Cell Biology Abstracts
288. The swollen cell phenotype of the swoA mutant of Aspergillus nidulans. Gretel M. Abramowsky, Patrick J. Westfall and Michelle Momany. University of Georgia, Botany, Athens, Georgia, USA.
Temperature sensitive swo (swollen) mutants of Aspergillus nidulans have been isolated. They represent eight genes that may be involved in polarity establishment, polarity maintenance and hyphal morphogenesis. One of these mutants, swoA, grows isotropically at restrictive temperature, and is unable to produce a germ tube. When shifted from restrictive to permissive temperature, multiple normal sized germ tubes emerge. Conversely, a shift from permissive to restrictive temperature halts elongation and isotropic swelling begins. Currently, further characterization and cloning experiments of swoA are being conducted.
289. Isolation of the rad12 gene of Coprinus cinereus. Sonia N. Acharya1, Marilee Ramesh2, and Miriam E. Zolan1. 1Indiana University, Biology, Bloomington, Indiana, USA. 2Department of Biology, Indiana University at Purdue, Indianapolis.
The Zolan lab has identified four Coprinus cinereus genes necessary for DNA repair and meiosis, rad3, rad9, rad11, and rad12. rad11 has been shown to be the Coprinus homolog of Saccharomyces MRE11, a gene involved in DNA double-strand break repair and meiosis. In both yeast and humans, Mre11 forms a complex with the Rad50 protein. I cloned the Coprinus homolog of RAD50 using degenerate PCR. rad50 maps to the rad12 locus; RFLP mapping revealed 0/115 recombinants therefore I am testing the hypothesis that rad50 is rad12. Cosmids containing rad50 will be used in transformation experiments to test for rescue of the rad12mutant phenotypes of radiation sensitivity and defective meiosis. Electron and light microscopy of surface-spread nuclei have shown that rad12mutants show prophase I defects in chromosome condensation and, depending on the mutant, varying degrees of synaptonemal complex formation. I will examine homolog pairing in the mutants using fluoresence in situ hybridization.
290. Intergeneric Fragmented DNA transfer from stress tolerant Bacillus sp. to improve some economic traits in S. cerevisiae. Kamel A. Ahmed1, Mohamed H. Hamoda1, Tahany M. El-Kawokgy2, and Nivien A. Abosereih2. 1University of Cairo, Dept. of Genetics, Cairo, Egypt. 2National Res. Centre, Microbial Genetics Lab, Egypt.
Fragmented naked DNA was isolated from two local Bacillus sp. strains. one is tolerant to 99% methylated alcohol and the second tolerated 30% Nacl. Each DNA was transfered to four genetically different haploid S. cerevisiae strains. The frequency of yeast transformants was found to vary between different recipient strains. Some alcohol tolerance transformants were able to tolerate 22% alochol compared to the level of 13% in the original recipients. In the case of Nacl transformants,their tolerance reached 18% while the tolerance level of recipients did not exceed 10%. 22% alcohol tolerant transformants or 18% saline tolerant ones were found to have higher growth rate, higher wheat meal fermentation ability than their original parents. When crossed to their original parents resulted duploids were also higher in their growth rate and fermentation ability than their original parents constructed duploids. Attempts were carried out to allocate tolerance genes.
291. Motor proteins involved in nuclear migration in the filamentous ascomycete Ashbya gossypii. C. Alberti-Segui, R. Altman, F. Dietrich, and P. Philippsen. University of Basel, Microbiology, Basel, Switzerland.
In order to follow nuclear movement within the hyphae during fungal growth, an in-frame GFP fusion to the histone H4 has been carried out which resulted in a strong fluorescent labeling of nuclei. Video fluorescence microscopy and time-lapse studies revealed an active traffic of nuclei within the hyphae, including continuous oscillations, frequent mitotic events, by-passing of nuclei and movement through septa. This dynamic behaviour results in a uniform distribution of nuclei along the hyphae. To better characterize nuclear movement, we also measured some dynamic parameters. Under time-lapse condition (minimal medium, 24 °C), we estimated the nuclear velocity to be 0.3 µm per minute which was similar to the hyphal tip extension, whereas oscilation velocity could reach 4 µm per minute. By random sequencing of three thousand Ashbya DNA clones, we identified five homologs to S. cerevisiae genes coding for molecular motors that have been shown to be involved in nuclear migration (4 kinesin-like proteins and 1 dynein heavy chain). PCR targeting based on homologous recombination has been used to delete the corresponding genes. Deletion of two of the kinesin-like proteins did not affect to an observable degree nuclear distribution or migration but caused morphogenic and sporulation defects. The mutant that showed the most severe defect in nuclear migration was the dhc1 mutant for which the whole dynein heavy chain gene has been removed. In contrast to the dynein mutant of Aspergilus nidulans where nuclei fail to move into the germ tube, we observed clumping of nuclei at the tip of the hyphae.
292. sodVIC is an alpha-COP-related gene
which is essential for establishing and maintaining polarised growth in Aspergillus
nidulans. Susan J. Assinder1, Susan L.
Whittaker1, Kelly J. Milward1, and John H. Doonan2.
1University of Wales, Bangor, Biological Sciences, Bangor, Gwynedd, UK.
2John Innes Centre, Cell Biology, Norwich, UK.Strains of Aspergillus
nidulans carrying the conditional-lethal mutation
sodVIC1 (= Stabilisation Of Disomy) are defective in
nuclear division and hyphal extension. The mutation affects both the establishment and the
maintenance of polar growth, since mutant spores do not germinate at restrictive temperature and
pre-existing hyphae stop growing upon upshift. The defect is reversible within the first 3-4 hours
at restrictive temperature but longer periods of incubation are lethal due to cell lysis and
morphological abnormalities. There is no evidence for a specific cell cycle lesion, suggesting the
existence of a feedback mechanism whereby hyphal extension is co-ordinated with nuclear
partitioning. The wild-type sodVIC gene has been cloned
from a chromosome VI-specific cosmid library and its product exhibits strong homology to the
alpha-COP subunit of the coatomer complex involved in the secretory pathway in yeast and
higher organisms. Molecular disruption of the gene is lethal, indicating that SodVIC
is essential for growth in A. nidulans. A codon-modified form of the gene for green
fluorescent protein (GFP), adapted for expression in plants, has been used as a vital reporter for
protein location in A. nidulans. Translational fusions which target GFP to the Golgi
or endoplasmic reticulum have been used to study the effect of the
sodVIC1mutation on protein trafficking.
293. Role of homologs of yeast bud1, bud2 and cla4 in hyphal
morphogenesis of the filamentous fungus Ashbya gossypii.
The cotton pathogen Ashbya gossypii is a promising model system for studying hyphal morphogenesis in filamentous fungi. Its haploid genome of 8.85 Mb encodes no more than 4500 proteins which is one third less than encoded by the Saccharomyces cerevisiae genome. Gene manipulations by PCR-based gene targeting work as efficiently in this fungus as in S. cerevisiae. The hyphae of Ashbya gossypii show apical growth, lateral branching and dichotomous tip branching. Sequence analysis of random genomic clones (with the help of Novartis Ltd) revealed several homologs to S. cerevisiae morphogenesis genes. Interestingly, only few of the identified Ashbya genes lack homology to S. cerevisiae. In order to characterize the role of homologs of yeast morphogenesis genes in Ashbya, we analysed as a first step phenotypes of deletion mutants. Agbud1 mutants grow slower than wildtype. Agbud2 mutants show increased lateral branching and decreased dichotomous tip branching. Agcla4 deletant strains develop much thicker hyphae and do not form septa. A detailed description of these phenotypes will be provided. In addition, 17 Ashbya genes lacking homology to S. cerevisiae were deleted; no morphogenetic defects were observed.
294. Analysis of homologous gene targeting in hyperrecombinant mutants of Aspergillus nidulans. Patricia J. Ayoubi, and Rolf A. Prade. Oklahoma State University, Microbiology and Mol Gen, Stillwater, OK, USA.
A number of molecular genetic tools and methods have recently been developed for filamentous fungi which rely on genetic recombination and integration of donor DNA into a chromosomal locus. Genetic recombination is a fundamental cellular process ubiquitous among living organisms and underlying most classical genetics techniques. However, the efficiency of more modern homology-based gene targeting techniques in filamentous fungi such as Aspergillus nidulans can be affected by many factors and often results in complex ectopic integration events. In sexually reproducing eukaryotic organisms, homologous recombination is required for pairing of homologs and ensures proper chromosome segregation. Sexual development in A. nidulans involves the development of specific sexual structures and probable gene expression patterns different from vegetative mycelia. Further, gene products required for sexual development likely include products involved in meiotic homologous recombination. For example, meiosis-specific RecA homologs have been identified in yeast (Dmc1) and other fungi, plants and animals. Interestingly, examination of sequences upstream of the uvsC transcription start site (a gene of A. nidulans homologous to dmc1 from yeast) reveals a putative MluI cell cycle box. In addition, uvsC cDNA is detectable in sexual structure-specific ESTs but not in asexual-specific ESTs. Combined, these observations indicate a strong correlation between homologous recombination and sexual development in A. nidulans. In an effort to better understand homologous recombination and possibly improve homology-based gene targeting rates in A. nidulans, we have compared the frequencies of gene targeting by circular and linear plasmid DNA constructs in various A. nidulans hyperrecombinant and DNA repair mutants.
295. A Polo-like kinase in Aspergillus nidulans: implications for regulation of mitosis and development. Catherine L. Bachewich and Stephen Osmani. Penn State College of Medicine, Weis Centre for Research, Danville, PA, USA.
Aspergillus nidulans has proven to be an excellent model organism for investigating aspects of general cell cycle regulation and development. In particular, our understanding of the regulation of mitosis was advanced by determining the presence and function of major mitotic regulatory factors, some of which for the first time, in this organism. The Polo-like kinases are a relatively new family of cell cycle regulators implicated in various stages of mitosis, as well as cytokinesis and septation. Despite their widespread occurrence, ranging from yeast to human cells, their precise functions and regulation are not clear. To date, Polo-like kinases have not been described in any filamentous fungus. In order to progress our understanding of mitosis and development in Aspergillus and higher organisms, and to elucidate potential functions and interactions of Polo-like kinases with other mitotic factors, such as NIMA kinase, we investigated the presence of a Polo-like kinase in Aspergillus. A full length clone was isolated, using RACE PCR with primers designed against sequences in Aspergillus EST's with similarity to conserved regions of the Polo sequence. The genomic clone was recovered from chromosome 7 of a cosmid library. The genomic and cDNA clones were similar in size, at approximately 3.8 kb. Sequencing the genomic clone revealed high similarity to the Polo-like kinases across the amino-terminal, catalytic domain, and within the carboxy-terminal "Polo box", a conserved region defining Polo-like kinases. Manipulation of the gene, including overexpression at various stages of the cell cycle and molecular deletion, are underway to help determine the function of the gene and its contribution to cell cycle progression in Aspergillus.
296. Mechanisms of replication of circular fungal mitochondrial plasmids and mitochondrial DNA. Dipnath Baidyaroy1, and Helmut Bertrand2. 1Michigan State University, Botany & Plant Pathology, East Lansing, Michigan, USA. 2Michigan State University, Microbiology, East Lansing, Michigan, USA.
Fungal mitochondrial plasmids can be of three major groups: (1) linear and usually encoding a DNA polymerase and an RNA polymerase, (2) circular with a gene coding for a reverse transcriptase, and (3) circular with a novel DNA polymerase gene. We are trying to characterize the replication mechanisms of the two types of circular plasmids by analyzing replication intermediates using two-dimensional gel electrophoresis. Contrary to previous studies (Maleszka, 1992, Biochem. Biophy. Res. Comm. 186:1669-1673), we found that the plasmids are present in vivo as multimeric circles instead of linear molecules of heterogenous sizes. In addition, we have obtained data indicating that the Mauriceville plasmid of Neurospora crassa, despite containing only a reverse transcriptase gene, replicates by three different mechanisms: (1) reverse transcription, (2) replication from an internal origin, and (3) a rolling circle mechanism which effectively generates numerous multimeric forms, some as big as octamers (~28.8 kb), of the unit length plasmid. On the other hand, the pCRY1 plasmid of Cryphonectria parasitica and the Fiji and LaBelle plasmids of Neurospora, all of which are assumed to replicate by the action of their own DNA polymerases, seem to replicate by a rolling circle mechanism. Mitochondrial DNA-derived circular plasmid-like elements were also observed to replicate via rolling circle mechanisms in C. parasitica. Collectively these observations suggest that replication in fungal mitochondria is predominantly mediated through the rolling circle mechanism. Supported by USDA grant 95-37303-1785 and MAES Project No. MICL01662.
297. Transmission of a mitochondrial plasmid across vegetative incompatibility barriers in Cryphonectria parasitica. Dipnath Baidyaroy1, Jonathan M. Glynn2, and Helmut Bertrand2. 1Michigan State University, Botany & Plant Pathology, East Lansing, Michigan, USA. 2Michigan State University, Microbiology, East Lansing, Michigan, USA.
Previous studies have shown that exchange of mitochondrial DNA (mtDNA) occurs between the hyphae of vegetatively compatible strains in Cryphonectria parasitica, but little is known about the asexual transmission of mitochondrial elements among incompatible strains. In this study, we have assessed the effect of five vegetative incompatibility (vic) genes on the horizontal transmission of the mitochondrial plasmid pCRY1. Out of the five genes tested, an allelic difference between the donor and the recipient strains at only one locus strongly inhibited the transmission of pCRY1. Two genes allowed transmission in one direction (from one specific allele to the other; only two alleles are known for each vic locus) while the remaining two genes allowed transmission in both directions. However, the extent of transmission varied from one gene to another. The significance of this study is that none of the vic genes completely inhibited the horizontal transmission of pCRY1. In most combinations, at least one recipient isolate acquired the plasmid. The plasmid was also successfully transferred between two highly incompatible strains by protoplast fusion. In every case, the plasmid invaded and stably colonized the recipient after being transmitted. This data suggests that, in nature, mitochondrial genetic elements can be transmitted irrespective of karyotic compatibility or incompatibility and can behave like infectious elements. This infectious behavior suggests that mitochondrial plasmids might be effective as biological control agents of C. parasitica in nature, provided they elicit mtDNA mutations like some of the Neurospora plasmids. Supported by USDA grant 95-37303-1785 and MAES Project No. MICL01662.
298. Identification and Characterization of the Neurospora crassa opsin, NOP-1. Jennifer A. Bieszke1, Edward L. Braun2, Laura E. Bean 3, Seogchan Kang4, Donald O. Natvig 3, Elena N. Spudich1, John L. Spudich1, and Katherine A. Borkovich1. 1University of Texas Medical School-Houston, Microbiology , Houston, TX, USA. 2The Ohio State University, Plant Biology, Columbus, OH, USA. 3University of New Mexico, Biology, Albuquerque, NM, USA. 4Pennsylvania State , Plant Pathology, University Park, PA, USA
Opsins are seven-transmembrane helical apoproteins that form light absorbing pigments
upon binding retinal. Previously, genes encoding opsins had only been identified in animals and
the archaea. Here, we report the characterization of an opsin gene, nop-1, from the
eukaryotic filamentous fungus Neurospora crassa. The NOP-1 protein sequence is
81.1% identical to archaeal opsins in the retinal-binding pocket. Evolutionary analysis revealed
clear homology between NOP-1 and several fungal opsin-related proteins; thus, NOP-1 is a
plausible link between archaeal and visual opsins. Expression of the nop-1 gene is
highest under conditions that favor conidiation and is also positively influenced by the presence
of light. A role for NOP-1 in conidiation is supported by the light-dependent conidiation
phenotype of nop-1 strains in the presence of the mictochondrial
H+-ATPase inhibitor oligomycin. The NOP-1 protein was overexpressed in the
methylotrophic yeast, Pichia pastoris, in order to study its photochemistry. Static
absorbance spectroscopy showed NOP-1 could bind all-trans retinal
(lmax> = 534nm). In addition, flash photolysis was used to show NOP-1
undergoes a photochemical reaction cycle. The results demonstrate that NOP-1 is an opsin
capable of binding and photocycling retinal in a manner most similar to archaeal rhodopsins, and
whose function is important for the conidiation process in N. crassa.
299. New insights into meiotic recombination from study of the am locus in
Neurospora.
Analysis of the segregation of flanking markers amongst recombinants in heteroallelic repulsion-phase crosses has been used to generate maps of the am locus. While three studies are in close agreement concerning the order of alleles within the locus, there is conflict over the orientation of the locus with respect to flanking markers. Alignment of the allele maps with respect to flanking markers was based on the relative frequency of the two classes of prototrophic progeny where flanking markers are recombined. Fincham's (1967) data are in general consistent with the 5' end of the gene being centromere distal, Smyth's (1973) data are consistent with the opposite orientation and Rambosek and Kinsey's (1984) data suggest no specific orientation. Molecular data confirm Fincham's orientation and because different combinations of flanking markers were used in each study, we speculated that the differing conclusions concerning the orientation of this locus may result from incidental crossing over in the flanking intervals (Bowring and Catcheside 1995). The segregation of molecular flankers located approximately 4 kb distal and 6 kb proximal of the am locus confirm this. Among prototrophic recombinants from a cross heteroallelic am1am6 , 93% of crossovers were in the intervals flanking am and thus not directly associated with the recombination event at this locus (Bowring and Catcheside, 1996). We have now shown that there is a clustering of crossovers immediately distal of am in prototrophic recombinants suggesting that recombination events in am promote crossovers nearby. This appears to be the first demonstration of negative interference reaching statistical significance.
300. SepH: A ser/thr protein kinase required for septation in Aspergillus nidulans. Kenneth Seward Bruno1, Jennifer L. Morrell2, John F. Marhoul3, and John E. Hamer1. 1Purdue University, Biological Sciences, West Lafayette, IN, USA. 2Vanderbilt University, Dept. of Cell Biology, Nashville , TN, USA. 3Novartis Crop Protection, Fungal Targets Group, RTP, NC, USA.
The filamentous fungus Aspergillus nidulans forms septa in order to partition portions of an extending germ tube thus cellularizing the resultant mycelia. Septum formation is temporally and spatially regulated by nuclear positioning and cell cycle progression. sepH1 was identified in a conditional mutant screen for cytokinesis mutants. At restrictive temperature sepH1 mutants polarize a germ tube and undergo many rounds of nuclear division but fail to form septa. Septum formation is reversible in sepH1 mutants when they are returned to permissive temperature, but remains blocked if nuclei are prevented from traversing mitosis. SEPH is a 165kD ser/thr protein kinase. SepH is 42% identical and 62% similar to cdc7, a gene required for septum formation in S. pombe. Targeted disruption of the sepH gene results in a strain (seph::argB) which is unable to form septa at any temperature. Interestingly, the disruption strain displays a thermosensitive-lethal phenotype with cells accumulating defects observed as aberrant nuclear morphology at 42 C. A screen for suppressors of this thermosensitve phenotype has yielded a pool of seph::argB/mutant strains that are viable at 42 C and capable of forming septa. Termed BSH for by-pass sep H, the phenotype of these suppressors may offer some insight into sepH function. Bsh167 is temperature sensitive and produces uninucleate sub-apical cells. This phenotype is strikingly similar to that of a strain highly expressing the S.pombe cdc7 gene under the control of the inducible AlcA promoter. SepH and cdc7 represent an emerging class of protein kinases involved in cytokinesis.
301. Monitoring of cytosilic Ca2+ levels in vivo in Emericella (Aspergillus) nidulans expressing Apoaequorin.Hong Cao and Diana C. Bartelt. St. John's University, Biological Sciences, Jamaica, NY, USA.
The focus of our research has been the A. nidulans calmodulin-dependent protein kinase (ACMPK) which can serve as both a target and a transducer of calcium-dependent signalling. We have shown that ACMPK is encoded by a single copy gene and is essential for viability. The molecular mechanisms by which Ca2+ and ACMPK control growth are unknown. The ability to measure changes in Ca2+ concentration in vivo during growth, cell division, and in response to external stimuli would aid in the identification of Ca2+-mediated cellular processes. Several laboratories have developed the approach of creating transgenic organisms that express apoaequorin which can be targeted to various intracellular compartments. Treatment such organisms with coelenterazine reconstitutes functional aequorin within the cytosol and subcellular organelles of their cells, thereby producing luminescence whose light emission directly reports the internal Ca2+ concentration. We have constructed strains of A. nidulans that express apoaequorin with a view toward studying Ca2+ fluxes in vivo as a function of cell division cycle, development and responses to external stimuli. Luminescence requires incubation of the cells with coelenterazine and is calcium-dependent. In both defined and rich media to which no calcim has been added, cytosolic Ca2+ levels are maintained below 50 nM. Characterization of the strains will be presented (Supported by N.I.G.M.S.).
302. Isolation and characterization of the gene encoding G-alpha protein homolog from Aspergillus nidulans. Mi-Hee Chang, and Kwang-Yeop Jahng. Chonbuk National University, Biological Sciences, Chonju, Chonbuk, South Korea.
Heterotrimeric G proteins, consisting of alpha, beta and gamma subunits, are implicated in major signal transduction pathways governing the various cellular functions in eukaryotic organism. To study the role of G protein in differentiation of the filamentous fungus A. nidulans, we isolated ganA gene encoding Galpha protein homolog from A. nidulans by PCR amplification using degenerate oligonucleotide primer and analyzed its nucleotide sequence. Open reading frame of ganA containing 361 amino acids is highly related to alpha-subunits of heterotrimeric G proteins in other organisms. DNA fragment containing ganA cDNA could partially complement gpa1 point mutation of S. cerevisiae. By targeted integration we made deletion mutations of ganA in which the internal fragment was replaced by the argB gene. We also constructed dominant-activating mutation and overexpression clone of ganA. However these mutations showed no significant phenotypes in asexual or sexual development. From these mutants, we are determining the cellular function of ganA in development and growth. This work was supported by grants for Research for Genetic Engineering from KRF.
303. Double strand break repair in Coprinus cinereus. W. Jason Cummings, Chris Johnson, and Mimi Zolan. Indiana University, Biology Department, Bloomington, IN 47405, USA.
Using the basidiomycete fungus Coprinus cinereus as an experimental system, we have isolated mutants, representing four distinct genetic loci, which exhibit elevated sensitivity to ionizing radiation and are also defective in meiosis. Epistasis analysis has demonstrated that these genes, called rad 3, 9, 11 and 12, are part of a single gamma-radiation survival pathway. In Saccharomyces cerevisiae, members of the RAD52 epistasis group illustrate that mutants exhibiting the dual phenotypes of hypersensitivity to ionizing radiation and defects in meiosis are compromised in their ability to repair DNA double-strand breaks. Since the C. cinereus rad mutants demonstrate these dual phenotypes, we are developing an assay for monitoring the repair of a single DSB formed by the rare-cutting endonuclease I-SceI. Strains containing integrated versions of a substrate construct and a transcriptionally regulatable endonuclease construct are used in this assay. The substrate construct contains repeated copies of a hygromycin resistance gene which, following cleavage, can be repaired either by homologous recombination or by an end-joining mechanism. This assay will allow us to asses the efficiency of DSB repair in C. cinereus, and will be used to determine the extent to which the mutants in our collection are deficient in DSB repair. In addition, the assay will be used to obtain insight into the specific mechanisms of repair that may be affected in rad mutants by testing the efficiencies of both homology-dependent repair and end-joining repair processes. We will present results obtained using the described assay in C. cinereus.
304. Gene map of chromosome I of Ashbya gossypii. Fred S. Dietrich1, Sylvia Voegeli1, Tom Gaffney2, Christine Mohr1, Corinne Rebischung1, Rod Wing3, Steve Goff4, and Peter Philippsen1. 1University of Basel, Applied Microbiology, Basel, BS, Switzerland. 2Novartis, Research Triangle Park, NC, USA. 3Clemson University, Genomics Institute, Clemson, SC, USA. 4Novartis, La Jolla, CA, USA.
We have identified the complete set of genes on Chromosome I of the filamentous ascomycete Ashbya gossypii. The genome size of A. gossypii is 8.8Mb divided into seven chromosomes. The smallest chromosome, chromosome I, is 680kb in length and encodes 28 tRNAs, 1 structural RNA, and 378 proteins. Also identified are the centromere and telomeres. More than 95% of the chromosome I genes share homology with genes in the public databases, particularly with S. cerevisiae. The gene map, which is based on 2-3 fold sequence coverage spanning the chromosome, reveals that nearly all genes are found in blocks of synteny between S. cerevisiae and A. gossypii. These blocks of synteny contain up to 63 genes when the genome duplication in the S. cerevisiae lineage is taken into account. Examination of the gene order indicates that a combination of more than 20 interchromosomal and intrachromosomal exchange events have occurred since the divergence from a common ancestor of S. cerevisiae and A. gossypii. Many of the exchange events, which can be recognized as break points between blocks of synteny, appear to have occurred at tRNA genes.
305. Cleavage membrane development in Allomyces
macrogynus. Karen Elizabeth. Fisher, and Robert W. Roberson.
Arizona State University, Plant Biology, Tempe, AZ, USA.The zoosporangia of Allomyces
macrogynus are coenocytic cells containing approximately 24 to 36 nuclei. During
zoosporogenesis, membranes develop which sequester nuclei and cytoplasm into uninucleate,
uniflagellate zoospores of approximately equal volume. We have documented the development
of cleavage membranes using standard epifluorescence and laser scanning confocal microscopy
in living zoosporangia stained with the lipophilic styryl dye, FM 4-64. During initial stages of
zoospore formation, FM 4-64 staining was observed only at the plasma membrane. Within 5 to
10 minutes, discrete sites of intense fluorescence were detected along the plasma membrane.
From the regions, membranous elements developed and ramified through the sporangial
cytoplasm. These membranes eventually converged after approximately 30 to 40 minutes,
forming an elaborate network of interconnected membrane sheets, which divided the cytoplasm
into hexagonally shaped zoospore initials. Zoosporangia treated with cytoskeletal inhibitors (i.e.
nocodazole, cytochalasin D [CD]) displayed irregular membrane development, generating
multinucleate, multiflagellate zoospores of disproportionate volumes. Results were most extreme
in zoosporangia treated with CD, an inhibitor of actin polymerization. These sporangia exhibited
little cleavage membrane development, forming only 2 to 4 large, multinucleate, multiflagellate
zoospores. Observations indicate that cleavage membranes originate at the plasma membrane and
their proper development and function requires an intact actin cytoskeleton.
306. Characterisation of Magnaporthe grisea NTH1/PTH9: a gene encoding a
neutral trehalase enzyme with a role in pathogenicity
The neutral trehalase encoding gene, NTH1/PTH9, was identified previously in the rice blast fungus Magnaporthe grisea using insertional mutagenesis. Strains carrying an insertion within the gene show reduced pathogenicity toward susceptible grasses as well as a consistently reduced trehalase activity in conidial extracts. Targeted deletion of NTH1/PTH9 in a rice pathogen of M. grisea also gives rise to strains with reduced pathogenicity and reduced conidial trehalase activity. Accumulation of trehalose coupled to increased trehalose turnover has been shown to play a role in response to varied stresses in yeast. Consistent with this view the NTH1/PTH9 gene is transcriptionally activated in response to hyper-osmotic stress and contains two closely spaced STRE elements within its promoter. This trancriptional induction is independent of the OSM1 MAP kinase, a homologue of Saccharomyces cerevisiae HOG1p. We have recently obtained a clone of a putative M. grisea trehalose-6-phosphate synthase encoding gene which is also expressed in response to hyper-osmotic stress. Molecular genetic and biochemical analysis together with cytological studies of infection structure formation and function are being employed with a view to ascertaining possible roles for trehalose metabolism in pathogenesis.
307. Expression of mitogen-activated protein kinase, Mkp1, of Pneumocystis carinii restores resistance to inhibitors of cell wall synthesis in slt2delta-deficient S. cerevisiae and may regulate expression of FKS2, a component of the glucan synthesis complex. Deborah S. Fox1, and George Smulian2. 1University of Cincinnati, Pathology and Mol. Med., Cincinnati, Ohio, USA. 2University of Cincinnati, Internal Medicine, Cincinnati, Ohio, USA.
Signal transduction pathways are important in the adaptive response of microbes to their environment. The MAP kinase Mkp1 of P. carinii has been shown to complement the slt2delta defect in the cell integrity pathway of S. cerevisiae. Mutants of the cell integrity pathway display increased sensitivity to a variety of compounds including caffeine, which influence some facet of cell wall synthesis since a variety of cell integrity MAP kinase cascade mutants with cell wall construction defects are sensitive. Overexpression of mkp1 in slt2delta-deficient S. cerevisiae reduced the toxic affect of caffeine to near wild type (SLT2+) levels. The expression of FKS2, a subunit of beta 1,3-glucan synthase, is regulated by the activation of the transcription factor, RLM1, a target of Slt2p. RLM1 is able to reverse the caffeine sensitivity phenotype when overexpressed in a cell integrity pathway defective strain and rlm1delta-deficient mutants are sensitive to caffeine. The observations that mkp1 expression results in the restoration of resistance to inhibitors such as caffeine demonstrates that Mkp1 is activated upon exposure to conditions which stimulate the cell integrity pathway of S. cerevisiae and suggests that Mkp1 may interact with one or more downstream targets of Slt2p, including Rlm1. The examination of the ability of Mkp1 to control the expression of FKS2 via the activation of Rlm1 will determine the role of Mkp1 in the regulation of S. cerevisiae cell wall synthesis and will provide a pivotal tool for the examination of cell wall synthesis within P. carinii.
308. Isolation and characterization of the mre11 gene of Coprinus cinereus. Erin E. Gerecke, and Miriam E. Zolan. Indiana University, Biology, Bloomington, Indiana, USA.
We are interested in understanding the functions of genes involved in DNA repair and meiosis. The Zolan lab has identified mutants defining at least four genetic loci, rad3, rad9, rad11, and rad12, which are necessary both for survival following gamma irradiation and for meiosis. The rad11-1mutant is highly sensitive to gamma radiation and fails to produce spores; most meiotic cells fail to undergo the first meiotic division. We have cloned the rad11gene; it is a homolog of MRE11, a gene involved in DNA repair and meiosis in numerous organisms, including yeast and humans. The mutation present in rad11-1 (mre11-1) generates a stop codon approximately midway through the predicted amino acid sequence. Surface-spread nuclei of the mre11-1 mutant have been examined using light and electron microscopy. During prophase I of meiosis, chromosomes show defects in condensation, and the synaptonemal complex appears abnormal, although short regions of apparent synapsis have been identified in the majority of nuclei. Using fluorescence in situ hybridization (FISH) on spread nuclei, we have demonstrated that pairing of homologous chromosomes is reduced, but not abolished, in mre11-1 meiotic nuclei. For a single locus on chromosomes 8 and 13, approximately 75% of nuclei demonstrated pairing for at least one of the two chromosomes, but only 30% showed pairing for both chromosomes. At least one of the two chromosomal loci examined is paired in approximately 50% of the nuclei.
309. Phospholipid biosynthetic mutants of Neurospora crassa. Marta Goodrich-Tanrikulu. Calgene LLC, Biochemistry, Davis, CA, USA.
Previously, four choline-requiring mutants of Neurospora crassa have been
identified genetically. Two of these, chol-1 and chol-2, have been
characterized biochemically, but chol-3 and chol-4 have not. The
chol-1 and chol-2 mutations affect the biosynthesis of the phospholipid
phosphatidylcholine. In addition, two other mutations, inl and un-17, are
known to affect phospholipid biosynthesis. The inl mutant has also been
characterized biochemically, and cannot synthesize inositol phospholipids. Phospholipid
biosynthesis is impaired in the un-17 mutant, which fails to grow above 34 C, but
the affected steps of phospholipid biosynthesis have not been identified. In this study,
phospholipid biosynthesis in the biochemically uncharacterized mutants chol-3, chol-4
and un-17 is examined.
310. A Glucoamylase::GFP fusion to study protein secretion in Aspergillus
niger.
Aspergillus niger is used as a host for heterologous protein production, however, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a similar mechanism to other eukaryotes with proteins destined for secretion being directed to the hyphal tip. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A.niger glucoamylase (GLA:499) in order to monitor protein trafficking through the secretory pathway. Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A.nigergenome. Confocal and fluorescence microscopy has shown that GLA::sGFP fusion protein is fluorescent in A.niger and appeared to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence was apparent and immunogold labelling of sGFP confirmed that sGFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::sGFP was only detected in culture filtrates of young mycelia grown in a soya milk medium. Current work is focusing on the disruption the secretory pathway by secretion inhibitors.
311. Dissection of the calcineurin signal transduction pathway in Cryptococcus neoformans. Jenifer M. Gorlach, and Joseph Heitman. Duke University, Genetics, Durham, NC, USA.
We demonstrated that calcineurin (Cn), a calcium/calmodulin-dependent protein phosphatase, is required for growth at 37 degrees C, tolerance to lithium, and pathogenesis of Cryptococcus neoformans (Odom et al. 1997, EMBO J 16:2576-2589). In this study, we took a yeast two-hybrid approach to identify genes encoding proteins which interact directly with Cn. In the two-hybrid screen, a protein was found that could interact with CNA1, the catalytic subunit of Cn, from C. neoformans, S. cerevisiae, and mouse. This 30 kD calcineurin A-binding protein (CBP1) shares only a small amount of sequence similarity with DSCR1, a protein transcribed from a gene located in the Down Syndrome Critical Region. The first 21 kD of the protein is required for binding to CNA1. CBP1 binding to CNA1 is dependent upon the presence of the CNB1 regulatory subunit of Cn, but does not interact with the calcineurin regulatory subunit alone in the two-hybrid assay. The Cn inhibitor FK506 disrupts the CNA1-CBP1 interaction and this inhibition is dependent upon the presence of an FKBP12-FK506 complex. A CBP1-GFP-tagged protein was constructed for in vivo binding assays and localization in C. neoformans. Calcineurin A co-immunoprecipitates with the CBP1-GFP fusion protein in extracts from CNA1 wild-type cells but not from cna1 mutant cells that lack calcineurin A. The subcellular localization of CBP1 under standard and stress growth conditions is being determined. In parallel, gene replacement experiments were conducted to identify a role of CBP1 in C. neoformans. To date, no observable phenotype has been found for a cbp1 mutant strain. Phenotypes for strains overexpressing CBP1 are currently being investigated.
312. A mutation within the catalytic domain of COT1 kinase confers changes in the presence of two COT1 isoforms in Neurospora crassa.Rena Gorovits1, Oshrat Prohpeta1, Mikhail Kolot2, and Oded Yarden1. 1The Hebrew University of Jerusalem, Plant Pathol. & Microbiol, Rehovot, Israel. 2Tel Aviv University, Biochemistry, Ramat Aviv, Israel.
Neurospora crassa grows by forming spreading colonies. cot-1 belongs to a class of N. crassa colonial temperature-sensitive (cot) mutants, and encodes a Ser/Thr protein kinase. We have mapped the cot mutation to a single base change resulting in a His to Arg substitution at amino acid 351, which resides within the catalytic domain. Antibodies raised against COT1 detected and immunoprecipitated a predominant 73-kDa polypeptide in N. crassa extracts, whose abundance was constant under all growth conditions tested. An additional, lower MW, COT1 isoform (67-kDa) present in the wild-type, was not detected in cot grown at the restricitve temperature. Similarly, this isoform was not detected in cot-3 or cot-5 strains, when grown at restrictive temperatures. Apparent changes in the phosphorylation state of P150Glued (encoded by ro-3, a suppressor of cot) and evidence of in vitro physical interactions between COT1 and calcineurin indicate a functional linkage between the kinase, phosphatase and cytoskeletal motor protein.
313. The role of SEPB in the Aspergillus nidulans DNA damage response. Scott E. Gygax, and Steven D. Harris. University of Connecticut Health Center, Microbiology, Farmington, CT , USA.
Temperature sensitive mutations in the Aspergillus nidulans sepB gene
dramatically perturb chromosomal DNA metabolism. At restrictive temperature, these
mutations cause; i) elevated levels of mitotic recombination and chromosome non-disjunction, ii)
progressive delays in nuclear division, and iii) inhibition of septum formation. Molecular
characterization of the sepB gene demonstrated that it encodes a predicted protein
possessing five consensus WD-40 repeats and a motif shared with DNA polymerase
alpha. We are currently employing a variety of approached to characterize the role(s) of
SEPB in chromosomal DNA metabolism. Here, we report observations that suggest that
SEPB is involved in DNA repair.
At the semi-permissive temperature, the
sepB3 mutation caused enhanced sensitivity to DNA alkylating agents such as
MMS. Further analysis showed that the DNA damage checkpoint is retained in
sepB3 mutants, but MMS-induced mutagenesis is virtually abolished.
Furthermore, levels of sepB transcript were slightly higher in MMS-treated cells,
and this increase depended upon UVSB function. Phenotypic characterization of double
mutants revealed the existence of genetic interactions between sepB3 and a number
of other mutations that affect the response to DNA damage (i.e. uvsB110,
uvsD153, musN227, and musP234). Notably, the
increased levels of mutagenesis caused by the uvsC114 and musN227
mutations were diminished by sepB3. Collectively, our observations suggest
that SEPB may be part of a DNA damage regulon that is required for induced
mutagenesis.
314. Transposable elements in Podospora anserina. Andrea Hamann, Frank Feller, and Heinz D. Osiewacz. J.W. Goethe University, Botanical Institute, Frankfurt, 60439 , Germany.
During the course of investigations aimed to elucidate the distribution and function of repetitive sequences in the genome of Podospora anserina, we identified mini- and microsatellite sequences as well as sequences related to class I and class II transposons. Repa, a repetitive sequence that previously was reported to result from the recombination between long terminal repeats (LTR) of an unidentified complete retrotransposons, was found to be present in about 15 copies in the genome. This sequence turned out to be a highly informative probe for the discrimination of different geographical races. One clone was selected containing two intact LTRs and an open reading frame (ORF) coding for a putative protein with significant homology to reverse transcriptases. However, this ORF contains a number of stop codons indicating that the selected element is inactive. A second element, Pat 1, was identified encoding a putative transposase with homology to Pot3, a class II transposable element of Magnaporthe grisea. The corresponding sequence is present in about 25 copies in the genome of P. anserina and appears to be rather conserved in the 18 investigated races of this ascomycete. Acknowledgment: The experimental work was supported by a grant of the Deutsche Forschungsgemeinschaft (Bonn, Germany).
315. Cloning and characterization of a subtilisin/Kex2p-like endoprotease gene of Aspergillus nidulans. Kap-Hoon Han, Sung-Min Ju, Dong-Min Han, and Won-Sin Kim. Wonkwang University, Division of Life Science, Iksan, Chonbuk, South Korea. Limited endoproteolysis of inactive precursor proteins at sites marked by paired or multiple basic amino acids is a widespread process by which biologically active peptides and proteins are produced within the secretory pathway in eukaryotic cells. The identification of a novel family of endoproteases homlologous with bacterial subtilisins and yeasts Kex2p has accelerated progress in understanding the complex mechanisms underlying the production of the bioactive materials. Many distinct proprotein convertases of this family have been identified in mammalian species and non-mammalian eukaryotes. We have cloned and characterized the Aspergillus nidulans subtilisin/Kex2p-like endoprotease gene using the amplified 540bp DNA fragment which was identified from a Blast search of the dbEST database with the mouse frurin. Sequence analysis revealed greatest homology to the catalytic domain of subtilisins, Kex2p and furin that extends over ~330 amino acids is highly conserved among the eukaryotic proprotein convertases.
316. The Roles of UVSB and MUSN in the DNA Damage Response in Aspergillus nidulans. Amy F. Hofmann, and Steven D. Harris. University of Connecticut Health Center, Microbiology, Farmington, CT, USA.
The DNA damage response (DDR) is a protective mechanism that prevents the transmission
of a damaged genome to successive generations. In the filamentous fungus
Aspergillus nidulans, the DDR encompasses several events, including, i) arrest of
nuclear division, ii) inhibition of septation, iii) induction of mutagenesis, and iv) transcription
induction of DNA damage-inducible genes. Phenotypic analysis of
uvsB110 mutants has revealed that UVSB is required for each of the aspects
of the DDR Molecular characterization of UVSB demonstrates that it is a member of a
PI-3 kinase subfamily that includes proteins implicated in cell cycle checkpoint function in
species ranging from yeast to humans. Notably, mutations in a human homologue, ATM,
are responsible for a hereditary cancer-prone syndrome known as ataxia telangiectasia.
Mutations in another A. nidulans gene,
musN, can partially suppress the damage sensitivity of uvsB110
mutants. Furthermore, the musN227 mutation suppresses some of the
specific defects observed in uvsB110 mutants, including the failure to inhibit
septation and induce mutagenesis. In addition, musN227 causes a
slight increase in uvsC transcription in uvsB110 mutants.
Surprisingly, musN227 does not restore DNA damage-dependent nuclear
division arrest to uvsB110 mutants. Together, these results suggest that
MUSN may function to promote recovery from the DDR.
317. Regulation of cell separation in Ustilago maydis.Helge
Hudel and Michael Boelker. University of Marburg, Biology, Marburg, Hassia,
Germany.
The phytopathogenic fungus Ustilago maydis exhibits a dimorphic life style. Haploid sporidia grow yeast-like by budding and are non-pathogenic. Only the filamentous dikaryon is able to induce tumors in maize plants. To identify genes that are involved in the regulation of morphogenesis and dimorphism mutants that exhibit aberrant morphologies were generated by UV and REMI mutagenesis. Mutants in two genes, don1 and don3, have been isolated that fail to separate cells after nuclear division and septum formation. The don1gene encodes a guanine exchange factor (GEF) specific for members of the Rho/Rac/Cdc42 family, the don3 gene codes for a member of the STE20 like kinases that are known to be activated by Rho/Rac proteins. The Ras-like GTPase Cdc42 interacts with both Don1 and Don3 and is thus most likely involved in this regulatory cascade. The Don1 protein contains, in addition to the GEF domain, a pleckstrin homology region and a potential Zn finger. Using fusion proteins of the Don1 Zn finger domain with GFP we could demonstrate that the fusion protein is localized to the vacuole membrane. The protein is also found to be accumulated at the septum before cell separation occurs. The implications of this localization for the function of Don1 will be discussed.
318. Analyses of structures andras-superfamily of Neurospora crassa. Sayoko Ito-Harashima1, Mayumi Sugisaki2, Takashi Ichihara2, Yasushi Matsui3, Akio Toh-e3, and Hirokazu Inoue2. 1 Institute of research and Innovation, Biotechnology, Kashiwa, Chiba, Japan. 2Saitama University, Regulation Biology, Urawa, Saitama, Japan. 3University of Tokyo, Tokyo, Japan.
ras and ras-related genes encode small G proteins that are highly conserved in a wide variety of organisms. The NC-ras gene is one of the ras homologs of N. crassa. We cloned genomic DNA fragment of the NC-ras gene and analyzed its structure and function. NC-ras ORF was interruped by three introns and the positions of introns were completely conserved between NC-ras and Aspergillus nidulans Aras. The strains overexpressed the NC-ras gene showed abnormality in life cycle. They were female sterile in sexual cycle. In vegetative growth phase they showed morphological abnormalities, and the defects were partially suppressed by addition of theophylline, an inhibitor of phosphodiesterase (a degradation enzyme of cAMP). These data suggest that the NC-RAS protein may be involved in the signal transduction pathway mediated by cAMP. Recently we also isolated another member of ras-superfamily, sar-1. The sar-1 is a homolog of Saccharomyces cerevisiae SAR1, which is involved in the intracellular vesicle transport. It has been elusidated that Sar1 protein is essential for the formation of transport vesicles from the ER membrane in yeast. N. crassa SAR1 protein showed 60-90% identity with SAR1 homologs of other organisms at the predicted amino acid level. Northern blot analyses indicated that the sar-1 was constitutively expressed during vegetative growth phase. We are now examining the functional relationship of sar-1 homologs between N. crassa and S. cerevisiae.
319. Molecular and genetic analyses of the nimO initiator of DNA synthesis in Aspergillus nidulans. Steven W. James, Karen E. Messner, and Maureen C. Miller. Gettysburg College, Biology, Gettysburg, PA, USA.
The nimO gene of Aspergillus nidulans shares structural and
functional homology with the budding yeast G1/S regulator, Dbf4, being required
both for initiation of DNA synthesis and efficient progression through S phase; and involved in a
checkpoint linking DNA synthesis with mitosis (James et al., accepted; 19th FGC). The gene is
essential, because strains containing alcA::nimO+ as their only copy of
nimO become completely ethanol-dependent. Since the greatest similarity between
nimO and Dbf4 lies in their C-termini, nimO function was
investigated using a series of alcA-driven C-terminal truncations. nimO
alleles lacking as much as 244 C terminal amino acids partially rescued nimO18
ts-lethality when expression was strongly induced, by permitting vegetative growth but not
asexual development. However, these truncated alleles could not complement a deletion of
nimO, indicating that the C-terminus is essential and suggesting that the
nimO18 protein and truncated polypeptides somehow interact. Dbf4p triggers
DNA synthesis by activating the Cdc7p kinase and escorting it to targets at origins of replication.
If nimO is a Dbf4 homolog, it should associate with its cognate
Aspergillus partner. We isolated and sequenced an apparent Aspergillus homolog of
Cdc7 (Cdc7Asp), and we have identified two new loci,
snoA and snoB, as partial suppressors of nimO18.
Preliminary studies using Cdc7Asp suggest that it may complement
snoA suppressors, and molecular and genetic studies are underway to ascertain if (1)
snoA encodes Cdc7Asp, and if (2) nimOp and Cdc7pAsp physically
associate to control DNA synthesis. (Supported by NSF-RUI# MCB 95-07485).
320. uvsF function, effects on recombination, and interaction with other Aspergillus
cell-cycle/repair mutants cell-cycle/repair mutants.
We are interested in the mechanisms of mitotic recombination in Aspergillus nidulans. DNA repair-defective mutants from several epistatic groups affect this process in various ways. Such genes may have different primary function and produce very different patterns of recombination, as identified for two hyperrec types, uvsF and uvsB. uvsF201 is defective in nucleotide excision-repair (NER) and shows increased mitotic crossing over and UV mutagenesis. Its gene sequence is homologous to human/yeast RFC1 genes which code for the largest of five subunits of replication factor C. RFCp is essential for DNA replication and for NER, and cdc44-1 of yeast is cold-sensitive lethal for RFC1. Surprisingly, uvsF201 grows well, but is highly mutagen sensitive and produces synthetic lethals with many different repair mutations. When uvsF cDNA in a yeast vector was expressed in cdc44-1 cells, Western blots showed that the expected Aspergillus protein was synthesized, but no interspecies "complementation" was found. However, as the uvsF201 point mutation can be complemented by uvsF gene fragments, the CDC44 gene in Aspergillus may yet rescue uvsF201. -- The closely adjacent gene to uvsF, which is a homologue of organelle ribosomal protein L16 genes, is transcribed from a common promoter region in opposite direction. The two genes may therefore be co-regulated, as claimed for such cases in yeast. The second hyperrec mutant, uvsB, grows poorly and the pattern of mitotic recombination in uvsB diploids is uniquely abnormal, differing significantly from uvsF and from controls. The uvsB gene was recently found to be homologous to rad3 of fission yeast and shown to be required for DNA repair as well as cell-cycle checkpoint control (S. Osmani, personal communication).
321. Hyphal morphogenesis and cell survival in Aspergillus nidulans. Susan G. W. Kaminskyj. Univ. Saskatchewan, Biology, Saskatoon, SK S7N 5E2, Canada.
Aspergillus nidulans hyphae grow by tip extension, and are later divided by crosswalls into actively growing tip and quiescent basal cells. Basal cells can branch, resuming growth after forming a new tip. Apical growth in tip cells, and growth arrest in basal cells, depend on the function of "hypercellular" genes, identified using temperature sensitive mutants. All hyp ts strains can complete their asexual life cycle at 42°C and produce viable conidia, suggesting that these genes are not essential. Intriguingly, if hypA or hypB function is perturbed by shifting 28°C-grown germlings to 42°C, basal cells are remodeled to assume the restrictive phenotype, but tip cells die. For hypA, only, tip cell survival appears to be related to cell cycle stage. About 20% of control tips survive upshift, and this is increased three-fold by treatment with hydroxyurea. These results suggest that hypA is a nuclear-cycle related growth switch for hyphal development. hypA has sequence similarity to genes in budding and fission yeasts, and a cosmid containing the fission yeast ortholog was able to complement the hypA defect. Current efforts focus on defining the upshift phenotypes and proving homology between the A. nidulans hypA, and the yeast genes.
322. Cloning and functional analysis of the gene coding a PLC homologue in Neurospora crassa. Yuko Kawahashi1, Mayumi Sugisaki1, Akio Toh-e2, and Hirokazu Inoue1. 1Saitama University, Faculty of Science , Urawa, Saitama, Japan.2Tokyo University, Faculty of Science, Bunkyo-ku, Tokyo, Japan.
Phosphoinositide-specific-phospholipase C (PLC) is responsible for the production of two second messengers in a signal transduction pathway. PLCs are usually divided into three types (beta,gamma,delta) based on size, immunological reactivity and amino acid sequence. Each isoform has X and Y regions which are highly conserved and regarded as catalytic regions in PLCs. We obtained a short DNA fragment by PCR using primers based on the two conserved amino acid sequences in the X region. Using the PCR product as a probe, we screened cosmid genome libraries by colony hybridization and isolated a genome DNA fragment of 2.6 kb. We sequenced this fragment and found it to be homologous to the X region. But, it was not the total gene coding a PLC protein; the Y region and the carboxy terminal sequences were deleted. PLC mutants made by RIP were highly sensitive to MBC (carbendazim),TBZ (thiabendazole), and thiophanate-methyl,which are chemical agents blocking polymerization of beta-tublin. This indicated that the PLC homologue of N.crassa is involved in organization cytoskelton. In addition , the mutants showed a sensitivity to H2O2 , suggesting that the homologue is related to oxidative stress-stimulated signal transduction pathway.
323. Comparative study intra and extracellular pectinases produced by P. frequentans.Cristina Yoshiko Kawano, Maria Angélica dos S.Cunha Chellegatti, Suraia Said, and Maria José Vieira Fonseca. Universidade de São Paulo, Ciências Farmacêuticas, Ribeirão Preto, São Paulo, Brasil.
Microbial pectinesterase and depolymerases (hydrolases and lyases), catalysing the degradation of pectic polysaccharides, play important role in the invasion of plant tissues by phytopathogens, and in the spoilage of fruit and vegetables. Furthermore theses enzymes are widely used in the bioconversion as adjuncts to cellulases and/or hemicellulases in treatment of cellulosic biomass and also in biopulping processes for clarifying fruit juices and liquefying fruits and vegetables. The pectic enzymes are synthesized and secreted from intact cells into the surrounding medium by fungi, bacteria and some yeasts. The fungus Penicillium frequentans was selected among 100 fungi isolated from soil in Brazil because of its ability to produce high levels of extracellular pectinases in solid medium using citrus pulp pellets.This fungus synthesized eleven PGs (polygalacturonases) and two PEs (pectinesterases) when grown in liquid culture supplement with pectin.These enzymes were separated and detected in polyacrylamide gels containing pectin and sodium polypectate (NaPP). Seven PGs and the two PEs were secreted in the medium while four PGs were not secreted. Among the secreted PGs, the endo-PG (10 band) and exo-PGs (5 band) were the enzymes secreted in highest levels. All secreted PGs bound to Con A and their secretion and/or enzymatic activities were inhibited by TM (tunicamycin), except the constitutive and inducible endo-PG (band 10). The affinity on ConA and TM effect studies suggested that the secreted endo- and exo-PG differed by level and process of glycosylation. The exo-PG was characterized as a N-glycoprotein while the endo-PG, probably, is an O-glycoprotein. The PGs (3 and 4) were neither bound to ConA or secreted and their enzymatic activities were inhibited by TM suggesting that they are, probably, N-glycoprotein with complex oligosaccharides of type three and tetraantennary. The order not secreted and unbound to Con A PGs (6 and 8) were not inhibited by TM. These enzymes presented, chromatographic characteristics and effects to TM, similar to endo-PG (band 10), since these PGs could be unglycosylated or/and aggregate forms of the endo-PG (band 10).
324. Genomics of Mycosphaerella graminicola. Gert H.J. Kema, Trudy B.M. van den Bosch, Cees Waalwijk. DLO-Research Institute for Plant Protection, Mycology & Bacteriology, Wageningen, Gelderland, The Netherlands.
Mycosphaerella graminicola, a heterothallic bipolar ascomycete, is the causal agent of Septoria tritici leaf blotch disease of wheat. This disease is currently considered to be Europe's most important wheat disease and therefore gains a lot of interest from the agro-industry. We have recently developed the first linkage map of the M. graminicola genome with over 300 AFLP and over 100 RAPD markers and including several biological markers such as the MAT gene and an avirulence locus. A BAC library was constructed of the avirulent parental strain of this M. graminicola mapping population as a starting point for a sequencing project on this fungus using the latest high troughput sequence technology. The current status of the project will be presented.
325. Mapping the Mycosphaerella graminicola genome. Gert HJ Kema1, Stephen B Goodwin2, Sonia Hamza3, Els CP Verstappen1, Jessica R. Cavaletto2, Theo AJ van der Lee4, Marjanne Hagenaar-de Weerdt1, Peter JM Bonants1 and Cees Waalwijk1 1DLO-Research Institute for Plant Protection [IPO-DLO], P.O. Box 9060, 6700 GW Wageningen, The Netherlands; 2Purdue University, Dept. of Botany and Plant Pathology, 1155 Lilly Hall, West Lafayette, IN 47907-1155, USA; 3Institut National Agronomique de Tunisie (INAT), 43 Av. Charles Nicolle, 1082 Tunis El Mahrajène, Tunisia; 4Wageningen Agricultural University, Dept. of Phytopathology, Binnenhaven 9, 6709 PD Wageningen, The Netherlands.
We have developed a crossing protocol to obtain a mapping population of M. graminicola, the causal agent of septoria tritici leaf blotch disease of wheat. The parental strains are highly contrasting for their avirulences towards a number of wheat cultivars, characteristics that appear to be under monogenic control. Progenies could therefore be used to map avirulence- and MAT-loci and to identify linked markers in order to start map-based cloning strategies to isolate these genes. A F1 progeny of IPO323 [avirulent, MAT1-1] and IPO94269 [virulent, MAT1-2] was isolated and 68 isolates were used to construct a linkage map using AFLP and RAPD technology. Using a LOD threshold value of 4, 410 of the 439 markers showed linkage to at least three other markers, eventually resulting in 26 linkage groups. In addition, we conducted bulked segregant analyses, which resulted in two additional markers that perfectly linked with the avirulence locus. AFLP markers that mapped on the same linkage group as the avirulence locus were used as probes to identify the chromosome on pulsed-field gel electrophoresis [PFGE] blots. Similarly, a heterologous MAT probe was used to identify the chromosome carrying the MAT locus. Although the map indicated that both genes were located on small linkage group, these probes hybridized to much larger chromosomes on PFGE filters, which was also confirmed by marker-derived SCARS on chromosome bands excised from PFGE gels. This strategy is used to relate linkage groups to specific chromosomes in order to establish a physical map of the genome. Acknowledgment Part of this project is funded by EU-BIOTECH PL096-352
326. Disruption of the gene encoding the catalytic subunit of the vacuolar H+-ATPase causes severe morphological changes. Ryan Kendle, Emma Jean Bowman, and Barry Bowman. University of California, Santa Cruz, Biology, Santa Cruz, CA, USA.
Fungal vacuoles are important as storage organelles for basic amino acids and polyphosphate and for maintaining cytosolic homeostasis for protons and calcium ions. To function properly the vacuoles depend upon the activity of the vacuolar H+-ATPase located in the vacuolar membrane. The enzyme pumps protons into the organelle, acidifying the interior and generating a proton gradient used to drive transport of solutes across the membrane. We have inactivated the V-ATPase of Neurospora crassa by RIPing vma-1, the gene encoding the catalytic subunit. The resultant vma-1 null strains are deficient in multiple stages of development. They grow slowly, exhibiting dichotomous branching rather than an apical dominant pattern typical of hyphal fungi. They produce no aerial hyphae or conidia and when crossed with each other from helper strain backgrounds produce no asci or ascospores. The null strains are unusually sensitive to medium pH above 6.0, to heavy metals such as zinc, and to high salt concentrations. Two strains with no detectable V-ATPase activity were characterized. Sequence analysis revealed that one is heavily RIPed and unable to make a protein product, while the other is lightly RIPed and is expected to produce a protein product. Interestingly, ascospores from the first strain rarely germinate, but those from the second strain germinate like wild type.
327. On the ultrastructure of cell wall in null pigmentation mutant of Aspergillus nidulans. Jeong-Mi Kim1, Kwang-Yeop Jahng1, Dong-Min Han2, and Yun-Shin Chung3. 1Chonbuk National University, Biological Sciences, Chonju, Chonbuk, South Korea. 2Wonkwang University, Molecular Biology, Iksan, Chonbuk, South Korea.3Washington University, Genetics, Washington, St. Louis, U.S.A.
To investigate the effects of cell wall on the pigmentation and branching in Aspergillus nidulans, the ultrastructure and cytochemical composition of cell wall in wild type (FGSC4) and npgA1 mutant (no pigmentation through the whole life cycle, WX17) were examined. Scanning electron microscope (SEM) showed that the hyphal surface and branching in WX17 were rough and poorly formed, in contrast to those in FGSC4 showing smooth and well developing, respectively. Transmission electron microscope (TEM) and carbohydrate staining showed that the hyphal wall of FGSC4 layered as H1, H2, H3, and H4 were mainly composed of protein, chitin, B-glucan, and the complex of chitin and B-glucan respectively. However the hyphal wall of WX17 was lacking H1 and H3 layers suggesting that the defect in pigmentation and hyphal branching of npgA1 mutant might be due to the lack of the B-glucan layer in the cell wall. To study the function of the gene npgA in constructing cell wall structure, we isolated DNA fragment that was able to complement npgA1 mutation from the genomic library of FGSC4. From its nucleotide sequence, we found that there was an open reading frame (ORF) in which the amino acid sequence was homologous to that of SLC1 gene which encoded fatty acyltransferase in Saccharomyces cerevisiae. The fragment was also capable of complementing the defect in a 1-acyl-sn-glycerol-3-phosphate-acyltransferase, encoded by the gene plsC, of Escherichia coli. From these results, it could be concluded that the npgA1-complementing element might play a role in constructing cell wall structure or depositing pigment.
328. Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L.Kohtaro Kirimura, Masashi Yoda, and Shoji Usami. Waseda Institute of Science and Technology, Applied Chemistry, Shinjuku-ku, Tokyo, Japan.
A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible
for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric
acid producing-fungus Aspergillus niger WU-2223L was cloned and expressed in
Escherichia coli as host strain. Synthetic primers were designed from the conserved
nucleotide sequences of the alternative oxidase genes from plants and a yeast. The 210 bp DNA
fragment was amplified by PCR with the primers using chromosomal DNA of A.
niger WU-2223L as template, and used to screen a cDNA library of A. niger.
One full-length cDNA clone, 1.2 kb, was obtained and sequenced to reveal that the clone
contained an open reading frame (ORF-AOX1) encoding a polypeptide deduced of 351 amino
acids. The predicted amino acid sequence exhibited 46%, 52%, and 48% homologies to the
alternative oxidases of Hansenula anomala, Neurospora crassa and
Sauromatum guttatum, respectively. In the 5'-terminus region of the ORF-AOX1, a
mitochondrial targeting motif was found. The whole open reading frame ORF-AOX1 was ligated
to plasmid pKK223-3 to construct an expression vector pKAOX1. E. coli
transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration,
and the expression was induced to two fold by addition of IPTG. These results indicated that the
ORF-AOX1 encodes an alternative oxidase of A. niger.
329. Human TNF-Alpha could be successfully produced in the filamentous fungus
Aspergillus niger heterologous protein expression-secretion system
The expression-secretion system of the GRAS filamentous fungus A. niger has proven to be capable of producing heterologous proteins at commercially interesting levels, however, it has also to be taken into account that it has not yet been applied for a wide range of heterologous protein products. Human tumor necrosis factor alpha (hTNF-alpha) monomer is a 17 kDa non-glycosylated protein, containing a single intra-molecular disulfide bond in its structure. In solution it exists as a compact, bell-shaped homotrimer, which is considered to be a biologically active form of this important cytokine with a wide range of biological activities. For heterologous expression of hTNF-alpha in A. niger the same strategies were used, which were proved to be the most successful for several other non-fungal proteins: a) gene-fusion with the A. niger glucoamylase GII form as a carrier-gene, with KEX2-like in vivo cleavage-site in between the genes; b) strong fungal transcription control regions and efficient secretion signals of the A. niger glucoamylase gene; and c) selection of high copy transformants due to the amdS selection marker in the protease-deficient host-strain A. niger AB1.13. No processed hTNF-alpha detected in the media or even in the cell, although specific mRNA of the expected size was observed. We could detect hTNF-alpha in the form of glucoamylase-fusion protein in the cell, regardless of the presence or absence KEX2-like in vivo cleavage-site. This glucoamylase-hTNF-alpha fusion-protein was N-glycosylated due to the glucoamylase GII part (O-glycosylation was not checked), mostly attached to the membranes, but not to the outside of the cell wall. Only 12% of hTNF-alpha added into maltodextrine production medium was proteoliticaly degraded after 24 hours, so degradation in the protease-deficient host-strain AB1.13 compared to N402 strain was not a problem. hTNF-alpha analogue LK 802 (Cys95/148, His107/108) was also expressed with carrier-gene strategy. Cys95/148 mutation is introducing an intermolecular disulfide bond so that a more compact and stable trimeric molecule is produced, and His107/108 mutation is introducing six histidine residues at the apical site of the molecule, providing an efficient separation method on metal affinity chromatography. However, the glucoamylase-hTNF-alpha analogue fusion-protein with both above mentioned mutations could not be successfully separated from the cell extract. In all these experiments hTNF-alpha synthetic gene optimised for E. coli bias was used, but when the gene was exchanged for natural human TNF-alpha cDNA, processed hTNF-alpha was successfully secreted into the medium. After the first screening of small amounts of transformants the yield was estimated to 13 mg/l culture.
330. Regulation of septum formation in Aspergillus nidulans by cyclin-dependent kinase activity. Peter R. Kraus, and Steven D. Harris. University of Connecticut Health Center, Microbiology, Farmington, CT, USA.
Strict regulation of cdk activity has been shown to be important for cytokinesis in a variety
of eukaryotic systems. In the filamentous fungus Aspergillus nidulans, the
cdk encoded by the nimX gene has been shown to be involved in regulating
cytokinesis. Genetic evidence suggests that the ability to separate mitosis and septum
formation depends upon a threshold level of NIMX activity. A transient inhibition of
NIMX activity by the activation of the DNA damage checkpoint pathway results in a significant
delay in septum formation. This supports the idea that germlings must reach a threshhold
level of NIMX activity at a certain point during interphase in order to initiate septum formation.
In order to identify additional components of the pathway regulating septum formation,
we have initiated a genetic screen for extragenic suppressors of the Ts nimT23
mutation, which results in inactivation of the NIMX-activating tyrosine phosphatase
NIMT. We have counter-screened 500 suppressors of the nimT23 mutation
for HU sensitivity in order to distinguish those mutants which may be defective in the checkpoint
pathways which respond to DNA damage or unreplicated DNA. We have isolated 7 HU
sensitive mutants which fall into three linkage groups (designated sntA,
sntB and sntC) and are distinct from alleles of nimX and
ankA (which encodes the A. nidulans homolog of S. pombe
Wee1p). snt mutations allow germlings to undergo septum formation before
the critical cell size is reached and in the presence of DNA damage generated by mutation
(sepB3) and by the bi-functional alkylating agent diepoxyoctane (DEO). The
snt mutations have different effects on cell cycle checkpoints and result in decreased
phosphorylation on Tyrosine-15 of NIMX. 331. The MAPKKK Ste11 regulates vegetative growth through a novel kinase cascade of
shared signaling components 332. Analysis of multiple alleles of Neurospora crassa ro mutants
Cytoplasmic dynein is a microtubule-associated, minus-end directed, mechanochemical enzyme, and the dynactin complex has been proposed to regulate the interaction of cytoplasmic dynein with membranous cargo. We have been using Neurospora crassa to genetically analyze cytoplasmic dynein and dynactin. Previously, we showed that ro-1, ro-3, and ro-4 encode cytoplasmic dynein heavy chain, p150Glued (the largest subunit of dynactin), and ARP1 (the most abundant subunit of dynactin), respectively. The availability of multiple independent alleles of ro genes encoding subunits of cytoplasmic dynein and dynactin provides us with the opportunity to do a detailed analysis of protein interactions and specific functions within the motor complex. The largest subunit of dynactin, p150Glued, is not present in a strain deleted for ro-4; however, p150Glued is present in a ro-4(E8) strain. The ro-4(E8) mutant is altered for localization of both cytoplasmic dynein and dynactin, with both complexes colocalizing with microtubule tracks at hyphal tips. The ro-4(E8) allele contains a point mutation predicted to affect a surface residue in a conserved region of ARP1. In the ro-4(E8) mutant the interaction of cytoplasmic dynein/dynactin with membranous cargo is partially impaired. Western analysis of different ro-1 mutants revealed that each ro-1 allele affects the stability of RO1 and RO3 to a different degree. In a similar manner, each mutation in ro-3 affects the stability of RO1 and RO3 to a different degree. Our results suggests that both cytoplasmic dynein and dynactin are required for stable interaction with membranous cargo.
333. Identification of cell wall proteins of Pneumocystis carinii by 2D gel electrophoresis and MALDI-TOF mass spectrometry.Michael J. Linke, and A.George Smulian. University of Cincinnati, Infectious Diseases, Cincinnati, Ohio, USA.
Regulation of cell wall synthesis is critical in the adaptation to environmental changes and cell wall components are important antifungal therapuetic targets, however, few cell wall proteins of the pulmonary pathogen, Pneumocystis carinii, have been characterized. Most proteins identified thus far have been identified based on their immune recognition by the infected host. To identify additional structural proteins and other components of the Pneumocystis carinii cell wall, a soluble cell wall extract was obtained by the treatment of P. carinii organisms with zymolyase followed by centrifugation at 100,000 x g. Individual protein "spots" within this soluble cell wall extract were resolved by 2D gel electrophoresis and identified by Coomassie Brilliant blue staining. Spots were excised, destained and subjected to in gel trypsin digestion. Peptide fragments were extracted and analysed by MALDI-TOF mass spectrometry. Peptide fragment fingerprints were compared to SwissProt and NCBI protein databases using Protein Prospector software and potential homologues identified. Peptide sequence information will be obtained on signature peptides by MS analysis following Post Source Decay fragmentation and Collision Induced Dissociation to verify the identity of the protein matches. 2D gel electrophoresis followed by MALDI-TOF mass spectrometry should allow high throughput, rapid identification of cell wall components.
334. Modification of fungal N-linked carbohydrates. Marleen Maras, and Roland Contreras. Gent University and VIB , Molecular Biology, Gent, Flanders, Belgium.
The carbohydrate moiety of glycoproteins has been shown to have a role in antigenicity, solubility, targeting, cell-cell contact, homing and pharmocological behaviour. Often, the primary structures of the carbohydrates found on proteins, secreted by mammalian cells, is complex, heterogeneous and difficult to fractionate on the basis of this heterogeneity. In order to obtain proteins with well defined, more homogenous carbohydrate structures, procedures have to be worked out for their synthesis. High-mannose oligosaccharides were characterized on glycoproteins from certain filamentous fungi. These N-glycans were found to have simple structures, that can serve as precursors for the synthsesis of mammalian-like oligosaccharides. Mammalian-like hybrid oligosaccharides were synthesized on glycoproteins from the filamentous fungus Trichoderma reesei by in vitro enzymatic modification. Succes of this conversion was found to be dependent on the strain of Trichoderma used. In addition, preincubation of the fungal proteins with Aspergillus saitoi alfa 1,2 mannosidase substantially improved formation of the hybrid structure with one strain, while it did not with glycoproteins from another strain. With all strains used, it was found that alfa 1,2 mannosidase did not convert all oligosaccharides to the acceptor substrate for N-acetylglucosaminyl transferase I. This demonstrates that Trichoderma synthesizes a fraction of high mannose-type but not mammalian-like N-glycans. Therefore, further studies were directed to a detailed analysis of the N-lined glycans of the CBHI protein. NMR data were obtained for two major components and detailed studies were carried out for several minor components. In addition, we cloned an alfa 1,2 mannosidase from T. reesei Ity may be responsible for the processing of high-mannose fungal oligosaccharides to Man5GlcNAc2, which is substrate for 'in vitro' N-acetyl glucosaminyl transferase I. The gene was engineered in a Pichia pastoris expression plasmid. Upon transformation to the yeast, alfa 1,2 mannosidase activity was easily detected. Initial experiments to characterize oligosaccharides of the transformed Pichia strain showed formation of N-glycans, smaller than Man8GlcNAc2 that are never synthesized in the wild type yeast.
335. Programmed cell death in fungi: Heterokaryon incompatibility involves nuclear DNA degradation. Stephen M. Marek1, Jennifer Wu2, N. Louise Glass2, David G. Gilchrist1, and Richard M. Bostock1. 1Univ. California, Plant Pathology, Davis, CA, USA. 2Univ. British Columbia, Biotechnology Laboratory, Vancouver, BC, Canada.
Within species, fungi can fuse to form heterokaryotic mycelia if they possess identical heterokaryon incompatibility (het) loci. If fungi attempting heterokaryosis differ at one or more het loci, the fused cells self-destruct by a killing reaction process reminiscent of apoptosis in animal cells (Jacobson et al. FG&B; 23:45). In Neurospora crassa, ten such het loci have been identified in addition to the mating locus. The het locus encodes a single nonessential protein and controls specificity as one of three alleles, OR, PA, or GR. In this study, the cytology of transformants carrying incompatible and compatible combinations of het-c alleles and pairings between vector control transformants were examined using fluorescent DNA stains and terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL). Transformants carrying incompatible het-c transgenes were inhibited in growth and contained heavily degraded nuclear DNA in cells also displaying vacuolated or shrunken cytoplasms. Transformants carrying compatible het-c transgenes contained little or no degraded DNA. In pairings between incompatible control transformants, fusion cells frequently contained heavily degraded nuclei in disorganized cytoplasms. In pairings between compatible control transformants, fused cells rarely contained degraded nuclei. Cells killed by the incompatibility reaction often contained cytoplasmic and nuclear remnants that often persisted after adjacent cells grew new hyphae into the killed cells. The process by which heterokaryon incompatibility kills and disassembles cells and the extent to which it is genetically controlled can best be described as programmed cell death.
336. Characterization of a vacuolar Ca+2/H+ exchanger (CAX) of Neurospora crassa. Emilio C. Margolles-Clark, Stephen Abreu, and Barry J. Bowman. University of California Santa Cruz, Department of Biology, Santa Cruz, California, USA.
A gene encoding a vacuolar Ca+2/H+ exchanger (CAX) was obtained from the Neurospora Genomic Project. This transporter is an integral membrane protein, possibly containing 11 membrane spanning regions, as indicated by hydrophilicity analysis of its amino acid sequence. A strain containing a mutated CAX gene was generated by RIPing. CAX protein proved to be non essential for the survival of the organism. The effect of the mutation on growth in different Ca+2 concentrations at different pH conditions will be discused. Transport experiments showed that Ca+2 transport into vacuolar vesicles of the mutant strain is dramatically reduced. This indicated that the Ca+2/H+ exchanger plays a major role in the vacuolar internalization of the cation. The low Ca+2 transport background found in the mutant allowed the study of the properties of the transporter in the wild type strain. The kinetic data of the transporter as well as its inhibition by other divalent cations will be presented.
337. Lytic enzymes in Ustilago maydis: amplification and cloning of a 1,3-b-glucanase and an acidic protease. Alfredo D. Martinez-Espinoza, and Jose Ruiz-Herrera. CINVESTAV-Unidad Irapuato, Genetic Engeering of Plants, Irapuato, Guanajuato, Mexico.
Ustilago maydis causes the disease named common smut in maize. It is believed that lytic enzymes play a role in mating and pathogenicity. Preliminary evidence showed the presence and activity of glucanases and proteases. We have initiated the search for genes coding for these enzymes. Oligonucleotides were designed based on conserved sequences of 1,3-b-glucanases and proteases from several fungi and were used as primers for PCR. Regarding the glucanases, a 190 bp PCR fragment was amplified, cloned and sequenced. The sequence showed homologies ranging from 75 to 85 % with exo-1,3-b-glucanases from S. cerevisiae, Y. lipolytica, A. bisporus and C. albicans. The PCR fragment was used to screen a genomic library of U. maydis. Two positive clones were isolated, and found to contain inserts of 9 and 8 kb. Restriction digests and Southern blots showed differences between the clones. Two fragments were identified and subcloned. They were characterized by restriction and sequence analysis. Regarding the proteases, an 800 bp PCR product was amplified, cloned and sequenced. The sequence showed conserved regions of aspartyl-proteases. Library screening is currently under way. Gene disruption will determine the role of these genes in mating and pathogenicity in U. maydis. 338. Identification and analysis of clonable extragenic suppressors of the nimXcdc2F223L mutation of Aspergillus nidulans. Sarah Lea McGuire, Melanie D. Schrader, Brett W. Carter, Dana L. Roe, Chad D. Young, Sean P. Grace, Gene A. Lang, and Suzanne E. Wahrle. Millsaps College, Biology Department, Jackson, MS, USA.
The nimXcdc2 protein kinase of Aspergillus nidulans regulates progression of nuclear division during G1>, S, and G2> phases of the cell cycle. To identify genes which encode proteins that interact with nimXcdc2, we have generated a collection of strains with mutations that suppress the temperature-sensitive nimXcdc2F223L mutation. The suppressor strains were screened for the presence of an additional phenotype (cold- or drug-sensitivity) that could be used in cloning. Of 1500 suppressors isolated, 37 contained additional phenotypes. 14 suppressor strains with additional phenotypes have been crossed with a wild-type strain to determine if the suppressor mutations were intragenic or extragenic. This yielded two strains with extragenic suppressor mutations that cosegregate with the additional phenotype. These strains were designated MDS250 and CY17. Both suppressor mutations have been shown to be recessive by diploid analysis, and phenotypic analysis indicates that both mutations stop the cell cycle during interphase. The growth phenotype of CY17 contains aberrant nuclear and cytoplasmic morphologies that indicate deregulation of tyrosine phosphorylation of nimXcdc2. We are currently performing analyses to determine at which point during interphase the nuclear division cycle is halted in these strains. In addition, both suppressor mutations are being mapped to chromosome to facilitate cloning and the remaining 23 suppressor strains are being analyzed genetically. Supported by NIH grant GM55885.
339. Morphological and genetic characterisation of Hbr-2, a hyperbranching mutant of Aspergillus nidulans. Stephen Memmott, Sarah Pollerman, and Geoffrey Turner. University of Sheffield, Molec Biol Biotech, Sheffield, South Yorkshire, UK.
Fungal morphology plays a major role in the fermentation industry as it affects the physiological state of the culture. This leads to consequences for both process conditions and final product yield. Fungal growth proceeds via the two basic mechanisms of hyphal elongation and the production of branches either at the hyphal tip or as lateral branches generated from intercalary compartments. The factors which control the production and frequency of branches are known to be both environmental and genetic. In order to discover underlying genetic components involved in the branching of Aspergillus nidulans, temperature sensitive, hyperbranching mutants were isolated and phenotypically characterised using branching frequency, septation and nuclear distribution measures. In addition genetic analysis was used to identify 10 loci affecting branching frequency. One such mutation, designated hbrA2, was chosen and the affected gene was isolated through functional complementation. A chromosome specific library and a co-transformation system was used to isolate a fragment complementing hbrA2. A sequence database search has given promising leads as to the function of hbrA2. Characterisation of this fragment will be described. Attempts are now being made to disrupt hbrA, and to place the gene under the control of a regulated promoter to study further the role of this gene in fungal growth.
340. Apical growth in smco7 mutant of N. crassa. Yuko Mochizuki, and Tadako Murayama. Kanto-Gakuin Univ., College of Engineering, Kanazawa-ku, Yokohama, Japan.
Transformation of a morphological mutant, smco7, with the plasmid carrying NC-ras2, a ras gene homologue of Neurospora, resulted in the recovery of the wild type morphology and a deletion of one nucleotide was detected in the region corresponding to the N-terminal region of the putative ras protein homologue encoded by NC-ras2 of the smco7 mutant. The apical cells of smco7 were shorter, thinner, more fragile than those of the wild type. The smco7 mutation seemed to cause defects in cell wall synthesis at the apex. Te extracellular invertase and trehalase activities were much lower in smco7 than in the wild type. The extracellular enzymes may be transported to the apex and secreted there through the common mechanism in the transport and exocytosis of the cell membrane and cell wall precursors An actin inhibitor, Cytochalasin A (CA), considerably inhibited the hyphal growth, made hyphae thinner, and lowered the level of extracellular invertase after the shift of the mycelia to the medium containing CA in the wild type. These results suggest that the actin plays important roles in the apical growth and the production of extracellular enzymes and the ras protein plays some roles in the regulation of actin in Neurospora.
341. sepG, a gene required for septation in Aspergillus nidulans. Maria Victoria Montenegro-Ch, and John E. Hamer. Purdue University, Biology, West Lafayette, Indiana, USA.
Aspergillus nidulans is a multicellular, filamentous fungus that divides by the formation of a crosswall called a septum. Septum formation is temporally and spatially regulated by nuclear positioning and cell cycle progression. sep G was identified as a conditional mutant that causes an aseptate phenotype in cells that have completed several rounds of mitosis. The sep G mutant is unable to form septa but displays no defects in growth or nuclear division producing long multinucleate hyphae at the restrictive temperture. The sepG gene has been cloned by complementation of the mutant with a chromosome specific library of A. nidulans. Sequencing and further characterization of the gene is underway. sepG is likely to be part of a signaling pathway mediating actin ring formation, the first event of septum formation.
342. Chemotactic signals trigger a calcium influx in zoospores and hyphae of Phytophthora sojae. Paul F. Morris, and Mary S. Connolly. Bowling Green State University, Biological Sciences, Bowling Green, Ohio, USA.
Both the motile zoospores, and germinating cysts of Phytophthora sojae respond chemotropically to nM levels of the isoflavonoids excreted from soybean roots. The role of calcium in the cellular response to a host signal was investigated by loading hyphal germlings in a chemotaxis chamber with Calcium Green -1. Cytoplasmic fluorescence of hyphal germlings increased in response to mechanical stimulation or the addition of daidzein to the media. The increase in fluorescence was transient and reversible. Once the chamber was rinsed free of daidzein and the cells were allowed to recover, a second fluorescent signal could be produced by the addition of daidzein. Confirmation that the increase in Calcium Green fluorescence was due to an influx of exogenous calcium demonstrated by x-ray microanalysis of individual encysted zoospores. Zoospores exposed to 10 mM calcium and daidzein at the time of encystment formed cysts that contained more calcium than zoospores exposed to only to calcium. The addition of exogenous calcium or daidzein to zoospores at the time of their encystment altered the development fate of cysts and the relative magnitude of calcium stores in the cells. Low levels of calcium reserves inhibited spore germination and enhanced the release of a zoospore from the cyst. High levels of calcium were associated with cyst germination. Isoflavones in the presence of at least 0.5 mM calcium also promoted cyst germination in cells fated to release a second zoospore, presumably by generating a transient increase in cytosolic calcium.
343. Mutants related to cyclic AMP cascade in N. crassa. Tadako Murayama. Kanto-Gakuin Univ., College of Engineering, Kanazawa-ku, Yokohama, Japan.
The wild type grew to be filamentous, whereas adenylyl cyclase mutant, cr-1, grew to be colonial on the solid medium. However, the wild type also grew to be colonial on the solid medium containing sorbose. The cr-1 strain grew to be much smaller colony than the wild type on this medium. High number of large colonies appeared in single colonies from 2-weeks old conidia of cr-1 mutant. The strains from these large colonies grew to be filamentous on the medium without sorbose, though they still showed some form of morphological abnormality. Genetic analysis showed that all 5 strains from the large colonies had original cr-1 mutation and suppressor mutations. The suppressor mutants had the mutations in the different genes and showed different types of morphological abnormality. Some of the suppressor single mutants contained the increase amount of cyclic AMP (cAMP). These mutants did not or scarcely formed conidia and had elevated activity of trehalse. One of the suppressor mutants did not produce conidia and trehalase. Some of them showed abnormality in carotenoid formation. These results suggest that these suppresor mutant genes have some roles in the cyclic AMP cascade and transduce the signal related to the regulation of trehalase, carotenoid production, conidia formation.
344. Maize Ribosome-inactivating protein 1 has antifungal activity against Aspergillus flavus and Aspergillus nidulans. Kirsten Nielsen1, Gary A. Payne2, and Rebecca S. Boston1. 1NC State University, Botany, Raleigh, NC, USA. 2NC State University, Plant Pathology, Raleigh, NC, USA.
Seeds contain a variety of proteins that function primarily to provide storage reserves or defense functions against pests and pathogens. Maize ribosome-inactivating protein 1 (RIP1) is an abundant seed protein with a putative role in plant defense. RIP1 readily inactivates ribosomes from non-plant eukaryotes but has little activity against ribosomes from plant species. RIP1 was tested for antifungal activity in a microculture assay. Conidia were treated with RIP1 or control proteins and monitored for abnormal growth or morphology over time. Fungi from the genera Aspergillus exhibited a strong response to RIP1. Aspergillus flavus, a maize pathogen which produces aflatoxin B1, had an increase in hyphal branching after RIP1 protein treatment that was not observed in control protein treatments. Quantitative analysis confirmed that the branching phenotype was significant. The chitin binding dye Congo red was used to show that branched hyphae treated with RIP1 had only one actively growing hyphal tip whereas the few control hyphae with branching had multiple growing hypal tips. The genetically well characterized but non-pathogenic species Aspergillus nidulans was also tested in the microculture assay. A striking decrease in the number and growth of hyphae was observed when A. nidulans conidia were treated with RIP1 protein. The changes in growth of A. flavus and A. nidulans are consistent with maize RIP1 having antifungal activity.
345.Genetics of alkaloid biosynthesis in Claviceps purpurea: evidence for a gene cluster. Paul Tudzynski1, Katja Hölter1, Telmo Correia1, Claudia Arntz1, Nicolas Grammel² And Ulrich Keller ². 1Institut f. Botanik, Westf. Wilh.-Universitaet, Muenster, Germany. ²Max-Volmer-Inst. f. Biophys. Chemie and Biochemie, Techn. Universitaet. Berlin, Berlin, Germany
The production of ergot alkaloids by the grass pathogen Claviceps purpurea and related species is a well established biotechnological process. The biosynthetic pathway has been worked out in detail and several strains have been developed for production in submerged culture, yet there is still a deficit in detailed genetic analyses. Recently, we have cloned and characterized the genes for Dimethylallyltryptophansyntase (DMATS), catalizing the first step of the pathway, from two C. purpurea strains, a pathogenic field isolate (T5) producing alkaloids only in planta and a (mutant) submerse production strain (P1). A chromosome walking approach [1] showed that the DMATS-gene of strain P1 is part of an alkaloid gene cluster: one of the neighbouring genes encodes D-lysergylpeptide synthetase 1 (LPS1), a trimodular enzyme catalyzing a crucial step in the formation of peptide alkaloids [2].The existence of a gene cluster allows the identification of other genes involved in the pathway and therefore forms a basis for a detailed molecular analysis of this biotechnologically important biosynthetic pathway.[1] P. Tudzynski et al. 1998 Molec Gen Genet. in press [2] B. Riederer et al. 1996 J Biol Chem. 271: 27524-27530
346. Oxidative stress and senescence in Podospora anserina. H.D. Osiewacz, N. Averbeck, C. Borghouts, and E. Kimpel. Johann Wolfgang Goethe-Universität, Botanisches Institut, Frankfurt, Germany
Podospora anserina is an ascomycete with a limited lifespan. Lifespan is controlled by the environment and by mitochondrial and nuclear genetic traits. During the course of investigations to elucidate the molecular mechanisms involved in the control of lifespan, we cloned and characterized a nuclear gene that is able to complement the pleiotropic long-lived mutant grisea to wild-type characteristics. The cloned gene (termed Grisea) was found to code for a protein with significant identity to three yeast transcription factors. These are ACE1 and MAC1 of Saccharomyces cerevisiae, and AMT1 of the pathogenic yeast Candida glabrata. Most strikingly, the three yeast transcription factors control the expression of different genes belonging to the molecular apparatus that is involved in control of cellular copper homeostasis or is part of the defense system protecting the cell against oxidative stress. The detailed molecular characterization of GRISEA revealed that this fungal transcription factor is a functional homologue of MAC1. The most convincing evidence for this conclusion is the ability of the wild-type copy of grisea to complement the yeast mac1-1 deficiency mutant. Furthermore, a one hybrid system analysis revealed a modular structure of GRISEA that is similar to the structure of MAC1. Finally, the function of GRISEA becomes repressed by increased copper levels. A model will be presented that explains the phenotype of the long-lived P. anserina mutant as the result of an impaired copper homeostasis and by a reduction of oxidative stress. Acknowledgment: The experimental work was supported by a grant of the Deutsche Forschungsgemeinschaft.
347. Host- and biotope-specific populations of Claviceps purpurea. Sylvie Pazoutová, Marie Skvánová, Michaela Dolejsí, and Marek Linka. Czech Academy of Sciences, Institute of Microbiology, Prague, Czech Republic.
Claviceps purpurea occurs in Northern temperate regions and colonizes mainly grasses of pooid and arundinoid subfamily. Different authors tried to find the host specific populations or formae speciales in C. purpurea or chemoraces, producing different types of alkaloids. The genetical variability was assayed in over 70 isolates with defined locality and host plant using RAPD and EcoRI restriction polymorphism of the ITS1-5.8S-ITS2 region of rDNA. The type of alkaloid produced was assayed and the shape and dimensions of conidia and sclerotia were recorded, to enable the comparison with the groups postulated in older literature. Two groups were found, that shared distinct RAPD bands. The first group encompassed the isolates from field and open meadows, whereas the isolates from the second group were found in shady or wet habitats. Isolates of the second group are producing ergosine and ergocristine 1:1, the first group is more heterogenous. The average size of conidia in the second group is about 7-8 um, whereas in the first group, 5-6 um was mostly observed. The sclerotia of the second group tend to thin curved form. The second group combines isolates that were once denoted as C. microcephala and/or f. sp. natans P. arundinaceae. The host preferences of the sub-groups are not absolute, the biotop is more important. Without the use of RAPD or rDNA polymorphism, it is not possible to distinguish the sub-groups solely on the basis of morphological characters. That was probably the reason for the differences in specifity groups postulated by different authors.
348. Molecular phylogeny of Japanese Amanita species based on nucleotide sequences of the internal transcribed spacer region of nuclear ribosomal DNA. Takashi Oda, Chihiro Tanaka, and Mitsuya Tsuda. Kyoto University, Agriculture, Kyoto, Kyoto, Japan.
We constructed a molecular phylogenetic tree of forty specimens of Japanese Amanita species based on nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. By comparing this phylogenetic tree with the classification systems of Bas (1969) and Singer (1986) which are based on morphological characters and have been widely accepted, we examined the phylogeny of Japanese Amanita species. Our results supported the taxonomical treatment in subgenus level of Bas (1969) and Singer (1986) who divided the genus Amanita into two subgenera Amanita and Lepidella based on the amyloidity of spores. However, in section level, we suggested that subgenus Amanita should be divided into three sections (Amanita, Vaginatae and Caesareae). Moreover, our results showed the necessity to rearrange the disordered assemblage of members in the subgenus Lepidella. As for three subspecies of Amanita hemibapha and three varieties of Amanita vaginata, it is necessary to grade up their taxonomical ranks from subspecies / variety to species. It appears that Amanita pantherina and Amanita muscaria are extremely related phylogenetically. The molecular biological technique was proven to be sensitive for detecting taxa that are difficult to detect by morphological characters.
349. The structure of the vacuolar ATPase in Neurospora
crassa. Jack C. Reidling, Emilio Margolles-Clark, Karen Tenny, June
Pounder, Emma Jean Bowman, and Barry Bowman. UCSC, Biology, Santa Cruz, Ca, USAThe
filamentous fungus Neurospora crassa contains many small vacuoles. These
organelles contain high concentrations of polyphosphates and basic amino acids such as arginine
and ornithine. Because of their size and density the vacuoles can be separated from other
organelles in the cell. The ATP-driven proton pump in the vacuolar membrane is a typical V-type
ATPase. We have examined the size and structure of this enzyme by the technique of radiation
inactivation and by electron microscopy. The vacuolar ATPase is a large and complex enzyme
that appears to contain at least 13 different subunits. We have characterized the genes that encode
11 of these subunits, are currently characterizing a 12th, and are attempting to obtain a 13th. The
ultimate goal of our laboratory has been to understand the function and structure of these
subunits. One approach we are taking is to purify the enzyme to homogeneity for further
studies.
350. Biological research on prospects of cultivation of the giant puffball
(Langermannia gigantea).
Bringing giant puffball into cultivation is urged by its economical importance in many fields: it is an edible, tasty mushroom, the mass of mycelium can be used for feeding animals, furthermore it is a pharmacologically important species. Its requirements for soil types, the fenology and the environmental factors of fructification, and the coenological rules of its occurence have been examined for ten years in more than 81 habitats. More than 2,000 data, gained from soil components of 58 habitats, give a guide for preparing suitable substrate for the cultivation of Langermannia gigantea, and for choosing areas for extensive cultivation. The occurence of this species is not associated tightly with any type of vegetation, therefore the extensive cultivation is not affected seriously by certain countrysides. It needs shade in the initial period of fructification, but later become more resistant to direct sunlight. The Langermannia gigantea is exclusively saprophyte, even its facultative mikorrhiza connection is unlikely. Its spontaneous appearance in cultivated vinyards and orchards supports the possibility of its cultivation.
351. The Spitzenkorper of Allomyces macrogynus serves as a microtubule organizing center.Robert W. Roberson, and Dennis P. McDaniel. Arizona State University, Plant Biology, Tempe, AZ, USA.Elongating hyphae of higher fungi contain a well defined Spitzenkörper. The role(s) of the Spitzenkörper in fungal cell growth and morphogenesis have long been enigmatic. We have shown that hyphae of the zoosporic fungus Allomyces macrogynus (Chytridiomycota) contain a well defined Spitzenkörper and recent data confirmed its role as a microtubule organizing center (MTOC). Using indirect immunofluorescence methods and laser scanning confocal microscopy (LSCM), we have demonstrated that gamma-tubulin is a component of the Spitzenkörper in A. macrogynus. We also found that gamma-tubulin is associated with centrosomes. LSCM of the microtubule (MT) cytoskeleton under control conditions and upon recovery from MT depolymerization suggested that the Allomyces Spitzenkörper functions as a center for MT organization and nucleation. Further data will be presented elucidating the ultrastructural location of gamma-tubulin within the Spitzenkörper and in vivo dynamics of MT polymerization. That the Spitzenkörper of Allomyces regulates MT nucleation and distribution demonstrates its relevance in regulating intracellular motility and maintaining cytoplasmic organization during hyphal tip growth. There is presently no evidence that Spitzenkörper of higher fungi contain gamma-tubulin and their role as MTOCs remains speculative.
352. Isolation and characterisation of a cot-1 homologue from Aspergillus nidulans. Mehran Safaie, and Geoffrey Turner. University of Sheffield, Molec Biol Biotechnol, Sheffield, South Yorkshire, UK.
Mycelial growth in filamentous fungi results from polarised growth and hyphal branching. In addition to interest in the fundamental processes controlling this form of growth, colonial morphology is important in industrial fermentations. In order investigate the genetic basis of hyphal branching and growth polarity in the genetic model A. nidulans, we are isolating genes by complementation of mutants affecting branching frequency (see poster by Memmott et al.) and by isolation of homologues of genes from yeast or filamentous fungi. The cot-1gene of Neurospora crassa, encoding a serine-threonine protein kinase (Yarden et al. 1992, EMBO J. 11, 2159) is one of a limited number of characterised genes known to affect polar growth and branching frequency. Degenerate oligonucleotides designed against cot-1, and homologues from C. trifolii and S. cerevisiae were used to amplify a fragment from A. nidulans. Sequence analysis revealed marked identity with cot-1. Using the PCR amplified fragment as a probe, 3 related cosmid clones, assigned to chromosome V, were isolated from the chromosome sorted genomic library. Although 2 were adjacent on the physical map (Prade et al. 1997, PNAS 94, 14564) a third was localised elsewhere on V, suggesting mislocalisation of one or more of the cosmids. Sequence analysis of the entire gene revealed high sequence identity at in the C-terminal region, but considerable divergence in the N-terminal region of the derived protein sequence. Attempts are underway to disrupt the gene, and alter the level of expression by promoter exchange.
353. The effects of some phenolic compounds on the growth and morphology of Neurospora crassa. Suraia Said1, Juliano SimõesToledo1, John Page2, and Anthony J.F. Griffiths2. 1Universidade de Sao Paulo, Ciências Farmacêuticas, Ribeirão Preto, São Paulo, Brazil. 2UBC, Botany, Vancouver, BC, Canada.
After carbohydrates, phenolic compounds are the largest group of substances in wood. Phenolics are also present in the waste of many industries. Some natural phenolics like cinnamic acid are known as potent inhibitors of microbial germination. The effects of different phenolic compounds on growth and hyphal morphology of N. crassa were analysed, as well as some biochemical aspects of cultures growing in the presence of cinnamic acid. No effects on the size or morphology of the colonies were observed with most of the substances used. However, in the presence of ferulic or cinnamic acid the colonies were small and compact, their hyphae were more branched and there was an increase of precocious aerial hypha. Cinnamic acid (250 mg/L) reduced growth about 93% in the first 24 h of incubation and 56.3% after 144 h. In liquid cultures supplemented with cinnamic acid the fungus could grow after 48 h and at this time the concentration of cinnamic acid was 22% of the initial concentration. A malodorous compound was detectable in the cultures, identified as 1,2,3-trimethylbenzene. Activity of the enzyme aryl-aldehyde:NADP oxidoreductase was detected only in the culture growing in cinnamic acid presence. The results show that this natural phenolic changes the growth rate and the morphology of N. crassa, but after 48 h the fungus is able to induce at least one enzyme to degrade this inhibitor. Suppoted by: Natural Science and Engineering Council of Canada Grant 55695. Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil) Grant 201181/95-9. Fundação de Amparo a Pesquisa do Estado de São Paulo (Brazil) Grant 97/03824-0.
354. Isolation and analyses of Neurospora crassa genes encoding Rad50 and Rad52 homologues. Yoshiyuki Sakuraba, and Hirokazu Inoue. Saitama University, Faculty of Science, Urawa, Saitama, Japan.
DNA double-strand breaks (DBSs) induced by ionizing radiation or methylmethane
sulfonate (MMS) are thought to be repaired by recombinational repair. In Saccharomyces
cerevisiae, the RAD52 epistasis group genes are classified in this repair pathway. In
N. crassa, some mutants defective in recombinational repair have been isolated and
characterized. To further probe the mechanism of the recombinatinal repair pathway, we isolated
the genes that encode Rad50p and Rad52p homologues in N. crassa using a PCR
method.The DNA sequence of the PCR fragment of the RAD50Nc
gene matched with the upstream sequence of the wc-2 gene which is located on the right arm of
chromosome I. A KpnI-KpnI fragment which contains the
RAD50Nc gene was subcloned from the cosmid including the
wc-2 gene (The cosmid was kindly given from Dr. G. Macino), and the DNA
sequence was determined. The RAD50Nc protein deduced from the nucleotide
sequence has 29.9% identity to Rad50p (S. cerevisiae) . The uvs-6
mutation has been genetically mapped near the wc-2 locus. Sensitivities to UV,
MMS and histidine of the uvs-6 mutant were complemented by transformation with
the RAD50Nc gene fragment. This indicates that the uvs-6
gene encodes the Rad50p homologue protein. We also cloned
RAD52Nc gene from cDNA library and DNA sequence was
determined. The RAD52Nc protein deduced from the nucleotide sequence has
27.6% identity to Rad52p (S. cerevisiae). RFLP mapping using a PCR fragment as a
probe demonstrated that the RAD52Nc gene locates closed to the
ro-4 locus on the right arm of chromosome V. Both RAD50Nc and RAD52Nc
mRNA could not detect in normal growth condition by Northern hybridization, but the
transcription of these genes were induced after UV irradiation or MMS treatment.
355. Kinetic study of protein secretion in Aspergillus niger
Filamentous fungi are able to produce and secrete large amounts of proteins, but so far the mechanisms and kinetics of secretion have not been studied in details. In this study, pulse-chase experiments have been used since they allow quantitative study of protein production and secretion kinetics. Based on the experimental data the secretion of hen egg white lysozyme (HEWL) by A.niger has been modeled using first order kinetics.
356. Tagging genes and trapping promoters of the barley pathogen Pyrenophora teres. Wilhelm Schäfer, Frank Josef. Maier, and Anke Petra Lösch. University of Hamburg, Inst. of General Botany, Hamburg, Hamburg, Germany.
Pyrenophora teres (anamorph Drechslera teres) is the causal agent of barley net blotch, which is one of the most widely distributed foliar diseases of barley throughout the world. It was the most damaging pathogen of winter barley in Germany during the last two growing seasons. The interactions between the host plant and the pathogen are not understood. To make the fungus amenable to the methods of molecular biology, we started by developing a transformation system based on the resistance of transformed cells to the antibiotic hygromycinB. Based on this, transformation by restriction enzyme mediated transformation was established to identify genes by insertional mutagenesis. Vector configuration and restriction enzyme activity greatly influence the integration of the foreign DNA. Using optimized conditions, about 95% of all transformants showed single vector integration. The addition of a heat shock increases the number of transformants 2.5 fold, although the viability of the protoplasts decreases dramatically from 70% to 3%. Of several transformants, DNA flanking the integrated vector was cloned and sequenced. No deletions or rearrangements were detectable as compared to the genomic wild type sequence. To tag genes and simultaneously trap fungal promoters, we constructed a vector for REMI transformation which carries, after linearization, the promoterless reporter genes coding for ß-glucuronidase and luciferase at the ends. Therefore, promoter active genomic sequences can be tagged after vector integration irrespective of their direction of transcription. To test for virulence mutants, a petri dish assay with detached leaf sections was developed. In a second test, mutants with reduced virulence were spray inoculated on intact barley plants in the growth chamber. Tests for ß-glucuronidase and luciferase activity of fungal transformants in vitro and during plant infection were established. Out of 183 REMI transformants, 11 were clearly reduced in virulence, 94 showed promoter activity, with 12 plant inducible promoters.
357. A Neurospora linkage group VI specific sub-library. Thomas J. Schmidhauser. The University of Southwestern Louisiana, Biology, Lafayette, LA, USA.
We have used the chromosome assignments generated by the University of Georgia Neurospora genome project group to assemble a 222 clone sub-library of the Orbach/Sachs Neurospora genomic library representing cosmids with inserts specific to LGVI. The sub-library has been used to extend a chromosome walk from the crosspathway control-1 locus to the unknown-4 and lysine-5 loci. Sib-selection is being used to isolate selectable markers. Results include the isolation of a cysteine-2+ cosmid and failure to identify a cosmid complementing the unknown-23 mutation.
358. EST analysis of the entomopathogenic fungus Metarhizium anisopliae. Steven E. Screen, and Ray J. St. Leger. University of Maryland, Entomology, College Park, MD, USA.
The era of genomics has already begun. The technology for rapidly determining the entire catalogue of genes that constitute the repertoire of fungal physiological capacity, is now tried and tested. We aim to use this technology to sequence the genome of the most extensively studied entomopathogenic fungus M. anisopliae. The most efficient and cost effective means is first to catalogue the protein coding re