Posters II: Gene Expression/Genome Structure

Transposable elements are well analysed genetic traits in higher plants and animals. However, in filamentous fungi transposons were isolated only recently. Most of them appeared to be retroelements, i.e. employ a reverse transcriptase for propagation(1). Non-retroelement transposons are far less frequently described(2). This particular type of transposons has proven to be extremely useful for the development of transposon tagging systems, in both higher plant and animal systems(3). We have successfully attempted the isolation of a non-retroelement transposable element from the filamentous fungus Tolypocladium inflatum. As a result a 4100 bp repeated DNA element with 20 bp terminal inverted repeats and 8 bp target site duplications was identified. Hybridization to chromosomal restriction fragments indicates the element being present at about 20 different genomic sites. The transposon carries a large open reading frame. Its deduced amino acid sequence exhibits strong similarities to members of the eukaryotic Ac-like transposon family. The data presented prove that the repeated DNA which we named restless has properties of a transposable element. Currently the biological activity of restless is under investigation. (1) e.g.: Cambareri EB, Helber J, Kinsey JA (1994) Mol Gen Genet 242:658-665; (2) Daboussi M-J, Langin T, Brygoo Y (1992) Mol Gen Genet 232:12-16; (3) review: McDonald JF (1993) Curr Opin Genet Develop 3:855-864

2. Expression and targeted secretion of a mammalian thrombolytic protein using the gla-1 gene of Neurospora crassa

Expression vectors using the highly secreted glucoamylase-1 (gla-1) gene of Neurospora crassa were created for the targeted secretion of a mammalian thrombolytic protein (mTh) in N. crassa. The gla-1 ORF was fused in- frame to the mTh cDNA at various distances from the gla-1 translation initiation site. The expression vectors were characterized by the location of the fusion site in the gla-1 ORF. Construct 1) The fusion was created 37 amino acids into the gla-1 ORF to include the propeptide of the glucoamylase gene. Construct 2) Contained 100 amino acids of the gla-1 ORF. This construct included the gla-1 propeptide and the first/only intron of the glucoamylase gene. Construct 3) The mTh cDNA was fused at the final codon of the gla-1 gene, 625 amino acids from the translation initiation site. A kex-2 endopeptidase recognition site was inserted between the gla-1 ORF and the mammalian Thrombolytic cDNA to cleave the fusion protein prior to secretion. Here we compare the secreted product of the three different expression vectors.

3. Analysis of a polyketide gene cluster in Aspergillus nidulans

Sterigmatocystin, a carcinogenic polyketide, is the product of a lengthy biochemical pathway found in the Aspergillus spp., A. nidulans, A. flavus and A. parasiticus. Whereas the latter two species convert sterigmatocystin to aflatoxin in a two step process, A. nidulans makes sterigmatocystin as an end metabolite. We have identified a ~60 kb cluster of genes in A. nidulans whose products are involved in sterigmatocystin biosynthesis. This cluster contains at least 20 genes proposed to encode both enzymatic activities and a regulatory protein, AflR. At least 15 structural genes including a polyketide synthase, a and b subunits of a fatty acid synthase, several monooxygenases, dehyrogenases and reductases have been identified through sequence homologies and gene disruption studies. Transcripts of these genes are co-regulated with verA, a previously described ketoreductase necessary for sterigmatocystin biosynthesis. The regulatory gene, aflR, is located in the middle of the cluster and functionally conserved between A. nidulans, A. flavus and A. parasiticus.

4. Aflatoxin biosynthesis in Aspergillus parasiticus and A. sojae is regulated at transcription

Aflatoxin, the most potent naturally formed carcinogen, is a secondary metabolite of Aspergillus parasiticus and A. flavus. Two very closely related species, A. sojae and A. oryzae, are used in food fermentations and never produce aflatoxins. We previously established that many A. oryzae isolates lack at least one of the genes necessary for aflatoxin biosynthesis but that all A. sojae, A. parasiticus and A. flavus isolates tested had homology to these genes in Southern blot analysis. To determine if aflatoxin biosynthesis genes were being transcribed, we screened mRNAs from six A. sojae and A. parasiticus isolates by northern blot analysis using one regulatory gene (aflR) and six structural genes (pks, fas, nor, aad1, ver1 and omt1) involved in aflatoxin biosynthesis.

The A. parasiticus isolates that produced aflatoxin or were known to be blocked at the end of the pathway had homology to all of the genes tested. One A. parasiticus isolate that had lost its ability to produce aflatoxin in culture and one A. sojae isolate did not have homology with any of the genes tested. Two of the A. sojae isolates had regions of homology with the regulatory gene, and fas but did not have homology with any of the other structural genes. These results indicate that aflatoxin production is transcriptionally regulated in these two species.

5. Population genetic structure in Rhizobium leguminosarum biovar trifolii: a hierarchical analysis

To determine the relative importance of gene flow, founder effects, selection, and mode of reproduction in Rhizobia populations, fifteen allozyme loci were used to estimate diversity and analyze the population structure in a collection of 912 Rhizobium leguminosarum biovar trifolii isolates. Data sets, comparing different sampling levels (total sample, individual population samples, and plants within a population), geographic separation (northern populations vs. southern populations), and temporal separation (southern populations collected in 1992 vs. those collected in 1993) were analyzed. Total genetic diversity among all isolates is H=0.426, with 57.6% of the variation found within individual plants. Gene flow among plants within a population is low relative to gene flow among populations within a geographic or temporal grouping. Significant differences were observed in (i) allele frequencies among populations and among plants within populations, and (ii) the frequency distribution of the most widespread and the most abundant strains. Multilocus linkage disequilibrium was calculated and significant levels were observed in the total sample and in three of the eight populations.

6. Cloning and characterization of a gene encoding a putative RNA binding protein from Neurospora crassa

A series of cosmids located around pyr-4 on LGIIL were isolated in a chromosome walk from pyr-4. Northern analysis of a 19.0 kb region of one of the centromere distal cosmids identified a 3.0 kb mRNA which is relatively abundant in vegetatively grown mycelia. Four overlapping cDNAs corresponding to this message were isolated; the largest of these incomplete cDNAs was 1.8 kb. A database search using BLAST revealed significant homology between the cDNA and a broad class of RNA binding proteins. RNA binding proteins are a diverse set of important proteins including poly(A) binding protein and regulators of developmentally specific splicing. Results of one conventional RIP (repeat induced point mutation) mutagenesis experiment suggest this gene may be essential. Sequencing of a genomic clone is in progress.

7. Molecular analysis of the negative regulatory gene scon-2, a beta-transducin homolog in Neurospora crassa

Sulfur uptake and assimilation in Neurospora crassa is accomplished via a complex regulatory circuit encompassing a set of sulfur-related structural genes and a set of trans-acting regulatory genes. These sulfur regulatory genes include cys-3+, which encodes a bZIP (basic region-leucine zipper) transcriptional activator, and the negative regulatory sulfur controller gene scon-2+. The scon-2+ gene encodes a polypeptide of 650 amino acids (molecular weight 72.2 kD) belonging to the expanding b-transducin family of eukaryotic regulatory proteins. Specifically, SCON2 contains six repeated Gb-homologous domains spanning the C-terminal half of the protein. Additionally, SCON2 exhibits an amino-terminal domain that potentially defines a new subfamily of b-transducin homologs. Expression of the scon-2+ gene has been examined using Northern hybridization and gel mobility shift analysis. Northern blots of scon-2+ mRNA indicated reduced expression of scon-2+ in a cys-3 deletion strain. Gel mobility shift assays uncovered four CYS3 binding sites within the scon-2 promoter. Taken collectively, this data suggests the presence of a control loop within the N. crassa sulfur regulatory circuit involving CYS3 activation of scon-2+ expression.

8. Quantitation of Phanerochaete chrysosporium mRNAs in soil by reverse transcription polymerase chain reaction

Analysis of fungi in complex substrates has been hampered by inadequate experimental tools for assessing physiological activity and estimating biomass. We report a method for quantitative assessment of specific fungal mRNAs in soil. The method was applied to complex gene families of Phanerochaete chrysosporium, a white-rot fungus widely used in studies of organopollutant degradation. Among the genes implicated in pollutant degradation, two closely-related lignin peroxidase (LiP) transcripts were detected in soil. The pattern of LiP gene expression was unexpected; certain transcripts, abundant in defined cultures, were absent in soil cultures. Relative transcript levels of several closely related cellobiohydrolases genes and the b-tubulin gene were also measured. The approach is applicable other systems including the laccase transcripts of Trametes hirsutas. The method will aid in defining the role of specific genes in complex biological processes such as organopollutant degradation, in developing strategies for strain improvement, and in identifying specific fungi in environmental samples.

9. Comparative phylogenetic and functional analysis of frequency (frq) homologs: support for a transcriptional regulatory role

The putative amino acid sequence of the Neurospora crassa frq protein contains sequences suggesting it is a nuclear transcription factor. Using PCR, we have cloned frq homologs from other filamentous fungi including Chromocrea spinulosa, Leptosphaeria australiensis, Podospora anserina and four additional Neurospora species. Alignment of the Chromocrea and Leptosphaeria sequences with those previously published for Neurospora and Sordaria shows that the former are about 50% identical to the latter and to each other. There are both regions of near total divergence and highly conserved regions including a predicted helix-turn-helix structure that may act as a DNA-binding domain. Amino acids at positions altered in the frq mutants are conserved among all species. Sequences consistent with frq being a transcription factor are generally conserved; most predicted post-translational modification sites are not. Transformation of the Neurospora frq9 mutant with the Chromocrea homolog rescued the pigmentation defect of the mutant but not the circadian defect; transformation with the Leptosphaeria homolog failed to rescue either phenotype.

10. The ylo-1 gene of Neurospora crassa encodes a putative polypeptide with strong similarity to mammalian class 3 aldehyde dehydrogenases

We have completed DNA sequence analysis of a Neurospora 2.3 kilobase (kb) genomic fragment containing ylo- 1+ as well as of a 1.7 kb ylo-1 cDNA. Northen analysis indicates a single ylo-1 gene specific transcript of approximately 2.0 kb. ylo-1 transcript accumulation is not photoregulated in mycelia or during conidiation; transcript levels remain constant during macroconidiation suggesting constitutive expression. Comparison of the genomic and cDNA sequences identified a 1602 base pair open reading frame. The putative ylo-1 open reading frame encodes a putative polypeptide of 60 kilodaltons. A database search revealed similarities between Ylo-1 and members of the aldehyde dehydrogenase family. The ylo-1 gene product is thought to catalyze the conversion of torulene or g- carotene to neurosporaxanthin a C35 apo-carotenoid that is unique to the fungi. The highest degree of similarity was observed between Ylo-1 and class 3 mammalian aldehyde dehydrogenases. Two amino acid residues placed in the active site of class 1 aldehyde dehydrogenase from human liver, Cys-302 and Glu-268 are highly conserved in the ALDH family of enzymes. This Cys residue is the only Cys residue conserved in all known ALDH structures. Both residues are conserved in Ylo-1.

11. Regulation of expression of the albino-1 (al-1) and al-2 genes of Neurospora crassa during macroconidiation

The albino genes of Neurospora encode enzymes essential for carotenoid biosynthesis. The al-1 gene encodes a phytoene desaturase while the al-2 gene encodes a phytoene synthase. The levels of al-1 and al-2 transcripts change dramatically in response to light and development during the formation of the major Neurospora crassa asexual spore (macroconidia). al-1 and al-2 mRNAs accumulate in response to a developmental cue, a specific stage of conidiation, irrespective of lighting conditions. During conidiation light induces accumulation of al-1 and al-2 gene specific transcripts at each stage tested; the photoinduced increase in albino gene transcript levels was not observed in two Neurospora mutants, wc-1 and wc-2, that are defective in all physiological photoresponses. Mutations in the genes fluffyoid, fld, and fluffy, fl, block conidiation at distinct early stages, both mutations reduce photoindependent albino gene transcript accumulation under conditions that promote conidiation.

12. Development of species-specific probes complementary to the 18S rRNA sequence of the yeast-like fungus Aureobasidium pullulans

Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonizes leaf surface and has potential as a biocontrol agent of pathogens. In an effort to develop specific probes for A. pullulans, we isolated genomic DNA from 12 strains of A. pullulans and from 16 different fungi. A 578-bp region within the 18S rRNA gene was PCR- amplified and sequenced. We also PCR-amplified the full length 18S rRNA gene from all the fungi tested. Southern blot hybridizations were performed to test the specificity of different probes. Multiple alignments of the 578-bp region of the 18S rRNA gene among all the A. pullulans strains showed few nucleotide differences. Rodotorula rubra, a phylloplane yeast, had the most closely related sequence to A. pullulans. On the basis of a single nucleotide mismatch, a 17-mer was identified which differentiated the 12 A. pullulans strains from R. rubra. Another 17-mer probe hybridized to A. pullulans strains but not to the 16 other fungi tested. We also identified a 17-mer highly specific for Cladosporium herbarum. An additional 100 phylloplane fungal isolates are being tested by Southern blot hybridization. These probes have potential in monitoring and quantification of fungi in leaf surface and other microbial communities.

13. Cloning of genes palB and palF of Aspergillus nidulans

Mutations in genes palB and palF increase acid phosphatase staining and decrease alkaline staining when mutant strains are grown at pH6.5. On this basis, it is possible that these genes may be involved in external pH- regulated gene expression, thus becoming an important target to be characterized at the molecular level. Alleles palB7 and palF15 are recessive and strains palB7 and palF15 do not grow at alkaline pH nor in medium with b-glycerolphosphate as the sole source of phosphate. Therefore, we have cloned the palB and palF genes by complementation using a chromosome-specific cosmid library of Aspergillus nidulans. The palB gene was subcloned as a 3.5 kb fragment with full complementing activity and which recognized a 3.0 kb mRNA. Furthermore, this fragment complemented the palE11 mutation, supporting the genetic analysis which suggested that palB and palE are the same gene. The palF gene was subcloned as a 5.0 kb fragment that fully complemented the palF15 mutation. An ~4.0 kb mRNA was identified using a internal fragment from this subclone. Two step gene replacement experiments ruled out the possibility that we had cloned supressors for the mutations palB7 and palF15. Financial support: CNPq, FAPESP, CAPES, FINEP and BID-USP

14. Glucose repression of xylanase formation by Trichoderma reesei regulates xyn1 but not xyn2 gene expression and is mediated by the Cre1 gene product

The filamentous fungus Trichoderma reesei secretes two xylanases (XYNI and XYNII) into the medium; when grown on xylan as carbon source. No xylanase activity occurs in the supernatants of glucose cultures, whereas lactose cultures exhibit xylanase activity (lactose promotes carbon catabolite derepression). Highest levels are obtained on xylan and xylose. Northern blot analysis of xyn1-and xyn2-mRNA formation as well as the use of the E. coli hygromycin B phosphotransferase-encoding (hph) gene as a reporter system for the xyn1 and xyn2 promoters document that transcription of one of the two xylanase-encoding genes (xyn1) is regulated by carbon catabolite repression. The low constitutive transcription of xyn2 on glucose contrasts with the absence of XYNII in the culture filtrates, and suggests a further regulation of XYNII formation following transcription. The 5' upstream sequence of both genes show several nt-motifs homologous to the postulated 5'-SYGGRG-3' consensus for binding of the carbon catabolite repressor Cre1 (CreA). Using a protein fragment containing the zinc-finger domain of T. reesei Cre1, prepared by expression as a GST fusion protein in E. coli (Strauss et. al., manuscript in preparation), these sequences were demonstrated as being functional in vitro in gel retardation assay and methylation protection foot printing techniques. Cre1 was shown to bind to several motifs present in xyn1 and xyn2. However only the xyn1 promoter contains a consensus form of an inverted repeat. In vivo deletion of 4 central nt of this motif resulted in the expression of the xyn1-hph fusion on glucose at a level comparable to that observed under carbon catabolite derepressing conditions. The functionality of this fragment was strengthened further by inserting it into the xyn2 promoter which lacks this motif. This resulted in a strong repression of the constitutive expression of the xyn2-hph fusion. Neither the loss of carbon catabolite repression of xyn1 nor the gain of carbon catabolite repression of xyn2 affected the induction of transcription of both genes by xylose and xylan. Based on these results we postulate that the transcription of xyn1 is repressed by glucose via Cre1 but that induction by xylose bypasses this repression. We also postulate that in T. reesei the single consensus motif for Cre1 binding is not sufficient to mediate carbon catabolite repression in vivo.

15. Cloning and transformant analysis of the period-2 (prd-2) clock gene of Neurospora crassa

prd-2 is a recessive clock mutation that lengthens the period of the circadian conidiation rhythm from the wildtype value of 21.5 hours to about 25.5 hours at 25 C. Genetic mapping of prd-2 localized the gene to the right arm of linkage group V between lys-2 and am. A chromosome walk in the Volmer-Yanofsky cosmid library in the genetically determined region of prd-2 yielded a set of cosmids spanning about 150kb. These cosmids were tested in a transformation assay for the ability to complement prd-2 and restore wildtype rhythmicity. Some of the cosmids showed partial rescue of the mutant phenotype shortening the period by up to 2 hours in approximately 20% of the primary transformants. Analysis of homokaryotic microconidial isolates showed that the suppressed phenotype is stable and not an effect of heterokaryosis. Analysis of transformants from overlapping cosmids localized the suppressing DNA to a 4kb region.

16. Karyotype mapping of Aspergillus ficuum SRRC 265 acid phosphatase genes

Four Aspergillus ficuum (niger) SRRC 265 phosphate-repressible phosphatase genes have been cloned (aphA, phoA, phyA and phyB). Several of these enzymes are economically important because they can degrade phytic acid. All four enzymes are secreted at high levels when A. ficuum SRRC 265 is grown under conditions of limited phosphate. This suggests their genes may share a common regulatory system. The chromosomal location is unknown for any of these extracellular acid phosphatase genes. To determine if any of these acid phosphatase genes are clustered we have started to map their chromosomal location by CHEF gel electrophoresis and Southern analysis. Taxonomically A. ficuum and A. niger are very similar and A. ficuum is widely considered a synonym of A. niger. We have found that the electrophoretic karyotype of A. ficuum has the same eight linkage groups as A. niger. A series of A. niger tester strains with introduced chromosomal size variations was obtained to verify chromosome assignment of the acid phosphatase genes.

17. Manganese superoxide dismutase activity in Neurospora crassa

As is observed in almost all aerobic eucaryotes, the filamentous fungus Neurospora crassa possesses two different forms of superoxide dismutase, one that is mitochondrial and has manganese as a co-factor (MnSOD), and another that is cytosolic and has copper and zinc as co-factors (CuZnSOD). Superoxide dismutase scavenges the superoxide anion, O2-, which is both toxic and ubiquitously generated during aerobic metabolism. Our laboratory previously isolated and sequenced sod-1, the gene that encodes CuZnSOD, and constructed a strain bearing a null allele of sod-1. We recently isolated a gene that appears to encode mitochondrial MnSOD and designated this gene sod-2. Sequence analysis of sod-2 suggests the possibility that an alternate pathway of expression of this gene may result in a cytosolic MnSOD activity, in addition to mitochondrial activity. We are examining sod-2 transcript levels and MnSOD activity in sod-1 mutant strains, and in copper-starved wildtype strains, in an attempt to establish if MnSOD activity levels or subcellular locations are altered in response to the reduction or elimination of CuZnSOD activity. We are also attempting to construct a mutant strain of N. crassa that lacks MnSOD activity. This strain will be useful in evaluating the contribution of MnSOD activity to the ability of an obligate aerobe to tolerate oxidative stress, and in confirming the mitochondrial location of the gene product.

18. Phenotypic analysis of the Neurospora crassa mei-3 mutant

The N. crassa mei-3 gene appears to encode a homolog of the E. coli RecA protein, one function of which is recombinational DNA repair. The mei-3 mutant is sensitive to ultraviolet irradiation, other mutagens and histidine at 39oC. There is evidence that the mei-3 gene product is induced under conditions of superoxide-mediated stress. A portion of the present study derives from our efforts to construct N. crassa strains carrying mutant alleles for both mei-3 and sod-1. The sod-1 gene encodes CuZn superoxide dismutase, a scavenger of toxic superoxide radicals. The sod-1 mutant exhibits a high spontaneous mutation rate, presumably as a result of increased superoxide-mediated stress. The mei-3 sod-1 double mutant was constructed to determine whether mei-3 has a role in either the repair of oxidation damaged DNA or the exacerbation of superoxide-mediated mutagenesis. During the construction of the mei-3 sod-1 strain, which requires a crossover on linkage group IL, we isolated progeny that exhibited extreme temperature sensitivity, even in the absence of mutagens. We are currently investigating the molecular basis of some of the known mei-3 mutations and the nature of temperature sensitivity. Analysis of crosses between mei-3 mutants and strains containing conventional markers on linkage group I will determine if the temperature sensitive phenotype is tightly linked to mei-3 or other linkage-group I genes. Transformation experiments are being conducted to ascertain if the wild type mei-3 gene can rescue the temperature sensitive phenotype. We also intend to sequence several mutant mei-3 alleles to elucidate the molecular basis of the mei-3 phenotype. Finally, we are attempting to disrupt the mei-3 gene using the RIP (repeat-induced point mutation) process in order to determine if (a) the known mei-3 phenotype can be reproduced and (b) if mei-3 null mutants are viable.

19. Altering fatty acid composition in Neurospora crassa

The fungal and plant kingdoms synthesize a large diversity of fatty acids. The fatty acid composition of membrane and storage lipids largely determines their properties. Neurospora crassa has many advantages for studying the biosynthesis of fatty acids, including the availability both of an extensive collection of mutants in fatty acid and membrane lipid synthesis, and of vectors for the expression of Neurospora and foreign genes. We are utilizing Neurospora for studying effects of mutations (ufa, pfa) in the fatty acid desaturation pathway, effects of transformation with fatty acid desaturases, and the synthesis of lipids with unusual fatty acids, such as ricinoleate.

20. Cloning and characterization of the carboxypeptidase Y homologue from Aspergillus niger

Aspergillus niger is a filamentous fungus used industrially for the production of secreted proteins, and has the capacity to secrete proteins in the gram per liter range. As part of our efforts to begin understanding the high secretory capacity of A. niger, we have decided to clone genes coding for proteins that are known to be localized to specific organelles in the secretory pathway. To obtain a marker for the vacuole (lysosome), we have cloned the gene coding for carboxypeptidase Y (CPY). In Saccharomyces cerevisiae, it has been demonstrated that CPY is localized to the vacuole where it is processed to the mature form. Based on regions of homology found between the S. cerevisiae CPY and other carboxypeptidases, degenerate oligonucleotides were used to PCR amplify a genomic fragment from A. niger. The nucleotide sequence of a 600 bp amplification product was determined and shown to contain an open reading frame of 200 amino acids having ~69% identity to the S. cerevisiae CPY protein. The PCR product was used as probe to screen both genomic and cDNA libraries of A. niger. Both genomic and full length cDNA clones of the A. niger CPY homologue have been isolated and characterized at the molecular level.

21. Development of Fusarium graminearum as a novel host for heterologous protein expression

Fusarium graminearum has been utilized as a commercial human food source in the UK for nearly ten years (A.P.J.Trinci. Mycol. Res. 96:1-13). The apparent absense of both plant pathogenicity and toxin production, and the well studied fermentation characteristics of this strain made it a good candidate as a host for heterologous protein secretion. In preliminary studies, we observed that F. graminearum secretes a low background of extracellular proteins. This feature is desirable in an expression system since it facilitates product recovery. As a first step, a transformation system was developed using the amdS gene of A. nidulans. We next examined the capacity of the fungus to express a trypsin-like protease gene from a closely related Fusarium species. This enzyme is transcribed as a pre-pro enzyme which requires processing by a maturing enzyme for protease activity. Production of this protease in Aspergillus has been problematic due to inefficient processing and proteolytic degradation. A vector containing both the amdS gene and the protease gene was constructed and transformed into the fungus. The enzyme was expressed and processed correctly. The regulation of expression of the protease gene was discussed.

22. Characterization of albino mutants of Neurospora crassa

The albino genes of Neurospora encode enzymes essential for carotenoid biosynthesis. The al-1 gene encodes a phytoene desaturase while the al-2 gene encodes a phytoene synthase. The al-1 and al-2 genes are closely linked on the right arm of linkage group I of Neurospora. Hunts for mutants defective in carotenogenesis have identified over twenty albino mutants that map to the right arm of linkage group I. However, these mutants have not been identified as alleles of al-1 or al-2. We have used DNA mediated transformation complementation analysis to assign these alleles to locus. Phytoene desaturases (pds) are common to all carotenogenic organisms including all photosynthetic organisms. Primary sequence analysis of carotenoid desaturases has established at least two classes of enzymes. Bacterial/fungal pds catalyze the conversion of phytoene to neurosproene, or more commonly, lycopene. Plant pds desaturates phytoene through a single intermediate, phytofluene, to z-caroteneprotein. A second plant carotenoid desaturase, z-carotene desaturase (zds) acts on z-carotene yielding first neurosporene then lycopene. Protein homology plots suggest that the bacterial/fungal pds are more closely related to plant zds then the plant pds. We present sequence analysis of alleles of Al-1 the Neurospora pds.

23. Characterization of genes affecting dimorphism in Candida albicans

Candida albicans is an opportunistic human pathogen responsible for deep seated and systemic mycoses in susceptible individuals. Fungal infections have been on the rise in recent years and antifungal research has taken on renewed interest. Pathogenesis by C. albicans has been linked to the ability to switch between a budding and filamentous growth form. We are using several molecular techniques including differential display to identify genes involved in regulation of dimorphism in C. albicans. Differential display detects a significant number of mRNAs uniquely expressed in cultures within the first few hours of germ tube formation when compared to budding cells. These transcripts are being further characterized by sequence analysis and northern blots. Analysis of the genes for these mRNAs may reveal promotor motifs that co-regulate hyphae specific genes.

24. In vivo analysis of transcriptional regulation of the Aspergillus nidulans creA gene mediating glucose repression

The creA gene of A. nidulans encodes a wide domain regulatory protein mediating glucose repression of a multitude of enzymes responsible for utilisation of less favoured carbon sources. CreA is a DNA binding protein of the C2H2 Zn finger class which binds specifically to consensus target sites composed of 5'-SYGGRG 3'. Cis acting mutations in the consensus binding sites of target genes (e.g. prnd 22) or mutations in the CreA binding domain (e.g. creAd1) lead to glucose derepression. Complete deletion of creA, however, results in lethal phenotype, which suggests an additional function of the protein beyond carbon catabolite control. Little is known about the signals mediating carbon catabolite repression by CreA. To this end we have started an investigation on the regulation of creA gene transcription. Northern blot analysis of creA mRNA revealed a complex expression profile: addition of glucose to a carbon derepressed mycelium of A. nidulans results in a transient stimulation of creA transcript formation ("early stimulation"), followed by partial repression. This effect is not observed in a creAd1 mutant strain. This data are consistent with the presence of a perfect tandem repeat of the SYGGRG consensus sequence, which has recently been shown to be functional in vivo in the genes of the proline cluster and the ethanol regulon of A. nidulans. In vicinity of this CreA target, also a putative binding site for the positive acting wide domain regulatory protein AreA, a member of the "GATA factor" proteins, which mediates nitrogen metabolite repression, is found. That this motif might be functional in vivo is supported by the finding that "early" stimulation of glucose mediated transcription is prevented in the presence of ammonia. Additional evidence is given by the observation that the steady state level of creA transcript on proline is constant at all sample times (corresponding to the derepressed level on low fructose) in mycelia pregrown on nitrate but needs an adaption time for derepression of around 10 minutes when pregrown on ammonia. We have moreover identified a putative "cAMP Responsive Element" (CRE) within the 5' regulatory region of creA. The in vivo function of those motifs are currently under investigation. To prove the involvement of the proposed target sites for activator/repressor binding in vivo, we have applied the in vivo genomic footprinting method using ligation mediated PCR. Data from these experiments will be presented and discussed with respect to the interaction of signals regulating creA expression.

25. Cloning and characterization of the signal recognition particle 54 kDa protein homologue from Aspergillus

Aspergillus oryzae and Aspergillus niger secrete large amounts of protein in submerged cultures. Because of this characteristic, they have been used industrially to produce heterologous proteins of commercial interest. In order to begin understanding the high secretory capacity of these organisms, we are cloning homologues of known components in the yeast and eukaryotic secretory machinery. The gene coding for the signal recognition particle (SRP) 54 kDa subunit has been cloned from several organisms including the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Alignment of the cloned SRP54 homologues identifies areas of strong homology between the proteins. These areas were used to design oligonucleotide primer sets for use in PCR experiments in attempts to amplify the SRP54 homologues from A. oryzae and A. niger. The resulting amplification product from A. oryzae contained an open reading frame of 498 bp. The predicted amino acid sequence of the PCR product was found to be 71% homologous to the SRP54p from S. cerevisiae. This PCR product was subsequently used to retrieve the full length genomic and cDNA clones from A. niger libraries.

26. Regulation of the extracellular acid protease(s) of Aspergillus nidulans

The proteases of Aspergillus species have become an area of great interest, mainly due to the economic importance of the hydrolytic enzymes produced by filamentous fungi. Although early studies of A. nidulans confirmed the existence of enzymes from the neutral and alkaline classes of endo-proteases (1), the methods used were unsuitable for the detection of acid proteases, therefore it was unknown if proteases of this class were produced by A. nidulans. Using a heterologous probe generated from the A. niger pepA gene encoding an acid protease (2), we have isolated a structural acid protease gene (prtB) from a lambda library of genomic A. nidulans DNA. Preliminary sequence data shows that prtB is also similar to an A. oryzae acid protease of the same class (3). Enzyme assays, polyacrylamide gel electrophoresis, and RNA blots have been employed to study the regulation of the structural protease genes of A. nidulans, which are expressed in response to carbon, nitrogen, or sulphur nutrient- limiting conditions. Although the alkaline protease gene (prtA) has been shown to be expressed under acid pH conditions (pH 3.5), preliminary studies suggest that the acid protease(s) may not be expressed under neutral and alkaline conditions. (1) Cohen BL (1973) J. Gen. Microbiol. 77:521-528. (2) Berka RM et al (1990) Gene 86:153-162. (3) Gomi et al (1993) Biosci. Biotech. Biochem. 57(7):1095-1100.

27. Electroporation-based transformation of freshly harvested conidia of Neurospora crassa

This transformation method was optimized for yield and flexibility rather than efficiency. Conidia were harvested from 7- to 28-day old cultures. All cell manipulations were done in 1M sorbitol solution. After three washes by centrifugation, a suspension of 2.5X10(9) cells/ml was prepared. One to 5 ug of linearized plasmid DNA was added to 100 ul of conidial suspension. A 40 æl aliquot of the cell suspension was placed in the bottom of a 0.2 cm gap electroporation cuvette. An InvitroGen Electroporator II with a voltage gradient of 7.25 kV/cm and settings of 71 uF and 200 ohms gave good results. Transformation to histidine prototrophy (his-3) or hygromycin resistance was demonstrated in a variety of strains of N. crassa. Prototrophic transformants were selected by direct plating on minimal plates containing 1M sorbitol. A pour plate method was used to select for hygromycin resistance in which the bottom agar contained hygromycin and no sorbitol and the top agar contained no hygromycin and 1M sorbitol. An input of 10(8) cells routinely yielded 200 to 1000 transformed cells. Co-transformation with DNA constructed for expression of heterologous proteins occurred in up to 30% of selected transformants. Transformants have been found to be stable and capable of production of significant amounts of heterologous proteins in N. crassa.

28. New tools for gene cloning and chromosome assignment studies of cloned genes in Aspergillus niger

The development of an improved gene cloning strategy by complementation of mutant alleles in A. niger is described. The strategy is based on the use of a fungal autonomously replicating pyrG vector, pAB4-ARp1. With this vector, a 10-100 fold increase in transformation frequency was obtained compared to integrative vectors. With pAB4- arp1, also the transformation frequency of a cotransformed plasmid is increased. A. niger transformants containing pAB4-ARp1 are mitotically unstable, but cotransformed plasmids strictly cosegregated with the autonomously replicating vector, as a result of recombination between both vectors. The use of pAB4-ARp1 in gene cloning was demonstrated by the cloning of two linkage group (LG) VII specific A. niger genes (nicB, lysF) with an A. niger gene library and pAB4-arp1. Furthermore, a method is described for assignment of cloned genes to Aspergillus niger chromosomes/linkage groups. With the cloning of a LG VII specific A. niger gene, all of the 8 LGs could be assigned to a chromosomal band in the electrophoretic karyotype of A. niger. The electrophoretic karyotype reveals 5 distinct bands, of which 3 consist of equally sized chromosomes. Using a set of A. niger strains with introduced chromosomal size variation, unambiguous assignment of cloned genes using CHEF-Southern analysis was demonstrated. The availability of these tester strains obviates the need of isolating or constructing mutant strains for the purpose of chromosome assignment of cloned genes.

29. Carbon catabolite-mediated regulation of genes encoding NAD-specific glutamate dehydrogenase and Hsp80 of Neurospora

The genes encoding the NAD-specific glutamate dehydrogenase (gdh-1) and heat shock protein 80 (hspe-1) are controlled through a common carbon catabolite repression mechanism. In addition, they are individually subject to regulation by specific stimuli, i.e. heat shock (hspe-1) and the substrate inducer, glutamate (gdh-1). Expression of these two genes was monitored during growth of cells under (a) carbon source starvation, (b) optimal conditions for gdh-1 induction and (c) heat shock. Northern blot analysis using DNA fragments of cloned genes, and protein blot (Western) analysis with HSP80- and GDH-specific polyclonal IgG and GDH enzymatic activity measurements were conducted to assess gene expression. While hsp80 gene is expressed at high levels during growth under starvation conditions, it appeared to be down-regulated by the presence of glutamate. GDH is also expressed at a high level upon carbon source depletion but its expression is enhanced further by the presence of glutamate. Exposure to heat shock, subsequent to the derepression treatments, resulted in the cessation of gdh mRNA synthesis as well as degradation of pre-existing messages. The elevated expression of hsp80 may be attributable to an increased demand for molecular chaperoning activity under starvation conditions.

30. Evidence for trans-inactivation of aflatoxin biosynthesis gene expression in diploids of Aspergillus flavus

Aspergillus flavus produces the decaketide aflatoxin that is a potent carcinogen. Strain 649 does not produce aflatoxin, and diploids formed by parasexual crosses between strain 649 and aflatoxigenic strains do not produce aflatoxin, indicating the dominate nature of the mutated afl-1 locus in strain 649. In metabolite feeding experiments, the diploids did not convert three intermediates of the aflatoxin pathway. Northern analysis indicated the aflatoxin biosynthesis genes nor, ver1, and omt1 were not expressed in diploids grown in medium conducive for aflatoxin production; however, there was low level expression of the regulatory gene aflR. A large (>150 kb) region that includes a cluster of genes involved in aflatoxin biosynthesis was found deleted from the genome of strain 649. Pulsed-field gel electrophoresis of chromosomes from strain 649 and the aflatoxigenic strain 86 indicated a larger (6 Mb) chromosome in strain 649 than the apparent homologous (4.9 Mb) chromosome in strain 86. The larger sized chromosome in strain 649 suggests that a rearrangement may have occurred in addition to the deletion These data suggest that a trans-sensing mechanism in diploids is responsible for the dominant phenotype associated with the afl- 1 locus in strain 649.

31. The expression of porcine relaxin in Neurospora crassa

This study was to determine if cDNA encoding porcine relaxin inserted into the genome of Neurospora crassa will be transcribed, the mRNA translated, and the protein post-transcriptionally modified by host cells. Relaxin, a member of the insulin family, is involved in parturition, uterine accommodation and sperm motility. Constructs for expression of relaxin were made using combinations of promoter sequences (tubulin and mtr), signal sequences (relaxin and chymosin), and the authentic porcine relaxin sequence. Neurospora host cells were transformed with these constructs. Media from transformed cell lines was screened for the production of relaxin by immunoassays. Transformants producing anti-relaxin antibody positive material were tested by Southern analysis for multiple integration sites. The relaxin protein was verified by reverse-phase HPLC followed by immunoassays on pooled HPLC fractions. This protein appears to be produced in a mature form and secreted into the media at levels of up to 45 ug/liter.

32. The family of AMA elements in Aspergillus nidulans

The AMA1 element was first characterized as a genomic sequence from A. nidulans which promotes extrachromosomal maintenance of plasmids in this fungus. The sequence is a perfect quasipalindrome (foldback element) of at least 7 kb, and it is represented by a single copy per genome, as demonstrated by hybridization with the unique central spacer. This complete copy is located on chromosome IV but several genomic copies of single arms are located on different chromosomes, the pattern of chromosomal location being highly variable between wild isolates. Several clones bearing sequences homologous to AMA1 (AMA-elements) were isolated from a genomic library. They share up to 95% sequence homology and differ mostly by AT-GC transitions, which suggests that they may have evolved from AMA1 by methlyation-induced mutations. None of the clones so far characterized other than AMA1 is arranged as a foldback element. All members of the family enhance transformation efficiency and produce phenotypically unstable transformants. However, in all the transformants (except those generated by the complete AMA1 element) transforming DNAs are probably rearranged and multimerized. We discuss possible models for extrachomosomal maintenance of AMA-bearing vectors.

33. MILD R.I.P.: an ideal tool to obtain leaky mutants from essential genes in N. crassa

The al-3 gene codes for Geranylgeranyl Pyro phosphate Synthase (GGPPS), an enzyme involved in the biosynthesis of carotenoids and of diverse prenylated compounds. The only two al-3 mutants isolated so far have a leaky albino phenotype, i.e. their carotenoid content is reduced. We used the R.I.P. (repeat-induced point mutation) mechanism to obtain new mutants of the al-3 gene upon introduction of a 1765bp fragment of the gene. Using phenotypic selection based on the colour of the mycelium to screen R.I.P. products, we obtained a much lower frequency of al-3 mutants than expected (only 0.3%), all with a leaky phenotype ranging from light orange to light yellow. We analysed the nucleotide sequence of the endogenous a1-3 gene from five mutants obtained by R.I.P. following cloning of a PCR-amplified product. In all the mutants, R.I.P. had introduced very few mutations (from two to four). Studies carried out suggest that al-3 is an essential gene and therefore heavily mutagenized ascospores do not survive. The data presented here show that varying degrees of R.I.P. exist and therefore the R.I.P. mechanism may have an intrinsic ability to introduce a highly variable number of mutations. We suggest that the mild mutagenetic effect of R.I.P. on duplicated sequences may represent a useful in vivo method of mutagenesis, to obtain functional mutant alleles of essential genes in Neurospora crassa.

34. The impact of an antisense transcript (aflRas) on the expression of aflatoxin biosynthesis pathway genes and the pathway-specific regulatory gene aflR

Aflatoxins are toxic and carcinogenic polyketides produced by Aspergllus flavus and A. parasiticus, which contaminate many important food crops. Aflatoxin biosynthesis is regulated by the gene aflR, however, the mechanisms by which this gene regulates pathway genes and is itself regulated are currently unknown. During the cloning of aflR, cDNA clones were isolated which represented the complementary strand of the regulatory gene. To determine if an antisense transcript is produced in vivo and the impact of such transcripts on aflatoxin regulation, we will determine the temporal expression of these two genes. Total extractable RNA will be isolated from a time- course study of A. flavus cultures grown on aflatoxin inducing and non-inducing media. Transcripts representing aflR and (aflRas) will be identified by Northern analysis and reverse transcription-polymerase chain reactions. RT- PCR is a highly sensitive, strand-specific RNA detection assay. We will also use RT PCR to determine the expression of pathway genes. If aflR is regulated in part by aflRas, we would expect to see differential expression of these two genes and an impact on the expression of the aflatoxin biosynthesis pathway genes.

35. Cloning and characterization of TB3, a kinase-encoding gene from Colletotrichum trifolii expressed during hyphal elongation

Colletotrichum trifolii is a fungal pathogen which incites anthracnose of alfalfa. To initiate research on molecular communication in this fungus a kinase-encoding gene (TB3) and the corresponding cDNA were cloned and sequenced. The deduced amino acid sequence of TB3 showed high homology to a Neurospora crassa, serine/threonine protein kinase, cot-1, required for hyphal elongation. Importantly, the carboxyl terminal catalytic domains of TB3 and cot-l are highly conserved but the amino terminal regions are divergent. Southern hybridizations showed that TB3 is part of a gene family in Colletotrichum trifolii. Furthermore a TB3 homolog is present in a related fungus Colletotrichum gloeosprioides f. sp. aeschynomene. We have also developed a procedure for isolating large numbers of germinating conidia and appressoria of Colletotrichum trifolii. Northern analysis indicated that TB3 is expressed in several fungal cell types. TB3 expression was highest in germinating conidia and vegetative mycelia and lowest in conidiating mycelia. These data show that TB3 is functionally similar to cot-1 and is important for hyphal elongation.

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