Margaret E. Katz, Martin McLoon, Stephen Burrows and Brian F. Cheetham, University of New England, N.S.W., Australia.
We have determined the DNA sequence of a 1.2 kb fragment from the alkaline protease (Alp) gene of two variant A. fumigatus strains, NSW3 and FRR1266. NSW3 was isolated from an ostrich in a project aimed at developing a method to diagnose aspergillosis in farmed ostriches. NSW3 failed to yield a product in our initial PCR detection method. Southern blot analysis suggested that NSW3 differed from the majority of ostrich isolates of A. fumigatus. FRR1266 is an environmental isolate which, like NSW3, originated in New South Wales, Australia. In a survey of A. fumigatus strains from 6 continents, FRR1266 was reported to differ significantly from the other strains in isoenzyme, RAPD and RFLP pattern (Rinyu et al.(1995) J. Clin. Microbiol. 33:2567-2575). The DNA sequence of the NSW3 and FRR1266 Alp gene was compared with the sequences of a second ostrich isolate (QLD1) and the published Alp gene sequences of 3 human A. fumigatus isolates. The results showed that the 3 human isolates and QLD1 were virtually identical but NSW3 and FRR1266 differ from the others by >5% of the nucleotides that were analysed. The two variant strains differ but are more closely related to each other than to "standard" strains of A. fumigatus. Partial characterisation of the 18S rRNA genes of NSW3 and FR1266 revealed no differences between the variants and a standard strain. Differences in the restriction enzyme cutting sites within the 1.2 kb Alp gene fragment can be used in RFLP analysis of environmental and clinical isolates of A. fumigatus to determine the distribution of the genetic variants.
Regulation of extracellular protease production in Aspergillus nidulans
Margaret E. Katz, Patricia vanKuyk, Pam Flynn and Brian F. Cheetham, University of New England, N.S.W., Australia.
Work by ourselves and others suggests that the extracellular proteases of
Aspergillus are regulated in response to five environmental signals:
carbon, nitrogen and sulphur metabolite repression, pH control and induction by
protein. We have studied the regulation of extracellular proteases in A.
nidulans by identifying putative regulatory genes through genetic analysis
and by characterisation of protease structural genes. Using several different
strategies, we have isolated mutants with altered protease enzyme levels. Two of
the 6 genes identified by genetic analysis have been cloned. Two classes of
xprG mutants have been isolated-a mutant with increased levels of
extracellular protease and mutants with a protease-deficient phenotype.
The xprG gene is very tightly linked to (or is an allele of) the
sarB gene. Mutations in sarB suppress a gain-of-function
mutation in the areA nitrogen control gene. We have isolated and
characterised two protease structural genes-prtA, a gene encoding a
serine (alkaline) protease and prtB, a gene encoding an aspartic (acid)
protease. An alkaline protease-deficient strain, constructed by gene replacement,
has been used to show that the prtA gene product is the most abundant
protease under a wide range of pH conditions. A 60 bp sequence in the 5' region
of the A. nidulans prtA gene is highly conserved in A. oryzae, A.
flavus and A. fumigatus. This high degree of sequence
similarity suggests that this region is important in the regulation of the
alkaline protease genes. Gel mobility shift assays have been used to identify
proteins that bind to this conserved sequence. Experiments to isolate the
genes that encode these DNA-binding proteins, using southwestern screening of
cDNA expression libraries, are in progress.
48. Characterisation of the cAMP-dependent protein kinase catalytic subunit gene from the fungus Aspergillus niger.
Mojca Bencinal,2, H. Panneman2, G.J. Rujiter2, M. Legisal, J. Visser2. 1National Institute of Chemistry, Ljubljana, Slovenia; 2Wageningen Agricultural University, Section Molecular Genetics of Industrial Microorganisms, Wageningen, The Netherlands.
The pkaC gene encoding the catalytic subunit of cAMPdependent protein kinase (PKA-C) was isolated from the filamentous fungus Aspergillus niger. An open reading frame of 1440 bp, interrupted by three short introns, encodes a polypeptide of 480 amino acids with calculated molecular mass of 53,813 Da.
The deduced catalytic core of PKA-C of A. niger shows extensive homology with the PKAC isolated from all other eukaryotes. The cAMP-dependent protein kinase catalytic subunit from A. niger has a 126 amino acids extension at the N-terminus compared to the PKA-C of higher eukaryotes that, except for the first 15 amino acids which are homologous to the Magnaporthe grisea PKA-C, shows no significant similarity to the N-terminus extension of PKA-C of other lower eukaryotes.
The cloned pkaC was used for transformiation of A. niger leading to increased levels of pkaC mRNA and PKA-C activity. Transformants overexpressing pkaC are phenotypically different with respect to gowth, showing a more compact colony morphology, accompanied by more dense sporulation. A number of transformants also showed a strongly reduced or complete absence of sporulation. The overexpression of pkaC in A. niger did not severely affect fungal viability but resulted in unstable transformants.
49. Fruitbody formation of Aspergillus nidulans tryptophan auxotrophic mutants.
Sabine E. Eckert, Christoph Wanke and Gerhard Braus, Georg-August-University Gottingen, Department of Microbiology Grisebachstrasse 8, D-37077 Gottingen, Germany
The filamentous fungus Aspergillus nidulans is a simple model organism for development and cellular differentiation. The molecular basis of its fruit body formation and the production of ascospores is largely unknown. We have started an analysis of the interplay between sexual development and metabolic pathways. We tested tryptophan requiring mutants of Aspergillus nidulans (Ref. 1, 2) for fruit body formation. The tryptophan mutants trpA, trpB, trpC and trpD are deficient in the structural genes for the biosynthetic enzymes of each step in the formation of tryptophan from chorismate. In addition, all mutant strains are unable to form fruit bodies on standard minimal and complete medium (Ref. 3).
Here we report that the strains carrying the mutations in the trpA, trpC and trpD locus can form cleistothecia on medium supplemented with indole, the strain trpA also on medium containing anthranilate, and all strains including the trpB strain on medium with 3-indolylacetic acid (heteroauxin). Therefore we conclude that the tryptophan biosynthetic pathway and the development of fruit bodies in Aspergillus nidulans are connected either by an intermediate or a derivative.
trpA, trpD trpD, trpC, trpC trpB
Chorismate ------ > anthranilate --- > --- > ---> [indole] -- > tryptophan
References:
1. Roberts, C. F. (1967): Genetics 55, 233 - 239
2. Hutter, R. and demoss, J.A. (1967): Genetics 55, 241- 247
3. Timberlake, W. E. (1990): Annual Review of Genetics 2, 3 - 24
50. Suppressors of a temperature-sensitive allele of nimTcdc25 in Aspergillus nidulans.
Dorothy B. Engle, Xavier University
In order to identify new genes involved in cell cycle control, we are making extragenic suppressors that compensate for a mutation in a known cell cycle gene. The nimT gene encodes a homolog of fission yeast cdc25 phosphatase. The phosphatase is required for entry into mitosis; it participates in the activation of the cyclin-dependent kinase (NIMX in Aspergillus) that triggers mitotic events. Extragenic, or second-site, suppressors of known mutations frequently encode proteins that interact with the original known protein.
The mutant allele nimT23 causes a temperature-sensitive arrest in G2 phase. We mutagenized a nimT23 strain, selected for survivors at restrictive Temperature (44 C), and distinguished revertants from extragenic suppressors by crossing to wild type. Chromosome mapping by diploid breakdown indicates that we have uncovered at least three different genes; they are located on chromosomes I, II and VI. The suppressor genes on chromosome VI are distinct from the bimE gene, another cell cycle gene located on VI. Further characterization of the suppressors, including dominance testing, is underway.
(Supported by NIH AREA grant GM49480-OlAl)
51. The construction of vectors and strains for efficient gene disruptions in Aspergillus fumigatus.
V. Gavrias, K. Thiede, N. Iartchouk, J.-A. Saxton, C. Hitchcock , W.E. Timberlake and Y. Koltin. ChemGenics Pharmaceuticals Inc., One Kendall Square, Cambridge , MA 01239. Pfizer Pharmaceuticals, Central Research Division, Pfizer Limited, Sandwich, Kent CT13 9NJ, UK.
The increasing medical significance of Aspergillus fumigatus as an opportunistic pathogen illustrates
the need for the development of genetic tools applicable to this organism. To clone A. fumigatus
genes coding for a variety of auxotrophic markers, we transformed Saccharomyces cerevisiae with
an A. fumigatus cDNA library constructed in a yeast expression vector. By complementation of S.
cerevisiae auxotrophies we isolated cDNA clones coding for HIS3, ADE2 and LEU2 A. fumigatus
homologs. Sequence analysis of these clones indicates a high degree of similarity between the
analogous proteins. Isolation of genomic clones by hybridization enables the construction of
auxotrophic strains for efficient gene disruptions in A. fumigatus as well as making this organism
more amenable to genetic manipulations.
52. Cloning of a gene that complements a camptothecin-sensitive mutant of Aspergillus nidulans.
Gustavo H. Goldman, G.C.M. Bruschi, C. C. de Souza & M.H.S. Goldman, FCFRP and *FORP, Universidade de Sao Paulo, Brasil.
Topoisomerases are enzymes that modify and regulate the topological state of DNA. Camptothecin is an anti-topoisomerase I drug. The filamentous fungus A. nidulans can grow on a high concentration of this drug but its topoisomerase-I is camptothecin-sensitive, Cellular sensitivity to the lethal action of this drug may be influenced by many factors that affect cleavage complex formation, its processing into permanent damage, or the cellular response to the permanent damage. We are interested in identifying additional factors of cellular sensitivity to camptothecin others than topoisomerase I. Towards this end, we decided to isolate camptothecin-sensitive mutants of the filamentous fungus A. nidulans. In one of these mutants, sca299 (Sensitivity to camptothecin) hypersensitivity to camptothecin is cosegregating with sensitivity to EMS, MMS, actinomycin, and 4-NQO. Topoisomerase-I assays indicate that its topoisomerase-I activity is comparable to that of the wild type. We have cloned by DNA-mediated transformation the gene that complements the mutant sca299. Additional work will focus on further genetic characterization of this gene.
Financial support by FAPESP, CNPq-Brazfl and ICGEB-UNIDO
53. Genetic characterization of cycloheximide-sensitive mutants of Aspergillus nidulans.
Gustavo H. Goldman, C. C. de Souza, M. Hiraishi, C. H. Pellizzon & M. H. S. Goldman*, FCFRP and *FOR Preto, Universidade de Sao Paulo, Brasil.
During the last decades, the incidence of fungal infections has dramatically increased. A. nidulans is a non-pathogenic fungus with a powerful genetic system that provides an excellent model system for studying different aspects of drug resistance in filamentous fungi. As a preliminary step to characterize genes that confer pleiotropic drug resistance in Aspergillus, we decided to isolate cycloheximide-sensitive mutants of A. nidulans. The rationale for this approach is to identify genes whose products are important for drug resistance by analyzing mutations that alter the resistance/sensitivity status of the cell. Fifteen cycloheximide-sensitive scy mutants of A. nidulans were isolated and genetically characterized. Some of the scy mutants showed a complex phenotype of resistance or sensitivity to different drugs and/or stress conditions. Genetic analysis of six of them has defined six linkage groups designated pdr (pleiotropic drug resistance)A-F. One of these mutants, pdrD, shows cosegregation to cycloheximide, hygromycin, osmotic, low pH, and t-butyl hydrogen peroxidesensitivity. Fluorescent microscopy and transmission electron microscopy indicate that the vacuolar system of this mutant is fragmented. Additional work will focus on the cloning and characterization of the gene that complements this mutant.
Financial support by FAPESP, CNPq-Brazil and ICGEB-UNIDO
54. Fungal Gene Disruption by Transposon Mutagenesis in E. coli.
L. Hamer & J.E. Hamer, Dept. Biology, Purdue University, West Lafayette IN 47907.
Application of gene knock-out technology in filamentous fungi by homologous recombination
generally requires initial construction of a disruption vector. This vector is composed of parts of the
gene flanking an appropriate marker gene. We have constructed 2 tn5-containing plasmids, pLH1
and pLH3, specialized towards mutagenesis of fungal genes from the filamentous fungi Aspergillus
nidulans and Magnaporthe grisea. pLHl contains the argB marker gene, and pLH3 contains the Hyg
marker gene, functional in A. nidulans and M. grisea, respectively. We show that the sepA gene from
A. nidulans cloned in E. coli can be readily mutagenized by pLH1. We have made multiple disruption
derivatives of the sepA clone with the concomitant introduction of the argB gene. Using a 3.8 kb
pBR322 derived backbone, hops into the ~ 6 kb gene were observed at a frequency of 0.25. The
exact position of the disruption can easily be determined, due to the presence of SP6 and T7 priming
sites at the ends of the intervening tn5/argB cassette. Because the disruption is confined by an
insertion, the entire sepA gene provides the targetting sequence for later in vivo disruption of the sepA
gene. In addition to speeding up the process of gene disruption, pLH1 and pLH3 are useful means
of integrating marker genes in the plasmid backbone.
55. sepA, a New Member of an Conserved Family of Genes Involved in Cytokinesis.
L. Hamer, S Harris*, K Sharpless*, J.E. Hamer. Dept. Biology, Purdue U, W Lafayette IN 47907; *Dept. Microbiol, U Conn Health Ctr, Farmington CT 06030.
Cytokinesis in Aspergillus nidulans is an actin-dependent process coordinated with mitosis. Ts-
mutations in A. nidulans sepA result in defects in both cytokinesis and polarized growth. At
restrictive temperature, sepA Ts- mutants form multi-nucleate hyphae, which fail to undergo cell
division and exhibit abnormal branching patterns. Temperature shift experiments suggest that the
sepA gene product acts late during cell division. sepA has been cloned, and shown to encompass the
previously described figA gene; thus figA is a truncated allele of sepA. Sequence analysis reveals that
sepA is a member of a conserved family of genes, whose products appear to function in actin-dependent processes. In particular, sepA shares significant blocks of homology with genes involved
in cytokinesis: S. cerevisiae BNI1, S. pombe CDC12, and D. melanogaster diaphanous. sepA deletion
mutants displays depolarized growth and delays in septation. Our results suggest that sepA may play
a role in mediating actin-polymerization, such as at the division site, and that sepA is required for
maintenance of cellular polarity.
56. A role for the NIMA kinase in linking spore polarization to cell cycle progression in Aspergillus nidulans.
Steven Harris, Department of Microbiology, University of Connecticut Health Center, Farmington, CT 06030-3205.
Aspergillus nidulans forms dormant conidia which contain a single nucleus arrested in G1 phase of the cell cycle. Nuclei re-enter the cell cycle during the process of condial germination. At the same time, the spore establishes a polarized axis of growth. Spore polarization was found to occur shortly after the completion of the first mitotic division in germinating conidia. Results described here suggest that two distinct mechanisms function to coordinate spore polarization with cell cycle progression.
Examination of the kinetics of polarization in a number of Ts mitotic mutants revealed that bimE mutants, which exhibit high NIMA kinase activity, fail to polarize. In addition, over-expression of the nimA gene also delays polarization in a dose-dependent manner. Finally, expression of a non-degradable version of the NIMA kinase completely inhibits polarization. These results have led to a model whereby NIMA kinase activity, which is required for entry into mitosis, also functions to inhibit spore polarization. Destruction of NIMA upon mitotic exit relieves the inhibition and allows polarization to proceed, thus linking it to the completion of the first nuclear division. Further experiments have shown that Ts mutants which fail to accumulate high levels of NIMA kinase must still progress through S phase before polarizing. Furthermore, Ts mutations which cause an irreversible arrest in S phase prevent polarization in a nimA-independent manner. These results suggest that an additional mechanism exists which prevents premature spore polarization during the first S phase in germinating conidia (when the NIMA kinase is normally inactive).
57. A novel zinc finger in the Aspergillus nidulans nimO gene is required for asexual development.
Steven W. James, Bryan A. Kraynack, Scott D. Wade, and Brett M. Forshey, Gettysburg College.
The nimO predicted protein of Aspergillus shares 29% identity with DBF4, a budding yeast G1/S
regulator that controls DNA synthesis through its association with origins of replication and the
G1/S-specific CDC7 kinase. nimO also appears to control DNA replication, because ts-lethal nimO18
mutants are unable to synthesize DNA, and override normal checkpoint controls by progressing into
mitosis with 1C DNA. nimO and DBF4 are most conserved in a C-terminal region containing a single,
novel Cys2-His2 zinc finger-like motif. In budding yeast this zinc finger is nonessential, as removal
of the C-terminal -200 amino acids only mildly slows growth. Similarly, alcA-driven expression of
a truncated nimO allele lacking the C-terminal 209 amino acids restored normal vegetative growth
to nimOI8 cells at restrictive temperature, with the striking exception that these strains were unable
to form conidia. The zinc finger appears specifically necessary for asexual development, because the
same aconidial phenotype was obtained by substituting one or both of the critical histidine residues
of the zinc finger with glutamine. The zinc finger mutants could initiate the developmental pathway
leading toward asexual reproduction, but aborted uniformly after producing conidiophores with
several metullae. These results suggest a dual role for nimO in controlling DNA synthesis and
asexual differentiation, and set the stage for biochemical and cell biological analyses of zinc finger
function.
58. Keeping in shape: Aspergillus uses hypA-E to regulate morphology.
S. Kaminskyj and J Hamer. Dept Biol Sci, Purdue U, W Lafayette IN 47907-1392.
Fungal hyphae use polarized wall deposition for two morphological processes: tip growth and septation (cytokinesis) which defines subapical cells. Septation is dispensable for vegetative growth in A. nidulans, but essential for conidiation. In A. nidulans and other systems, cytokinesis is triggered temporally and spatially by mitotic nuclei. However, A. nidulans cells typically contain three or more nuclei, so there must be a mechanism determining which mitotic nuclei trigger cytokinesis.
Conditional ts- mutations defining genes named hypA-E grow slowly and have short cells and abnormal hyphal morphologies. hypA-E are dispensable for nuclear cycle progression and conidiation: all strains produce macroscopic colonies and viable spores after 3d incubation at 42 C. Thus, whilst hypA-E dramatically affect multiple facets of A. nidulans morphology regulation, they are not essential for viability or asexual reproduction.
In A. nidulans, subapical cells have a reversible nuclear cycle arrest. This is relieved by branching or by loss of hypA-E function due to temperature upshift. The latter induces apolar enlargement of subapical cells followed by mitosis and insertion of additional septa. Thus, A. nidulans seems to use hypA-E dependent cell volume control as a component of nuclear cycle regulation in subapical cells. However, double mutant analysis showed that hypA did not interact directly with nimA, nimE, nimT, nimX, bimE.
A cosmid complementing the hypA defect in co-transformations was identified after genetic mapping
showed linkage to sepA and lysF on chromosome IR. The complemeting region was subcloned a 1.8
kb SacI fragment. Sequence analysis suggests that hypA is a novel gene, and that A. nidulans uses
previously unknown mechanisms to reaulate hyphal morphology and cytokinesis.
59. The gene product of hapC is a subunit of AnCP/AnCF, a CCAAT-binding protein in Aspergillus nidulans.
M. Kato, A. Aoyama, F. Naruse, Y. Tateyama, P. Papagiannopoulos1, M. A. Davis1, M. J. Hynes,1 T. Kobayashi, N. Tsukagoshi. Nagoya University, Nagoya 464-01,Japan. 1 University of Melbourne, Parkville, Victoria 3052, Australia.
CCAAT sequences in promoter regions of many fungal genes such as the taa (Taka-amylase A) and amdS (acetamidase) genes play important roles in the determination of expression levels. We have shown that an Aspergillus nidulans CCAAT-binding protein (factor), AnCP/AnCF recognizes CCAAT sequences in several genes in A. nidulans. The hapC gene, an A. nidulans counterpart of the yeast HAP3 gene, was isolated and used to obtain hapC disruptants. No CCAAT binding activity was detected in nuclear extracts from a hapC disruptant. Taken together with the high similarity of HapC to the HAP3 protein this result suggests that the hapC gene product is a subunit of AnCP/AnCF.
To examine whether or not HapC is a component of the AnCP/AnCF complex, a recombinant MalE-HapC fusion protein was produced in E. coli and purified. AnCP was denatured in the presence of MalE-HapC, renatured, and used for gel shift assays. The shift band corresponding to a taa promoter-AnCP complex disappeared and a new band with lower mobility was observed. When anti-MalE antiserum was added to the binding reaction, the band was supershifted. These results indicate that the MalE-HapC fusion protein was functionally incorporated into the AnCP/AnCF complex bound to the CCAAT containing sequence. This clearly demonstrates that the hapC gene encodes a subunit of AnCP/AnCF.