Posters III: Gene Expression/Genome Structure

1. Molecular cloning of cDNA encoding Neurospora crassa wall-associated proteins.

Dorothy McColl and P.J. Vierula, Biology Department, Carleton University, Ottawa, Ontario, Canada. K1S 5B6.

A significant fraction of the cell walls of the filamentous fungus, Neurospora crassa is thought to be proteinaceous. To study this cell component, we have been analysing proteins which are found to co-purify with the cell walls. Using antisera raised against a crude cell wall preparation as a probe, several clones have been isolated from a cDNA expression library. A partial sequence of one of these clones has revealed homology to a thiamine- regulated protein in yeast. Results of the characterization of this gene were presented.

2. The Aspergillus nidulans catA locus encodes a developmentally regulated catalase.

Rosa E. Navarro(1), Mary A. Stringer(2), Wilhelm Hansberg(1), William. E. Timberlake(2) and Jesús Aguirre(1). (1)Instituto de Fisiologío Celular-UNAM, Apdo. Postal 70-242, 04510 México, D.F. (2)University of Georgia, U.S.A.

The Aspergillus nidulans CAN5 cDNA clone was isolated as a developmentally regulated transcript. Here we present the complete sequence of the corresponding gene. Comparison of this gene with sequence data bases shows significant identity to know catalases and has been denominated catA (developmental catalase). CHEF gel electrophoresis and hybridization of this gene with a chromosome-specific cosmid library showed that catA is in chromosome III. Northern Blot analysis indicates that catA is regulated during sporulation and is accumulated in spores. Catalase activity determination using native gel electrophoresis demonstrated that there are at least two catalases in A. nidulans; one catalase has low molecular weight and is induced during the first hours of development (0-25 h) whereas the other catalase (higher molecular weight) is accumulated preferentially in spores. Disruption of the catA gene abolished the major catalase activy present in spores without affecting the activity associated to growth.

3. Molecular and functional characterization of the specifically regulated aox gene of Penicillium chrysogenum.

Edith Schreiner, Klaus Holzmann and Helmut Schwab. Institut für Biotechnologie, Arbeitsgruppe Genetik, TU Graz. A-8010 Graz, Austria.

A specifically regulated gene, dependent on the physiological state and on external pH conditions, was cloned from Penicillium chrysogenum. A full length cDNA clone as well as the corresponding genomic clone of this gene were sequenced. The isolated gene shows strong homology to alcohol oxldases of methylotrophic yeasts at both the amino acid and nucleotide level. In order to investigate the function of this gene in Penicillium chysogenum the aox gene was overexpressed in Escherichia coli. However, no alcohol oxidase activity could be measured with lysates of recombinant E. coli. Therefore the aox coding region was fused to the strong Aspergillus nidulans gpd promoter. Transformation of Penicillium chrysogenum with this construct resulted in strains that strongly overexpress the aox gene. We are currently analysing these transformants. In addition, studies to analyse thc promoter region and its regulatory signals are in progress.

4. Characterisation of creA alleles in Aspergillus nidulans.

Robert Shroff and Joan Kelly. Department of Genetics, University of Adelaide, South Australia, 5005.

Carbon catabolite repression is a wide domain regulatory mechanism that, in the presence of readily metabolised carbon sources, acts to repress the expression of enzymes involved in the breakdown of alternative carbon sources. Genetical and biochemical analysis in Aspergillus nidulans has identified a number of genes involved in carbon catabolite repression. The dominance properties and the non hierarchical heterogeneity demonstrated by creA alleles suggest that this gene is a negatively acting repressor protein. The creA gene has been cloned. Theoretical translation of the DNA sequence shows the presence of a Cys2-His2 zinc finger DNA binding motif and an alanine rich region, both of which are consistent with the suggested function of CREA (Dowzer and Kelly, 1991). In addition, a comparison of the creA gene between A. nidulans and A. niger shows a small, highly acidic region and a sequence of 42 identical amino acids. This sequence shows a significant degree of similarity to a region found in RGRl - a protein involved in carbon catabolite repression in Saccharomyces cerevisiae (Drysdale et al. 1993). Using a PCR- SSCP approach, sequencing and phenotypic analysis we have characterised a number of creA alleles. The mutations fall into two broad groups. (1) Mutations disrupting the zinc finger binding domain; and (2) nonsense or frameshift mutations leading to truncation of the resulting protein. Some phenotypic characteristics are specific to the two classes of mutations. Dowzer, C. and Kelly, J. (1991) Mol. Cell. Biol. 11:5701-5709 Drysdale et al. (1993) Gene 130:241-245

5. Starvation stress and development in Aspergillus nidulans.

Isaac Skromne, Olivia Sanchez and Jesús Aguirre. Instituto de Fisiolog¡a Celular-UNAM, Apdo. postal 70-242, 04510 México, D.F.

Expression of the A. nidulans brlA gene plays a fundamental role in the switch from vegetative growth to asexual reproduction Using a media shifting protocol to induce submerged sporulation and brlA-lacZ as an expression marker, we have found that carbon and nitogen starvation stress induced brlA transcription to different degrees. Glucose starvation induced brlA rapidly to high levels and resulted in spore formation on reduced conidiophores, whereas nitrogen starvation induced brlA gradually to lower levels and sporulation occured to a lesser extent from more complex conidiophores. No clear qualitative differences between the two brlA transcripts were found in these starvation conditions, suggesting that the different patterns of sporulation could be explained by quantitative expression differences. Non-repressing carbon sources such as glycerol, acetate and arabinose were as effective as glucose in preventing brlA mRNA accumulation, suggesting that glucose effects on brlA expression could be explained as a response to nutrient starvation, rather than by carbon catabolite repression. When mycelia was not shifted to starvation conditions, sporulation was not observed in standard minimal medium even after glucose was exhausted, unless medium was buffered. This and other results suggest that strong deviation from external neutral pH partally prevented brlA full induction and/or function.

6. Aflatoxin pathway gene cluster map in Aspergillus parasiticus and Aspergillus flavus.

J. Yu(1), P.-K. Chang(2), M. Wright(1), J. W. Cary(1) , D. Bhatnagar(1) , T.E. Cleveland(1) , J.E. Linz(4) and G.A. Payne(3). (1)USDA/ARS, Southern Regional Res. Ctr. New Orleans, LA, (2)Tulane Univ. New Orleans, LA, (3)NCSU Raleiph, NC, (4)MSU East Lansing, MI

Aflatoxins are potent carcinogens produced by the fungi Aspergillus flavus and A. parasiticus. Aflatoxins are synthesized by condensation of acetate units; their biosynthesis is estimated to involve at least sixteen different enzymes. Genes encoding four of these enzymes including pksA, nor-1, ver-1, and omt-1, as well as a gene aflR, which regulates transcription of the pathway genes, have been cloned in our laboratories. We report here the organization of the aflatoxin pathway genes and a regulatory gene on the Aspergillus parasiticus and A. flavus chromosome. By determining overlapping mapped regions of the inserts in cosmid and lambda clones, the aflatoxin pathway genes were located and were shown to reside within a 60 kb DNA fragment in the order of pksA, nor-1, aflR, ver-1 and omt-1 in both A. parasiticus and A. flavus. This order is coincident with the order of the activities encoded by these genes in the biosynthetic pathway, with the exception of the regulatory gene, aflR, which is located between the nor-1 and ver-1 genes. Transcriptional mapping of the region between the ver-1 and the omt-1 gene revealed two additional transcripts which may be involved in the late steps of aflatoxin biosynthesis.

7. A putative recombination specific site in Penicillium chrysogenum.

Francisco Fierro, S. Gutiérrez, F.J. Fern ndez, K. Kosalkova, J. Casqueiro and J.F. Martín. Dpto. Microbiología, University of León, 24071 León, Spain.

Four non-producer mutants of penicillin were analysed by Southern hybridization. All of them lack the entire penicillin biosynthetic gene cluster. By chromosome walking of the parental strains the borders of the deleted region were determined and sequenced. The sequence of the junction zone resulting from the deletion indicates that the deletion has occured at the same point in the four mutants, within the hexanucleotide TGTAAT. This short sequence also appears at the borders and the junction zone of an amplified genomic region containing the penicillin biosynthetic genes in overproducing strains. These findings support the theory that both, the deleted and the amplified region, have arised as a consequence of recombination processes induced by treatment with mutagenic agents (probably as a part of a repair mechanism for mutation damage) and that the sequence TGTAAT is a hot spot for recombination.

8. Promoter replacement studies of the cefG gene show that its expression is limiting for cephalosporin biosynthesis in Acremonium chrysogenum.

F.J. Fernández, J. Velasco, F. Fierro, A.T. Marcos, S. Gutiérrez and J.F. Martin. Area of Microbiology, University of León, 24071 León, Spain.

The conversion of deacetylcephalosporin C to cephalosporin C is inefficient in most Acremonium chrysogenum strains. The cefG gene, which encodes deacetylcephalosporin C acetyltransferase, is expressed very poorly in A. chrysogenum. Introduction of additional copies of the cefG gene with its native promoter did not produce a significant increase of the steady state level of the cefG transcript. Replacement of the native promoter by some strong promoters from other genes and other sources led to a very high steady state levels of cefG transcript and to increased deacetylcephalosporin C acetyltransferase activity in the transformants. Cephalosporin production was increased two- to three-fold in A. chrysogenum C10 transformed with constructions in which the cefG gene was expressed from some of these promoters due to a more efficient conversion of deacetylcephalosporin C to cephalosporin C, as shown by HPLC analysis of the intermediate and the final product. Promoter optimization studies may be, therefore, of great interest to increase antibiotic yields.

9. The recombinator cog in Neurospora crassa is within highly polymorphic DNA.

P. Jane Yeadon and David E. A. Catcheside, Flinders University, Adelaide, Australia.

The St Lawrence ST74-OR23-1VA and Lindegren Y8743 strains of Neurospora crassa are derived from different wild collections and have dissimilar cog alleles. The allele in Lindegren, cogLa (previously designated cog+), is a more efficient recombinator than cogS74A and cogEa (previously cog), respectively the alleles in St Lawrence and Emerson a. Restriction fragment length polymorphisms (RFLP) and sequence polymorphisms (SP) were used to map the difference between cogLa and cogS74A to a region that extends from 2.3 to 3.2 kb 3' of the his-3 coding sequence. The DNA sequence from 400 bp 3' of his-3 to 120 bp 3' of the cog-region in these strains was found to be homologous but to diverge by 3.5%. The differences include single base pair changes, short insertion/deletions, differences in the length of poly-T tracts and three longer sequences (101, 20, and 15 bp) present only in St Lawrence. Southern analysis of other laboratory strains revealed four major and several minor variants of this region. All strains examined were descendants of Lindegren A, Lindegren a, Abbott 4A and Abbott 12a and it is clear that each of these progenitors collected from the wild population had a different variant of the cog region. Sequence divergence of this degree seems remarkable, even for an intergenic region, for fully interfertile strains of a single species.

10. Genome organization and strain compatibility in Trichoderma harzianum.

I. Gomez-Caballero(1), I. Chet(2), and A. Herrera-Estrella(1). (1)Genetic Engineering Dept. Centro de Investigación y Estudios Avanzados. U. Irapuato. P.O. BOX 629, Irapuato, Gto. Mexico. (2)Faculty of Agriculture, The Hebrew University of Jerusalem, Israel.

The soil fungus Trichoderma harzianum is a mycoparasitic fungus well known for its use as a biocontrol agent of phytopathogenic fungi. Among other factors, Trichoderma produces a series of antibiotics and secretes cell wall degrading enzymes. In an attempt to understand fungus-fungus interactions we are studying both the responses of Trichoderma harzianum to other fungi and to itself. Recent studies on the mechanism of action of Trichoderma spp. have led to the conclusions above mention, however, it is still unclear how does Trichoderma protect from its own lytic enzymes and antibiotics. And it is known that different species of the genus attack each other. First we established the electrophoretic karyotype of ten different T. harzianum isolates collected around the world and localized a series of genes in the chromosomal bands. From these experiments five different karyotypes were clearly distinguishable. The localized genes always corresponded to the same band when the strains had the same karyotype. Further studies were made using RAPDs, from these data the use of five different oligonucleotides led to the same conclusions as the karyotype and gene assignment. The use of an extra set of three oligonucleotides allowed us to distinguish within isolates sharing karyotype. Confrontation experiments in different growth conditions led us to the conclusion that isolates with the same electrophoretic karyotype have compatible interactions and that those with different karyotype give in-compatible interactions. We conclude from these data, first that there is a high karyotype variation within the T. harzianum species group aggegate (Rifai), that there is a self respect signal encoded in its genome, and that the high variation in karyotype maybe due to mistaken classification because exactly the same karyotype is found in very distant places.

11. Cloning, characterization and expression of pepF and pepG, genes encoding serine carboxypeptidases from Aspergillus niger.

J.P.T.W. van den Hombergh(1), G. Jarai(2), F.B. Buxton(2) and J. Visser(1). (1)Agricultural University Wageningen, section Molecular Genetics of Industrial Organisms, Dreijenlaan 2, NL-6703-HA Wageningen, The Netherlands. (2)Ciba Geigy AG, dept. Biotechnology, CH4002 Basel, Switzerland.

Aspergillus species, and in particular A. niger, are increasingly used as hosts for the expression and secretion of large quantities of homologous and heterologous industrial enzymes. Yields of the target proteins vary considerably and one cause of low yields is recognized as being proteolytic degradation of the heterologous proteins by host proteases. The observed problems in heterologous expression in fungi combined with the industrial applications of proteases have increased the interest in fungal proteases. We have cloned a gene encoding a serine carboxypeptidase, pepF, from Aspergillus niger. The sequences were identified in a lambda genomic library using a synthetic oligodeoxyribonucleotic probe, based on the N-terminal sequence of PEPF. Nucleotide sequence data from pepF genomic and cDNA revealed that it is composed of four exons of 199, 283, 277 and 881 bp. Three introns which interrupt the coding sequence are resp. 53, 69 and 59 bp in length. The sequence of the pepF gene codes for a polypeptide of 530 amino acids. Directly downstream from the putative start codon lies a sequence that is removed by proteolytic cleavage at a monobasic site (Lys52). The cloning of another carboxypeptidase (CPDI) encoded by the pepG gene was also reported. Northern blot analysis of total cellular RNA extracted from A. niger cells indicate that the pepF gene is transcribed as a single 1.8-kb mRNA. In addition we show that the expression the pepF gene in A. niger N400 is regulated by a number of factors including nitrogen and carbon repression, specific induction and the pH of the culture medium.

12. Guest: a small inverted repeat transposable element in Neurospora crassa.

P. Jane Yeadon, Amanda H. Taylor and David E. A. Catcheside, Flinders University, Adelaide, Australia.

Amongst the sequence differences between the St Lawrence 74A and Lindegren strains is a 101 bp insert in St Lawrence 2.0 kb 3' of the histidine-3 locus in linkage group I. This insert is flanked by a 3 bp direct repeat of a sequence present in Lindegren, has terminal inverted repeats (TIR) and shares features with several known transposable elements. At 98 bp, it may be the smallest transposable element yet found in eukaryotes. There are multiple copies of the TIR in the Neurospora genome, similar but not identical to the one sequenced. PCR amplification of Neurospora genomic DNA using 26 bp of the TIR as a single primer, gave products of discrete sizes ranging from 100 bp to about 1.3 kb, suggesting that the element isolated (Guest) may be a deletion derivative of a family of larger transposable elements. Two further iterations of Guest have been mapped in St Lawrence 74A, one to each of linkage groups III and V. Guest appears to be the second DNA intermediate transposable element reported for fungi and the first in Neurospora.

13. Novel method for differentiating populations and subpopulations of fungi.

S. Akkaraju, D. Steineker, and M.H. Perlin. University of Louisville, Louisville, KY.

Often it is useful to be able to track populations of organisms, both for ecological examination of host/pathogen interactions as well as for other questions in population genetics. Other approaches to these questions have employed DNA fingerprinting techniques such as RFLP analysis, Southern hybridization, and RAPDs. These methods can be time-consuming, expensive and technically demanding. We have developed a method which is quicker, technically simple and inexpensive. The method utilizes analyses of total DNA thermal denaturation profiles. As such, it has the advantage of detecting and utilizing microheterogeneities throughout the test organisms' genomes. By modifying the parameters for analyses one can differentiate species, species hybrids, and even parental lineages. To date, this method has been used by us to distinguish Cuban crocodiles from Cuban/American hybrids and to correctly predict familial relationships for alligators and red-winged blackbirds. The anther smut, Ustilago violacea, infects flowering species in the Carnation family. It has been suggested that the 70-or-so host species define "races" of the fungus, i.e., populations or subpopulations of the fungus are delimited by which host species they are able to infect. Fourteen different sporidial isolates from a total of 5 different host species were analyzed by DNA thermal profiling. Analyses were performed using total genomic DNA, mitochondrial DNA, and purified PCR products from each strain. In many cases, the cladograms produced from such analyses reflected known population differences. This suggests that the method provides a quick and simple alernative for characterizing different populations of fungi.

14. Characterization of the arg-12 UORF reveals complex regulatory coordinaton with a gene upstream of the arg-12 UORF.

Gloria E. Turner and Richard L. Weiss. University of California, Los Angeles

Ornithine carbamoyltransferase is encoded by the arg-12 gene in Neurospora crassa. The gene has been sequenced and encodes a polypeptide of 363 amino acids that has extensive homology to the mammalian and fungal OCTase's. Primer extension analysis revealed multiple transcription starts between nucleotides -344 and -259 relative to the initiator methionine at nucleotide 1. On the arg-12 sense strand an UORF begins at nucleotide -1345 and ends at nucleotide -710; it has a putative 65bp intron. The antisense strand has an overlapping UORF that starts at nucleotide -710 and ends at nucleotide -1357; it has a putative 96bp intron. The putative introns are also overlapping. Developmental northerns probed with sense and antisense RNA from the UORF show opposite expession patterns. The antisense probe hybridized to 2 different transcripts whose sizes are 3.6 kb and 3.0 kb. Expression of these transcripts is maximal at 8 hours. The sense probe hybridized to 3 transcripts, 3.6, 3.0 and 1.5 kb whose expression is maximal in conidia except for the 1.5 kb transcript whose expression is maximal at 8 hours; this expression, however, is much reduced relative to the antisense message levels for this time point and all time points examined. The larger transcripts are not detected at 8 hours by the sense probe. A 2.0 kb DNA fragment upstream of the UORF has been cloned. Developmental northerns and northerns using polyA RNA revealed hybridization to a 3.6 kb transcript. This mRNA is highly expressed in polyA RNA made from stationary phase mycelia. The identity of this gene and the role of the UORF in controlling expression of arg-12 and the unknown gene are being investigated.

15. A new locus for osmotic-sensitive mutants of Neurospora crassa.

Scott M. Buntin, Sara Neville Bennett, and Wayne A. Krissinger. Georgia Southern University, Statesboro, GA 30460.

SS-788, an osmotic-sensitive mutant of Neurospora crassa, was isolated following UV irradiation of wild type, 74A, conidia and a filtration-concentration procedure in minimal medium containing 3% NaCl. In contrast to typical osmotic mutants, the morphology of SS-788 resembled that of wild type. On medium containing 2% NaCl, hyphal elongation of SS-788 was reduced by about 80%, and there was no growth on medium containing 4% NaCl. Genetic analysis located the new os locus of SS-788 on LGVI, linked to ad-1 and trp-2. This is the first osmotic- sensitive mutant reported in this linkage group. It is proposed that the os locus in SS-788 be designated os-9.

16. Genetic and morphological analysis of KT-27, a new vinclozolin-resistant, osmotic-sensitive mutant of Neurospora crassa.

Regan M. Challinor, Wayne A. Krissinger, and Sara Neville Bennett. Georgia Southern University, Statesboro, GA 30460.

Several mutants which exhibited resistance to the dicarboximide fungicide, vinclozolin, were isolated. following UV irradiation of Neurospora crassa wild-type, 74A, conidia. These mutants were also found to be osmotic sensitive, consistent with previously reported work (M. Grindle. 1984. Trans. Br. Mycol. Soc. 82:635). Grindle also reported allelism of several of his vinclozolin-resistant mutants to the os-1 locus. Genetic analysis of our isolate, KT-27, also supports allelism to os-1. KT-27 exhibits altered hyphal morphology and pigmentation as compared to wild-type.

17. Isolation and characterization of UV-induced mutations affecting regulation of Neurospora arg-2.

Michael Freitag, Nelima Dighde and Matthew S. Sachs, Oregon Graduate Institute, Portland, Oregon

The N. crassa arg-2 gene encodes the small subunit of arginine-specific carbamoyl phosphate synthetase. Translational and transcriptional components are involved in negative regulation of arg-2 by arginine. A 24-codon upstream open reading frame (uORF) has been implicated in arginine-specific negative regulation. We transformed the N. crassa arg-12s pyr-3 strain with an arg-2 hph reporter gene containing the uORF. A transformant containing a single ectopically integrated copy of this reporter gene was able to grow on medium containing the antibiotic hygromycin B, but not when the medium contained both hygromycin and arginine. Forty seven mutants were isolated following UV mutagenesis that were able to grow on media containing both hygromycin and arginine. One mutant whose phenotype genetically cosegregated with the arg-2 hph fusion gene and which showed constitutive expression of hygromycin phosphotransferase was characterized. The sequence of the fusion gene revealed a missense mutation at codon 12 of the arg-2 uORF predicted to change Asp to Asn. Other mutations altered the expression of both the fusion gene and the endogenous arg-2 gene, suggesting functional changes in one or more trans-acting factors that affect expression of both genes directly, or which have roles in determining intracellular levels of arginine.

18. Translational regulation of arg-2 and cpc-1 in response to changes in amino acid availability in Neurospora crassa.

Zongli Luo, Michael Freitag, and Matthew Sachs, Oregon Graduate Institute, Portland, Oregon

N. crassa arg-2 specifies the small subunit of arginine-specific carbamoyl phosphate synthetase, and it is the only component in the arginine biosynthetic pathway known to be negatively regulated by arginine. Moreover, arg-2 is positively regulated in response to limitation for many different amino acids through a mechanism known as cross- pathway control. Neurospora cpc-1 specifies a transcriptional activator important for cross-pathway control. Arg2p synthesis rates, Arg2p levels, arg-2 mRNA levels and the distribution of arg-2 mRNA in polysomes in wild-type N. crassa cells were examined under different conditions of amino acid availability. The amount of cpc-1 mRNA and its distribution in polysomes were also examined. Both translational and transcriptional components appeared to contribute to negative regulation of arg-2 in response to arginine and positive regulation of arg-2 and cpc-1 in response to amino acid limitation. The arg-2 and cpc-1 transcripts contain upstream open reading frames whose features suggest they have roles in translational control.

19. Demonstration of the inducible and constitutive forms of aryl hydrocarbon hydroxylase (cytochrome P450) in Neurospora crassa

D.G. Freitag and M. Kapoor. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4

New therapeutic agents must be thoroughly investigated before approval is given for human consumption. One aspect of this research is to evaluate the manner in which drugs are metabolized oxidatively and whether the products are active. Traditionally, such information has been obtained from animal models. However, with growing concern over animal testing, acceptable alternatives are being explored. A logical approach is to utilize fungi containing cytochrome P450 enzyme systems, analogous to those found in human cells. One such microorganism is the filamentous fungus, Neurospora crassa, which we found to possess an inducible and constitutive form of the cytochrome P450 aryl hydrocarbon hydroxylase. This was demonstrated by the biosynthesis of the 3-hydroxy metabolite of Benzo(a)pyrene, in crude mycelial extracts. Enzymatic activity was determined fluorometrically in uninduced and induced 16- and 96-h mycelium. For induction, 1 mg/ml of Benzo(a)pyrene was added to the cultures at 24 h. Results of our experiments showed enzymatic activity in the uninduced 16-h cells as well as the 96-h induced mycelium. No activity was detected in 96-h uninduced cultures. N. crassa was chosen as a model for studying oxidative biotransformation reactions because it is a genetically and biochemically well-characterized organism, and genomic and cDNA libraries are available. This will permit us to study the expression of genes encoding these enzymes, thereby facilitating direct comparisons with mammalian systems.

20. Identification of NIT4 binding sites within the nit-3 promoter.

Sarah Evans and George A. Marzluf, Ohio State University, Columbus, Ohio.

The expression of nit-3 and nit-6, structural genes in Neurospora crassa which encode nitrate and nitrite reductase, respectively, requires limitation of primary nitrogen sources and induction by means of nitrate. Transcriptional activation of these genes also requires the action of NIT2 and NIT4, positive regulatory proteins. NIT2 is a global acting regulatory protein, also needed for expression of a variety of other nitrogen metabolizing enzymes, while NIT4 is a pathway specific regulatory protein. NIT4 contains 1090 amino acids with a putative Cys6 zinc cluster DNA binding domain, similar to that of GAL4, found near the amino terminus. A NIT4/betaGAL fusion protein has been expressed and purified, and its DNA binding specificity determined. Two NIT4 binding sites of varying strengths in the nit-3 promoter were identified through mobility shift and DNA footprinting experiments. The stronger site contains the palindromic sequence TCCGCGGA, while the weaker site contains the related sequence TCCGTGGC. Related sequences are found in the nit-6 promoter. Further examination of this octomeric binding site and its flanking sequences is currently being done.

21. Developmental expression of Neurospora crassa HSP80.

Tara L. Lozon, Y. Yijayaraghavan, and M. Kapoor, University of Calgary Calgary, Alberta, Canada.

The heat shock response of Neurospora crassa, has been studied previously and the production of hsp70 and hsp80 transcripts, encoding two major heat shock proteins (HSPs), has been demonstrated to occur upon heat shock for 1 hour. With Northern blot analysis, we have demonstrated that not only is there an increase in the transcription of hsp70 and hsp80 under conditions of externally applied stress but an increase when the cells undergo an internal stress. Internal stress can be caused by active mycelial growth or by conidial germination. The expression of HSP80, under internal and external stress conditions was assessed by protein blot analysis using polyclonal anti-HSP80 IgG preparations and by monitoring hsp80-specific mRNA levels by Northern blot analysis. Sporulating 2-day and 4-day old cultures, subjected to heat shock, showed an increase in both the mRNA and protein levels compared to the same states that were not heat-shocked. However, the non-shocked 15-hour-old mycelial cells showed and elevated hsp80 mRNA and protein level over the heat-shocked 2 and 4-day cultures. Furthermore, there was an increase of hsp80 mRNA and protein levels in non-shocked germinating conidia. It is possible that HSP80 is required for the growth and germination, as a molecular chaperone to assist accurate protein folding and assembly.

22. The detection of a stress inducible DNA-binding protein in Neurospora crassa.

Patricia M.J. Ouimet and M. Kapoor, University of Calgary, Calgary, Alberta, Canada.

Heat shock protein (Hsp) gene promoters are typically silent until activated by various stresses especially high temperature. Induction depends upon a specific DNA sequence, the heat-shock element (HSE). As an initial step to clarifying this activation mechanism, a Neurospora crassa factor which requires DNA binding activity upon stress induction has been identified. For our analysis, the Neurospora radiolabelled HSP70 promoter fragment (Cr27-Cr16) containing the putative HSE(s) was utilized to screen Southwestern blots of control (14 hr, 28 C) and heat-shocked (48 C, 1 hr) N. crassa mycelium. Cr27-Crl6 was found to interact specifically with a polypeptide of approximately 60 kDa in both samples but stronger binding was witnessed in heat-shocked extracts. In addition we have demonstrated that oxidative stress was able to induce binding of this protein to HSE sequences. Neurospora cells grown in varying plumbagin concentrations, an oxygen radical producing compound, were also subjected to Southwestern analysis with Cr27-Cr16 probe of hsp70 gene. Once again, the 60 kDa protein was found to bind more strongly in these samples than in control mycelium. Preliminary purification of this protein was conducted using ammonium sulfate precipitation, ion exchange chromatography and resolution on size-fractionation columns. This heat and oxidative stress activated DNA-binding protein is probably the heat shock transcription factor (HSF) of N. crassa.

24. Generation of minichromosome variability during meiosis in Glomerella graminicola.

Jeffrey A. Rollins and Robert M. Hanau. Dept. of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907

Pulsed-field electrophoresis and Southern hybridization analysis were employed to establish electrophoretic karyotypes and to study sequence relationships, genetic makeup and meiotic stability of minichromosomes in six isolates of Glomerella graminicola. Three to five minichromosomes ranging in size from approximately 400 kb to 1.2 Mb were present in each isolate. No two isolates had identical minichromosome profiles. Southern hybridization analysis with four minichromosome-specific clones revealed that each clone hybridized with no more than one chromosome per isolate, and in some cases failed to hybridize with any chromosome. The organization of these sequences also varies among isolates. Sequences found on the same minichromosome in one isolate were found on two minichromosomes in another isolate. Variation in minichromosomes among isolates is, therefore, the result of structural rearrangements and either sequence deletions or additions. The presence of transcriptionally active sequences residing on minichromosomes was established by the isolation of a cDNA clone which hybridized to a single minichromosome in each isolate. Karyotype analysis of tetrad progeny recovered from controlled crosses revealed nonparental chromosomes. Hybridization analysis and identification of reciprocal, nonparental homologs in tetrad progeny indicated that sequence deletions and recombination between homologous chromosomes with size polymorphisms were responsible for generation of nonparental chromosomes. We conclude that minichromosomes within G. graminicola isolates are not genetically inert. They share sequence homologies, undergo meiotic recombination resulting in the generation of novel sized chromosomes, and contain transcriptionally active sequences.

25. Homologues of aflatoxin biosynthetic genes in Aspergillus oryzae.

A.J. Watson(1), D.B. Archer(1), S. Seal(2) and J.E. Linz(3). (1)Department of Genetics and Microbiology, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. (2)Natural Resources Institute, Central Avenue, Chatham Maritime, Kent ME4 4TB, U.K. (3)Dept. of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824-1224, U.S.A.

Aspergillus oryzae is a non-toxigenic species used in food production and industrial enzyme production. A. oryzae is not known to produce aflatoxins but is closely related to the toxigenic species Aspergillus flavus and Aspergillus parasiticus. We have shown by Southern blotting using stringent hybridization conditions that A. oryzae contains sequences that hybridize to known structural and regulatory genes that encode enzymes in the aflatoxin biosynthetic pathway. Expression of these 'pseudogenes' did not occur under conditions known to induce aflatoxin biosynthesis in the toxigenic species. Sequences from A. oryzae have been cloned and characterized by sequence analysis. We report on the sequences obtained and discuss the possible reasons for A. oryzae being non-toxigenic.

26. Promoter elements required for arabinose induction of the beta-galactosidase (bgaS) gene from Penicillium canescens.

I.V. Nikolaev and Yu P. Vinetski, Research Institute for Genetics and Selection of Industrial Microorganisms, Moscow 113545, Russia.

A secreted beta-galactosidase from Penicilium canescens is an inducible enzyme and sugar beet pulp has been shown to be the most efficient inducer. Low molecular weight carbohydrates present in the culture fluid during fungal growth on sugar beet pulp were separated and tested for the ability to affect betaGal formation. The fraction capable of inducing the synthesis appeared to contain mostly L-arabinose. The inducing role of L-arabinose was also confirmed in transfer culture experiments. Besides beta-galactosidase, a number of other carbohydrolases were revealed in the P. canescens culture fluid upon arabinose induction, thus implying the existence of a common regulatory mechanism of expression of the whole set of genes coding for secreted proteins. To locate functional elements within the upstream region of the bgaS gene, promoter deletion analysis was initiated. A series of 5' non-coding deletion mutants were constructed and their effect on the bgaS gene expression was examined in Aspergillus nidulans, which was used as a host for gene regulation studies. The area required for arabinose induction is localized to a fragment of 300 bp, between -735 and -435 bp upstream of the ATG codon. A palindromic sequence 23 bp long containing a putative CREA binding site was observed within this fragment. Site specific mutagenesis of this motif resulted in a decreased level of the bgaS gene expression instead of expected release from carbon catabolite repression.

27. Developmental factors are necessary for the production of toxic secondary metabolites in Aspergillus nidulans.

Robert A.E. Butchko(1), Nancy P. Keller(1), Jenny Wieser(2), Thomas H. Adams(2). (1)Dept. Plant Path. and Micro. and (2)Dept. Biology, Texas A&M University, College Station, TX, 77843

Aflatoxin (AF) and sterigmatocystin (ST) are biochemically related toxic secondary metabolites produced by a number of aspergilli. A putative transcription factor, aflR, containing a binuclear zinc binding domain, has been found to regulate the AF pathway of A. flavus and A. parasiticus. Using a construct which places aflR under the control of an inducible promoter, we have previously shown that expression of aflR is sufficient to induce the expression of genes encoding enzymes necessary for the biosynthesis of ST in A. nidulans thus indicating that aflR is functionally conserved between Aspergillus spp. A number of A. nidulans mutants defective in development have been described that do not conidiate properly, and produce no or little ST. To determine if there is a link between development and secondary metabolism, we crossed the inducible aflR construct into the developmental mutants. This allows the induction of the aflR gene to test whether or not it is sufficient for the induction of pathway genes. Preliminary evidence indicates that the expression of aflR is sufficient to induce pathway genes in some, but not all, of the developmental mutants. This result suggests that the role of aflR in activating ST biosynthesis requires elements that are also needed for development.

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