Paulo C. Alves, Margarida Duarte, Teresa Almeida and Arnaldo Videira. Instituto de Ciencias Biomedicas de Abel Salazar da Universidade do Porto
Complex I is a multi-subunit structure of the inner mitochondrial membrane that couples electron transfer from NADH to ubiquinone with proton translocation from the matrix to the inter membrane space of the organelle. The enzyme is assembled from many nuclear-coded and a few mitochondrially-synthesized polypeptides. We are concentrating in the disruption of specific complex I subunits, by the generation of repeat-induced point mutations in their genes, to study several aspects of the biogenesis and function of the enzyme. As a step toward the isolation of mutants, two new genes were cloned and mapped in the Neurospora crassa genome by RFLP analysis. Several mutants have been obtained and we will describe the strategy that is being used to induce novel mutants. In addition, an analysis of complex I assembly has been performed in the stopper mutants of Neurospora. In some strains, assembly of complex I is not detected and the peripheral arm of the enzyme is accumulating.
34. Expression and secretion of human antibody light and heavy chains in Neurospora crassa.
Stephen Buczynski, David Schneck, Doug Vann, Elie Kato, and W. Dorsey Stuart, University of Hawaii, Office of Technology Transfer and Economic Development /Neugenesis Corp. (Joint Project)
A proprietary strain of N. crassa has been transformed by electroporation with plasmid vectors containing cDNAs for either a human kappa light chain or a human gamma heavy chain. The expression vectors contained the promoter and signal sequence of the gla-1 gene of N. crassa fused in frame with the cDNA of either antibody chain followed by the gla-1 terminator region. Prototrophic or antibiotic resistant co-transformants were screened for secretion of individual antibody chains by ELISA. Stable homokaryotic transformants were then grown in a liquid culture medium optimized for high expression of the gla-1 promoter and low protease secretion. Extracellular media from production cultures was harvested. The heterologous protein product was fractionated using either cation exchange or affinity chromatography. The engineered protein product was validated by ELISA and Western blot methods. DNA integration into the host cells was characterized by Southern blot analysis. Current production levels are in the nanograms per milliter level for both kappa and gamma. Increased production levels will be achieved by designing upgraded expression cassettes, mutagenesis and selection for higher producing strains. In addition, strategies are being developed for using this system to produce intact human IgG antibody molecules.
35. Secretion of Trichoderma reesei beta-glucosidase by Saccharomyces cerevisiae.
Chris Cummings and Tim Fowler, Genencor Int. Inc., 180 Kimball Way, South San Francisco, CA 94080.
An intronless form of the bgl1 gene encoding an extracellular b-glucosidase from Trichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL1 promoter. Transformation of a yeast strain with this vector resulted in transformants that produce and secrete active beta-glucosidase into the growth medium. Additionally, active recombinant b-glucosidase protein was shown to be localized predominantly in the periplasmic space by using a p-nitrophenyl beta-D-glycoside hydrolysis assay against fractionated yeast cells. The apparent size of the recombinant enzyme was 10-15 kDa larger than that of the native form. Treatment of the recombinant beta-glucosidase with endoglycosidase-H indicated the apparent increase in size is due to N-linked glycosylation.
36. sepB, an Aspergillus nidulans gene that regulates the initiation of cytokinesis.
Steven Harris(1) and John Hamer, Dept. of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392. (1)current address: Dept. of Microbiology, University of Connecticut Health Center, Farmington, CT 06030-3205.
We have undertaken a combined genetic and molecular analysis of septum formation in Aspergillus nidulans to determine how septa are assembled and to elucidate the mechanisms that spatially and temporally coordinate this assembly with other cell cycle events. Temperature-sensitive sepB mutants are members of a novel class of A. nidulans mutants that appear to be specifically defective in the coordination of septum formation with nuclear division. At restrictive temperature, sepB mutants complete the first three rounds of nuclear division but arrest prior to initiating septum formation. Although these mitoses occur with kinetics similar to wild-type, the resulting nuclei exhibit an aberrant morphology, appearing elongated and multi-lobed. In addition, a colony sectoring assay was utilized to demonstrate that sepB mutants exhibit defects in chromosome segregation. These results suggest that sepB functions to maintain the fidelity of chromosomal DNA metabolism. Results from temperature shift experiments demonstrate that sepB functions early in septum formation. Moreover, failure to perform this function leads to the cessation of cell growth and nuclear division as well as the rapid onset of lethality. Molecular analysis of the sepB gene reveals that it is essential and possesses homology to a Saccharomyces cerevisiae gene involved in chromosomal DNA metabolism. We propose that the sepB gene product is involved in maintaining the fidelity of chromosome segregation while playing an essential role regulating the initiation of cytokinesis.
37. Translation, sexual reproduction and longevity in Podospora anserina.
Philippe Silar(1), Benedicte Gagny(1) and Marguerite Picard(2). (1)Centre de Genetique Moleculaire du CNRS, 91198 Gif sur Yvette cedex, France. (2)Inst. de Genet. et Microbiol. de l'Univ. de Paris-Sud, C.N.R.S. URA 1354, Bat. 400, 91405 Orsay cedex, France.
Mutations in components of the translation machinery modifying the accuracy of the decoding process have numerous phenotypes affecting various aspects of the life of P. anserina, especially life span. It is not clear what is responsible for the drastic longevity changes in the strains carrying one such mutation. We have undertaken an extensive analysis of high fidelity mutations (antisuppressors that antagonize informational suppressors) in the translation elongation factor EF-1a encoded by the AS4 gene. Antisuppressive properties of the AS4 mutations are highly variable depending on the suppressor used and the target mutation suppressed, precluding any classification on this basis. On the opposite, vegetative and reproductive abilities of the mutants are inversely correlated with their longevities. The mutants also present an additional inability to eject spores out of the perithecia. Sequences of the AS4 mutations reveal structural modifications similar to the yeast EF-1a suppressor mutations. The analysis suggests that a global modification of translation, and not the production of more accurate proteins, is responsible for the life span extension of the mutant strains. We are now investigating the biochemical parameters of translation altered in these mutants and we have started an analysis of the other accuracy genes, i.e. the omnipotent suppressor loci su1 and su2 and the genes coding for ribosomal proteins.
38. An estimation of the number of nuclear genes interfering with the senescence syndrome of Podospora anserina.
Philippe Silar and Michelle Rossignol. Centre de Genetique Moleculaire du CNRS, 91198 Gif sur Yvette cedex, France.
Vegetative growth of P. anserina is accompanied by modifications of the mitochondrial DNA, leading to the death of the apical cells (senescence). The kinetics of modification is under the genetic influences of both mitochondrial and nuclear genomes. In order to estimate the number of nuclear genes involved in the control of life span, longevities of mutant strains were measured. Some of the mutants used have already been obtained on several other criteria unrelated to senescence, allowing us to uncover relations between various physiologic processes and mitochondrial DNA stability. The other mutants were constructed by insertional mutagenesis with a vector which integrated randomly and did not modify longevity by itself, allowing us to precisely estimate the proportion of the genome involved in life span control. Preliminary results suggest that life span is affected by modification of many processes. Indeed, mutations in some amino-acid biosynthetic pathways, in the cytoplasmic translation machinery and many mutations altering the morphology do modify life span. Consequently, a very large number of genes seem to be involved: among the random insertion mutants, an average of about 5% have a modified longevity. Taking in account that half of the genome is non coding and that our system can detect only a subset of the genes controlling longevity, more than 10% of the genes are involved in life span control in P. anserina.
39. Isolation and characterization of an Achlya ambisexualis cDNA and expression of a Hsp60 transcript population during antheridiol-induced differentiation.
Chai Chen and Julie C. Silver. University of Toronto, Scarborough Campus.
Previous and ongoing studies from this laboratory have shown that several different Hsp70 and Hsp90 chaperone/heat shock proteins and their mRNAs are up-regulated during antheridiol-induced hyphal differentiation in the oomycete Achyla ambisexualis. Hsp60 (GroEL) is a chaperone which mediates protein folding under normal physiological conditions, within,the mitochondrial matrix. This chaperone is also a heat shock protein. We have isolated a cDNA encoding Achlya Hsp60 and characterized the expression of Hsp60 transcripts and protein during antheridiol-induced hyphal differentiation. The levels of both were found to increase markedly with heat shock and with hormone-treatment of the mycelium. These results suggest a role for mitochondria during steroid hormone- induced hyphal branching and differentiation. (Supported by NSERC)
40. Cloning and characterization of Aspergillus nidulans cysB and cysC genes involved in cysteine synthesis.
Jacek Topczewski, Marzena Sienko, Andrzej Paszewski, Polish Academy of Science, Institute of Biochemistry and Biophysics, Department of Genetics, ul. Pawinskiego 5a, 02-106 Warsaw, Poland
In A. nidulans three genes, cysB, cysC and cysE have been identified which control the last step in cysteine synthesis. The wild type cysB, coding for cysteine synthase, was cloned by complementation of a cysB mutation using our own cosmid gene library. The gene and its cDNA copies were sequenced revealing one intron at 5' end. The amino acid sequence of the enzyme shows a considerable similarity to cysteine synthases from E. coli and plants. Cysteine synthase activity in some transformants was found several-fold higher than in the wild type stains and correlated with a high resistance to sulfide. Studies of cysB expression are in progress. The cysC gene was localized on cosmid W5C10 from pWE15/pLORIST2 library from FGSC, by complementation of cysC- mutation. The transformants, in contrast to cysB+ ones, show no significant difference in the cysteine synthase level as compared to the wild type. The 5kb EcoRI/EcoRI fragment of the insert, caring the cysC gene, was subcloned. Preliminary data on the sequence analysis was presented.
41. Effects of mislocalization of ornithine transcarbamylase from the mitochondria to the cytosol in Neurospora crassa.
Terri L. Moulds and Richard L. Weiss. University of California, Los Angeles.
Regulation of metabolic pathways is accomplished in various ways. The role of compartmentation in regulation of the arginine biosynthetic pathway in Neurospora crassa is being investigated using ornithine transcarbamylase (OTC) as a model system. OTC catalyzes the formation of citrulline from ornithine and carbamyl phosphate in the mitochondria. Both substrates can cross the mitochondrial membrane and OTC's location is different in closely related organisms. Thus, the location of OTC may affect more than the arginine biosynthetic pathway. To mislocate OTC to the cytoplasm, a plasmid containing the coding region but lacking the sequence which encodes the mitochondrial targeting sequence was transformed into an arg-12 mutant that makes a truncated OTC. The transformants are arginine prototrophs but have reduced growth rates. Southern analysis indicated ectopic integration of the transformed DNA. OTC activity is found only in the cytoplasm but the specific activity is lower than wild type. The "mislocalization" construct has also been introduced into a strain that has a disruption in the arg-12 locus. This recipient strain has no OTC activity and no detectable protein in the mitochondria. The effect of a cytoplasmic OTC in this and other mutational backgrounds is being investigated to determine the role of compartmentation in the control of amino acid metabolism.
42. The processing pathway of a polyprotein precursor of two mitochondrial enzymes in Neurospora crassa.
Lilian E. Parra and Richard L. Weiss. University of California, Los Angeles.
In Neurospora, the mitochondrial arginine biosynthetic enzymes N-acetylglutamate kinase (AGK) and N-acetyl-g-glutamyl-phosphate reductase (AGPR) are made from the cleavage of a 96 kDa cytosolic polyprotein precursor. The precursor consists of a leader peptide followed by the two protein domains which are separated by a connector region. In order to identify sites of cleavage, elements involved in recognition by the processing protease, and the function of the polyprotein precursor, we have introduced several mutations in the region that connect the two protein domains. Processing has been analyzed by in vitro import assays with purified mitochondria and radiolabeled precursor. Analysis of processing and protein function in vivo has been performed by transformation of mutant precursors into AGK-AGPR- strains. We found a consensus arginine at position -2 from the cleavage site of the leader and at positions -2 and -3 from the N-terminal amino acid of the distal AGPR in the connector region of the polyprotein. This suggested that the same protease(s) involved in cleavage of the leader sequence may be involved in cleavage of the precursor into two mature proteins. Processing of a precursor in which arginine residues at positions -2 and -3 were changed to G and P identified a second processing site since processing yielded a wild type AGK and a larger form of the distal AGPR. Both proteins were functional as they complemented an AGK- AGPR- mutant. The mutant AGPR protein was microsequenced and the alternative cleavage site in the precursor was identified. This second cleavage site is located 20 amino acids upstream from the N-terminus of the AGPR and has an arginine residue located 3 residues upstream from the site of processing. Mutations that replace this arginine with a glycine or a proline have been obtained and its role on processing is being analyzed. Comparison of amino acid sequences in the vicinity of wild type and alternative processing sites revealed the presence of a threonine residue 2-3 amino acids downstream of the cleavage sites. The role of this residue on the mechanism of proteolytic cleavage is currently under investigation.
43. Pseudoreversion analysis of bimD6 in Aspergillus nidulans identifies a gene coding for a chromosome scaffold protein.
Cydne L. Holt and Gregory S. May, Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030
A pseudoreversion analysis of the bimD6 mutation was undertaken to identify additional genes that function in the bimD pathway. Seven extragenic cold sensitive cs- suppressors of bimD6 heat sensitivity hs- were identified among 1505 revertant colonies. The cs- phenotype of all the suppressors was recessive and they fell into six complementation groups. Genetic mapping of the suppressor mutations has led us to conclude that we have identified three and possibly four extragenic suppressor genes which exhibit complex genetic interactions. These genetic interactions include both linked complementation and unlinked noncomplementation, suggestive of physical associations. We have cloned one of the suppressor genes sudA by complementation of its recessive cs- phenotype. Sequence analysis of sudA has shown that it is a member of a rapidly growing family of proteins termed DA-box proteins because of conserved aspartate and alanine residues found in their carboxyl terminal globular domain. Structurally DA-box proteins are very similar and have two globular domains separated by a region of putative coiled-coil sequence. DA-box proteins function in chromosome condensation, segregation and gene regulation. One member of the family, dpy-27 of C. elegans, is involved in X chromosome compensation and therefore has an apparent chromosome specific function. These results suggest that bimD functions in chromosome segregation and structure in co-operation with DA-box proteins. Our suppressor genetic analysis suggests that there are two and possibly three other genes that also function in this pathway with bimD and sudA.
44. Hsp30-defective mutants of Neurospora crassa show conditional intolerance for high temperature.
Nora Plesofsky-Vig and Robert Brambl, The University of Minnesota, Saint Paul
The alpha-crystallin-related heat shock proteins are produced by all eukaryotes, but the role of these proteins in thermoprotection remains unclear. To investigate the function of one of these proteins, we disrupted expression of the single-copy hsp30 gene of Neurospora crassa, using repeat-induced point mutagenesis, and we generated and characterized mutant strains that were deficient in hsp30 synthesis. These strains could grow at high temperature and they acquired thermotolerance from a heat shock. However, the hsp30-defective strains proved to be extremely sensitive to the combined stresses of high temperature and carbohydrate limitation, enforced by the addition of a non-metabolizable glucose analogue. Under these conditions, their survival was reduced by 90% compared with wild- type cells. This sensitive phenotype was reversed by reintroduction of a functional hsp30 gene into the mutant strains. The mutant cells contained mitochondria from which a 22 kDa protein was readily extracted with detergents, in contrast to its retention by the mitochondria of wild-type cells. Antibodies against hsp30 coimmunoprecipitated a protein also of approximately 22 kDa from wild-type cells. Results of this study suggest that hsp30 may be important for efficient carbohydrate utilization during high temperature stress and that it may interact with other mitochondrial membrane proteins and function as a protein chaperone.
45. Cytochrome c oxidase in Neurospora crassa contains myristic acid covalently linked to subunit 1.
Alexei O. Vassilev, Nora Plesofsky-Vig, and Robert Brambl, The University of Minnesota, Saint Paul
Radiolabel from [3H]myristic acid was incorporated by Neurospora crassa into the core catalytic subunit 1 of cytochrome c oxidase, as indicated by immunoprecipitation. This modification of the subunit, which was specific for myristic acid, represents an uncommon type of myristoylation through an amide linkage at an internal lysine, rather than an N-terminal glycine. The [3H]myristate, which was chemically recovered from the radiolabeled subunit peptide, modified an invariant Lys324, based upon analyses of proteolysis products. This myristoylated lysine is found within one of the predicted transmembrane helices of subunit 1 and could contribute to the environment of the active site of the enzyme. The myristate was identified by mass spectrometry as a component of mature subunit 1 of a catalytically active, purified enzyme. This is the first identification of a mitochondrial inner-membrane protein that is post-translationally modified by a fatty acylation.
46. Cloning and characterization of a functional homologue of the S. cerevisiae sar1 gene from Aspergillus niger.
Gerrit Veldhuisen, Martijn Fiers and Cees A.M.J.J. van den Hondel. Dept of Molecular Genetics and Gene- technology, TNO Nutrition and Food Research Institute, PO Box 5815, 2280 HV Rijswijk, The Netherlands.
To initiate research towards a systematic analysis of the mechanism of protein targeting and secretion in filamentous fungi, the cloning of various genes encoding secretion-related GTP-binding proteins was undertaken. Cloning of these genes was carried out using heterologous hybridisation with cloned S. cerevisiae / pombe genes as probes. The successful cloning of the SAR1-related A. niger sarA gene is described. Further characterisation of the cloned gene was carried out by functional complementation of S. cerevisiae sar1 and sec12 mutants with a full length cDNA copy of the sarA gene in a yeast expression vector. Using gene-replacement strategies and a (conditional) mutant version of the cloned gene, A. niger secretion mutants will be generated.
47. Repressible phosphate/cation symporters in Neurospora crassa.
Wayne K. Versaw and Robert L. Metzenberg, University of Wisconsin-Madison.
The filamentous fungus Neurospora crassa posseses two non-homologous, high-affinity, phosphate permeases, PHO-4 and PHO-5. We have isolated separate null mutants of these permeases which has allowed us to study the remaining active transporter in vivo in terms of phosphate uptake and sensitivity to inhibitors. The specificity for the co-transported cation differs for PHO-4 and PHO-5, suggesting that these permeases employ different mechanisms for phosphate translocation. Phosphate uptake by PHO-4 is stimulated 85-fold by the addition of Na+, which supports the idea that PHO-4 is a Na+/phosphate symporter. PHO-5 is unaffected by Na+ concentration, but is much more sensitive to elevated pH than is PHO-4. Presumably, PHO-5 is a H+/phosphate symporter. Na+- coupled symport is usually associated with animal cells. The finding of such a system in a filamentous fungus is in harmony with the idea that the fungal and animal kingdoms are more closely related to each other than either is to the plant kingdom.
48. Extragenic suppressors of the nudAl cytoplasmic dynein mutation that blocks nuclear migration in Aspergillus nidulans.
Gustavo H.Goldman and N. Ronald Morris*, FCFRB Universidade de São Paulo, Brazil and *Department of Pharmacology, UMDNJ, USA.
Cytoplasmic dynein is a larqe molecular weight protein complex that functions as a microtubule dependent, negative end directed, "motor". Mutations in nudA which encodes the heavy chain of cytoplasmic dynein, inhibit nuclear migration in A. nidulans. We isolated and characterized extragenic suppressors of the nudAl mutation preparatory to the identification of other protein that interact with the cytoplasmic dynein heavy chain. Genetic analysis of 19 revertants has defined at least 5 extragenic suppressors of nudAl (snaA-E). All the sna mutations, except one, were recessive in diploids homozygous for nudAl and heterozygous for sna mutations. To characterize the nuclear migration phenotype in the sna mutants, conidia of one representative of each complementation group were germinated, fixed and stained with DAPI. The sna mutants display partial suppression of the nudA1 nuclear migration defect. The conidiophore morphology appeared abnormal in all the sna mutants except snaB76, and is linked to the extragenic suppressor mutation.
49. Search for gene(s) that biodegrade TNT in Phanerochaete chrysosporium.
M.M. Jackson and S.K. Dutta, Depts. of Biology, and Genetics & Human Genet., Howard Univ., Washington DC.
It is known that the white rot fungus Phanerochaete chrysosporium has the ability to degrade TNT (2,4,6- trinitrotoluene) (Fernando et al.: Appl. Microbiol. 56:1666-1674, 1990) but there is no report of actual isolation of this gene(s). The search for these gene(s) in this fungus responsible for TNT biodegradation has been an on going study in our laboratory. Preliminary studies indicate that this fungus can degrade TNT comes from growth studies using differential media. The fungus was grown in low starvation dose of five carbon source which consist of glucose, verathal alcohol, ammonium tartrate, sodium acetate, and tween-80 which act as cometabolites and 25 ppm TNT. Recently our laboratory has isolated TNT gene sequence from the bacterium Pseudomonas aeruginosa strain (Dutta et al., 1994, unpublished). We are currently performing hybridization studies by use of dot blots and southern blots for evidence of TNT degrading gene(s) present in P. chrysosporium. We are developing two strategies of approaches in going about isolating the gene: hybridization studies using known TNT gene probe and construction of cDNA library which are ongoing projects in our laboratory. (Supported in part by the U.S. Department of Army).
50. Isolation and characterization of monomorphic mutants of Ustilago maydis
A D. Martinez-Espinoza, C. Leon, G. Elizarraraz and J. Ruiz Herrera. Centro de Investigacion y Estudios Avanzados- Unidad Irapuato IPN. Irapuato Mexico.
U. maydis is a basidiomycete responsible for the corn smut or "huitlacoche". The fungus has a saprophytic phase, growing as haploid, yeast-like cells (sporidia). Sporidia of compatible mating type are able to fuse to form an invassive dikaryotic mycelium. We have developed conditions which promote the dimorphic transition of haploid cells in vitro. Using this procedure for selection we have been able to isolate about 20 mutants which are unable to grow as mycelium under our experimental conditions. These mutants have been characterized based on their cell and colonial morphology in different media. Most of the mutants were found to be recessive when crossed with the wild type, bE or bW cells of the opposite sex. By means of reciprocal crosses among mutants of opposite mating type, strains have been allocated into at least four complementation groups. In addition, mutants have been characterized based on their pathogenic reaction. All mutants recessive for morphogenesis in vitro resulted recessive in their pathogenic behavior. Non-complementing mating pairs of mutants were non-pathogenic when inoculated into corn seedlings. Accordingly, these mutants define genes involved in mycelial growth and pathogenicity other than b.
51. The aglF gene encoding a secreted alpha-galactosidase in Aspergillus niger.
Anders Kassow(1), Ana M. Mateo Rosell(1), Peter J. Punt(1), Carsten Hjort(2) and Cees A.M.J.J van den Hondel(1). (1)TNO Nutrition and Food Research, Department of Molecular Genetics and Gene Technology, PO Box 5815, NL-2280 HV Rijswijk, The Netherlands. (2)Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark.
Filamentous fungi of the genus Aspergillus are widely used for the production of fermented foods, organic acids and enzymes. Due to their capacity to secrete large amounts of proteins these organisms are often chosen for the production and secretion of homologous and heterologous proteins. However, not much is known about the secretion machinery in filamentous fungi. We plan to use alpha-galactosidases as reporters to study secretion in Aspergillus niger. A. niger has been shown to have at least six different enzymes with alpha-galactosidase activity. Depending on growth conditions most of the alpha-galactosidase activity is found in the growth medium. Two genes (aglA and aglN) encoding proteins with alpha-galactosidase activity were previously cloned from A. niger. aglA has sequence homology to other eucaryotic alpha-galactosidase encoding genes, whereas aglN shows homology to an alpha-galactosidase encoding gene from E. coli. We have now cloned the gene (aglF) responsible for one of the extracellular alpha-galactosidase activities as well as part of an additional gene likely to be encoding another alpha-galactosidase. The DNA sequence of aglF shows homology to the previously cloned aglA gene as well as other eucaryotic alpha-galactosidase encoding genes. CHEF/Southern analysis assigned the gene to A. niger chromosome II. Some data on the expression of the gene was be presented.
52. Growth pulses in Aspergillus niger wild type and an apical branching mutant.
Cristina G. Reynaga-Peña and Salomon Bartnicki-Garcia. University of California, Riverside.
Video enhanced microscopy made it possible to make precise measurements of fungal growth and to discover that fungal hyphae do not elongate steadily but grow in alternating pulses of fast and slow growth (1). We have made a detailed analysis of pulsed growth in Aspergillus niger wild type strain, and a temperature-sensitive mutant capable of apical branching at the restrictive temperature. Hyphae from either strain showed pulses of similar frequency (9- 11 pulses per min) despite substantial differences in elongation rate. The apical branching mutant allowed us to analyze growth pulses in adjoining branches from the same hypha. The pulses in these closely connected hyphal branches were of similar frequency but were not synchronous (fast and slow growth periods did not occur simultaneously). The similarity in pulse frequency suggests that hyphal growth is largely regulated by a mechanism common to all hyphae in a mycelium, while the lack of pulse synchrony suggests a certain degree of growth independence for each hypha. Presumably, the final events in the discharge of wall-building vesicles responsible for apical growth is controlled locally at each growing point. (1) Lopez-Franco, R., S. Bartnicki-Garcia and C.E. Bracker. 1994 Proc. Natl. Acad. Sci. USA. 91:12228-12232.
53. Heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori.
I.A. van Gemeren(1), A. Beijersbergen(2), W. Musters(2), R. Gouka(3), C.A.M.J.J. van den Hondel(3), C.T. Verrips(1, 2). (1)Department of Molecular and Cellular Biology University of Utrecht NL. (2) Unilever Research Laboratory Vlaardingen NL. (3)TNO Nutrition and Food Research Rijswijk NL.
Filamentous fungi are used as hosts for the commercial production of heterologous proteins because they have an enormous capacity for secretion. The production levels of many heterologous proteins are however rather low compared with the high levels which are obtained with homologous proteins such as glucoamylase. This study has been started to sort out the bottlenecks in the production of heterologous proteins and subsequently to identify the factors involved in these rate-limiting pathways. Therefore the effect of different pro- and pre- sequences and the presence of multiple copies of the gene on the production of heterologous proteins is studied. The cutinase protein from Fusarium solani pisi is used as a model for the production of heterologous proteins. A synthetic copy of the cutinase cDNA was constructed and expressed under the control of the endoxylanase II expression signals from A. awamori. Four different constructs were used to test the effect of different pre- and pro-sequences. A single copy of these expression cassettes was integrated at the pyrG locus of A. awamori. Shake flask induction experiments revealed that the pre-sequences used were equally efficient in the production of extracellular cutinase. The absence of a pro-sequence however resulted in a two-fold increase in extracellular cutinase. To study the effect of multi copy gene expression one of the constructs was integrated in multiple copies into the genome of A. awamori. There was no linear correlation between copy number and extracellular cutinase production but the amount of active enzyme produced correlated with the level of cutinase specific mRNA. These data and the fact that a relatively small amount of cutinase was found inside the cell suggest that there is no limitation in the secretion of this protein by A. awamori.
54. Plasma Membrane H+-ATPase in differentiation of Neurospora.
Tatiana A. Belozerskaya, A.N. Bach Institute of Biochemistry RAS., Moscow, Russia.
N. crassa hyphae were found to be differentiated in respect of the membrane potential, input resistance and electrical coupling between adjacent cells. Energetic cooperation via ionic fluxes through septal pores was revealed in 3-4 apical hyphal cells. The functioning of such cell ensembles appears to be under genetic control. Transport processes of plasma membranes of the cells composing fungal hyphae are influenced by blue light which also controls gene expression during differentiation of reproductive structures Light-induced membrane electrogenesis and photoinduced gene expression are probably controlled by a common photoregulating mechanism. Plasma membrane hyperpolarization in the illuminated hyphal cells appears to be energy-dependent and thus connected with the functioning of H+-ATPase. As in the case of gene expression, it is apparently mediated by cAMP. Except that membrane electrogenesis seems not to be a necessary connecting transduction link from the photoreceptor to genome. Synchronization of the reactions of individual hyphal segments showing a profound electrophysiological heterogeneity seems to be the functional signifigance of plasma membrane electrical responses to light.
55. Characterization of a class II type hydrophobin of Trichoderma reesei.
T. Nakeri-Setälä, N. Aro and M. Penttilä. VTT Biotechnology and Food Research, Finland.
We have earlier reported the isolation of T. reesei genes highly expressed on glucose-containing media. One of the genes isolated has been identified as encoding a novel hydrophobin, hfb1. It is a small hydrophobic protein of 97 amino acids with a single sequence and eight cysteine residues arranged in a conserved pattern. The hydrophobin of T. reesei shows strongest similarity to cryparin of Cryphonectria parasitica (59%) and cerato-ulmin of Ophiostoma ulmi (52%) both acting as wilt toxins causing pathogenicity of these fungi. It belongs to the class II type of hydrophobins (J. Wessels, pers. comm.). We have also detected a hydrophobin mRNA in T. harzianum used in biocontrol of plant pathogenic fungi by using the T. reesei hfb1 as a probe in Northern analysis. The HFB1 protein of T. reesei was isolated from culture medium by precipitation induced upon freezing and from mycelium based on its SDS-insolubility and further dissolution in TFA. The isolated proteins reacted with polyclonal antibodies obtained against the HFB1 protein expressed in E. coli and where confirmed as the product of the hfb1 gene by N-terminal sequencing.
56. A new role in pseudohyphal growth. An Aspergillus nidulans abaA homologue?
V. Gavrias and W.E. Timberlake, Myco Pharmaceuticals, One Kendall Square, Cambridge, MA 02139
A typical feature of many pathogenic fungi is their ability to interconvert between a yeast and filamentous growth pattern. A characteristic of such dimorphic nature is the production of morphologically distinct cell types through alterations in the polarities and patterns of cell divisions. Aspergillus nidulans and Saccharomyces cerevisiae are providing insights into the mechanisms controlling such morphogenesis in that as well as offering tractable genetic sytems they both undergo a yeast like filament interconversion. Using these two organisms as tools to study this biological phenomenon, we are investigating the possibility of analogous developmental pathways being shared by these apparently morphologically divergent organisms. Forced expression of the A. nidulans developmental transcription activator gene abaA in S. cerevisiae results in enhanced filamentous growth of a diploid strain, which suggested the existence of a similar developmental pathway in yeast as well as the presence of an AbaA homologous protein. To further investigate such protein we constructed null mutants TEC1, whose gene product has been suggested to share a DNA binding domain with AbaA, was thus an attractive candidate for such a protein. By disrupting this gene and investigating the ability of a homozygous tec1/tec1 strain to form pseudohyphae we determined that TEC1 is necessary for such morphology. This would suggest that a new pathway responsible for this growth pattern has been uncovered in S. cerevisiae, and it is anticipated that this may be the analogous pathway involved in the A. nidulans developmental process.
57. Drug efflux transporters, determinants of fungal pentamidine susceptibility.
Yi Li, Gabriele Ludewig, and Chuck Staben. University of Kentucky.
Pentamidine inhibits the growth of many taxonomically diverse fungi during respiratory growth. Inhibition occurs only at much higher pentamidine concentrations during fermentative growth. The accumulation of pentamidine by fungi appeared to determine this pattern of inhibition. The facultatively aerobic fungi that we tested, Saccharomyces cerevisiae and Candida albicans, accumulated pentamidine only when grown on nonfermentable carbon sources. An obligate aerobe, Dipodiascus uninucleatus, accumulated pentamidine on all carbon sources. At least one fungus, Rhodotorula mucilaginosa, with a high resistance to pentamidine did not accumulate pentamidine from the medium. Two of the pentamidine resistant mutants of S. cerevisiae that we have isolated showed different patterns of pentamidine accumulation. One mutant (YLS4) failed to accumulate pentamidine, a second mutant (YLS2) accumulated normal amounts of pentamidine. The mutation responsible for resistance in mutant YL54 was in the SGE1 gene. SGE1 encodes a member of the bacterial multidrug resistance efflux protein family. Disruption of the SCE1 gene increased the cell's susceptibility to pentamidine. These results indicated that drug transport is a major determinant of pentamidine susceptibility in fungi and that specific efflux transporters have a role in normal susceptibility as well as in resistance mechanisms.
58. Cryptococcus neoformans can use inositol as an energy source.
Susan E. Ramos, Merceditas Castillo, and Lisa S. Klig, Department of Biological Sciences, California State University, Long Beach, CA 90840
Cryptococcus neoformans is an encapsulated fungi that infects immunocompromised hosts. Unlike the systemic infections with most fungal pathogens, C. neoformans infections usually localize to the central nervous system (CNS), a region of high inositol concentration. One distinguishing trait of C. neoformans is its capacity to use inositol, a key cellular metabolite, as a sole carbon source. Most other microbes cannot use inositol as a source of energy for growth. The first step of inositol catabolism, in higher eukaryotes, is the conversion of inositol to glucuronic acid. This reaction is catalyzed by inositol oxygenase. Inositol oxygenase activity has been demonstrated in crude lysates of C. neoformans. Moreover, this enzyme has been shown to be regulated in response to the carbon source in the growth media. Growth of C. neoformans in media with inositol instead of glucose or glucuronic acid as the carbon source results in higher specific activity of inositol oxygenase. In addition, inositol oxygenase specific activity appears to increase immediately after a shift of carbon source from glucose to inositol. Furthermore, the activity of this enzyme appears to decrease as cells enter stationary phase. To dissect inositol metabolism in C. neoformans, mutant strains were isolated that grow when glucose is provided as the carbon source but do not grow in media with inositol as the carbon source. Studies of the inositol catabolic pathway in C. neoformans will contribute to understanding overall metabolism of this organism, and may provide insight into the biochemical basis of its localization and pathogenicity.
59. Binding of fibrinogen to rodletless Aspergillus nidulans is impaired.
R.G. Washburn, M. Parta, Y. Chang, N.C. Julian, and K.J. Kwon-Chung. Dept of Medicine, Bowman Gray School of Medicine, Winston-Salem, NC, and National Institute of Allergy and Infectious Diseases, Bethesda, MD.
Human fibrinogen is known to bind to A. fumigatus conidia, and there is evidence for specific fibrinogen receptors on their surfaces. We wished to study the effect of an intact hydrophobin rodlet layer on fibrinogen binding to A. nidulans conidia. We therefore compared quantitative binding of l25I labeled fibrinogen to the following isolates: FGSC 26 (wild-type, intact hydrophobin rodlets), RMS025 (rodletless), and Tx 5 (Rod A+, RMS025 transformant). Incubation mixtures contained 2 x 10(7) conidia and l25I-fibrinogen (100,000 cpm). Binding results were as follows:
A. nidulans Isolate l25I-fibrinogen bound, cpm (Mean +/- SEM. n=3) FGSC 26 6,327 +/- 291 RMS025 1,643 +/- 32 Tx5 3,562 +/- 82Thus bindinq of 125I-fibrinogen to the rodletless strain, RMS025, was significantly reduced compared to FGSC 26 and Tx 5 (p < 0.05, Students paired t-test). These data suggest that an intact hydrophobin rodlet layer is required for optimal fibrinogen binding.
60. Cloning and characterization of Aspergillus nidulans genes coding for homocysteine synthesizing enzymes.
Marzena Sienko, Jacek Topczewski, Andrzej Paszewski, Polish Academy of Science, Institute of Biochemistry and Biophysics, Department of Genetics, ul. Pawinkiego 5a, 02-106 Warsaw, Poland.
In A. nidulans there are two enzymes which synthesise homocysteine: cystathionine beta-lyase and homocysteine synthase coded for by metG and cysD genes, respectively. The first enzyme belongs to the main pathway of homocysteine synthesis while the second one represents the alternative pathway of methionine and cysteine synthesis, which seems to occur only in fungi and yeast. Both genes have been cloned by complementation of appropriate mutants using a plasmid gene library and have been sequenced. The metG gene, the first eukaryotic cystathionine beta-lyase gene cloned, contains an intron sequence at its 5' end and codes for a polypeptyde of 407 amino acids. In the cysD gene, five introns were found and their positions were identified through the analysis of appropriate cDNA fragments synthesized by PCR. A 437 amino acid sequence of homocysteine synthase deduced from DNA sequence shows 63% similarity with its S. cerevisiae counterpart. Both genes show homology to the family of genes coding for transsulfuration enzymes, such as human, rat, and yeast cystathionine g-lyases, yeast homocysteine synthase, and both the cystathionine gamma-synthase and cystathionine beta-lyase from E. coli.
61. Septation and nuclear distribution in Aspergillus nidulans.
Tom Wolkow and John E. Hamer, Purdue University, W. Lafayette, IN 47907
The intercalary compartments of Aspergillus nidulans hyphae maintain a uniform size of approximately 395 um (Trinci and Morris, 1979). This size is determined by the positioning of septa during cytokinesis. In many organisms the position of the dividing nucleus determines the location of cytokinesis. Thus we investigated the role of nuclear positioning in determining septal placement in A. nidulans. Nuclear positioning was altered by growing three temperature sensitive nuclear distribution mutants, nudA, nudC and nudF, at semi-permissive temperature (39 C). Nud+ strains maintained an evenly spaced distribution of nuclei when grown at this temperature. In contrast, nuclei in the nud- strains clustered throughout the length of the hyphae. The nud- strains also displayed aberrant septal positioning. Although each nud- strain maintained an overall average intercalary compartment length close to 395 um, individual lengths varied considerably from this average, causing nud- intercalary lengths to differ significantly from nud+ intercalary lengths. An experiment following septal kinetics demonstrated that the mechanics of septation is normal in nud strains, suggesting that none of the nud gene products are directly involved in septation. Examination of double mutants made between nud- and sep- (septation deficient temperature sensitive mutants) revealed nuclear clumping in the absence of septa at 39 C. We conclude that nuclear distribution affects the process of septation in A. nidulans.
62. Why search for integrin homologs in fungi?
Susan G. W. Kaminskyj and John E. Hamer, Purdue University, West Lafayette, IN 47907
Fungi are extolled as model organisms for studying the structure and function of metazoan eukaryotes, such as ourselves. As well, features of animal cells have been used successfully to predict homologous structures and/or functions in fungi. Cells in both systems appear to be regulated by protein components in the substrate to which the cell is attached: the cell wall and extracellular matrix, respectively. Consistent with this, nonstructural fungal cell wall components vary with developmental stage. Cytoskeleton-substrate attachment and signalling proteins, such as integrins, are well characterized in animal cells where integrin-matrix protein docking regulates cell form and proliferation. Integrin and matrix protein homologs in fungi and oomycetes have been identified by immunological crossreactivity and by inhibiting normal cell function with exogenous peptides containing integrin-matrix protein binding sequences. Integrins have recently been implicated in host cell attachment and pathogenesis by Candida, so these proteins' role in fungal biology is also medically relevant. However, our present understanding of the roles and regulation of integrins in fungi is rudimentary and wants first for their cloning and molecular characterization. Aspergillus nidulans contains a 156 kDa protein which crossreacts with antiserum to a conserved cytoplasmic beta-integrin domain on western blots. Using immunofluorescence, this antiserum stains peripheral patches. We are using this crossreactivity to attempt cloning a fungal integrin homolog from cDNA expression libraries.
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