytophthora infestans. Mami Kaneko and Masatoki Taga. Department of Biology, Faculty of Science, Okayama University, Okayama, Japan.

Although mitotic chromosome observation is the most basic measure of genome analysis in many eukaryotes, it has not been exploited for P. infestans. We developed a method to visualize mitotic chromosomes of P. infestans with the germ tubes from encysted zoospores. A cyst population produced by vigorously shaking zoospores was incubated to germinate on a glass slide for 2 h at 21C in 1% glucose, then the medium replaced with PDB containing both 0.02% colchicine and 0.07% colcemid to resume incubation for further 18-24 h, and finally treated with 1 mM cycloheximide for 1 h. The slide was immersed in the methanol:acetic acid (78:22) fixative, flame-dried, and fluorescence-stained with DAPI. Chromosome specimens thus prepared were mostly spread outside the burst germ tubes, making clear visualization of individual chromosomes possible under a microscope. Chromosomes were composed of the distinct, coiling two sister chromatids, and significantly long and slender. Chromosome counting with some Japanese and European strains suggested that chromosome number may not exceed 18 for each strain, but further work is necessary to draw an unambiguous conclusion. Fluorescence in situ hybridization with a part of 28S rRNA gene as a probe revealed the distal position of rDNA on the two chromosomes in a nucleus of a certain European strain, indicating that ploidy level of the strain is diploid.

592. The fungal gibberellin biosynthetic pathway : gene clusters and evolution. Christiane Bömke, Stefan Malonek and Bettina Tudzynski. Westfälische-Wilhelms-Universität Münster, Institut für Botanik, Schlossarten 3, D-48149 Münster, Germany

Gibberella fujikuroi is a species complex of 9 sexually fertile mating populations (MP-A to MP-I) and at least 36 anamorphic Fusarium species. One of these species, Fusarium fujikuroi (MP-C), is famous for its ability to produce high amounts of gibberellins (GAs). In the past years we identified and characterized all seven GA biosynthetic genes organized in a gene cluster. The seven enzymes catalyze 13 biosynthetic steps due to the multifunctional character especially of the four cytochrome P450 monooxygenases. Comparison of GA biosynthesis in plants and F. fujikuroi disproved the hypothesis of horizontal gene transfer between higher plants and fungi and confirms the suggestion that both organisms have evolved the GA biosynthetic pathways independently during evolution. We analyzed 50 species of the species complex and found out that almost all of them contain the entire GA biosynthetic gene cluster (90-98% sequence similarity), but do not produce any GAs. Interestingly, none of the so far analyzed Fusarium species outside the G. fujikuroi species complex, e.g. F. graminearum, contain any GA biosynthetic genes. On the other hand, fungi not closely related to the genus Fusarium, such as Sphaceloma and Phaeosphaeria, produce GAs. Therefore, it arises the question of the evolutionary origin of fungal GA biosynthetic gene clusters. We identified parts of a putative GA gene cluster in the genomes of fungal species not known as GA producers, such as M. grisea and B. cinerea.

593. A Genetic Selection for Circadian Output Pathway Mutations in Neurospora crassa. Michael W. Vitalini, Louis W. Morgan, Irene J. March and Deborah Bell-Pedersen. Center for Biological Clocks Research and Program for the Biology of Filamentous Fungi, Department of Biology, Texas A&M University, College Station, Texas 77843

 To identify components of the circadian clock in Neurospora crassa, we have carried out a genetic selection to isolate mutations that alter the expression of clock-controlled gene-1 (ccg-1). The promoter of ccg-1 was fused to the mtr gene to create plasmid pCCG1M. The mtr gene encodes a neutral amino acid permease that allows for both positive and negative selection. Loss of MTR function can be selected for based on resistance to the amino acid analog p-flourophenylalanine (FPA). Gain of MTR function can be selected for based on growth of tryptophan auxotrophs on high arginine/low tryptophan (TA) media. The pCCG1M plasmid was transformed into a bd;frq¹⁰;mtr;trp-2 strain. Levels of ccg-1 mRNA fluctuate randomly but remain elevated in a frq-null (frq¹⁰) strain as a result of the loss of a functional FRQ-based circadian oscillator. The pCCG1M transformed strain, CCG1M, displayed the predicted Mtr+ phenotype: growth on TA media, but not on FPA. Eighty mutant strains were isolated from selective media (FPA) following UV mutagenesis of CCG1M. Analysis of some of these mutations indicates that they reside in an output pathway downstream of the FRQ oscillator. The oscillator components FRQ and WC-1 accumulate with a normal periodicity in the mutant strains; whereas genes known to require a functional FRQ oscillator for rhythmic expression (ccg-1 and ccg-2) are arrhythmic in the mutant background.

594. Changes and Resources at the Fungal Genetics Stock Center. Kevin McCluskey, Sheera Walker and Mike Plamann. Fungal Genetics Stock Center, School of Biological Sciences, University of Missouri, Kansas City, Kansas City, MO.

Since the last Fungal Genetics conference, the FGSC has moved from the University of Kansas Medical Center to a newly remodeled laboratory at the University of Missouri, Kansas City. The FGSC move was largely uneventful and we have offered nearly uninterrupted service to our community. The new lab includes space for new resources housed at the FGSC including the many gene libraries from different genome projects. Among these are Neurospora, Aspergillus, Fusarium, Magnaporthe cosmid, fosmid and/or BAC libraries that are end-sequenced and mapped onto their respective genomes. Additional libraries are expected for genomes being sequenced at the Broad Institute. The FGSC has also added numerous cDNA and genomic library pools from different fungi.
With the funding of the Neurospora program project, the FGSC anticipates the addition of all the knock-out mutants as they are generated. Other systematic knock-out proposals have included the FGSC in the planning and proposal stages. As ever, the FGSC depends on the support of the fungal genetics community. If there is something that we could do, please do not hesitate to ask.

595. Isolating yeast mutants that affect mtDNA maintenance: a tool for teaching genetics and molecular biology to undergraduates. Stephan G. Zweifel. Department of Biology, Carleton College, Northfield, MN.

One of the challenges of designing an undergraduate laboratory course is to instill an appreciation of research science, yet still be able to pitch the course to students with limited laboratory experience. With its well-defined genetics and molecular biology, Saccharomyces cerevisiae is an ideal workbench for a student’s first advanced laboratory course. In the sophomore level Genetics course at Carleton College, students are given the task of identifying genes responsible for the maintenance of mitochondrial DNA (mtDNA). After an introduction to basic microbial techniques, the project begins with EMS mutagenesis and a screen for temperature sensitive mutants. The experiment is a replica-plating screen based on a yeast cell’s ability to grow on a non-fermentable carbon source only if functional mtDNA is present. The concepts of fermentation and respiration also provide an excellent opportunity to introduce biochemical pathways. Potential mutants are further characterized through classical genetic methods, asking such questions as: dominant or recessive, allelic to known mtDNA maintenance genes, located in the nucleus or the mitochondria. In the second half of the semester, students employ a series of cytological and molecular techniques to confirm mtDNA loss. DAPI staining and fluorescence microscopy, Southern blotting, and PCR analysis are used for the physical detection of mtDNA. The course also lends itself to an introduction to Bioinformatics with the investigation of known genes involved in mtDNA maintenance.

596. Surprising behaviour of G1 and mitotic cyclins in Ashbya gossypii. Katrin A. Hungerbuehler, Peter Philippsen and Amy S. Gladfelter. Biozentrum, University of Basel, Klingelbergstr. 50, CH-4056 Basel, Switzerland

The cell cycle process has been conserved throughout eukaryotes and requires temporally regulated expression, localization and degradation of cyclins and regulatory proteins. We are studying this cell cycle machinery in A. gossypii, a filamentous ascomycete, which is multinucleated and displays asynchronous mitoses such that neighboring nuclei are in different cell cycle stages.

The five A.gossypii cyclin genes have been deleted. Three cyclin genes are essential but the mutant strains were able to go through several rounds of nuclear division until they died. Localization studies showed that G1 and mitotic cyclins are nuclear but present during all cell cycle stages. However this localization alone cannot explain asynchrony of nuclear division.

One way nuclei could behave independently is if key cell cycle factors are trapped within nuclei. If nuclear sequestration is the basis for asynchrony, then how do translated proteins find the nucleus from which they were transcribed? We are investigating whether nuclear cell cycle proteins reenter their transcriptional mother nucleus. For mitotic cyclins, we showed that nuclei remain independent although proteins transcribed from one nucleus can diffuse to and enter neighboring nuclei.

Given that cyclins are freely diffusing and appear to be relatively constant in abundance, we asked whether cyclins are degraded in A.gossypii. A set of cyclin destruction box mutants was built, missing sequences important for degradation of cyclins in other systems. These mutants were viable and showed no growth defect unlike in other cells where stabilized cyclins led to an arrest in mitosis.

These data support the existence of a novel, nuclear autonomous division cycle in which cyclin levels do not cycle.

597. The Formin Homology Protein AgBni1 is involved in tip-splitting in the filamentous fungus Ashbya gossypii. Hans-Peter Schmitz, Andreas Kaufmann, Michael Koehli, Philippe Laissue, and Peter Philippsen. Biozentrum, University of Basel, Klingelbergstr. 50, 4056 Basel, Switzerland

From yeast to human, Formin Homology Proteins are known nucleators of actin filaments. In order to nucleate actin cables, formins are commonly activated by Rho-type GTPases, small proteins that can act as molecular switches.

We have shown by deleting the Ashbya gossypii formin AgBNI1 that this gene is crucial for hyphal morphogenesis and formation of an intact actin cytoskeleton. Germinating spores deleted for AgBNI1 form potato-shaped cells which lack actin cables. In contrast, germlings expressing an activated form of AgBni1, in which the auto-inhibitory domain is deleted, develop into young mycelia with extensive tip-splitting prior to emergence of lateral branches – a behavior not observed in wildtype mycelium. Four out of the seven Rho GTPases found in the Ashbya gossypii genome interacted with AgBni1 in a two-hybrid assay and are thus potential regulators of the latter. Mutant alleles coding for constitutively activated Rho GTPases were introduced into the genome. Presently, we test which of these alleles may cause a tip-splitting phenotype similar to the one observed with an activated allele of AgBNI1.

598. Visualization of organelles in the filamentous ascomycete Ashbya gossypii. Dominic Hoepfner, Tineke van den Hoorn, Claudia Birrer, Ivan Schlatter, Philippe Laissue, Hans-Peter Schmitz, Philipp Knechtle, and Peter Philippsen. Biozentrum University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Schwitzerland

Organelle inheritance and dynamics are not well understood in filamentous fungi. While in single cell organisms most organelles are passed on to daughter cells by mechanisms including division prior to or during mitosis, filamentous fungi do not produce daughter cells and thus may not require elaborate organelle inheritance mechanisms. Hyphal tips of filamentous fungi continuously elongate thereby enlarging the cytoplasmic volume, and most organelles may only elongate with the growing tip. We use the model fungus A. gossypii to test whether organelles divide or elongate during hyphal growth and to visualize their dynamics. Previous studies on nuclear dynamics using a histone-GFP fusion revealed division kinetics comparable to S. cerevisiae and extensive oscillations at all nuclear cycle stages (Alberti-Segui et al. J. Cell Science 114, 975). We constructed and analyzed amino- or carboxy-terminal GFP or CFP fusions to A. gossypii homologs of the following S. cerevisiae organelle-specific proteins: Sec63 for ER, Cox4 for mitochondria, Tfp1 for vacuoles, Sec4 for secretory vesicles, Abp140 for actin cables, Cap1 and Cap2 for actin patches, Tub1 for microtubules, and CFP fused to the peroxisomal targeting sequence PTS1. We are now in the process of combining all fluorescence data in a 3D model of organelles and cytoskeletal structures in growing A. gossypii hyphae.

599. Septins and Mitotic Regulators in the Filamentous Ascomycete Ashbya gossypii. Hanspeter Helfer and Amy S. Gladfelter. Biozentrum, University of Basel, Klingelbergstr. 50, CH-4056 Basel, Switzerland

Septins are evolutionary conserved proteins with essential functions in cytokinesis, and more subtle roles throughout the cell cycle. Much of our knowledge about septins originates from studies with S.cerevisiae, where they form a ring-like protein scaffold at the mother-bud neck. For example, the cell cycle regulators Hsl1 and Hsl7 are recruited to this septin ring and subsequently inactivate the mitotic inhibitor Swe1, a protein kinase acting on the cyclin dependent kinase (CDK), Cdc28. The genome of the filamentous ascomycete A.gossypii encodes homologues of the five S.cerevisiae mitotic septins and all cell cycle regulators including Hsl1, Hsl7, Swe1 and the phosphatase Mih1 which counteracts the protein kinase Swe1. Nuclei in A.gossypii hyphae divide asynchronously and we wanted to know whether this mode of division is spatially controlled by septins, acting as cortical markers to locally direct mitosis through regulation of Swe1. We investigated the phenotypes of septin mutants with respect to septin ring formation, nuclear density, potential induction of mitosis, branching and sporulation. Deletion of single septin genes leads to loss of septin rings and sporulation but none of the deletions tested had a major impact on nuclear division frequency. To determine whether the activity of AgCdc28 is spatially controlled by septins and interacting proteins, we have started experiments to follow AgCdc28 phosphorylation in both wild type and mutant strains. Ideally these experiments will provide insights into how conserved cell cycle regulators have evolved to function in cells with diverse shapes and number of nuclei.

600. Spitzenkörper and Polarisome of Ashbya gossypii. Michael Köhli (1), Philipp Knechtle (1), Kamila Boudier (1), Robert Roberson (2), and Peter Philippsen (1). Biozentrum University of Basel, Klingelbergstr. 50, 4056 Basel, Switzerland (1), School of Life Sciences, Arizona State University, Tempe AZ 85287, USA (2).

Ashbya gossypii is a filamentous ascomycete. Despite its close evolutionary relation to the yeast S. cerevisiae, A. gossypii exclusively grows by apical extensions and branching. The hyphae have a diameter of 3-4 micrometers and growth speeds can reach 0.2 mm/h. We are interested in the organization of the apical growth zone which, based on genome comparison with S. cerevisiae, most likely carries a very similar set of proteins known to maintain yeast polar growth. A Spitzenkörper is lacking in growing S. cerevisiae buds and, to date, the cytoplasmic organization of the hyphal tip in A. gossypii is unclear. Using video enhanced light microscopy we observed an oval, phase-dense body that almost completely filled the tip zone. The behaviour of this structure was followed and analyzed during steady state growth and tip branching.

Several GFP labelled A. gossypii proteins locate to growing tips, e.g. homologs of the S. cerevisiae polarisome component Spa2, the membrane associated polar landmark protein Rax2, or the signalling protein Boi1. Based on the shape and the dynamics of these components investigated by video fluorescence microscopy, we conclude that the presumptive A. gossypii Spitzenkörper and the polarisome are different functional units of the apical growth machinery.

601. Stability of pesticide resistance in Phytophthora infestans. Stefan Bosmans, Alfons J.M. Debets, J. Arjan G.M. de Visser and Rolf F. Hoekstra. Laboratory of genetics, Wageningen UR

Mutations that confer resistance to a pesticide are generally believed to infer a fitness cost when the pesticide is no longer used. Earlier work on several organisms suggests that this cost can be compensated by additional mutations, thus leading to a stable frequency of resistant genotypes in a population. This project aims to provide insight into the stability of genetic resistance in populations of the economically important pathogen Phytophthora infestans. We will generate genotypes that are resistant to one of the following fungicidal substances: metalaxyl, fluazinam, mancozeb, dimethomorph and cyazofamid. The resistant genotypes will be generated with repeated sub-culturing on media containing sub-lethal doses. The possible fitness consequence of the several mutations will be estimated by comparing the resistant genotypes with their ancestral genotypes. Additionally, a hypothesis about the role of heterokaryons in the stability of fungicide resistance will be presented.

602. Phenotypic analysis of a deletion of the Aspergillus nidulans homologue of yeast BRO1 shows involvement in the regulatory network that includes the CreB deubiquitination enzyme. Robin Lockington and Joan Kelly. School of Molecular and Biomedical Science, University of Adelaide, Adelaide, 5005, Australia.

A strain bearing a precise deletion of the A. nidulans creA gene, encoding the transcriptional repressor responsible for carbon catabolite repression, is viable, whereas a deletion of creA that extends into sequences downstream of creA is essentially lethal, indicating that sequences 3’ of creA are involved in the synthetic lethality. We have identified this downstream gene as the A. nidulans homologue of the yeast BRO1 gene, and named it broA. Precise deletion of broA results in a strain with pleiotropic defects including compact growth and pale conidiation on complete medium, impaired conidiation on minimal media, and impairment of the ability to utilize several carbon sources. When the broA deletion mutation was combined with a null mutation in the creB gene encoding a regulatory deubiquitinating enzyme, the broA deletion was found to suppress many of the effects of this mutation, implicating it as being involved in the CreB regulatory ubiquitination/ deubiquitination system involving the creD, acrB, creB, and creC gene products.

603. Sporulation in Stagonospora nodorum. Rohan Lowe, Peter Solomon, Richard Oliver. Australian Centre for Necrotrophic Fungal Pathogens, SABC, Murdoch University, W.A., Australia.

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. Very little is currently known about the molecular mechanisms required for pathogenicity of S. nodorum, despite its major impact on Australian agriculture. S. nodorum is a polycyclic pathogen. Rain-splashed pycnidia attach and colonise plant tissue and sporulate within 2-3 weeks. Several cycles of infection are needed to build up inoculum for the damaging infection of flag leaves and heads. Sporulation is therefore a critical component of the infection cycle of S. nodorum; our aim is to determine the genetic and biochemical requirements for sporulation in order to better understand the process.

An in planta cDNA library has been constructed using transcripts isolated from infected wheat tissue. The cDNA library will be screened for genes that are expressed during the onset of sporulation. Also, the plant/fungal metabolome has been investigated during the infection process using GC/MS. Wheat plants were infected with S. nodorum and polar metabolites extracted from the infected tissue. Changes in metabolite levels were monitored during the onset of sporulation. Key fungal metabolites identified include mannitol and trehalose. The concentration of both mannitol and trehalose increased dramatically late in the infection period. Overall sugar levels reduced as the infection progressed, while some amino acids were observed to increase in concentration. After considering both the in planta cDNA library and metabolite analysis, several candidate genes have been selected for mutagenesis in S. nodorum. 

604. Involvement of type 2B Ser/Thr phosphatase (Calcineurin) and PKA in sclerotial development of Sclerotinia sclerotiorum. Arye Harel, Rena Gorovits, Sally Bercovich, Oded Yarden. Department of Plant Pathology and Microbiology and The Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Sclerotia of Sclerotinia sclerotiorum are distinct pigmented, multihyphal structures which play a central role in the life and infection cycles of this plant pathogen. Type 2B Serine/Threonine phosphoprotein phosphatases (PP2B/calcineurin) have been demonstrated to regulate fungal cation homeostasis, morphogenesis, and pathogenesis.

To test the involvement of PP2B in sclerotial development we utilized a pharmacological and genetic approach. In the presence of the PP2B inhibitors Cyclosporin A (240nm) or FK506 (90nm), sclerotia formation was inhibited by 40 and 60%, respectively. S. sclerotiorum transformants harboring an inducible antisense PP2B expression construct exhibited near-normal hyphal elongation rates yet sclerotial formation was impaired.

As PP2B and cAMP-dependent protein kinase A (PKA) have been shown to be involved in at least one common pathway, we monitored changes in relative PKA activity levels during sclerotial development. Relative PKA activity levels increased during the white-sclerotium stage in the wild-type strain, while low levels were maintained in non-sclerotium-producing mutants. The changes in PKA activity that accompany sclerotial development in a distinct developmental phase manner and the requirement of PP2B for sclerotial development, indicate the involvement of these components, in addition to other signal transduction pathways, in the regulation of sclerotiogenesis.

605. On the evolution and ecology of fungal senescence. Marc F. P. M. Maas, Rolf F. Hoekstra and and Alfons J.M. Debets. Laboratory of Genetics, Wageningen University, The Netherlands

Filamentous fungi, being typical modular organisms, are not expected to age and die. Nonetheless there are some striking examples of aging or senescence in these fungi. Most, if not all of the cases thus far described are associated with instability of the mitochondrial genome and recent evidence has shown that this perhaps results from mitochondrial oxidative metabolism. Calorie restriction (CR) is the only life span extending regimen known that applies to all aging organisms, and also extends life span in P. anserina. Natural variation in the life span extending response of CR can in the latter species largely be explained by the presence of pAL2-1 homologous mitochondrial invertrons. The pAL2-1 homologues drastically reduce life span and are effectively similar to the structurally related plasmids pKALILO or pMARANHAR from Neurospora. Probably their effect is in the shadow of other aging factors and is drawn out of that shadow by CR, explaining why it has thus far been overlooked. We argue that fungal senescence is from an ultimate perspective, aging in the true sense of the word.

606. Agrobacterium Tumefaciens – Mediated Genetic Transformation of the Edible Straw Mushroom Volvariella volvacea. Tran Hoang Ngoc Ai, Hoang Quoc Khanh*. Institude of Tropical Biology, Vietnam Academy of sciences and Technology

  Agrobacterium tumefaciens is known to transfer parts of its tumor – inducing plasmid , the T- DNA, to plants, yeasts and filamentous fungi; therefore, we have used this system to transform germinating basidiospores and vegetative mycelium of the cultivated basidiomycete Volvariella volvacea.

   Plasmid pPK2 contains hygromycin hph gene (B phosphotransferase ) with the Aspergillus nidulans gpd promoter (glyceraldehyd - 3 - phosphate dehydrogenase ). Hph gene which is 1 kb long was designed between LB and RB of T-DNA in this plasmid DNA. Out of T-DNA is kanamycin resistant gene, used to select transformed bacteria.

   Spores of V. volvacea were collected on glass Petri dishes. The standard growth medium for V. volvacea was PDA and incubation was performed at 28oC.

   pPK2 was transformed into A. tumefaciens via electroporation. Bacterial cultivation was performed as describe in De Groot et al. ( 1998 ). For induction of virulence and T-DNA transfer, A. tumefaciens was grown on induction medium (IM) with 200 mM AS and, for negative controls, without AS. Selection for transformed mycelial colonies was performed on M-100 agar containing cefotaxime (500 mg/ml) and hygromycin B (70 mg/ml) and on CYM –R containing hygromycinB (70m/ml). Hph gene sequence of T-DNA which integrated genomic DNA of V. volvacea was checked by PCR. Analysis of transformants shows that the T-DNA integrates at random sites into the host genome and that the selection marker is stable during mitosis and meiosis.

607. Differential expression of chsE encoding a chitin synthase of Aspergillus nidulans in response to developmental status and growth conditions
Jeong Im Lee, Yoo Mi Rho, Beom Chan Park, Jeong Hwan Choi, Yeong Man Yu, Hee-Moon Park, and Pil Jae Maeng. School of Bioscience & Biotechnology, Chungnam National University, Daejeon 305-764, Korea

As one of the consecutive efforts to understand the role of the chitin synthase genes of Aspergillus nidulans, we analyzed the mode of expression of the chitin synthase gene, chsE, both by Northern blotting and by a vital reporter system with sgfpencoding a modified version of green fluorescent protein (sGFP). On a minimal medium containing 1% glucose as a carbon source, chsE was expressed at a considerable level in conidiophores and at a somewhat lower level in vegetative mycelia. During sexual development, chsE was strongly and specifically expressed in young cleistothecia and Hulle cells at early stage, however, was not expressed either in mature cleistothecial shells, in ascospores, or in Hulle cells. The level of chsE expression was increased to some extent by substituting sucrose or lactose for glucose contained in the medium as a sole carbon source, however, was slightly reduced by sodium acetate. While osmostress caused by high concentrations (up to 1.2 M) of KCl, NaCl, or sorbitol stimulated the expression of chsE, heat shock did not affect its expression at all. Consequently, it is suggested that chsE has quite a distinctive mode of expression and function from those of the other chitin synthase genes of this fungus, such as chsA, chsB, and chsC.

608. Oxidative damage of aconitase AcoA from Aspergillus nidulans regenerates the second function as a methylaconitase
Claudia E. Maerker, Matthias Brock. Institut für Mikrobiologie, Universität Hannover, Schneiderberg 50, 30167 Hannover, Germany

Aconitases are monomeric proteins, which are involved in the citric acid cycle as well as in the glyoxylate cycle. Beside this enzymatic function, the major aconitase of E. coli and cytoplasmatic aconitase of mammals serve as mRNA-binding posttranscriptional regulators, depending on the status of its iron-sulfur cluster.
We describe a new enzymatic function associated with the naturally occurring oxidative damaged cluster of aconitase AcoA from Aspergillus nidulans, most likely the [3Fe-4S]-form. The [4Fe-4S] cluster is associated with the aconitase activity, but oxidative stress or iron-chelating agents increases a different enzymatic activity, namely the methylaconitase activity. The methylaconitase is part of the propionate-degrading methylcitrate cycle and catalyzes the reaction of methyl-cis-aconitate to methylisocitrate. However, other aconitases, e.g. from pig heart or E. coli seem to bear both activities in the [4Fe-4S]-form. In order to get a strain producing high levels of aconitase we transformed the wild type with the acoA-gene. In crude extracts of the transformant strains, we observed a 3-fold increase of both enzymatic activities compared with the wild type. At the beginning of a typical purification, the ratio of aconitase to methylaconitase was 5:1. During the purification the aconitase looses the labile 4th iron and the ratio changed to 1:9 towards methylaconitase activity at the end of the purification. Under anaerobic reducing conditions with iron and sulphur the aconitase activity was reconstituted with simultaneously diminished methylaconitase activity. EPR- and UV-Vis-Spectra of different cluster-types are presented. Additionally, the concentration of [3Fe-4S]-clusters in a reactivated probe was estimated and correlated with the activity.

609. Expression of the Candida albicans genes PHR3 to 5 is pH-independent
Sabine E. Eckert¹, Werner J. Heinz², K. Haynes³, Bernd Hube⁴ and Fritz A. Mühlschlegel^{1 1} University of Kent, UK. ² University of Wuerzburg, Germany. ³ Imperial College London, UK. ⁴ Robert Koch-Institute, Berlin, Germany

The pathogenic yeast Candida albicans contains two previously described and studied PHR genes, which are required for cross-linking of beta-(1,3)- and beta-(1,6)-glucans in the cell wall. Both PHR1 and PHR2 expression are regulated by the transcription factor Rim101p according to the pH of the medium. A PHR1/PHR2 double deletion mutant is still viable, therefore we searched for more PHR genes. The C. albicans genomic database revealed the presence of three more homologues (PHR3 to 5) from the PHR family. The genomic ORF of PHR3 contains an intron and the C-terminus has an interesting diverging sequence, lacking a cysteine- or serine-rich stretch. The gene PHR3 was found to be expressed constitutively at a low level. Deletion of PHR3 did not result in a noticeable phenotype. PHR4 encodes an unusually long deduced protein of 641 aa and contains several spacer-like regions. PHR5 encodes a short protein with a reduced cysteine and serine box, transcription is upregulated in response to serum. The expression of PHR3, 4 and 5 is not dependent on the pH of the medium, which is conclusive with our findings that their promoter regions contain no Rim101p binding sites. We present an overview of the genes PHR3, PHR4 and PHR5, their expression under different environmental conditions, in silico analysis of ORFs and promoters, along with a phylogenetic tree of the known members of the Phrp family in different ascomycete fungi.