Posters I: Gene Expression/Genome Structure

To improve activity of cytochrome P450 enzymes in Aspergillus niger we have cloned and overexpressed the cytochrome P450 reductase encoding gene (cprA) from A. niger. The gene was overexpressed in strains having multiple copies of two fungal cytochrome P450 genes. The highly substrate inducible A. niger bphA gene encodes a cytochrome P450 involved in the conversion of benzoate to 4-OH-benzoate, the P. italicum P45014dm gene encodes lanosterol 14(alpha) demethylase. Transformants having multiple copies of both the P450 gene and the cprA gene had highly increased enzymatic activities as compared to transformants which had an identical cytochrome P450 gene copy number but only the wildtype cprA gene. This clearly shows that cprA expression levels can be limiting in cytochrome P450 overexpressing filamentous fungi. Induction of expression of the cprA gene by a number of generally used important inducers of mammalian cytochrome P450s was studied. None of these well known inducers had an effect on the expression levels of the cprA gene. However, the use of benzoic acid, the inducer of the highly regulated cytochrome P450 bphA gene, resulted in a 2-4 fold induction of CPR activity. This results clearly indicate the presence of co-induction of the two components of this P450 enzyme system. At present we are investigating the DNA elements involved in this co-regulation event.

2. Cloning of the ornithine transaminase gene from Aspergillus nidulans

In Aspergillus nidulans arginine can serve as a source of proline. It depends on the presence and inducibility of arginine catabolic enzymes, arginase and OTAse, which are coded by agaA and otaA genes, respectively. Both agaA and otaA genes are subject to nitrogen and carbon catabolite repression. In A. nidulans these two kinds of general control are mediated by AreA and CreA gene products. The otaA gene has been cloned by transformation of the A. nidulans with cosmid gene library. The gene contains two putative introns and codes for a 453aa long protein. The protein shows a high degree of homology to other known ornithine transaminases. There is evidence that otaA gene is controlled at the transcriptional level.

3. Gene expression studies in Acremonium chrysogenum: promoter analysis using reporter gene systems

Acremonium chrysogenum (syn. Cephalosporium acremonium) is the most important industrial producer of the beta-lactam antibiotic cephalosporin C. The pcbAB/pcbC and cefEF/cefG genes, encoding key enzymes of the cephalosporin C biosynthetic pathway, are organized in clusters in this fungal genome [1]. A detailed promoter analysis is required to understand the molecular mechanisms controlling cephalosporin C gene expression. The relative promoter strength of the above mentioned genes was initially evaluated by fusing the intergenic regions with reporter genes. We have developed a system that allows the simultaneous detection of promoter strength from two divergently orientated genes in Acremonium chrysogenum. This system is based on transcriptional fusions of pcbAB/pcbC and cefEF/cefG intergenic regions with the bacterial reporter genes lacZ and gusA. Quantitative tests of reporter gene expression revealed that the pcbC promoter expression is significantly stronger than the pcbAB promoter expression [2]. For this reason the pcbC promoter was chosen for a detailed deletion analysis. Similarly, the cefEF/cefG promoter region was also investigated in expression studies using chimeric reporter gene constructs. In addition, the reporter gene system described above is suitable for the investigation of unknown promoter sequences and we have developed a rapid test system for the detection of functional DNA sequences mediating promoter activity. [l] Smith DJ, Burnham MKR, Bull JH, Hodgson JE, Ward JM, Browne P, Brown J, Barton B, Earl AJ, Turner G (1990) EMBO J 9:741-747 and [2] Menne S, Walz M, Kuck U (1994) Appl Microbiol Biotechnol (in press).

4. Inviability among Aspergillus nidulans transformants containing multiple copies of the selectable marker pyr-4

Transformation of a haploid Aspergillus nidulans pyrG auxotrophic strain (1-85) with a vector containing the heterologous Neurospora crassa pyr4 gene (pGM32) resulted in strong growing transformants that could be classified into two groups based on morphology. The minority were morphologically very similar to the parental strain, easily purified and mitotically stable. The majority (10 times more frequent) appeared to be mitotically unstable and were subsequently shown to be heterokaryons that could not be resolved into homokaryons. The ratio of the two types of transformants depended on several parameters such as the protoplast preparation, addition of spermidine to the transformation mix, the conformation of the transforming vector and the concentration of DNA in the transformation mix. Genetic analysis of a random sample of both types of transformants indicated the difference was due to the number of copies of the integrated vector. The heterokaryons were shown to have a high copy number in a small proportion of the nuclei while the homokaryons had a low copy number in all nuclei. This suggests that multiple copies of pyr-4 are lethal. These results were obtained as part of a study investigating the effect of various parameters on the frequency of gene targeting in filamentous fungi using Aspergillus nidulans as a model organism. The well characterised loci of niaD and amdS were targeted using the pGM32 vector containing an internal fragment of the targeted gene. Disruption could be screened phenotypically by loss of function. Parameters investigated include (a) length of homologous DNA in the disruption cassette and (b) transcriptional status (on/off) of the targeted gene.

5. Disruption of hapC, the Aspergillus nidulans homologue of the Saccharomyces cerevisiae HAP3 gene, is lethal

DNA binding factors that recognise the core sequence CCAAT have been found in many eukaryotic systems and regulate a wide range of processes, including respiration and immunoglobulin production. The 5' regulatory region of the amdS gene of Aspergillus nidulans contains a CCAAT sequence which is required for setting the basal level of amdS transcription. Mobility shift studies have identified a factor in A. nidulans nuclear extracts which binds to this CCAAT sequence. The gene(s) encoding this factor have yet to be identified. In Saccharomyces cerevisiae the HAP3 gene encodes one component of a multisubunit complex which binds CCAAT sequences. Genes with homology to HAP3 have been isolated from a number of species. A search of the Genbank database has found an A. nidulans sequence with significant homology to the HAP3 gene adjacent to the previously cloned regulatory gene amdR. Sequencing of the remainder of this region has revealed a gene with extensive homology to HAP3. This gene has been named hapC. hapC cDNA clones have been isolated and sequencing has revealed three introns in the coding region of hapC and one in the 5' untranslated region. Disruption of hapC is lethal in haploids and this lethality can be reversed by providing a complete copy of the hapC gene in trans. This finding indicates that hapC is an essential gene in A. nidulans. The function of hapC in A. nidulans and its possible role in the regulation of the amdS gene is being investigated.

6. Activity of N-acetylglutamate synthase requires N-acetylglutamate kinase polypeptide in Neurospora

The arg-14 locus of Neurospora crassa encodes acetylglutamate synthase (AGS), which catalyzes the first step of arginine biosynthesis. AGS activity is also defective in some of arg-6 mutant strains. The arg-6 gene produces two mitochondrial enzymes, acetylglutamate kinase (AGK) and acetylglutamyl-phosphate reductase (AGPR), the second and the third enzymes of the same pathway. We have reexamined the three reported complementation groups of arg-6, A (AGK-, AGPR+), B (AGK+, AGPR-), and NC (AGK-, AGPR-). We found AGS+ members in both group A (K- R+ S+) and B (K+ R- S+), and identified two new AGS- groups, A' (K- R+ S-) and B' (K+R- S-). We also found a new AB group (K+ R+ S-) which complements group A, B, and arg-14, but not groups A', B', or NC. Existence of the extended groups indicates that arg-6 gene products play roles in AGS activity, although neither AGK nor AGPR enzyme activity is necessary for AGS enzyme activity. Transformation of a NC (K- R- S-) strain with the cloned arg-6+ gene restored AGS activity as well as AGK and AGPR activity. Transformation of an A' strain with pAKH4 that produces only the AGK polypeptide restored AGS activity. Thus, the AGK protein can activate AGS. Evidence for physical contact between AGK and AGS using the yeast two-hybrid system were described.

7. Conversion of Fusarium oxysporum pro-trypsin to active trypsin in Aspergillus oryzae by co-expression with a Fusarium metalloprotease

Proteases are often encoded as zymogens containing an N-terminal polypeptide pro-region that results in an in active form of the enzyme. A secreted trypsin from Fusarium oxysporum contains a five amino acid pro-peptide that is proteolytically processed to yield the active enzyme in Fusarium. When the gene encoding this pro-trypsin is expressed in the heterologous host, Aspergillus oryzae, processing of the pro-region is inefficient resulting in a mixture of pro-trypsin and active enzyme. We have purified a secreted metalloprotease from F. oxysporum based on its ability to process pro-trypsin to active enzyme in vitro. The corresponding gene has been cloned, and co- expressed along with the Fusarium pro-trypsin gene in the host, A. oryzae. The addition of the metalloprotease gene resulted in a 3.5-fold increase in the production of active trypsin. We conclude that the production of an enzyme activity from a heterologous host organism can be significantly enhanced by the co-expression of the pro-enzyme along with a "maturase" activity that has also been derived from the same source organism.

8. Extracellular polysaccharidases of Neurospora

Enzymatic studies have shown glucoamylase and a cellulase complex in Neurospora. Two genes, gla-1 (glucoamylase) and cbh-1 (cellobiohydrolase) have been cloned and sequenced. There is close sequence homology between these and equivalents from other filamentous fungi including the ascomycete-like deuteromycetes Trichoderma, Humicola, and the basidiomycete Phanetochaete. Constructs containing both gla-1 and cbh-1 have been made and transformed back into Neurospora, using hygromycin as the selectable resistance marker. Representative transformants have subsequently been crossed to an Oak Ridge wild-type, and meiotic progeny isolated. These progeny have been screened for RIP derivatives, by screening halo sizes on media containing starch (subsequently stained with iodine in potassium iodide) for gla-1 and carboxymethyl-cellulose (stained with Congo Red) for cbh-1. RIPed gla-1 and cbh-1 progeny have both been obtained. They have reduced or absent halos, poor growth on starch or cellulose respectively, and altered DNA sequence as detected by Southern blotting and analysis of restriction fragments. Representative gla-1 and cbh-1 mutants have been deposited in FGSC.

9. Molecular cloning of an initiation factor kinase (eIF-2alpha-kinase) of Neurospora crassa

Phosphorylation of eIF-2alpha, the a subunit of the eukaryotic initiation factor 2, is one of the best characterized mechanism of translational control in eukaryotic cells. Dependent on the organism eIF-2alpha kinases are activated by distinct stimuli, e. g. hemin deficiency, double-stranded RNA or amino acid starvation. We identified a Neurospora eIF-2alpha kinase sequence via homology with known eIF-2alpha kinases especially that of GCN2 in Saccharomyces cerevisiae. GCN2 is an essential control element in the mechanism of general control of amino acid biosyntheses. The Neurospora sequence contains - like GCN2 - in close proximity to the kinase domain a second domain with homology to histidyl-tRNA synthetases. According to the working model of A. Hinnebusch and collaborators the synthetase interacts with uncharged tRNAs as the signal for amino acid limitation which in turn activates the kinase. We intend to disrupt the gene to examine its function in Neurospora crassa.

10. Restriction endonuclease analysis of mitochondrlal DNA of Metarhizium anisopliae strains

The species Metarhizium anisopliae contains two recognized varieties, M. anisopliae var. anisopliae and M. anisopliae var. majus, which mainly differ in spore size. M. anisopliae var. anisopliae isolated from various geographical locations is currently being used for the biological control of insects. Mitochondrial DNAs from strains A4, A19, AL, E6, E9 and MT of M. anisopliae var. anisopliae, collected from several Brazilian States, were isolated and characterized by restriction-endonucleases. The banding pattern obtained was the same for all strains investigated, indicating that their mtDNA have the same molecular sizes (about 36 Kb) and the same restriction sites for the enzymes used. Mitochondrial DNA from a strain of M. anisopliae var. majus showed an electrophoretic pattern that differed markedly from that of var. anisopliae. Thus, the restriction pattern of mtDNA may serve as a specific marker in the identification of var. anisopliae or of some subpopulation of this variety. Financial Support: FAPESP, CAPES and CNPq.

11. Acid phosphatase occurs in multiple active forms in the mold Neurospora crassa

Acid phosphatase excreted by both pregC (a constitutive mutant for Pi-repressible phosphatases) and wild-type strains of the mold Neurospora crassa grown at pH 5.4 consists of multiple active forms, as demonstrated by isoelectric focusing (IEF). The enzyme components were visualized by activity staining using alpha-naphthylphosphate in the presence of Fast Garnet GBC salt and found to be predominantly acidic proteins with isoelectric points in the range of pH 4.0 to 7.2. In addition, the population of active isoforms excreted by pregC was altered as a function of time of growth and variations in extracellular phosphate concentration, suggesting that this constitutive mutant is still sensing Pi levels. Financial support: FAPESP, CNPq, CAPES and IDB-USP

12. Expression of yeast citrate synthase gene in Aspergillus nidulans

The coding region of the CIT1 gene for mitochondrial citrate synthase (CSl) from Saccharomyces cerevisiae was amplified by PCR and cloned into an Aspergillus nidulans expression vector, pAL4, downstream of the alcohol dehydrogenase (alcA) promoter to yield pALCSl. In addition, deletion of the N-terminal mitochondrial targeting signal from the CIT1 gene was performed by PCR and the mutant gene (CIT1delta) was cloned as the wild type CIT1 gene, yielding pALCSldelta. After transforming into A. nidulans, both constructs gave stable transformants that were phenotypically stable for several mitotic divisions. Southern blot analysis of the DNA from the transformants showed that both the CIT1 and CIT1 genes were successfully integrated into the chromosomes of A. nidulans. Western blot analysis and enzyme assay for citrate synthase activity proved that both the integrated genes are effectively expressed by the alcA promoter induced by ethanol or threonine.

13. Identification and characterization of alternative oxidase gene and its upstream regulatory sequence in Neurospora

Mitochondria of Neurospora, like certain other fungi, algae, protists and most plants, contain a cyanide- insensitive alternative pathway of respiration in addition to the standard electron transport chain. In Neurospora, the alternative oxidase is only active (or induced) when electron flow through the cytochrome chain is restricted. Thus the regulation of the alternative oxidase gene(s) is of particular interest as it may provide a model system for studying the mechanism of communication between mitochondria and nucleus. By using degenerate PCR primers, derived from the most conserved regions of alternative oxidase proteins from other species, we have isolated and sequenced both genomic and cDNA for the alternative oxidase of Neurospora and have mapped the transcription start site. We have also shown that the sequence of the Neurospora protein is homologous to that of other species and the induction of its expression is at the transcriptional level. Mutants deficient in the alternative oxidase contain mutations causing amino acid substitutions in the coding sequence of the alternative oxidase gene. We are currently attempting to identify the regulatory element(s) responsible for the induction of alternative oxidase.

14. Reverse transcriptase activity of an intron-encoded polypeptide of Podospora anserina

A number of group II introns from eukaryotic organelles and prokaryotes contain open reading frames for polypeptides with homology to reverse transcriptases (RTs). We have used a yeast system to express intronic RT- ORFs from eukaryotic organelles (1). This include the mitochondrial coxI intron il from the fungus Podospora anserina, the plastid petD intron from the alga Scenedesmus obliquus and the mitochondrial RTL gene from the alga Chlamydomonas reinhardtii (2). The ORFs were fused with the TYA ORF from the yeast transposon Ty to produce virus-like particles in the recipient yeast strains with detectable amounts of RT-like polypeptides. Analysis of the heterologous gene products revealed biochemical evidence that the P. anserina coxI intron encodes an RNA-directed DNA polymerase with properties typically found for RTs of mobile elements. The direct biochemical evidence of an intron-encoded RT supports the idea that RTs are involved in intron mobility.

(1) Muller et al. (1987), Mol Gen Genet 207:421-429
(2) Fassbender et al. (1994), EMBO J 13:2075-2083.

15. The cloning and characterization of the Neurospora crassa catalase gene, cat-1

The filamentous fungus Neurospora crassa is believed to possess at least three biochemically and genetically distinct catalases (Cat-1, Cat-2, Cat-3), which are expressed differentially during development. An effort to clone the Neurospora crassa genes was undertaken using a labeled fragment from the Aspergillus niger catalase-R (catR) gene to probe the Orbach-Sachs cosmid library of Neurospora crassa. Overlapping cosmids were identified as having sequences that hybridized to the catR gene. A 7.7 kb KpnI fragment containing the putative catalase gene was subcloned into pUC-18 and sequenced. Analysis of this sequence revealed a strong similarity with catR and other catalase genes. This fragment was found to contain the entire catalase structural gene as well extensive upstream and downstream regions. Multicent (RFLP) mapping has tentatively identified this new catalase gene as cat-1 (LG IIIR). We are attempting to confirm this by examining mRNA and enzyme levels in N. crassa cells at different developmental stages. Similarities in the structure and amino acid sequences of catalases of N. crassa, A. niger, and E. coli suggest the existence of a separate, and previously unrecognized, catalase sub-family.

16. Structure, inheritance, and transcriptional effects of Pce1, an insertional mutation of Phanerochaete chrysosporium lignin peroxidase gene lipI

An insertional mutation within a lignin peroxidase allele of Phanerochaete chrysosporium strain BKM-F-1767 has been characterized. The 1747 bp element, Pce1, lies immediately adjacent to the fourth intron of lipI2. Southern blots reveal the presence of Pce1- homologous sequences in other P. chrysosporium strains. Transposon-like features include inverted terminal repeats and a dinucleotide (TA) target duplication. Atypical of transposons, Pce1 is present at very low copy numbers (1-5), and conserved transposase motifs (Doak et al 1994 Proc. Nat. Acad. Sci. 91:942-946) are lacking. The mutation transcriptionally inactivates lipI2, and it is inherited in a 1:1 Mendelian fashion among haploid progeny. Thus, Pce1 is a transposon-like element which may play a significant role in generating ligninolytic variation in P. chrysosporium.

17. Cloning and molecular characterization of the genes encoding extracellular phenoloxidases from Myceliophthora thermophila and Scytalidium thermophilum

Genomic DNA fragments encoding extracellular phenoloxidases (laccases) have been isolated from two thermophilic fungi, Myceliophthora thermophila and Scytalidium thermophilum. By DNA and peptide sequencing techniques we have deduced the primary structure of these two genes and their polypeptide products. The gene encoding M. thermophila laccase is composed of seven exons (246, 79, 12, 70, 973, 69 and 411 nt) interrupted by six introns (85, 84, 102, 72, 147, and 93 nt). The coding region, excluding intervening sequences, is rich in guanosine and cytosine residues (65.5% G + C) and encodes a preproenzyme of 620 aa (22 aa signal peptide, 25 aa propeptide, mature laccase comprising 573 aa). The gene encoding S. thermophilum laccase is also GC-rich (60.8% G + C) and comprises five exons (243, 91, 70, 1054, and 390 nt) punctuated by four small introns (63, 58, 55, and 65 nt). Like the Myceliophthora enzyme, Scytalidium laccase appears to be synthesized as a preproenzyme (616 aa) with a 21 aa signal peptide and a propeptide of 24 aa. The deduced amino acid sequences of both laccases are approximately 60% identical to Neurospora crassa laccase. The sequence similarity is highest in regions that correspond to the four histidines and one cysteine which are involved in formation of the trinuclear copper cluster at the active site of the enzyme.

18. Cloning of the cpc-1/GCN4 homolog of Aspergillus niger

We have cloned the Aspergillus niger gene encoding the transcriptional activator protein responsible for the general control ("cross-pathway control") of amino acid biosynthesis. The cDNA clone regulated by a heterologous yeast promoter was able to complement a S. cerevisiae gcn4 mutant strain. The cDNA clone was used as probe to clone the corresponding chromosomal locus which we have named gcnA. The nucleotide sequence of both clones revealed significant similarity to the yeast GCN4 and the N. crassa cpc-1 gene. The gcnA protein contains an apparently degenerate leucine zipper motif with only a single leucine residue. Gene disruption experiments and the study of gene regulation are on their way.

19. Polyamines regulate the stability of ornithine decarboxylase mRNA in Neurospora crassa

Ornithine decarboxylase (ODC) initiates the synthesis of polyamines and is encoded by spe-1 gene. ODC is highly regulated by polyamines, but in contrast to other organisms, the variation of the synthetic rate of ODC in Neurospora crassa (5 to 7-fold) is correlated with a similar variation in the abundance of spe-1 mRNA. This correlation suggested that spe-1 mRNA abundance was transcriptionally controlled. Run-on transcription experiments, however, showed that the transcription rate of spe-1 mRNA does not vary in response to the polyamines. The stability of spe-1 messenger was then measured by using the transcriptional inhibitor thiolutin. The stability of spe-1 mRNA was different in the presence and the absence of polyamines, and the difference accounts adequately for the difference in the steady-state levels of spe-1 mRNA in the two conditions. The stabilization of spe-1 mRNA in the absence of polyamines was accompanied by an increase in the size of spe-1 mRNA polysomes. Decreasing the translational elongation rate with low concentrations of cycloheximide, leading to larger spe-1 mRNA polysomes, also led to a substantial increase of spe-1 mRNA abundance. This fact suggests the participation of protein synthesis in polyamine-mediated regulation of spe-1 mRNA turnover. (Supported by NIH Grant GM35120 to R. H. D.)

20. Removal of the 5' untranslated leader increases the abundance and ribosomal loading of spe-1 mRNA in Neurospora crassa

The spe-1 gene of Neurospora crassa encodes ornithine decarboxylase (ODC), a key regulatory enzyme in the synthesis of polyamines (putrescine, spermidine and spermine). Starvation for polyamines leads to concomitant increases in ODC activity and spe-1 mRNA abundance. The increase in spe-1 mRNA abundance in this condition is due to stabilization of spe-1 mRNA. The increase in spe-1 mRNA abundance is correlated with an increase in the size of spe-1 mRNA polysomes. The spe-1 mRNA has an unusually long (535 nucleotides) untranslated leader with no obvious secondary structure or open reading frame. The removal of nucleotides +9 to +486 of the leader also leads to an increase in spe-1 mRNA abundance and an increase in polysome size. Deletions show that this effect is localized to the first 315 nucleotides of the leader. However, substantial regulation of spe-1 mRNA turnover by polyamines remains in constructs lacking the leader. We now wish to know the extent to which the increases in spe-1 mRNA abundance following polyamine starvation and following removal of the spe-1 mRNA leader share mechanistic features, such as protection by ribosomes, and in what way these increases arise from distinct mechanisms. (Supported by NIH Grant GM35120 to R. H. D.)

21. Transformation of Neurospora crassa with the Aspergillus nidulans creA gene

The qa gene in N. crassa is subject to carbon catabolite repression in wild type. These studies have involved transformation with a plasmid which contains the creA gene which is involved in carbon catabolite repression in A. nidulans and the qa-2 gene from N. crassa as a selectable marker. Transformants were selected in a qa-2 mutant background. These transformants were crossed to met-7 (a very tightly linked gene to the qa cluster) to obtain homokaryotic isolates with duplicated copies of the qa-2 gene and the creA gene and its N. crassa homologues. Inactivation (RIPing) of the qa-2 gene was detected in tetrads when two isolates carrying the transformed DNA were crossed. These strains no longer had catabolic dehydroquinase activity as determined by enzyme assays. DNA from these tetrad isolates was digested with NdeII and Sau3A and the Southern blot was probed with a 32P labeled creA gene. These results indicated that the A. nidulans creA gene had been inactivated (methylated). Northern blot analyses of RNA's from these tetrad isolates grown on dextrose plus quinic acid (carbon repression conditions) and quinic acid (induction conditions) indicated that qa gene expression was still carbon repressed. These studies indicated that the mutant creA gene had no affect on relieving carbon repression of the qa gene cluster.

22. Hsp80 of Neurospora crassa as a molecular chaperone of cellular protein folding

The heat shock response of N. crassa is characterized by the rapid and transient production of a number of heat shock proteins (HSPs) along with the cessation of synthesis of a fraction of the normal cellular proteins. The most abundant stress protein, HSP80, is synthesized at high levels on exposure to heat shock or carbon source starvation. NAD-specific glutamate dehydrogenase of N. crassa is also induced by carbon-catabolite repression but gdh gene expression is switched off during heat shock. During growth under carbon starvation conditions, however, both gdh and hsp80 genes are expressed. To assess the role of HSP80 as a chaperone of cellular protein folding, denaturation/renaturation of NAD-GDH was investigated in vitro, in the presence and absence of HSP80. Purified preparations of HSP80 and GDH were employed in conjunction with guanidine hydrochloride as a protein denaturant. Our experiments show that the presence of HSP80 reduces the rate of GDH denaturation. In addition, renaturation of denatured enzyme is enhanced by HSP80, suggesting the capacity of this stress protein to act as a molecular chaperone.

23. Regulation of genes for amino acid biosynthesis in Magnaporthe grisea

Magnaporthe grisea is the causal agent of rice blast disease. To understand how this organism is able to infect plants it is important to determine which aspects of the biology of M. grisea differ from non-pathogenic fungi. We expect conservation of mechanisms for regulation of genes of primary metabolism in pathogenic and non-pathogenic fungi. However, data supporting this assumption are lacking. To examine control of amino acid biosynthesis, we have cloned the M. grisea homolog of Neurospora crassa arg-2. Control of N. crassa arg-2 expression is complex, the gene is transcriptionally regulated by cross-pathway control and translationally regulated by a cis-acting upstream open reading frame. We provide evidence that transcriptional and translational regulation of the M. grisea gene is likely identical to the regulation of the arg-2 gene of N. crassa.

24. Analysis of the Neurospora con-10 promoter

As an approach to understanding the regulation of gene expression in filamentous fungi, we are studying the promotor region of con-10 gene, a gene that is expressed preferentially during spore formation in Neurospora. Initial studies showed that there are at least four segments in the promotor that are involved in expression of con-10. Using site-directed mutagenesis, we have further defined the sequences that are involved in con-10 expression. The expression of con-10 is regulated not only by developmental signals such as conidiation, but also by environmental signals such as light. It appears that there are factors both of repression and of activation involved in con-10 expression. Our initial questions are 1) What are the promotor elements involved in repression and activation of con- 10? 2) Do different induction cues use common regulatory components? 3) Do developmental and environmental signalling pathways influence each other?

25. Isolation of the fluffy and fluffyoid genes of Neurospora crassa

Macroconidiation in Neurospora begins with the production of aerial hyphae from mycelia. The aerial hyphae undergo apical budding to form minor and major constriction chains. Septation of the procondial chain is followed by maturation and release of free conidia. Fluffy, a Neurospora conidiation mutant isolated in 1933 by Carl Lindegren, is blocked at the minor constriction stage. Fluffy does not form free conidia under the most permissive conditions for development. Fluffyoid, another conidiation mutant, is blocked at an earlier stage than fluffy. However, fluffyoid does produce abundant proconidial chains under carbon starvation. We have begun chromosome walking experiments to isolate the fluffy and fluffyoid genes.

26. Molecular dissection of NIT4 reveals multiple functional domains and a novel leucine- rich activation motif

NIT4 is pathway-specific transcriptional activator of nitrate assimilation in Neurospora crassa. NIT4 positively regulates nit3 and nit6 genes specifying nitrate reductase and nitrite reductase, respectively. To understand the mechanism by which NIT4 acts, we took several approaches to dissect the functional domains of the NIT4 protein. By fusing different NIT4 regions to the GAL4 DNA-binding domain, three transcriptional activation domains were identified by their ability to activate transcription in yeast. These three acitivation domains of NIT4 are scattered within the C-terminal 400 residues and are both structurally and functionally separable from the DNA-binding domain. A novel 28 residue leucine-rich activation domain was identified at the C-terminus of NIT4, which alone showed strong transcription activation potency. However, NIT4's full activity requires all three activation domains. A dimerization region of NIT4 was identified within residues 109-144. NIT4 homodimer formation was confirmed both by chemical crosslinking in vitro and with the yeast two- hybrid system in vivo. The importance of the internal region of NIT4 was shown by deletions which caused complete loss of function. Previous experiments have suggested that the NIT3 protein autogenouly controls its expression by binding to the NIT4 protein. A possible physical interaction between the NIT4 and NIT3 proteins was tested by yeast two-hybrid system as well as by the GST fusion. Neither experimental approach revealed any evidence for NIT4-NIT3 protein-protein interaction.

27. The frequency locus encodes a central component of the circadian clock, the level of which is rapidly reset by light

Based on its genetics, the frequency (frq) locus has been proposed as a key component in the cellular oscillator generating circadian rhythmicity (Dunlap, Ann. Rev. Physiol. 55:683-728, 1993). Several testable predictions have been made regarding the regulation of genuine components (state variables) of the clock, and frq encodes a factor satisfying all of these criteria (Aronson, Johnson, Loros, and Dunlap, SCIENCE 263:1578-1584, 1994). frq encodes a central component of a molecular feedback loop in which the product of frq depresses the level of its own transcript, resulting in a daily oscillation in the level of this frq transcript. Rhythmic frq mRNA expression is essential for overt circadian rhythmicity: constitutively elevated expression of FRQ-encoding RNA in a frq+ background results in arrhythmicity, and no level of constitutive expression is capable of rescuing normal rhythmicity in frq loss-of-function mutants. Step reductions in frq transcript levels at any time in such constitutively elevated strains sets the clock to a unique and predicted phase. Recent data (Merrow and Dunlap, EMBO J. 13:2257-2266, 1994) also show phylogenetic conservation of frq structure and function. Finally, it is now clear that light acts rapidly to increase the level of transcript(s) arising from frq, consistent with a model in which elevation of the level of frq transcript(s) in the cell is the initial clock-specific event involved in resetting of the clock by light. These data support a model where the Neurospora circadian clock consists of a negative feedback loop in which the product of the frq gene regulates the level of the transcript(s) of the frq gene.

28. The ccg-1 gene of Neurospora displays multiple levels of regulation including the clock, development, light and heat shock

A circadian biological clock controls several aspects of growth and development in the ascomycete fungus Neurospora crassa, including the timing of the initiation of conidiogenesis. This property has allowed genetic and molecular techniques to be used to study the circadian clock itself from two converging angles. One approach has been to examine the pathways whereby clocks act to control cellular metabolism and behavior. Initial efforts targeted the isolation of genes whose transcript levels are controlled by the clock. One of the genes, designated clock controlled gene-1 (ccg-1), identified by a subtractive hybridization procedure (Loros et al, Science 243:385-388, 1989) was shown by nuclear run-on analysis (Loros and Dunlap, Mol. Cell. Biol. 11:558-563, 1991) to have clock regulated transcription as the primary point of regulation. ccg-1 is a highly abundant gene, with a 535 nt mature transcript encoding a 71 aa peptide. Sequence analysis of ccg-1 genomic and cDNA clones showed it to be identical to a Neurospora gene, glucose repressible gene-1 (grg-1) (McNally & Free, Curr Genet 14:545-551, 1988). In collaborative work with Giuseppina Arpaia and Giusseppe Macino, University of Rome, we have found ccg-1 gene expression to be blue-light photo-inducible (MGG in press, 1995). Disruption of the ccg-1 locus produces no detectable phenotype and does not effect normal operation of the clock. We have shown by both transcript and protein analysis that ccg-1 is turned on early in development and is regulated by the aconidial-2 (acon-2) locus which, when mutated, blocks conidiation completely. This locus acts early in the developmental pathway at the decision point of commitment to development where aerial hyphal cells are forming constrictions on the way to budding off conidia. ccg-1 is now the earliest gene known to be turned on at the point of commitment in the Neurospora developmental pathway. In collaborative work with Stephen Free, State University of New York at Buffalo, Western analysis of the CCG-1 protein indicates CCG-1 to be present in undifferentiated hyphae in a time-of-day specific manner. Immunocytochemical localization shows extensive, non-nuclear staining of CCG-1 in the cytoplasm of aerial hyphae and conidiospores. Additionally, we demonstrate that ccg-1 is heat-shock inducible but shows limited similarity at the structural and sequence level to the diverse class of evolutionarily conserved small heat-shock and stress proteins. Deletion analysis of upstream ccg-1 sequences suggests that the clock regulatory elements lie near the start site of transcription and are distinct and separate from sequences conferring glucose and developmental regulation.

29. Promoter analysis of the Neurospora crassa circadian clock-controlled ccg-2 (eas) gene

The N. crassa ccg-2 gene encoding a fungal hydrophobin is transcriptionally regulated by the circadian clock. In addition, ccg-2 is positively regulated by light, and transcripts accumulate during asexual development. To sort out the basis of this complex regulation, deletion analysis of the ccg-2 promoter was carried out to localize the cis-acting elements mediating clock, light, and developmental control. A distinct positive clock element was localized to within a 45 nt region, just upstream of the TATA box. Using an unregulated promoter/reporter system we show that this element is necessary and sufficient for conferring clock regulation on the ccg-2 gene. We are currently using this element as a probe in gel-mobility shift assays to identify trans-acting clock factors.

30. Output and input: clock control, heat-shock and photo-blind mutants in Neurospora

A circadian biological clock controls several aspects of growth and development in the ascomycete fungus Neurospora crassa, including the timing of the initiation of conidiogenesis. This property has allowed genetic and molecular techniques to be used to study the circadian clock itself from two converging angles. One approach has been to examine the pathways whereby clocks act to control cellular metabolism and behavior. Initial efforts targeted the isolation of genes whose transcript levels are controlled by the clock. One of the genes, designated clock controlled gene-1 (ccg-1), identified by a subtractive hybridization procedure (Loros et al, Science 243:385-388, 1989) was shown by nuclear run-on analysis (Loros and Dunlap, Mol. Cell. Biol. 11:558-563, 1991) to have clock regulated transcription as the primary point of regulation. ccg-1 is a highly abundant gene, with a 535 nt mature transcript encoding a 71 aa. peptide. Sequence analysis of ccg-1 genomic and cDNA clones showed it to be identical to a Neurospora gene, glucose repressible gene-1 (grg-1) (McNally & Free, Curr Genet 14:545-551, 1988). In collaborative work with Giuseppina Arpaia and Giusseppe Macino, University of Rome, we have found ccg-1 gene expression to be blue-light photo-inducible (MGG in press, 1995). Disruption of the ccg-1 locus produces no detectable phenotype and does not effect normal operation of the clock. We have shown by both transcript and protein analysis that ccg-1 is turned on early in development and is regulated by the aconidial-2 (acon-2) locus which, when mutated, blocks conidiation completely. This locus acts early in the developmental pathway at the decision point of commitment to development where aerial hyphal cells are forming constrictions on the way to budding off conidia. ccg-1 is now the earliest gene known to be turned on at the point of commitment in the Neurospora developmental pathway. In collaborative work with Stephen Free, State University of New York at Buffalo, Western analysis of the CCG-1 protein indicates CCG-1 to be present in undifferentiated hyphae in a time-of-day specific manner. Immunocytochemical localization shows extensive, non-nuclear staining of CCG-1 in the cytoplasm of aerial hyphae and conidiospores. Additionally, we demonstrate that ccg-1 is heat-shock inducible but shows limited similarity at the structural and sequence level to the diverse class of evolutionarily conserved small heat-shock and stress proteins. Deletion analysis of upstream ccg-1 sequences suggests that the clock regulatory elements lie near the start site of transcription and are distinct and separate from sequences conferring glucose and developmental regulation.

31. Expression of cloned genes in Penicillium chrysogenum

The ability of industrial strains of certain filamentous fungi to secrete large quantities of protein continues to elicit interest in their potential for large scale homologous and heterologous protein production. Some, like Aspergillus and Neurospora, have been extensively used as model organisms for diverse transformation and expression systems. Although Penicillium chrysogenum is of great industrial importance, only a few attempts were made to use this organism for homologous or heterologous gene expression. One reason is, that in contrast to Aspergilllus no suitable promoter system was available until now. We have recently cloned the gene encoding a secreted phosphate- repressible acid phosphatase (phoA) of P. chrysogenum, which has all the features required as a basis for an efficient expression system. To develop a regulated secretion system for proteins in Penicillium, a fungal xylanase gene (xylP) and the gene encoding the human lipocalin 1 (LCN1) as model genes were fused to the phoA promoter and signal sequence. Synthesis of recombinant XylP and LCN1 in positive transformants carrying the corresponding constructs was regulated by inorganic phosphate (Pi) present in the medium, so that induction of the heterologous protein expression could be attained by lowering Pi concentration.

32. NRE, the major nitrogen regulatory protein of Penicillium chrysogenum, interacts with the promoter regions of nitrate assimilation and penicillin biosynthetic gene cluster

Nitrogen metabolite repression is a wide-domain regulatory system, which operates to ensure that a constant supply of nitrogen is readily available for growth in response to variable or rapidly changing environments. In Aspergillus nidulans and Neurospora crassa, the global nitrogen repression/derepression is mediated by the major positive control genes areA and nit-2, respectively. Because of the central position of nitrogen regulation and its effects upon the synthesis of secondary metabolites, we elected to investigate this regulatory mechanism in the filamentous fungus P. chrysogenum, a well known producer of the beta-lactam antibiotic penicillin. In a first step, the areA/nit-2 homologous gene from P. chrysogenum, nre, was isolated and shown to complement nit-2- mutants of N. crassa. nre encodes a protein of 862 amino acids and contains a single Cys2/Cys2-type zinc finger with an adjacent basic region. The overall amino acid sequences of AREA, NIT-2 and NRE show only 30% identity, but they display 98% identity in their zinc finger motif. We have expressed the putative DNA-binding region of NRE as a fusion protein with E. coli beta-galactosidase. Mobility shift assays and missing contact probing experiments indicated that NRE binds in a sequence-specific fashion to the intergenic promoter regions of the Penicillium nitrate assimilation and penicillin biosynthetic gene clusters.

33. In vitro reconstruction of fungal chromosomes and genomes. I. A physical map of the entire Aspergillus nidulans genome

Physical maps of fungal genomes provide new research strategies for a wide range of fundamental biological problems, for engineering the production of new pharmaceuticals, and for understanding the cause of fungal diseases in plants and animals. The physical maps reported here represent the in vitro reconstruction of entire chromosomes from recombinant DNA libraries and provide useful tools to clone genes, determine genomic structure, and study genome evolution. We report a 29 kb resolution physical map of the entire 31 Mb genome of Aspergillus nidulans reconstructed from a 5134 clone cosmid library using 648 probes. The maps are the result of a novel two-way ordering process in which overlapping clones (redundant) and non-overlapping probes (tiles) are ordered to span the entire genome. The physical map is composed of eight matrices with clones down the rows and probes across the columns, one for each chromosome. The redundant order of clones contains 4550 anchored clones (89% of the cosmid library) into 132 contigs with an average of 17 contigs per chromosome. The compressed map (providing a minimum tiling of all 8 chromosomes) is reduced 5-fold in redundancy and contains 1085 clones. By integrating the physical and genetic maps with chromosome and clone hybridization data, we found that repeated DNA sequences are non-randomly distributed along chromosomes in a way reminiscent of heterochromatic banding patterns on cytological maps in other eukaryotes.

34. In vitro reconstruction of fungal chromosomes and genomes. II. Assessing the statistical reliability of physical maps by bootstrap resampling

We provide a methodology for quickly ordering clones in a genomic library into a physical map and for applying a statistical tool known as the bootstrap to assess the statistical reliability of a clonal ordering. Each clone is assigned a binary fingerprint by one of a variety of experimental approaches to physical mapping. For example, the binary fingerprints might be generated by hybridizing a panel of m probes to a library of n clones. The resulting n x m binary data matrix, X, is input to a program, ODS_BOOTSTRAP, which utilizes the similarity in binary fingerprints of clones to construct a physical map. Under this particular implementation of bootstrap resampling, the m probes (or columns of the data matrix) are sampled randomly with replacement in the computer to generate a new n x m matrix, X*, from which a second physical map is constructed. The resampling process is repeated 100 or more times to generate 100 or more X* matrices. The resulting 100 or more physical maps are compared with the original physical map based on the original data matrix X by counting how often links in the original physical map reappear. These counts yield confidence measures for each link on the physical map. The procedure is illustrated with the physical map of Aspergillus nidulans chromosome IV.

35. In vitro reconstruction of fungal chromosomes and genomes. III. ODSTM, software and user interface for physical mapping

The ODS package provides computational and graphical tools for reconstructing physical maps. The software is appropriate for those engaged in chromosome walking, STS content mapping, and contig mapping by a variety of experimental approaches. The objective of the ODS software package is to couple physical mapping algorithms (e.g. Genetics 132:591-601 and PNAS 91:11094-11098) with a friendly user interface so that the software can be easily used by laboratory scientists. This work was based on the DNA fragment ordering problem described in Ordering DNA Sequences, CABIOS 9:215- 219. ODS orders DNA fragments based on the similarity of their fingerprints assigned to DNA fragments in a library by one of several experimental approaches. The algorithm has been used to map the entire Schizosaccharamyces pombe genome (PNAS 9:4461-4465), the entire Aspergillus nidulans genome, and a region of human chromosome 9 (Cytogenetics and Cell Genetics 64:120). The ODS software displays the maps graphically in a variety of formats and prints graphical displays of summary statistics to check mapping progress, the quality of a physical map, and whether or not underlying models of physical mapping are fitting the data. More information can be obtained by email to ODS@BSCR.UGA.EDU, by phone to (706) 542-9359, or by mail to Robyn Ansley, Genetics Department, Life Sciences Building, University of Georgia, Athens, GA 30602 USA.

36. In vitro reconstruction of fungal chromosomes and genomes. IV. A fungal Genome database (FGDB)

An object-oriented Fungal Genome Database (FGDB) system is being developed at the University of Georgia. (See Kochut, K.J., J. Arnold, J.A. Miller, and W.D. Potter (1993). "Design of an object- oriented database for reverse genetics." pp. 234-242. First International Conference on Intelligent Systems for Molecular Biology. AAAI Press. Menlo Park, CA). The goal of this project is to provide a software system to support reverse genetics in fungi. FGDB currently contains physical and genetic maps of all 8 Aspergillus nidulans chromosomes. FGDB is based on an object-oriented database management system, ONTOS. The system includes a user-friendly X-windows based interface. Unique features of the system in comparison to other genome database systems are the: (1) ability to create maps; (2) ability to edit maps; and (3) tools to integrate genetic and physical maps. Contig maps can be automatically created and then manually edited and fine-tuned within FGDB. In order to verify and to assist in the editing of contig maps, the user can integrate the genetic and contig maps. Currently, FGDB supports connections over the mnetwork via an X-Windows interface.

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