NEUROSPORA 2006 POSTER ABSTRACTS

BIOCHEMISTRY AND SECONDARY METABOLISM

1. Using metabolomics to identify the molecule that causes efflux of basic amino acids from the vacuole of Neurospora crassa.
Kelly A. Keenan, Michael Coords and Mia Carraturra, Richard Stockton College, PO Box 195, Pomona, NJ 08240, keenank@stockton.edu

The goal of this project is to both begin the identification of metabolites found in Neurospora crassa and to identify the metabolite that causes efflux of basic amino acids from the vacuole. Under conditions of nitrogen starvation, arginine is released from the vacuole and it has been shown that a metabolite is responsible for this. Metabolite extracts have been prepared from N. crassa grown under normal as well as nitrogen starvation conditions, derivitatized to protect reactive groups and then passed through the gas chromotograph-mass spectrometer (GC-MS)using the Feihn method which has yielded information on metabolites in several other organisms. Peaks from the GC are selected and the mass spectra are compared to the NIST library to identify the metabolites. Results so far indicate that there are peaks unique to the nitrogen starvation extract and the identification awaits confirmation by running standards. Such peaks are thought to contain the metabolite that causes efflux. Such metabolites will be tested in the efflux assay. Other metabolites found in both extracts are also being identified and should provide a starting point for list of metabolites.

2. The Mechanism of Ammonium Transport via Amt Proteins in Neurospora
Clifford Slayman and Alberto Rivetta Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520 clifford.slayman@yale.edu alberto.rivetta@yale.edu

On the basis of physiological data plus high-resolution X-ray data from bacterial proteins, several laboratories have concluded that ammonium transporters homologous with the E. coli AmtB protein actually function as channels for ammonia: so that each transit requires NH4+ to shed a proton into solution on the cis side of the membrane and NH3 to retrieve a proton from solution on the trans side of the membrane (P.N.A.S. 95:7030, 1998; Science 305:1587, 2004; P.N.A.S. 101:17090, 2004). In low-pH solutions (free NH3 scarce), then, this ammonia-channel mechanism should mimic ammonium/proton antiport: being electroneutral overall and counterflowing one proton for each NH3 transit. Electrophysiological data from Neurospora contradict these inferences*. During ammonium starvation, the Neurospora plasmalemma becomes increasingly susceptible to depolarization by test pulses of ammonium (0.01-0.1 mM). Susceptibility increases with a half time of ~15 minutes and yields maximal depolarization (from -200 mV to about –50 mV) with ~10 mM NH4+. The system is only about 10% as sensitive to methyl ammonium as to ammonium itself, and is unresponsive to K+ and to lipid-soluble cations such as TPMP+. Critical unanswered questions are which of the Amt homologues in Neurospora are synthesized/activated during ammonium starvation? which mediate the observed currents? and how do their “pore” structures differ from AmtB? There are four putative Amt genes in Neurospora: NCU03257.1, NCU05843.1 (MEPA), NCU06613.1, and B14A6.240. *Slayman, C.L., 1977. Energetics and control of transport in Neurospora. In Jungreis, A.M., et al., eds., “Water Relations in Membrane Transport in Plants and Animals.” Academic Pr. (New York), pp. 69-86.

CELL BIOLOGY

3. Live-cell imaging of trichogyne-macroconidium interactions in Neurospora crassa.
 Hsiao-Che Kuo and Nick D. Read. Institute of Cell Biology, University of Edinburgh, Rutherford Building, Edinburgh, EH8 3JH, UK

The initial stages of Neurospora crassa sexual reproduction involve a specialized ‘female’ receptive hypha (the trichogyne) which grows out from the ascogonium of the protoperithecium, and fuses with a ‘male’ cell (a conidium). We have analysed this process using live-cell imaging. On water agar medium, trichogynes first emerged from protoperithecia which were 20-25 um in diameter. Fringe hyphae grew out from the protoperithecial wall soon after. Trichogynes first responded chemotropically to macroconidia of opposite mating type ~6 h after adding macroconidia; macroconidium-trichogyne fusion took place ~4 h later. At 24°C trichogynes grew more slowly (0.25-0.63 um/min) than vegetative hyphae (5-20 um/min). More than one trichogyne grew out from a single protoperithecium and all of these trichogynes could home towards different or the same macroconidia. Even if a macroconidium had germinated it still attracted trichogynes although conidial germ tubes often stopped growing when trichogynes were in their vicinity. Furthermore, the trichogyne was sometimes attracted to the conidial germ tube rather than the conidium itself. This suggested that chemoattracting sex pheromone can be produced from germ tubes as well as from macroconidia. Male macroconidial nuclei migrated into the trichogyne and mitotic division of both male and female nuclei in the trichogyne was inhibited during at least the first 5 h following fusion. In addition, the female nuclei in the trichogyne immediately became immobilized following fusion and only the male macroconidial nuclei moved through the trichogyne towards and into the ascogonium.

4. Ontogeny of the Spitzenkörper in germlings of Neurospora crassa
C. L. Araujo-Palomares, E. Castro-Longoria, and M. Riquelme. Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada. Ensenada, Baja California, México. ecastro@cicese.mx

The Spitzenkörper (Spk) is a highly dynamic and pleomorphic complex located in the cell apex of filamentous fungi. In previous studies the structure and dynamics of the Spk have been analyzed in mature hyphae of filamentous fungi, both in main leading hyphae and branches. By enhanced phase-contrast high resolution video-microscopy we have analyzed the intracellular changes prior to the appearance of the Spk in germlings of Neurospora crassa. Observations were done from the initial stages of spore germination until a conspicuous Spk was observed in the apex of germ tubes. Before a Spk could be observed, the cell showed a uniform distribution of organelles such as nuclei, mitochondria and cytoplasmic granules. Once the germlings of N. crassa reached a length of approximately 150 µm the organelles were displaced towards the subapical region of the cell and a small exclusion zone (0.6 ± 0.3 µm) was formed at the apex. The position of this exclusion zone within the apex seemed to direct the germ tube growth direction, which was highly erratic. Thirty minutes after it first appeared, the exclusion zone started to become occupied by a dark-phase material that gradually concentrated into a light gray body that we called immature Spk. During this phase the presence of the Spk in the apical dome was not constant. Approximately 20 minutes later the Spk became more robust until acquiring its typical dark-phase appearance, at which point the growth direction of the germ tube became more unwavering

5. Characterization and transcriptional profiling of vib-1 in Neurospora crassa.
Karine Dementhon, Chaoguang Tian, Elizabeth Hutchison, and N. Louise Glass. Plant and Microbial Biology department, UC Berkeley, California, 94720

During vegetative growth, filamentous fungi produce heterokaryons by undergoing hyphal fusion. However, differences at the het-c haplotype, whose specificities are categorized into one of three major types, cause heterokaryon incompatibility (HI), resulting in programmed cell death and repression of conidiation and growth. Non-allelic interactions of polymorphic het-c and pin-c alleles are essential for nonself recognition and HI. HI is suppressed by mutations in vib-1, a putative transcription factor. Though mutations in vib-1 suppress the HI phenotype, vib-1 mutants exhibit irregular conidiation. GFP-tagged vib-1 has been shown to localize to the nucleus, and the signal intensity of vib-1::GFP increases in incompatible vs. compatible cells. These results indicate that vib-1 has an important role in HI. In order to investigate the function of vib-1, transcriptional profiling experiments were performed to identify targets of vib-1 regulation. When expression patterns of a vib-1 knockout strain were compared to those of a wildtype strain, more than 100 genes exhibited altered expression. Transcriptional profiling will also be used to compare a vib- 1 overexpression strain to a wildtype strain. Finally, the expression patterns of a vib-1 knockout in a compatible heterokaryon will be compared to those in an incompatible heterokaryon. By comparing vib-1 knockout strains in compatible vs. incompatible situations, differences in expression patterns can be attributed to the effects of the het-c and pin-c interactions. Transcriptional profiling experiments, in conjunction with data on the characterization of vib-1 mutants, will give important insights into the role of vib-1 during vegetative growth and HI.

6. Modifications of PCNA and DNA repair in Neurospora crassa.
Tsuyoshi Kawabata, Akihiro Kato, Keiichiro Suzuki, Hirokazu Inoue. Department of Regulation Biology, Faculty of Science, Saitama University, Saitama City, Japan.

Proliferating cell nuclear antigen (PCNA) is an auxiliary factor for DNA polymerases and is essential for DNA replication. It forms a heterotrimeric clamp of DNA. Some yeast PCNA mutant alleles exhibited sensitivity to mutagens such as UV and MMS, indicating that PCNA is related to DNA repair. Recently, it has been elucidated that PCNA is modified by ubiquitin in response to DNA damage and by SUMO at S- phase. In yeast, PCNA is monoubiquitinated by Rad6/Rad18 and polyubiquitinated by Rad5/Ubc13/Mms2. However, there are some differences between yeast and human in these modifications. Because of insufficiency of genetic studies, these precise molecular events remain unclear in higher eukaryote. Therefore, in order to clear how PCNA is modified in higher eukaryote, we analyzed modifications of PCNA in N. crassa. In this study, we made disruptant of Neurospora homologs of RAD5 and UBC13 (mus-41 and mus-46, respectively) and investigated roles of these genes in modifications of PCNA. These mutants were sensitive to mutagens as observed in yeast, but epistasis analysis showed that mus-41 and mus-46 have roles different from yeast. We created strains that express FLAG- or HA-tagged PCNA and observed PCNA modifications by western blotting. It was shown that PCNA is polyubiquitinated independently from mus-46 and mus-41 in N. crassa. It suggests existence of some unknown factors required for PCNA polyubiqiutination in N. crassa.

7. SO, a protein involved in hyphal fusion, localizes to septal plugs.
Andre Fleissner and N. Louise Glass. Department of Plant and Microbial Biology, University of California, Berkeley, CA

The so mutant of Neurospora crassa exhibits a pleiotropic phenotype, including lack of anastomosis, shortened aerial hyphae, and female sterility (1). The so gene is highly conserved in filamentous ascomycetes, but not present in ascomycete yeast species. The encoded protein contains a conserved WW-domain possibly involved in protein-protein interactions. In this study we show that SO-GFP fusion proteins localize to septal plugs. In filamentous ascomycetes septa get plugged in injured hyphae and at different stages during the fungal development, such as protoperithecia formation, aging, or cell death during vegetative incompatibility. While injured hyphae are sealed by Woronin bodies, other plugs seem not to contain this organelle. In contrast SO-GFP localizes to the different types of plugs mentioned above. To prove that this localization is Woronin body independent, we expressed so-gfp in the hex-1 mutant, which lacks Woronin bodies (2,3). Although hex-1 hyphae extensively bleed cytoplasm after injury they are eventually sealed in a Woronin body independent manner. SO-GFP localizes to these late forming plugs. However overexpression of so- gfp does not complement the hex-1 phenotype. The so mutant has no bleeding phenotype, and a double mutant of hex-1 and so does not show a more severe bleeding phenotype than hex-1 alone. We conclude that SO is part of septal plugs, that its localization to the plugs is Woronin body independent, and that its role is different from simply preventing the loss of cytoplasm.
(1) Fleissner et al., Euk. Cell 2005 (2) Jedd and Chua, Nature Cell Biol. 2000 (3)Tenney et al., Fungal Genet. Biol. 2001

8. Antibiotic Binding Sites and the Structure of the Vacuolar ATPase.
Emma Jean Bowman, Marija Draskovic, Molly McCall, and Barry Bowman. Dept. of MCD Biology, University of California, Santa Cruz, CA

The V-ATPase is a complex enzyme that functions as a rotary motor, transporting protons across membranes. Changes in V-ATPase activity appear to have a significant role in many human diseases, such as kidney malfunction, osteoporosis, and cancer. The macrolide antibiotics bafilomycin and concanamycin are potent inhibitors of V-ATPases. To determine where the drugs bind and how they inhibit, we selected mutant strains of Neurospora crassa that are resistant to these antibiotics. We also developed a method for site-directed mutagenesis to generate additional mutant forms of the enzyme. Approximately 20 amino acid residues were identified that, when mutated, resulted in a drug-resistant enzyme. All of the selected mutations were in subunit c, a small hydrophobic protein. Multiple copies of subunit c associate to form part of the “rotor,” which is embedded in the membrane. Using a prokaryotic homolog of the V- ATPase, a high-resolution structure for this sector of the enzyme was recently reported (T. Murata et al. Sci. 308:654-659). We used these data to construct a model of subunit c and the rotor of the N. crassa V-ATPase. The amino acid sequence of the N. crassa protein fit remarkably well to the model based on the E. hirae structure. Furthermore, the residues implicated in binding antibiotics were located in a pocket formed at the interface between two c subunits. The model appears to be an excellent representation of the structure of this part of the V-ATPase in N. crassa and other eukaryotes. Furthermore, the data strongly support the conclusion that the antibiotics inhibit by blocking the rotation of the c subunits.

9. A functional approach to the study of cellular morphogenesis in filamentous fungi.
Aleksandra Virag and Steven D. Harris, Plant Science Initiative, University of Nebraska, N234 Beadle Center, Lincoln, NE 68588- 0660 (sharri1@unlnotes.unl.edu)

Polarized hyphal growth is a defining feature of the filamentous fungi. Despite its importance to the fungal lifestyle, the molecular mechanisms underlying the establishment and maintenance of hyphal polarity remain poorly understood. Moreover, during asexual development, fungi such as Neurospora crassa switch to a budding mode of growth that may involve dramatic switches in the spatial and temporal patterns of cellular morphogenesis. Previous genetic screens have identified several genes required for cellular morphogenesis in N. crassa, including many novel genes that are unique to fungi. We reasoned that a systematic survey of the N. crassa knockout collection would be the most effective approach to identify the complete set of genes involved in cellular morphogenesis. As a preliminary study, we have screened a set of ~100 knockout mutants for defects in hyphal morphogenesis, branching patterns, and conidial morphogenesis. Despite the small size of our sample, we have identified at least two genes (NCU08741.2, related to Striatin/Pro11; NCU03043.2, related to krueppel protein) with previously unknown roles in hyphal morphogenesis, as well as others whose role is far more important than previously suspected (i.e., NCU00406.2, CHM1). We will describe our screening format and present results for each of the mutants we have analyzed.

10. Calcium and polar growth: The role of calcium transport proteins in organelles.
Barry Bowman, Marija Draskovic, Emilio Margolles-Clark, and Stephen Abreu. Dept. of MCD Biology, University of California, Santa Cruz, CA

Calcium, a key signaling molecule in all organisms, is often sequestered in intracellular compartments. In filamentous fungi a calcium gradient has been proposed to play a key role in polar grow. We identified five genes in Neurospora crassa that may encode organellar calcium transporters. Using information from yeast homologs, calcium is predicted to be transported into the vacuole by NCA-2 and NCA-3 (P-type ATPases) and CAX (a Ca+2/H+ exchanger). In the Golgi, calcium and/or manganese are transported by PMR (a P-type ATPase). Unlike S. cerevisiae, N. crassa has a homolog of the mammalian SERCA ATPase (P-type), named NCA-1; it may reside in the ER. We generated null-mutant strains for each transporter. In the wild-type strain the vacuoles contained high concentrations of calcium, at least 20 mM. Nca-2 and nca-3 mutant strains maintain high levels of calcium, but inactivation of the cax gene caused the complete loss of calcium from the vacuole. Wild type, nca-3 and cax strains grew normally in media with high concentrations of calcium (50-200 mM), but growth of the nca-2 mutant and of the nca-2 cax double mutant was strongly inhibited. The data were not consistent with a localization of NCA-2 in the vacuole, but instead suggested that in N. crassa NCA-2 may pump calcium out of the cell. Polar growth was affected only in the pmr mutant strain. Because the loss of PMR affects the function of the Golgi, it cannot be concluded that the polar growth defect is directly caused by mis-localized calcium. Thus, the results indicate that the vacuole is a major storage site for calcium; however, loss of vacuolar calcium does not affect polar growth or the ability to tolerate high concentrations of calcium in the medium.

11. Circadian Rhythms in Neurospora crassa: “after-effects” of the vvd mutation.
Stuart Brody, Kevin Schneider, Sabrina Perrino Division of Biological Sciences-0116, UCSD, La Jolla, CA. 92093

Vvd mutant strains, which over produce carotenoids, are known to be deficient in photo-adaptation. On certain media, strains containing vvd mutations and the band (bd) mutation will exhibit short (6 – 14 hours) periods in constant light (L/L) but relatively normal (22-23 hours) periods in constant darkness (D/D). After four days of growth in D/D, these strains were switched from D/D to L/L. The subsequent periods were 17 hours, 9 hours, 9 hours, etc., i.e. a relatively quick adaptation to the light. However, when these strains were switched from L/L to D/D, the periods were 8 hours (in L/L), then 9 hours,10,12,12,15, and finally 20 hours, i.e. a slow adaptation to the dark. Control (bd) strains immediately showed normal (22 hour) periods upon transfer from L/L to D/D or loss of rhythmicity from D/D to L/L. One possible explanation is that the “after-effects” could be due to some key clock component which becomes elevated in L/L (in vvd) and therefore drives the conidiation rhythm to a high frequency. It then decreases slowly (in D/D) and eventually the rhythm returns to a 23 hr period. Another possible explanation may be analogous to the removal of hamsters from L/L to D/D and the slow loss of “splitting” seen in L/L of their rhythms. The hamster splitting has been postulated to occur due to two oscillators, the E and M oscillators.

12. The response regulator, RRG-1, functions upstream of the OS-2 MAPK cascade.
Carol A. Jones, Suzanne Phillips, and Katherine A. Borkovich. Department of Plant Pathology, University of California, Riverside, 92521, carol.cjones@gmail.com

Two-component signaling pathways are found in a variety of organisms including bacteria, plants, and fungi. Two-component regulator systems regulate phosphotransfer between two components: a histidine kinase (HK) at a histidine residue and a response regulator (RR) at an aspartate residue. In Neurospora there are eleven predicted hybrid histidine kinases (HHKs), which contain a histidine kinase domain and a response regulator domain within the same protein. Phosphotransfer is believed to occur as follows. A conserved histidine residue in the histidine kinase domain is autophosphorylated in response to some environmental signal. That phosphate is then transferred intramolecularly to the aspartate residue in the response regulator domain of the HHK. From the RR domain in the HHK the phosphate is then transferred to a histidine residue in the histidine phosphotransferase (HPT) protein, and finally onto an aspartate residue on a separate RR protein (RRG-1 or RRG-2). The RR are involved in the activation of downstream signaling pathways such as mitogen-activated protein kinase (MAPK) cascades and/or in transcriptional regulation. Environmental signals that may activate two-component cascades include light, osmolarity, nutrient availability, and many other factors. Analysis of the rrg-1 gene replacement mutation shows that RRG-1 is involved in osmotic stress response, fungicide resistance, conidial integrity, and other factors. The rrg-1 deletion mutant and mutants in the downstream os-2 MAPK cascade have a reduced growth rate on salts and have resistance to certain fungicides. Also the phosphorylation status of OS-2, a downstream MAPK, is greatly increased by exposure of wild type Neurospora to salts or fungicide. The working model is that NIK-1/OS-1 and at least one other HHK function upstream of RRG-1 through the HPT protein. RRG-1 can then activate the OS-2 MAPK cascade in response to osmotic stress, fungicide treatment, and/or other stimuli.

13. Apical microtubule dynamic instability in Neurospora crassa hyphae
Maho Uchida¹*, Rosa R. Mouriño-Pérez² and Robert W. Roberson¹. ¹School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA ²Departamento de Microbiología. Centro de Investigación Científica y Educación Superior de Ensenada. Ensenada, B. C. Mexico *Correspondence: maho.uchida@asu.edu

Neurospora crassa exhibits one of the fastest hyphal growth rates among filamentous fungi. To assess the probable role of microtubules (MTs) in such rapid extension rate, we monitored the dynamic instability (DI) of GFP-tagged apical MTs using live-cell imaging methods. Results obtained in mature hyphae revealed that MT polymerization rates were twice as fast as those reported in Aspergillus nidulans, while MT depolymerization rates in both species were similar. Furthermore, MT polymerization rates in Neurospora were much faster than in other organisms thus far reported, including plant and mammalian cells. In order to address the influence of motor protein mutations on DI, GFP-tagged MTs were examined in ropy 1 and nkin strains. In these strains, MT polymerization rates were reduced by one half relative to the wild type. Significance of these results will be discussed relative to hyphal growth and regulation of MT dynamics.

14. Optical tweezer micromanipulation of Neurospora crassa.
Graham Wright^a,c, Jochen Arlt^b,c, Wilson Poon^b,c and Nick Read^a,c.  ^a Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Rutherford Building, Edinburgh, EH9 3JH, UK ^b School of Physics, University of Edinburgh, James Clerk Maxwell Building, Edinburgh, EH9 3JZ, UK ^c COSMIC - Collaborative Optical Spectroscopy, Micromanipulation & Imaging Centre, University of Edinburgh, James Clerk Maxwell Building, Edinburgh, EH9 3JZ, UK

Optical tweezers were first described in the 1980’s and have been implemented in a wide variety of research across many disciplines; however, the attention received from fungal biologists has been limited. In an attempt to correct the balance we have evaluated what can be achieved using optical tweezers to micromanipulate cells of the model filamentous fungus Neurospora crassa. Utilizing the properties of laser light, tweezers permit the non-invasive trapping and movement of cells and organelles. We have built a simple, safe and user-friendly optical tweezer system that can be mounted on a commercial microscope and is, importantly, easy to use for biologists who lack optics experience. The tweezers can be used synchronously with differential interference contrast, phase contrast and fluorescence microscopy to enable enhanced visualization and manipulation of living cells and organelles. The position and orientation of whole cells with respect to one another can be controlled and a variety of organelles (e.g. vacuoles and Woronin bodies) can be manipulated within cells. Inert beads can be used as tools for the measurement of forces generated by Neurospora hyphae, for the localized mechano-stimulation of cells or for the localized delivery of chemicals. We are presently using the optical tweezers as a powerful experimental tool to address a range of novel questions, using Neurospora as a model system.

15. Control of circadian clock gene expression by a frq-less oscillator in Neurospora crassa. Sanshu Li and Patricia L. Lakin-Thomas. Department of Biology, York University, Toronto, Ontario, Canada. sanshuli@yorku.ca

The fungus Neurospora crassa expresses a circadian rhythm of conidiation when growing on agar medium. Assays of rhythmic gene expression in Neurospora using liquid culture systems have been used previously to construct a model for the circadian oscillator involving the frq and wc genes. We have now assayed the expression of circadian clock-associated genes using the same agar medium on which the function of the clock is assayed. We have found synchronous peaks of FRQ and WC- 1 proteins, in contrast to the out-of-phase rhythms found in liquid, and we have found out-of-phase RNA rhythms of the clock-controlled genes ccg-2 and ccg-7, in contrast to the synchronous peaks in liquid. We have also assayed gene expression in the long-period choline-requiring mutant chol-1, and have found long-period rhythms in FRQ and WC-1 proteins and frq RNA, but circa-24 h rhythms in ccgs. Because the long-period chol-1 conidiation rhythm continues in frq and wc null mutants, we conclude that long-period frq and wc expression rhythms must be driven by a frq-less oscillator. Regulation of rhythmic gene expression depends on metabolic conditions, and in the more physiologically relevant conditions of solid agar medium, the FRQ/WC feedback loop may not be operating as proposed. This research is supported by Discovery Grant 250133-02 from NSERC.

16. Karyotyping of Neurospora crassa using synaptonemal complex spreads of translocation quadrivalents.
Benjamin C. Lu, University of Guelph, Department of Molecular and Cellular Biology, blu@uoguelph.ca

The purpose of the present research is (1) to establish the karyotype of Neurospora crassa using visualization of kinetochores in the synaptonemal complex (SC) spreads, (2) to assign each chromosome to a linkage group, and (3) to examine chromosome pairing and recombination nodules in quadrivalents. Two strains containing reciprocal translocations were used: T(I;II)4637, which involves linkage groups I and II, and alcoy, which contains three independent translocations involving I and II, IV and V, and III and VI. Visualization of kinetochores in the spreads requires the use of freshly prepared fixatives. Kinetochore locations and arm ratios were documented in all seven N. crassa chromosomes. The new information, based on kinetochore position, arm ratios, chromosome length, and quadrivalent analyses, enabled unequivocal confirmation of chromosome assignments to genetic linkage groups. Chromosome pairing in a translocation quadrivalent starts at the four terminal regions, and proceeds right up to the translocation break point. Recombination nodules are found in all four arms of quadrivalents. The ability to identify a specific chromosome to a genetic linkage group together with the ability to visualize recombination nodules and their locations will allow future cytological analysis of recombination events.

17. Mutational analysis of two components of the GPI-anchor transamidase complex.
Shaun M. Bowman, Amy Piwowar, Eric D. Arnone, and Stephen J. Free, Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260, email: free@buffalo.edu

We have used the RIP process to generate temperature-sensitive mutants in the Neurospora gpit-1 and gpit-2 genes. These two genes encode auxiliary components of the GPI-anchor transamidase, a multiprotein complex found in the ER membrane that transfers the GPI-anchor onto GPI-anchored proteins. It has been difficult to obtain structural data on the organization of the complex and how the different proteins function and interact with each other because the components are multipass transmembrane proteins and haven’t been individually purified. We have used a RIP mutational analysis to define regions within each of these two proteins that can be mutated without loss of the protein’s function at a permissive temperature. The analysis helps to identify regions within each of these two proteins that are critical for the activity of the proteins. For the most part, the sequences we define as being critical are sequences that are conserved during evolution.

18. GPI-anchored proteins are required for cell wall biosynthesis.
Shaun M. Bowman, Amy Piwowar, Mash'el Al Dabbous, John Vierula*, and Stephen J. Free, Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260 and *Department of Biology, Carleton University, Ottawa, Ontario, Canada, K1S 5B6 email: free@buffalo.edu

We have isolated and characterized Neurospora mutants affected in four of the genes involved in the biosynthesis of the GPI-anchor structure and demonstrated that the production of GPI-anchored proteins is required for normal cell wall biosynthesis. We show that mutants affected in GPI-anchoring produce a cell wall that has an altered protein and carbohydrate composition and grow in a tight colonial manner. The mutant cell walls are weak, as assessed by cell lysis assays, and are sensitive to the presence of calcofluor white and congo red, reagents that disrupt chitin biosynthesis. Proteomic analysis of the cell walls demonstrated the loss of a number of GPI-anchored proteins.

19. The STE20/germinal center kinase POD6 interacts with the NDR kinase COT1 to coordinate polar tip extension in a dynein/kinesin-dependent manner in Neurospora crassa
Stephan Seiler†, Nico Vogt†, Carmit Ziv§, Rena Gorovits§, and Oded Yarden§.
†Institut für Mikrobiologie und Genetik, Universität Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany. §Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Rehovot 76100, Israel.

20. Action of a Salivary Antimicrobial Peptide, Histatin 5, on Neurospora
Alberto Rivetta and Clifford Slayman Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520 alberto.rivetta@yale.edu; clifford.slayman@yale.edu

Human salivary glands make histidine-rich fungicidal peptides known as histatins, of which the best studied is histatin 5 (Hst5): DSHAKRHHGYKRKFHEKHHSHRGY. The normal target for histatins is Candida albicans (oral thrush), which is killed via a process of non-lytic cytodialysis. The surface-bound stress proteins Ssa1p and Ssa2p function as receptors for Hst5 (J. Biol. Chem. 278:28553, 2003), and the subsequent loss of small metabolites (incl. ATP) is mediated by deranged channel-like behavior of Trk1p (J. Biol. Chem. 279:55060, 2004), the protein primarily responsible for potassium accumulation in Candida. The high density of cationic charge on Hst5 is presumed to be essential to its entry and/or its intracellular binding. Hst5 also kills Neurospora, and that organism is more convenient for studying the rapid effects of Hst5 at the plasmalemma: esp., its influence on membrane voltage and resistivity. Under standard conditions, 0.06 mM Hst5 depolarizes the Neurospora plasma membrane, from about –200 mV to about –50 mV, with a time-course similar to that observed in response to 1 mM cyanide (J. Membr. Biol. 14:305, 1973). Depolarization is accompanied by a high-voltage leak that is consistent with the deranged channel behavior of Trk1p in Candida, and is kinetically consistent with deactivation of the plasma-membrane proton pump (Pma1p) by depletion of cytoplasmic ATP. The two most likely intracellular actions of Hst5 in Neurospora are thus i) disruption of the potassium transporter, Trk1p, and ii) uncoupling of mitochondrial oxidative phosphorylation. We are soliciting the collaboration of genetics laboratories interested in constructing pertinent gene- deletion strains of Neurospora.

21. The Ribosomes of Neurospora crassa.
Alan Radford, IICB, Faculty of Biological Sciences, The University of Leeds, a.radford@leeds.ac.uk

This describes the results of data-mining of the Neurospora crassa genome for the genes encoding the structural ribosomal RNA components and the polypeptide components of the cytosolic and mitochondrial ribosomes. For the rRNA genes, homologous Blast probes with known N. crassa sequences was used to identify the multiple copies. For the ribosomal polypeptide genes, the probe sequences were predominatly from Saccharomyces cerevisiae. For cytosolic ribosomes, 33 small subunit polypeptide genes, 44 large subunit polypeptide genes and 3 acidic polypeptide genes, mainly by homology with yeast. For the 18S, 5.8S and 28S rRNA units, 43 copies spread over circa 40 assembly 7 contigs have been found, compared to an earlier Southern blotting estimate of 100-200 copies. It thus appears that the nucleolus organiser region is significantly under-represented in genome assembly 7. For the 5S rRNA genes, 87 copies (including some apparent pseudogenes) spread over 42 assembly 7 contigs have been identified, within the limits of an earlier estimate. For mitochondrial ribosomes, 18 small subunit polypeptide genes (all except 1 nuclear-encoded) and 33 large subunit polypeptide genes (all nuclear-encoded) were identified.

22. Circadian Rhythms in Neurospora crassa: New fluorescent reporters.
Michael Ferry†, Stuart Brody‡, Jeff Hasty† † Department of Bioengineering, UCSD; ‡Division of Biological Sciences, UCSD

We have made new, novel fluorescent constructs useful for studying the circadian oscillator in N. crassa. These are fluorescent proteins that are not excitable or emit light at wavelengths below 520 nm since the WC-1 light receptor responds to light in this range. We are in the process of creating a fusion protein between the FRQ protein and the mCherry protein (excitation max: 587nm, emission max 610 nm) linked by a sequence designed to separate the domains of the fusion protein. We have also made constructs between mCherry and the rhythmic ccg2 promoter, and the inducible qa2 promoter and are currently targeting them to the His-3 locus of the FGSC # 9717 _ mus-51::bar+; his-3 mat A strain. To target the frq locus we have generated a construct with both positive (hygromycin resistance) and negative (fluorodeoxyuridine sensitivity) markers and have transformed the N. crassa strain ?mus-51::bar+ FGSC # 9718. We are currently targeting our FRQ-mCherry construct to the native frq locus of this strain by growing them on media containing fluorodeoxyuridine and selecting against the untransformed cells. These reporters will allow us to: 1). Create a mathematical model for this oscillator which can be tested quantitatively; 2) obtain new information about this oscillator (amplitudes of components, production, degradation rates, etc) that would be of general interest to the study of circadian oscillators and of specific application to the modeling of this system.

23. Genetic analysis of POL32 homolog in Neurospora crassa.
Keiichiro Suzuki, Shuuitsu Tanaka, and Hirokazu Inoue Dept. of Regulation-Biol., Fac. of Sci., Saitama Univ., Japan shtanaka@post.saitama-u.ac.jp

DNA translesion synthesis (TLS) is an important mechanism to avoid replication fork collapse by DNA damage. The POL32 gene of Saccharomyces cerevisiae encodes a non-essential subunit of DNA polymerase ƒÂ which is the major replicative DNA polymerase in eukaryotic cells. In S. cerevisiae pol32 mutant is deficient in UV-induced mutagenesis, suggesting Pol32 act in TLS. In this study, we present identification and characterization of the POL32-homolog in N. crassa. A knockout mutant of Nc POL32 is viable and sensitive to several DNA damaging agents. Epistasis analyses indicated that Nc POL32 belong to same epistasis group with mus-8 (RAD6 homolog), uvs-2 (RAD18 homolog), mus-41 (RAD5 homolog), and upr-1 (REV3 homolog, catalytic subunit of PolƒÄ), suggesting a role for Nc POL32 in post- replication repair (PRR). We also found that Nc pol32 mutant is deficient in UV-induced mutagenesis, indicating that Pol32 is required for UV-induced TLS. Further genetic analysis revealed that pol32 is lethal in combination with mutation in mus-9 (MEC1/ATR homolog), but not in combination with mutation in mus-21 (TEL1/ATM homolog). Surprisingly, we also showed that POL32 and TEL1/ATM belong to same epistasis group. These findings suggest that Nc POL32 plays a role in both TLS and checkpoint pathways.

24. Apical microtubule dynamics in Neurospora crassa and their role in rapid growth.
Maho Uchida^1*, Rosa R. Mouriño-Pérez² and Robert W. Roberson¹ . ¹School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA ²Departamento de Microbiología. Centro de Investigación Científica y Educación Superior de Ensenada. Ensenada, B. C. Mexico *Correspondence: maho.uchida@asu.edu

Neurospora crassa exhibits one of the fastest hyphal growth rates among filamentous fungi. To assess the probable role of microtubules (MTs) in such rapid extension rate, we monitored the dynamic instability of GFP-tagged apical MTs using live-cell imaging methods. Results obtained in mature hyphae revealed that MT polymerization rates were twice as fast as those reported in Aspergillus nidulans, while MT depolymerization rates in both species were similar. Furthermore, MT polymerization rates in Neurospora were much faster than in any other system studied thus far, including plant and mammalian cells. In order to address the influence of motor protein mutations on dynamic instability, GFP- tagged MTs were examined in ropy 1 and nkin strains. In these strains, MT polymerization rates were reduced by one half relative to the wild type. These results will be further analyzed with impending data obtained in slow growing germlings.

25. RHO-4 localization during conidial development.
Carolyn Rasmussen and N. Louise Glass 111 Koshland Hall Rm. 341 University of California, Berkeley cgrasmus@berkeley.edu

Proteins in the Rho family are small monomeric GTPases primarily involved in polarization, control of cell division and reorganization of cytoskeletal elements. We show that rho-4 loss-of-function mutants in Neurospora crassa lack septa. GFP-tagged RHO-4 was targeted to septa and to the plasma membrane. RHO-4 formed a ring at incipient septation sites that appeared to constrict with the formation of the septum. We studied RHO-4 localization during conidiation and in the conidial separation mutants csp-1 and csp-2. Interestingly, RHO-4 disappears from the septum during or after the formation of the second septum and becomes cytoplasmically localized. We show that cytoplasmic localization of RHO-4 in conidia is dependent on the negative regulator RDI-1, a predicted Rho guanine dissociation inhibitor. Further, rdi-1 mutants show greatly increased septation compared with a wild- type strain. Experiments to test direct interaction between RHO-4 and RDI-1 will be discussed.

26. Nutrient sensing G protein coupled receptors in Neurospora crassa.
Liande Li and Katherine Borkovich, Plant Pathology Department, UC Riverside

Neurospora crassa is able to utilize a wide variety of carbon and nitrogen sources. The N. crassa predicted G protein coupled receptor (GPCR) GPR-4 is similar to a group of carbon-sensing GPCRs characterized in yeasts. Levels of gpr-4 are greatly elevated during growth on a poor carbon source. gpr-4 deletion mutants have reduced mass accumulation compared to wild type when starved for carbon or when cultured on high levels of poor carbon sources and also undergo inappropriate asexual sporulation in submerged cultures. Epistasis analyses and two hybrid assays support interaction of GPR-4 with GNA-1 in vivo. Analysis of various facets of cAMP metabolism are consistent with a downstream signaling pathway at least partially dependent on cAMP levels. Our results support the hypothesis that GPR-4 is coupled to GNA-1 in a pathway that senses and regulates the response to carbon starvation and/or poor carbon sources in N. crassa.

DEVELOPMENTAL BIOLOGY

27. Origin and Significance of Vacuolar Proliferation during Nutrient Restriction
Clifford Slayman (1) and Tatiana Potapova (2) 1. Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520;
clifford.slayman@yale.edu 2. A.N. Belozersky Institute, Moscow State University, 119899 Moscow, Russia; potapova@genebee.msu.su

Vacuoles in fungi, especially in mycelial fungi, have long been known as polyfunctional, highly pleiomorphic organelles, whose appearance is strongly influenced by age and by nutritional status. A particularly striking example of age- and nutrition-linked morphological change was noted in 1968 by Robertson and Rizvi (Ann. Bot. 32:279, who found that in mature hyphae, “cells” lying more than ~1 cm behind the growing tips are often stuffed with a vacuolar “foam”. Similar pictures are generated by several hours of carbon starvation; and these vacuoles accumulate a variety of “cytoplasmic” dye probes (e.g., BCECF) presented as neutral esters (J. Exp. Biol. 196:419, 1994), and greatly enhance intracellular proton buffer capacity (D. Sanders & C.L. Slayman, unpublished expts.). As viewed in the light microscope (phase contrast or interference contrast), vacuoles begin to proliferate within a few minutes of the onset of carbon starvation, first forming a “bubble-wrap” against the hyphal plasmalemma, and then crowding the interior, as the number and size of vacuoles increases. After several hours of carbon starvation, the vacuolar foam can occupy more than 80% of the apparent intrahyphal volume, and the foam can be fused into large vacuoles by mechanical shock. Upon re-presentation of a carbon source, the whole morphological process reverses, with a roughly symmetric time-course. We are beginning an investigation into the biogenesis of this vacuolar proliferation, by means of the newly available Neurospora genomic microarrays.

29. Characterization of the bd mutation: filling the gap between ROS and RAS.
Luis F. Larrondo, Bill Belden, Alan Froehlich, Mi Shi, Chen-Hui Chen, Jennifer Loros and Jay Dunlap. Department of Genetics, Dartmouth Medical School, Hanover, NH-03755. U.S.A.

The bd mutation in Neurospora crassa has been an integral tool, for the last 40 years, in the study of circadian rhythms. This mutation confers a semidominant phenotype that allows the clear visualization of the conidiation (banding) pattern, which is circadianly regulated by the N. crassa clock. Using a SNP mapping strategy, developed as part of the Program Project, we have identified bd as a single point mutation in the ras1 gene, changing T79 to I. A genomic fragment generated from a bd strain and containing only the ras1 gene was able to confer the bd phenotype when transformed into a WT strain. Based on the extensive literature for Ras biology and on the hypothesis that hyperoxidant states trigger cell differentiation events (Hansberg, W. and Aguirre, J. J. Theor. Biol. 142:201-221; 1990), we have postulated that the bd mutation leads to an imbalance in ROS (Reactive Oxygen Species) levels, which in return, is responsible for the banding phenotype observed on race tubes. A variety of genetic and biochemical data based on gene replacements and physiological analyses of strains are consistent with this hypothesis. L.F.L. is a PEW Postdoctoral Fellow

GENE REGULATION

30.

31. SAD-2 is required for Meiotic Silencing by Unpaired DNA and perinuclear localization of SAD-1 RNA- directed RNA polymerase.
Patrick K.T. Shiu¹, Denise Zickler², Namboori B. Raju³, Gwenael Ruprich-Robert², and Robert L. Metzenberg⁴. ¹University of Missouri, Columbia, Missouri. ²Université Paris-Sud, Orsay Cedex, France. ³Stanford University, Stanford, California. ⁴California State University, Northridge, California.

A gene unpaired during the meiotic homolog pairing stage in Neurospora generates a sequence-specific signal that silences the expression of all copies of that gene. This process is called Meiotic Silencing by Unpaired DNA (MSUD). Previously, we have shown that SAD-1, an RNA-directed RNA polymerase (RdRP), is required for MSUD. We isolated a second gene involved in this process, sad-2. Mutated Sad-2^RIP alleles, like those of Sad-1, are dominant and suppress MSUD. Crosses homozygous for Sad-2 are blocked at meiotic prophase. SAD-2 colocalizes with SAD-1 in the perinuclear region, where siRNAs have been shown to reside in mammalian cells. A functional sad- 2⁺ gene is necessary for SAD-1 localization, but the converse is not true. The data suggest that SAD-2 may function to recruit SAD-1 to the perinuclear region, and that the proper localization of SAD-1 is important for its activity.

32. Investigation of two putative Neurospora crassa heat shock transcription factors.
Seona Thompson, Antonis Sotiriou, Nirvana Croft, Ray O’Keefe and Susan Crosthwaite. Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT. susan.k.crosthwaite@manchester.ac.uk

The heat shock response is a ubiquitous response to adverse high temperatures. At the onset of heat shock there is a rapid increase in the expression of heat shock proteins and inhibition of the production of normal non-stress proteins at both the transcriptional and post-transcriptional level. The induction of most heat shock genes is regulated by heat shock factor (HSF), a transcription factor displaying high functional conservation between organisms. Two open reading frames, NCU08512 and NCU08480, encoding putative HSFs are present in the Neurospora genome. We have deleted NCU08512 and preliminary data indicates that it is an essential gene. The single HSF encoded in the S. cerevisiae genome is also essential for viability. To find out if the putative Neurospora HSFs could function in yeast we transformed a diploid strain of S. cerevisiae heterozygous for the HSF gene with yeast expression vectors carrying NCU08512 and NCU08480 cDNA. Of note, several splice variants of NCU0840 are synthesized that encode altered forms of the predicted NCU0840 protein. The transformed yeast strain was induced to sporulate and our initial analysis of tetrads indicates that the Neurospora genes cannot functionally replace the yeast HSF. If NCU08512 and NCU0840 do indeed encode HSFs this result suggests that, like human HSF1, they may require different signals to their yeast counterpart for activation.

33. Mutations in a gene repressor or a glucose transporter result in sustained gene photoactivation in Neurospora.
Maria Olmedo, Laura Navarro-Sampedro and Luis M. Corrochano Departamento de Genetica, Universidad de Sevilla, Spain corrochano@us.es

The gene con-10 of Neurospora is expressed during conidiation and after illumination of vegetative mycelia. Photoactivation of con-10 is transient and disappears after two hours of light. The rco mutants were isolated by the abundant expression of con-10 in vegetative mycelia. The gene rco-1, encoding a putative gene repressor, and the gene rco-3, encoding a putative glucose sensor, are required for the repression of con-10 in vegetative mycelia. We have observed that rco-1 and rco-3 mutants have an enhanced and sustained photoactivation of con-10 and con-6, a phenotype they share with vivid mutants. The abundant photactivation of con-10 and con-6 in rco and vivid strains is best observed after five hours of light. The rco and vivid mutations do not alter the stability of the con-10 and con-6 mRNAs, suggesting that the sustained photoactivation is due to a high transcriptional rate that is not subjected to adaptation to light. The threshold of con-10 and con-6 photoactivation is significantly lowered in the rco-1 mutant, but remains unchanged in the rco-3 and vivid mutants. The circadian clock in these mutants does not seem to be altered. We propose that VVD, RCO-1, and RCO-3 participate in the mechanism responsible for transient gene photoactivation.

34. The dominant suppressor of repeat-induced point mutation (RIP) in the Adiopodoume strain of Neurospora crassa is linked to an allele for a variant catalytic subunit of DNA polymerase zeta.
Ranjan Tamuli, C. Ravindran and Durgadas P. Kasbekar Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.

Crosses involving the Adiopodoume strain of Neurospora crassa are defective for repeat-induced point mutation (RIP), a genome defense mechanism of fungi. Mapping studies suggested that the Adiopodoume strain contains a dominant suppressor of RIP (Srp) located proximal to mat on linkage group (LG) IL. Srp strains can occasionally lose the dominant RIP suppressor phenotype and became phenotypically intermediate or non-suppressor. Localization of crossovers with respect to molecular markers further narrowed Srp to a ~34 kb genomic segment that is ~26 kb proximal to mat. This segment contains the upr-1 gene, which codes for the catalytic subunit of the translesion DNA polymerase zeta (Pol z). The upr-1 allele of Adiopodoume contains several synonymous and non-synonymous mutations relative to the standard Oak Ridge (OR) allele, suggesting that it has undergone rapid evolution. Another translesion polymerase, Rev1, is encoded by the mus-42 gene on LG IL and a Pol z regulatory subunit is encoded by the mus-26 gene on LG IVR. The mus-26 and mus-42 genes do not show evidence for rapid evolution in the Adiopodoume strain. The upr-1, mus-26 and mus-42 gene products are not required for RIP. We suggest that dominant RIP suppression might result from the variant Pol z catalytic subunit interfering with some other DNA polymerase that is required for RIP. The other genes in the candidate region have either very few or no mutation.

35. Identification of Proteins Binding to the cis Regulatory STRE Element of the gsn Promoter. A Mass Approach. Freitas, F. Z. ; Bertolini, M. C. Departamento de Bioquímica e Tecnologia Química, Instituto de Química, UNESP, Araraquara, SP, Brazil.

Cells respond and adapt to environmental stressing conditions such as heat shock by modulating metabolic responses. In the yeast Saccharomyces cerevisiae, heat shock activates multiple stress related genes by the binding of the trans acting elements to the cis elements HSE (Heat Shock Element) and STRE (STress Responsive Element) leading to transcription activation. The gsn gene, which encodes the enzyme glycogen synthase in Neurospora crassa is repressed at transcriptional level when cells are exposed to high temperatures (heat shock). Analysis of the gsn 5'-flanking region showed the presence of multiple HSE elements and two STRE elements, which were demonstrated to be involved in the gene transcription regulation by EMSA (Electrophoretic Mobility Shift Assay). Our main purpose is to identify the protein(s) that bind to the STRE elements in order to study how these elements function in the regulation of gene transcription. Nuclear extract of heat-shocked mycelia was analyzed by SDS-PAGE and SouthWestern blotting, and fractionated by affinity chromatography. EMSA identified a protein fraction showing higher DNA-binding activity. To enrich this approach the crude nuclear extract was fractionated by SDS-PAGE, the fractions of molecular mass intervals were excised from the gel, crushed into a renaturation buffer and eluted from the gel. The fractions were analyzed by EMSA and the MW range was identified. In the same way, the nuclear proteins were fractionated by isoeletric focusing, and the pI of the active fractions was determined. All protein fractions were subjected to trypsin digestion, and the peptides were analyzed by mass spectrometry (MALDI). Supported by FAPESP, CAPES, CNPq and LNLS.

36. Two Circadian Timing Circuits in Neurospora crassa Cells Share Components and Regulate Distinct Rhythmic Processes. Zachary A. Lewis, Renato M. de Paula, Andrew V. Greene, Kyung Suk Seo, Louis W. Morgan, Michael W. Vitalini, and Deborah Bell-Pedersen Center for Research in Biological Clocks, Department of Biology, Texas A&M University, College Station, TX 77843

In Neurospora crassa, the frequency (frq), white collar-1 (wc-1), and wc-2 genes, and their corresponding proteins (FRQ, WC-1, and WC-2), comprise the core circadian FRQ-based oscillator that is directly responsive to light and drives daily rhythms in spore development and gene expression. However, physiological and biochemical data have suggested the existence of additional timing circuits in the cell that function in the absence of FRQ (collectively termed FRQ-less oscillators, FLOs). We previously identified an evening-peaking gene, W06H2 (now called clock-controlled gene 16 (ccg-16)), which is expressed with a robust circadian rhythm in cells that lack FRQ protein. Here we demonstrate that ccg-16 rhythms are generated by a temperature compensated FLO that, similar to the FRQ-based oscillator, requires functional WC-1 and WC-2 proteins for activity. Furthermore, we show that FRQ is not essential for rhythmic WC-1 levels. Our results are consistent with a model in which two circadian timing circuits exist within Neurospora cells, both of which require the WC proteins.

37. VVD’s role in entrainment of the Neurospora crassa circadian clock.
Mark Elvin and Christian Heintzen. Faculty of Life Sciences, Michael Smith Building, The University of Manchester, Manchester, M13 9PT, UK

The PAS/LOV protein VVD is a modulator of light responses in Neurospora. We have recently shown that VVD modulates clock resetting at dawn and dusk with implications for entrainment of the Neurospora circadian clock to light-dark cycles (Elvin et al., 2005, Genes Dev 19, 2593-2605). In vvd knockout strains the phase of clock-controlled conidiation is altered when compared to wild-type and a second round of conidiation is initiated. The cause for this second peak of conidiation in vvd knockout strains is unclear but may be due (amongst other possibilities) to a reduction in the threshold for clock-independent light- induced condiation, repression of conidiation, or unmasking of a second pacemaker that controls a second round of conidiation in light-dark cycles. Data will be presented that distinguish between these possibilities.

GENOMICS AND PROTEOMICS

38. Systems Biology of the Biological Clock.
W Dong, Y. Yu, C. Altimus, J. Griffith, M. Morello, L. Dudek, H.-B. Schuttler, & J. Arnold. Genetics Department, University of Georgia. arnold@uga.edu.

Genetic networks as models for the biological clock have been developed and successfully fitted to published data in the literature. A series of microarray experiments were performed on N. crassa liquid cultures placed in the dark for 48 h to measure RNA levels of all 11,000 N. crassa genes every 4 hours (13 time points) to refine the fitted ensemble of models. A total of 3594 genes were found to be oscillatory with a period between 16 and 30 hours. Among these, 3594 oscillatory genes, 1,835 (16.6%) genes were found to have a WHITE-COLLAR complex consensus binding site for inclusion in the genetic networks. Microarray results were validated by real-time PCR. Using several different methods of analysis the clock and its associated genes was placed in the context of the metabolic web. The data are being used to guide additional microarray experiments to refine the specification of the genetic networks describing the clock. As a further test of the genetic networks, light entrainment experiments were performed using race tubes to test genetic network light response. Work is supported by NSF QSB-0425762.

39. The Neurospora crassa e-Compendium.
Alan Radford, IICB, Faculty of Biological Sciences, The University of Leeds. a.radford@leeds.ac.uk

The initial "gene list" has developed, in both the number of genes listed and the level of sophistication, to the point where it has been named the "e-Compendium". The URL is http://www.bioinf.leeds.ac.uk/~gen6ar/newgenelist/genes/gene_list.htm. The e-Compendium continues the development of the gene database from Barratt et al (1954) Adv in Genet., Perkins et al (1982) Micro Revs, and Perkins et al (2001) The Neurospora Compendium into the era of the WwW. It provides a browsable and searchable database of genetic and molecular data about individual genes (gene symbol and name, map location, phenotype, gene product, sequence data, contig data, references etc.) with cross-links to sequence databases, Exapasy Enzyme, Pubmed, graphic images of morphological mutants, biosynthetic pathway diagrams etc.. Recent additions to the e-Compendium include a Java-based utility for compiling linkage maps from the genetic and physical data in the e-Compendium, and another utility for the extraction into a spreadsheet of all the contig data on genes in the database. New genes are added to the database and additional data added as the curator finds it or as individual workers submit new information. The e-Compendium is as up to date as you, the Neurospora community, make it. Please use the in-built utilities to submit new and amended information.

40. Comparative Genomic Hybridizations using the Genus Neurospora.
Luz B Gilbert¹, Lee Chae¹, Takao Kasuga¹, Jeff Townsend ², John W. Taylor¹, ¹Department of Plant and Microbial Biology, UC Berkeley, ²Department of Molecular and Cellular Biology, University of Connecticut, lgilbert@berkeley.edu

Comparative Genomic Hybridization (CGH) using DNA microarrays is becoming a popular method of determining phylogenetic relationships among individuals and even species. Although researchers praise the large-scale genome information output as an advantage of this technology for use in phylogenetic analysis, few have questioned the reliability of CGH to correctly determine evolutionary relationships. With the tools at my disposal for the filamentous fungus Neurospora: a genome sequence, a DNA microarray, and a well supported phylogeny for this genus, I had the ability to rigorously address the utility of CGH for phylogenetic studies using both experimental and simulated data.

41. High throughput mutation procedure for Neurospora genes
Luibov Litvinkova, Lorena Altamirano, Gyungsoon Park, John Jones, and Katherine Borkovich
Department of Plant Pathology, University of California, Riverside, CA

Deletion mutations have been made in annotated Neurospora genes using a high throughput procedure as part of an NIH-funded Program Project (P01). We have used hph marked KO cassettes created by yeast recombinational cloning techniques along with mus-51 and mus-52 deletion mutants as recipient strains for transformation. We have completed construction of of 225 Neurospora knockout mutants and the strains have been submitted to FGSC. Due to ascospore inviability, we were not able to generate homokaryotic deletion mutants for another 56 genes; heterokaryotic transformants for these 56 genes were submitted to FGSC. Homokaryons were isolated using the microconidiation procedure for seven of these genes; these strains are still in the mus deletion background. The list of submitted strains is available at the Neurospora genome project website (http://www.dartmouth.edu/%7Eneurosporagenome/knockouts_completed.html). At this time, 384 genes are in various stages of the knockout procedure. We have also developed informatics tools for tracking of genes during the knockout procedure. First, we have designed a program (http://borkovichlims.ucr.edu/southern/) that allows automated identification of the appropriate restriction enzyme to use during Southern analysis for confirmation of the gene replacement. Second, we have developed and implemented a Laboratory Information Management System (LIMS; www.borkovichlims.ucr.edu) for our gene knockout process. All plates and tubes used during the knockout procedure are labeled with barcodes and managed systematically. Use of these tools and our current progress in creating knockout mutants will be presented.

42. Proteomic Approaches to Mitochondrial Function. Richard A. Collins Department of Medical Genetics and Microbiology University of Toronto Toronto, Canada M5S 1A8

There are many examples of mitochondrial responses to changes in the functional or metabolic state of the cell. For example, inhibition of mitochondrial protein synthesis leads to the synthesis of alternative oxidase and to an increase in cytochrome c content. However, we have no global-scale information about the number and identity of proteins whose concentration changes in such a situation. We know anecdotally about the few proteins that we have chosen to examine, but does the cell respond by changing the amounts of a few proteins? Or a few dozen? Or a few hundred? To answer this question, I am determining what fraction of the mitochondrial proteome can be resolved, detected and quantified using a variety of fractionation, 2D gel, and mass spectrometry approaches. This will provide an experimental platform with which to investigate how the cell compensates for a variety of alterations in mitochondrial function.

43. Genetic analysis of cytoplasmic dynein in Neurospora crassa
Michael Plamann, David Madole, Robert Schnittker, and Elizabeth Wulff. 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, we have isolated over 50 DHC mutants of Neurospora that produce full-length proteins that are defective in function.  We have identified DHC point mutations in all domains within the dynein motor head.  To help define the mechanism(s) by which specific mutations lead to loss of dynein activity we have isolated revertants for a subset of DHC mutants.  We are now in the process of identifying the respective intragenic suppressor mutations in these revertants.

44. Cytoplasmic dynein, cytoplasmic streaming and nuclear movement in Neurospora crassa

 Michael Plamann, Robert Schnittker, Samuel Schowengerdt, and Omar Abdulla Almoghrabi. 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 is required for nuclear distribution in nearly all eukaryotes.  In germinated conidia of Neurospora, dynein null mutants have clusters of nuclei in distal regions while hyphal tips are often anucleate.  Using a nuclear-localized GFP constructed by Dr. Michael Freitag, we examined nuclear distribution in mature colonies of dynein mutants.  In contrast with germinated conidia, nuclear distribution is nearly wildtype in hyphae at the colony edge of dynein mutants.  Examination of these colonies revealed that there is very rapid streaming of cytoplasm and nuclei (up to ~50 mm/sec) from the interior of the colony to growing tips at the colony periphery.  Hyphal fusion is critical for the formation of interconnecting hyphal networks within the interior of colonies.  We found that Neurospora soft (so) mutants, which are blocked in hyphal fusion, lack cytoplasmic streaming.  Dynein, soft double mutants were constructed and found to grow very slowly and produce short aerial hyphae with no conidia.  The results suggest that cytoplasmic dynein is required for movement of nuclei in newly formed hyphae from germinating spores and young colonies, but nuclear movement in mature colonies results primarily from the bulk flow of cytoplasm. 

45.  withdrawn

OTHER

46. The mitochondrial ribosomal RNA genes are as identified earlier by other workers. S. Conway, P. J. Yeadon, F. J. Bowring and D. E. A. Catcheside
School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, South Australia, Australia

While the Neurospora genome appears to lack an msh4 orthologue, a TBlastn search, using msh4 sequences from Saccharomyces cerevisiae, Homo sapiens and Mus musculus, identified an msh4 candidate on contig 3.27. This putative gene, which has an ATPase domain and a DNA-binding mismatch repair domain typical of MutS family members, was deleted by split-marker replacement. In crosses of deletion strains, sporogenesis is delayed and there is a four- to six-fold reduction in fertility. These defects also occur in heterozygotes showing that the gene is subject to meiotic silencing. The frequency of crossing over in the intervals flanking his-3 is halved and, as is the case for yeast msh4 mutants, residual crossovers show no interference, suggesting that we have knocked out Neurospora msh4. Interestingly, allelic recombination at his-3 appears to be elevated in the mutant, a result that was not predicted using the yeast msh4∆ phenotype as a model.

47. Novel Linear Vector Imparts High Stability to “Unclonable” DNA Sequences.
Ronald Godiska¹, Vinay Dhodda, Valerie Gilbert¹, Nikolai Ravin², and David Mead¹. ¹Lucigen Corp., Middleton, WI. ²Centre BioEngineering RAS, Moscow, Russia

We have developed a novel linear E. coli cloning vector incorporating transcription-free cloning capabilities. This vector showed unprecedented ability to maintain large AT-rich inserts (>10-20 kb), as well as di-, tri-, and tetra-nucleotide repeats. These inserts were not stable in conventional plasmids. Torsional strain inherent to supercoiled plasmids can induce localized melting and generate secondary structures, which are substrates for deletion or rearrangement by resolvases and replication enzymes. For example, tandem repeats and palindromic sequences are highly unstable, presumably due to cleavage of hairpin structures or to replication slippage across the secondary structures. Most plasmid vectors also induce strong transcription and translation of inserted fragments, and they allow transcription from cloned promoters to interfere with plasmid stability. As a result, many DNA sequences are deleterious or highly unstable, leading to sequence “stacking”, clone gaps, or a complete inability to construct libraries, especially from AT-rich genomes or toxic cDNAs. These problems appear to be greatly diminished with the transcription-free, linear vector. This vector showed numerous other advantages in cloning, such as the ability to clone inverted repeats, a significantly lower bias in size of fragments cloned, a low background of non-recombinants, and simple construction of large-insert libraries.

48. Nonself recognition and Programmed Cell Death in Neurospora crassa
Karine Dementhon, Isao Kaneko, Julie Welch and N. Louise Glass Plant and Microbial Biology department, UC Berkeley, California, 94720. email: karine@berkeley.edu

Filamentous fungi are capable of undergoing hyphal fusion with each other to form a vegetative heterokaryon (genetically different nuclei in a common cytoplasm). However, if individuals undergoing hyphal fusion differ in allelic specificity at any one of a number of heterokaryon incompatibility loci called het, the heterokaryotic cell is rapidly compartmentalized and destroyed by a programmed cell death reaction. This phenomenon is called heterokaryon incompatibility (HI). This nonself recognition is believed to reduce the risk of transmission of infectious elements such as mycoviruses and debilitated organelles throughout fungal populations, and to restrict resource plundering between individuals. In Neurospora crassa, we determined that het-c HI requires non-allelic interactions with pin-c, a locus closely linked to het-c. HET-C is a plasma membrane protein and PIN-C encodes a HET domain protein. Isolates from populations fall into one of three allelic specificity groups for the het-c locus: het-c(1), het-c(2) and het-c(3) (previously called Oakridge, Panama and Groveland, respectively). They differ by a highly variable domain (specificity domain) which determine allelic specificity. Pin-c alleles in isolates of alternate het-c specificity are extremely polymorphic. Pin-c alleles can also be classified into 3 groups: pin-c(1) , pin-c(2) and pin-c(3), which are in severe linkage disequilibrium with het-c. Similar to het-c, pin- c alleles show trans-species polymorphisms. Together, our data suggest that balancing selection operates at het-c/pin-c haplotype to maintain allelic diversity for nonself recognition and HI in N. crassa.

49. Systems Biology of the Quinic Acid Cluster in Neurospora crassa
Allison Koch and Dr. Jonathan Arnold of the University of Georgia

The quinic acid (qa) gene cluster, in Neurospora crassa, encodes the organism’s pathway for utilizing quinic acid (QA) as a sole carbon source.  When N. crassa is grown on sucrose medium, the quinic acid gene cluster is inactive.  When the organism is shifted to a medium that has quinic acid instead of sucrose, the qa cluster genes are activated.  To see the changes in expression of these genes, samples were taken at eight time points after the shift to quinic acid from 0 to 8 hours.  The RNA samples from these time points were analyzed with micro array chips, which gave the precise measurements of the RNA levels of each of the 11,000 genes in the genome.  A total of 549 genes respond to a shift to quinic acid, and 164 of these have a QA-1F binding site.  These data are being used to evaluate a genetics network describing: (i) qa gene cluster regulations; (ii) QA metabolism; (iii) catabolic repression by glucose or sucrose

50. Protein interaction studies of the Neurospora crassa G_i alpha homolog, GNA-1.
Sara Martinez and Katherine Borkovich. Department of Plant Pathology, University of California, Riverside, CA 92521

Heterotrimeric G protein signaling pathways are important for eukaryotic cells to respond to environmental stimuli. Heterotrimeric G proteins consist of a triad of proteins named G alpha, G beta, and G gamma. In pathogenic filamentous fungi, these proteins are important for virulence. In the non-pathogenic filamentous fungus, Neurospora crassa, there are three G alpha subunits (GNA-1, GNA-2, and GNA-3), one G beta subunit (GNB-1), and one G gamma subunit (GNG-1). It has been previously reported that GNA- 1 is involved in female fertility, vegetative growth, and stress response. Such a broad impact of this protein suggests that there are multiple proteins with which it interacts. A cDNA library was made using RT-PCR to make cDNA and yeast recombination to make a library in the yeast two-hybrid AD vector (Clontech). The RNA was obtained from 16hr submerged culture tissue and 6 day old SCM plates. This library was screened with GNA-1 using a mating strategy, and several interesting interactors were identified. This poster describes the techniques used for the screen and follow up experiments on some of the interesting hits. Identifying novel downstream effectors of GNA-1 will give insight as to how eukaryotes respond to stimuli, and also give us a clue as to how GNA-1 affects such a broad range of tissues types in Neurospora crassa.

51. Meiotic recombination in Neurospora
F. J. Bowring, P.J. Yeadon and D. E. A. Catcheside. School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, South Australia, Australia

Our knowledge of meiotic recombination is based upon detailed analysis of a small number of organisms. The genome project coupled with recent advances in methodology makes a comparable analysis of the process in Neurospora feasible. We are currently using fusion-PCR/split-marker deletion to examine the role of several genes in Neurospora recombination. We are also trying to develop a visual recombination reporter system using histone-GFP fusions targeted to a recombination hotspot.

52. Activities and Research at the Fungal Genetics Stock Center.
Kevin McCluskey, Sheera Walker and Mike Plamann. School of Biological Sciences, University of Missouri- Kansas City.

In 2005 the FGSC distributed nearly two times as many strains as ever before. We sent out 2125 fungal strains to 138 different recipients in thirty-one different countries. Of these, 1509 were Neurospora strains. We have also distributed significant numbers of cloned genes and library clones. Not including strains from the functional genomics program, we added 406 new strains to the collection. 277 of these were Neurospora strains. Six were other organisms that have been sequenced or will be soon.
We have also added a number of new resources to the FGSC web-site including all back issues of the Aspergillus newsletter. We also built an interface that allows us to link between online methods and existing indices.
In our continuing effort to identify the function of temperature sensitive unknown lesions, we have complemented two different unknown genes. One of these, un-16, had been previously identified. Nevertheless, we have attempted to develop this gene as a selectable marker for transformation in Neurospora.
The FGSC is supported by the US National Science Foundation, Grants  0235887 and 0603830

53. The Neurospora crassa community genome annotation project.
Heather M. Hood¹, Matthew R. Henn², Dave DeCaprio², James E. Galagan², Bruce W. Birren², Jay C. Dunlap³, & Matthew S. Sachs^1
1Oregon Health & Science University, Beaverton, OR 97006; ²Broad Institute of MIT & Harvard, Cambridge, MA 02141; ³Dartmouth Medical School, Hanover, NH 03755.

In order to produce a more accurate Neurospora protein-coding gene catalog, we need to include the wealth of information about gene structure and function that is contained within the scientific community. Automated gene calling predicts 10620 putative genes in Release 7. As of this conference, a web-based community annotation resource is released. Using this site, community annotators will be able to add a variety of information that automated annotation cannot capture to Release 7, which will be incorporated into the database. These data include gene symbol, gene name, and protein product. Incorrect gene models also can be fixed or alternative transcripts can be appended to the predicted gene. Gene product function can be assigned using Gene Ontology terms along with the corresponding literature citation. Annotators can also indicate where the gene or its product is expressed using the Fungal Anatomy Ontology. To ensure accuracy, all community annotations will be manually inspected and curated. Integrating these community annotation data into the Neurospora genome annotation will greatly improve this resource.
 

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