Secondary Metabolism

109. Aspergillus flavus mutant strain 241, blocked in aflatoxin biosynthesis, does not accumulate aflR transcript.

Matthew P. Brown and Gary A. Payne, North Carolina State University, Raleigh, NC 27695

Eleven non-allelic aflatoxin biosynthesis mutants in A. flavus have been mapped to linkage groups (LG), 10 to LG VII, which includes genes in the aflatoxin biosynthetic cluster, and one in strain 241 to LG 11. The absence of a colored intermediate in strain 241 and the physical separation of its mutation from the biosynthetic cluster, suggest that the mutated gene may be regulatory in nature. An analysis of the RNA transcripts from strain 241 (tan, afl-4, pdx6 indicates that aflR message is absent from this strain. Genetic analysis demonstrated that the mutation in 241 is not due to a non-functional copy of aflR. Transformants containing an aflR::GUS construct failed to exhibit -glucuronidase activity, indicating that the aflR promoter is not activated in this strain. Transformants provided with aflR message, by a construct expressing aflR via a heterologous promoter, were restored in both aflR transcript and aflatoxin biosynthesis. These data allow us to putatively identify the afl-4 mutation as an upstream regulatory gene controlling the transcriptional activity of aflR Strain 241 exhibits no abnormalities in development or morphology, thus distinguishing it from developmental mutants which have been shown to also be affected in aflatoxin biosynthesis. A cosmid genomic library is being screened in strain 241, and progress in the cloning of the regulatory gene mutated in strain 241 by complementation will be presented.

110. A visual screen to detect Aspergillus nidulans mutants defective in aflR regulation.

Robert A.E. Butchko1, Thomas H. Adams2 and Nancy P. Kellerl. lDepartment of Plant Pathology and Microbiology and 2Department of Biology, Texas A&M University, College Station, TX

Aspergillus nidulans possesses a 60 kb gene cluster containing 25 coregulated transcripts involved in the biosynthesis of the mycotoxin sterigmatocystin (ST). The sixth gene in the cluster, aflR, is a positively acting transcription factor required for the expression of most, if not all, of the remaining 24 cluster genes (called stc genes for sterigmatocystin cluster). aflR activity can be visually assessed in A. nidulans TSS40 (wA, methGl, biA1; stcE::argB) because a stcE mutation results in the accumulation of norsolorinic acid (NOR, an orange colored intermediate in the ST biosynthetic pathway) rather than ST. Chemical mutagenesis of TSS40 has resulted in ~100 mutants unable to produce NOR. Genetic analysis of the mutants show that some contain mutations that are linked and some unlinked to the ST cluster. The linked mutations could represent lesions in one of four cluster genes required to produce NOR (aflR, stcA, stcJ and stcK) whereas the unlinked mutations potentially represent an aflR trans-acting regulatory factor. We are characterizing mutants in each class through genetic complementation and isolation of the gene(s) involved in the defect.

111. Development of a vector for introducing a deletion in the aflatoxin blosynthetic pathway of Aspergillus parasiticus and A. flavus.

J.W. Cary, N. Barnaby, B. Montalbano, K.C. Ehrlich, and D. Bhatnagar. USDA-ARS, Southern Regional Research Center, New Orleans, LA 70124.

Transformation of A. parasiticus RHNL with a norA disruption construct fortuitously gave rise to a transformant that was found to have undergone an approximate 6 kb deletion of a region of the aflatoxin biosynthetic pathway. The deletion event resulted in the partial and complete loss of the known aflatoxin biosynthetic genes avnA, ver-1, and orf2 (a homolog of A. nidulans stcS) respectively. Due to the loss of function of the avnA gene in the deletion mutant, it was unable to further metabolize averantin, and therefore this metabolite was the main polyketide found in the product mixture. However, besides averantin, methylaverantin, represented 16% of the recovered product mixture. Utilizing PCR techniques a vector has been constructed that was used to introduce this deletion into A. parasiticus or A. flavus via homologous recombination. One area of intense interest has been in the development of biological control agents to reduce levels of aflatoxin contamination in field crops. A. flavus strains in which the aflatoxin pathway deletion has been introduced should make for excellent biocontrol agents with respect to control of aflatoxin contamination of susceptible crops.

112. Characterization of a gene involved in singlet oxygen resistance in Cercospora nicotianae.

M. Ehrenshaft, A.E. Jenns, and M.E. Daub. NC State University.

Singlet oxygen is an extremely toxic form of activated oxygen against which cells have few effective defense mechanisms. Photoactivated compounds that produce singlet oxygen (photosensitizers) are of diverse chemical structure and are ubiquitous in nature. Numerous Cercospora species synthesize cercosporin, a polyketide photosensitizer, which is toxic at micromolar concentrations to many other fungi, most bacteria, virtually all plants and even mice. Cercospora fungi, however, can accumulate up to 1 mM cercosporin in culture without measurable effect, and are also resistant to other, diverse singlet oxygen-generating photosensitizers. Using functional complementation of mutants sensitive to cercosporin and other photosensitizers, we have isolated a clone that appears to confer singlet oxygen resistance to C. nicotianae. Sequence analysis revealed an open reading frame encoding a protein of 343 amino acid residues. Data base searches found several protein sequences with 55-75% homology to this predicted polypeptide. None of the homologous sequences have a known function. Two are from higher plant expression libraries while the rest are open reading frames derived from large-scale sequencing projects. Studies are underway to determine if our gene is regulated or expressed constitutively and if it can be expressed in other organisms to confer cercosporin and/or singlet oxygen resistance.

113. Effects of deletions in the aflR promoter on overproduction of averantin by Aspergillus parisiticus.

Kenneth C. Ehrlich, Beverly G. Montalbano, Deepak Bhatnagar, and Thomas E. Cleveland, Southern Regional Research Center, ARS USDA, New Orleans, LA 70179.

The gene, aflR, encodes a CYS6-zinc clusters sequence-specific, DNA-binding protein that appears to activate all of the genes in the aflatoxin biosynthesis gene cluster. Previously, we found that A. parasiticus containing extra copies of aflR gave reddish-orange pigmented colonies resulting mainly from nine-fold higher yields of averantin and other pigmented metabolites. We hypothesized that this overproduction resulted from self-activation by AFLR. We, therefore, prepared for transformation of A. parasiticus niaD-containing plasmids where the region from -220 to -12 bp upstream of the aflR translation start site was mutated using overlap extension PCR. Transformants in which this entire region was deleted had normal levels of metabolite production, indicating that they contained an intact copy of aflR that was not affected by the mutant copy. When only a portion of the palindrome, TTAGGCCTAA, identified previously as an AFLR autoregulatory site, was deleted, clones with this altered copy of aflR had one-fourth the level of averantin as did clones with an extra copy of intact aflR, but still almost 2-fold more than the untransformed strain. Clones in which two other dyad symmetric sites were deleted gave averantin yields equal to those of wild-type transformants, suggesting that these sites are not involved in autoregulation of aflR expression.

114. Role of polyamines in sporulation and mycotoxin formation in Aspergillus nidulans.

Guzman de Pena, D. and Ruiz-Herrera, J. Departamentos de Ingenieria Genetica y Genetica y Biologia Molecular. Unidades Irapuato y Mexico, D.F. Centro de Investigacion y Estudios Avanzados del IPN. Mexico.

The importance of polyamines during cell growth and differentiation in eucaryotes has been substantiated, although their precise mode of action remains unknown. In A. nidulans there occurs an increase in the levels of the enzymes involved in polyamine synthesis during spore germination . Similar results have been observed during cell differentiation in other fungal systems. Addition of the ornithine decarboxylase (ODC) competitive inhibitor diaminobutanone (DAB), leads to the inhibition of differentiation in these systems

Previously we have decribed (Fung. Gen. Biol., in press) that DAB inhibits both sporulation and aflatoxin biosynthesis in Aspergillus parasiticus. With these data we have proceeded to analyze the probable role of polyamines in sporulation and sterigmatocystin biosynthesis in A. nidulans FGSC A26, and brlA1-, and puA- (ODC-) mutants. We observed that blocking the synthesis of polyamines by the ODC competitive inhibitor DAB, inhibited sporulation of the wild type strain, and the formation of aerial mycelium in brlA-. Growth of puA with limiting concentrations of putrescine did not inhibit vegetative growth, but severely affected sporulation. Under all these conditions, synthesis of sterigmatocystin was inhibited, and addition of putrescine restored the normal phenotype in all cases. We then proceeded to analyze the effect of DAB on the transcription of brla in A. nidulans. It was observed that DAB inhibited sporulation and sterigmatocystin formation. RNA was purified from both cultures and hybridized under stringent conditions with a brlA fragment from pBS2.5. The results showed that accumulation of the brlA transcript, was inhibited when the fungus was grown in the presence of DAB. According to these results we may suggest that polyamines play a role in sporulation and the regulation of mycotoxin formation in A. nidulans, at a step previous to the execution point of brlA.

115. Cometabolic degradation of methyl tert-butyl ether (MTBE) by the filamentous fungus Graphium after growth on gaseous n-alkanes and diethyl ether.

L.K. Hardison, M.R. Hyman, and L.M. Ciuffetti. Dept. of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97330-2902.

Ether-bonded compounds are in general recalcitrant to biological transformation. In this study we demonstrate that the filamentous fungus Graphium sp. can utilize diethyl ether as a growth-supporting substrate, and that the gasoline oxygenate methyl tert-butyl ether (MTBE) can be rapidly degraded by mycelia grown on diethyl ether or gaseous n-alkanes. Graphium is one of the few eukaryotic organisms known to grown on gaseous n-alkanes (ethane, propane, n-butane); the initial oxidation of these substrates is thought to be catalyzed by an inducible cytochrome P450 enzyme. Several observations lead us to conclude that in Graphium gaseous n-alkanes, MTBE, and diethyl ether are oxidized by the same enzyme, a putative cytochrome P450, and that MTBE oxidation is a cometabolic process. First, acetylene and other unsaturated gases are potent inhibitors of growth on both n-alkanes and diethyl ether, and also inhibit MTBE degradation in n-alkane- and diethyl ether-grown mycelia. Second, MTBE and n-butane (or diethyl ether) are competitive substrates, although the presence of a growth-supporting substrate is required for sustained MTBE degradation. In contrast, MTBE is not degradad by mycelia grown on non-specific media. The maximal rate of MTBE degradation we have observed is ~10nmoles MTBE/h/mg dry weight. We have detected two major products of MTBE degradation, tert-butyl formate (TBF) and tert-butyl alcohol (TBA). Kinetic studies demonstrate that TBF is the first detectable product of MTBE oxidation and that TBF is hydrolyzed both biotically and abiotically to yield TBA. Our studies suggest a potential role for n-alkane-utilizing fungi in the bioremediation of MTBE and diethyl ether-containing solvent contamination.

116. Regulation of Mycotoxin Biosyntheis in Aspergillus spp.

J. K. Hicksl, J.-H. Yu2, N.P. Kellerl, and T.H. Adams2. Dept. of Plant Pathology and Microbiology l and Dept.of Biology2, Texas A&M University College Station, 77843.

Several species of the fungal genus Aspergillus, including Aspergillus nidulans, produce sterigmatocystin (ST) which also serves as the penultimate precursor in the aflatoxin (AF) biosynthetic pathway. Because AF and ST are among the most toxic, carcinogenic, and mutagenic compounds produced in nature, contamination of commercial food crops by these toxins results in significant health and economic losses. We are interested in finding means by which toxin synthesis is controlled during the fungal lifecycle and have found that flbA and fluG, two genes required for asexual sporulation in A. nidulans, are also needed for ST biosynthesis. In addition, overexpression of flbA in submerged culture causes both activation of conidiation and ST production. We have shown that FlbA functions in negatively regulating FadA, the alpha subunit of a heterotrimeric G-protein that functions in stimulating a proliferative growth pathway. Thus, a common link between sporulation and ST biosynthesis is a requirement for endogenous growth control. We have found that this requirement for growth control in secondary metabolism is conserved in the closely related A. parasiticus AF pathway. Recently, we have identified and begun characterizing several putative A. parasiticus flbA homologues.

117. DNA polymorphisms in sterigmatocystin biosynthetic pathway genes of Aspergillus nidulans, A. rugulovalvus and a related taxon.

Maren Klich, Ed Mullaney and Catherine Daly, USDA/ARS/SRRC, New Orleans LA.

Stergimatocystin is a mycotoxin blosynthetically similar to aflatoxin. Sterigmatocystin is produced by over 20 Aspergillus species. Several of these species have teleomorphs in the genus Emericella, including an as yet poorly described taxon which has morphological affinities with A. nidulans and A. rugulovalvus which both also have sexual states in Emericella. One strain of this new taxon is a patent strain improperly described as A. nidulans var. roseus. Strains of all three of these taxa have been isolated from cotton field soils in the desert southwest of the US, indicating that the species have ecological similarities. Morphological and physiological studies have shown that this new taxon is more closely related to A. rugulovalvus than A. nidulans. To help establish the molecular affinities of this new taxon in the important area of mycotoxin biosynthesis, we compared RFLPs of total DNA of a number of strains of each taxon utilizing previously cloned genes from the aflatoxin/sterigmatocystin biosynthetic pathway.

118. Regulation of iron homeostasis in Ustilago maydis .

Leong, S. A.1,2, Zhao, Q.2,3, Yuan, W.2, An, Z.2#, Gentil, G.2*, Mei, B.2+, Budde, A.1^ and J. Markley.3.

USDA/ARS PDRU,1 Department of Plant Pathology,2 Department of Biochemistry,3 University of Wisconsin, Madison.

Present address: #ChemGenics, Inc., Boston, MA; *University of Montreal; +Promega, Inc., Madison, WI; ^USDA Barley and Malt Lab, Madison, WI.

Regulation of cellular iron homeostasis requires a careful balance between uptake of adequate iron for metabolism and restriction of iron uptake such that deleterious iron-catalyzed free radicals are not generated in vivo. In response to iron starvation Ustilago maydis produces the cyclic peptide siderophores ferrichrome and ferrichrome A (Budde, A. D. and S. A. Leong, 1989, Mycopathologia 108:125-133). Two clustered genes, sid1 and sid2, are required for ferrichrome siderophore biosynthesis. sid1 encodes ornithine-N5-oxygenase (Mei, B., Budde, A. and S. A. Leong, 1993, Proc. Natl. Acad. Sci. U. S. A. 90:903-907) while sid2 encodes a ferrichrome peptide synthetase.

Regulation of sid1 has been studied by GUS reporter analysis and in vitro electrophoretic gel mobility shift analysis (An, Z., Mei, B., Yuan, W. M. and S. A. Leong, In Press, EMBO J). As expected, GUS activity was regulated by the availability of iron. Sequences required for iron-mediated regulation of sid1 were identified by deletion and site-directed mutagenesis. Two GATA motifs located 1.6 kb upstream of the transcription initiation site were required to mediate repression of sid1. Previous studies have led to the cloning of urbs1, a putative repressor of sid1 (Voisard, D., Wang, J., McEvoy, J., Xu, P. and S. A. Leong, 1993, Mol. Cell. Biol. 13:7091-7100). Electrophoretic gel mobility shift analysis using these motifs as well as GAT mutant derivatives as DNA probes indicated that Urbs1 binds specifically to the GATA sequences of sid1. Mutation of the C-terminal finger motif but not the N-terminal finger motif of Urbs1 significantly reduced DNA binding activity. Efforts are underway to purify Urbs1 from U. maydis and to determine how Urbs1 senses intracellular levels of iron in cells. In vitro DNA binding and in vivo reporter gene analysis has revealed that siderophores are not corepressors of Urbs1. As with the bacterial homologue Fur, we anticipate that iron may interact directly with Urbs1 to mediate repression of target genes.

119. A CCAAT binding protein (PENR1) regulates all Aspergillus nidulans structural genes required for biosynthesis of the secondary metabolite penicillin.

Olivier Litzka, Katharina Then Bergh and Axel A. Brakhage. Lehrstuhl fur Mikrobiologie, Universitat Munchen, MariaWard-Str. la, D-80638 Munchen, FRG.

The -lactam antibiotic penicillin is a classical secondary metabolite produced by some filamentous fungi, e. g. by Aspergillus nidulans. The penicillin biosynthesis is catalysed by three enzymes which are encoded by the following three genes: acvA (pcbAB), ipnA (pcbc) and aat (penDE). The genes are organised into a gene cluster. AcvA and ipnA are bidirectionally oriented, separated by an intergenic region of 872 bp. The aat gene lies 825 bp downstream of ipnA. Promoter analysis using deletion constructs, band shift and methyl interference analyses led to the identification of two CCAAT containing DNA elements, one of which is located between acvA and ipnA, and the other one in the aat promoter region. These elements were specifically bound by a protein (complex) designated PENR1, for penicillin regulator. In addition to the CCAAT sequence, some of the neighbouring nucleotides were also required for binding of PENR1. Specific deletion of the PENR1 binding site between acvA and ipnA led to a 10-fold increase of acvA expression and, simultaneously, to a reduction of ipnA expression to about 30%. Mutagenesis of the CCAAT core sequence in the aat promoter resulted in a four-fold reduction of aat-lacZ expression. Hence, PENR1 represents a major regulatory protein involved in the regulation of all penicillin biosynthesis genes. Formally, the CCAAT sites mediate a negative effect on acvA, but positive effects on both ipnA and aat expression.

Then Bergh et al. (1996) J Bacteriol 178:3908; Litzka et al. (1996) EJB 238:675

120. Biodegradation of the polyketide toxin cercosporin.

Thomas K. Mitchell and Margaret E. Daub, North Carolina State University, Raleigh, N.C.

Cercosporin is a photoactivated polyketide toxin synthesized by many species in the genus Cercospora. Cercosporin produces singlet oxygen when photoactivated, and production of cercosporin is highly correlated with the ability of these pathogens to cause disease on a wide range of hosts. The only resistance known to cercosporin is restricted to the fungi that produce it. In an attempt to identify genes which may have utility in engineering resistant plants, we have searched for bacteria able to degrade the toxin. We screened 160 isolates of bacteria representing 11 genera. Isolates from 4 genera produced cleared zones when grown on solid media containing cercosporin (which is normally red in color), suggesting that they are able to degrade cercosporin. Degradation of cercosporin was confirmed by toxin extraction from bacterial cultures grown in cercosporin containing medium. A pigmented compound extracted from liquid cultures containing cercosporin has been isolated from thin layer chromatography plates and is believed to be the breakdown product of cercosporin. This compound and the kinetics of its production are currently being characterized. Isolation of the gene necessary for the detoxification of cercosporin is proceeding. Once isolated, we intend to mobilize it into tobacco tissue to create plants capable of degrading cercosporin, rendering them resistant to Cercospora diseases.

121. Rapid prescreen for antitumor activity with mutant B. subtilis assay.

Rosanna Pena-Muralla. Department of Biology, Ateneo de Manila University, Loyola Heights, Quezon City, Philippines.

In studies aimed at discovering new compounds with biological activity from microorganisms the choice of a suitable prescreen is important to eliminate those species with little or no activity and focus detailed studies on potentially promising isolates. Mechanism based screens are best used as leads to identify species with bioactive compounds. In our research, mushrooms and filamentous fungi from diverse sources were examined as novel sources of antiumor substances. A microbial prescreen with supersensitive mutant strains of Bacillus subtilis was successful in screening large numbers of cultures. The assay was highly specific and selective for cytotoxic activity. This was confirmed with in virto mammalian cell toxicity assays on murine cell lines. The mutant assay was easy to perform and results were readily available within 24-hours.

122. Aflatoxin production in yeast.

Rafael Prieto and Charles P. Woloshuk, Purdue University, West Lafayette, Indiana.

Aflatoxins are polyketide-derived secondary metabolites, produced by Aspergillus parasiticus and Aspergillus flavus, that are highly toxic, mutagenic and carcinogenic in animals and are suspected carcinogens in humans. The final enzymatic step in the aflatoxin biosynthetic pathway is the conversion of O-methylsterigmatocystin (OMST) to aflatoxin by an oxidoreductase. Transformants of A. flavus strain 649WAF2 containing a 3.3 kb genomic DNA fragment and the aflatoxin biosynthesis regulatory gene aflR converted exogenously supplied O-methylsterigmatocystin to aflatoxin Bl. A gene, Ord1, corresponding to a transcript of about 2 kb was identified within the 3.3 kb DNA fragment. The promoter region contained a putative AFLR binding site and a TATA sequence. The nucleotide sequence of the gene revealed an open reading frame encoding a protein of 528 amino acids with a deduced molecular mass of 60.2 kDa. The gene contained six introns and seven exons. Ordl encodes a cytochrome P450-type monooxygenase, and the gene was assigned to a new P450 gene family named CYP64. Heterologous expression of the ordl open reading frame in Saccharomyces cerevisiae under the transcriptional control of the yeast galactose-inducible gall promoter, resulted in the ability to convert O-methylsterigmatocystin to aflatoxin Bl. The data indicate that Ord1 is sufficient to accomplish the last step of aflatoxin biosynthesis.

123. Analysis of a Gibberella fujikuroi mutant deficient in a hydroxylation step of fumonisin biosynthesis.

Robert H. Proctor, Anne E. Desjardins, and Ronald D. Plattner. USDA/ARS National Center for Agricultural Utilization Research, Peoria, Illinois.

Gibberella fujikuroi mating population A is a pathogen of maize and sorghum and produces a family of toxins known as fumonisins that are associated with a number of mycotoxicoses. The B-type fumonisins consist of a 20-carbon-long backbone with two tricarboxylic acid moieties and one amino, two methyl, one to three hydroxyl groups. We have employed UV mutagenesis to study the genetics of fumonisin biosynthesis in G. fujikuroi. Three hundred forty-three strains derived from conidia that survived UV exposure were analyzed for their ability to produce fumonisins. Nine of the survivors failed to produce fumonisins and one (uv26) only produced fumonisin B3, which lacks the hydroxyl group at carbon atom 5 (C-5). Wild-type strains of the fungus produce fumonisins B1 and B2, which are C-5 hydroxylated, as well as B3. Genetic analyses revealed that the mutation segregates as a single locus and is linked to the fum2 and fum3 loci (previously identified in natural variants), the RAPD marker OPA16, and a putative aldehyde dehydrogenase gene on chromosome 1 of G. fujikuroi. In addition, fum3- and the uv26 mutation may be allelic because both confer the same fumonisin phenotype and no recombinant progeny were recovered from a cross between strains carrying these two markers. Currently, we are attempting to complement the uv26 mutation by transformation with cosmid libraries constructed with DNA from wild-type strains of G. fujikuroi.

124. Characterizing the interaction of Aspergillus parasticus and its host, peanut.

Frances Trail, Department of Botany and Plant Pathology, Michigan State University, East Lansing MI 48824

We hypothesize that there are specific factors in resistant lines of peanut that repress the invasion of Aspergillus parasiticus into the peanut pod and the synthesis of aflatoxin in the pod. The timing of induction and shutdown of expression of these resistance factors may be responsible for the resistance breakdown observed under certain field conditions (most notably drought and high temperatures). We seek to identify the natural resistance mechanisms present in the developing peanut pod that are important to limiting fungal growth and aflatoxin production initially by detecting the timing and location of known resistance genes in infected pods. Several peanut lines are now available that appear to have differing resistance mechanisms as observed in the field. As yet uncharacterized resistance mechanisms affecting aflatoxin production and infection can be detected by use of genetically engineered strains of A. parastiicus and by observation of infection at the cellular level. Once resistance mechanisms have been identified that are effective, their expression may be optimized through genetic engineering so that the resistance is protective over a longer period of time.

125. Isolation and Regulation of Genes of the Gibberellin Biosynthesis Pathway in Gibberella fujikuroi.

Bettina Tudzyski Thorsten Voss. Pia Linnemannstons and Veronika Homann. Westfilische Wilhelms-Universitat, Institut fur Botanik Schlossgarten 3, 48149 Munster, Germany.

The rice pathogen Gibberella fujikuroi accumulates large amounts of gibberellic acid (GA3) and some other gibberellins which induce the superelongation disease on infected rice seedlings. The biosynthetic pathway for gibberellins has been established from the identification of intermediates and by using mutants affected in the gibberellin biosynthesis. However, genetics of gibberellin formation and regulation of this biosynthetic pathway are not well understood until now. Therefore, we first isolated some of the versatile genes of the central isoprenoid pathway which are involved in the biosynthesis of sterols, carotenoids and gibberellins. So far, the genes coding for HMG-COA reductase, FPP- and GGPP syntheses were isolated and characterized. The sequence comparison with analogous genes of other fungi and plants showed that the isoprenoid pathway must be highly conserved.

Beside those genes from the central terpenoid pathway, we are trying to isolate the most important genes from the gibberellin-specific part of the pathway such as the kaurene synthetase-. the kaurene oxidase-and the C20-oxidase genes. On the basis of sequence alignment of the recently isolated corresponding plant genes, we designed specific PCR-primers in order to amplify, parts of the corresponding Gibberella genes. Additionally, REMI mutagenesis and differential cDNA screening were used to isolate structural or regulatory genes of the gibberellin pathway. Furthermore, in order to understand the nitrogen and carbon repression of gibberellin formation on molecular level, the corresponding major regulatory genes, areA, nmr (nitrogen ) and creA (carbon) were isolated. Since we could show by Northern analyses that the recently isolated genes are expressed constitutively, the target genes for both of the different regulation types should be in the more specific part of the gibberellin pathway e.g. in the kaurene synthetase gene coding the formation of the first intermediate with the specific gibberellin sceleton.

126. Plasmid tagging of paxilline biosynthetic genes in Penicillium pasilli.

Carolyn Young1, Yasuo Itoh2, Richard Johnson1, and Barry Scott1. 1Molecular Genetics Unit, Department of Microbiology

and Genetics, Massey University, Palmerston North. 2Department of Biology, Shinshu University, Matsumoto, Nagano, 390 Japan.

Penicillium paxilli is a filamentous fungi that produces large quantities of the tremorgenic mycotoxin, paxilline, in submerged cultures. This compound is of particular interest as it is an important intermediate in the biosynthesis of several other mycotoxins, including lolitrem B, that is produced by Acremonium endophytes in association with perennial ryegrass. We are interested in cloning some of the genes involved in paxilline biosynthesis with the aim of understanding the regulation of this pathway in both P. paxilli and A. lolii.

One of the strategies we are using to clone genes involved in paxilline biosynthesis is that of pAN7-1 plasmid tagging (Itoh et al. 1994; Itoh and Scott 1994). Using a monoclonal based ELISA assay for the detection of paxilline, 600 single-spore purified pAN7-1 transformants of P. paxilli were screened for paxilline production and one Pax- mutant, YI-20, was identified. Molecular analysis of this mutant showed that pAN7-1 had integrated at a single site but was present in 4 - 6 copies in tandem array. Rescue of sequences flanking the point of integration and subsequent molecular analysis revealed that YI-20 consisted of an extensive deletion and a chromosomal rearrangement between chromosomes V and VI. Gene disruptions at the chromosome V locus resulted in the generation of two additional Pax- mutants CY-2 and CY- 102. Both of these new mutants were the result of single crossovers and had deletions extending beyond the mapped region but did not have chromosomal rearrangements. The extent of the deletion in these three Pax- mutants is currently being mapped by chromosome walking.

127. Aflatoxin biosynthetic pathway gene cluster.

J. Yul, P.-K. Chang2, J.W. Caryl, D. Bhatnagarl, T, E, Cleveland1, N. P. Keller3, J. E. Linz4, G. A. Payne5, C. P. Woloshuk6,

and F. S. Chu7 1USDA/ARS/SRRC, New Orleans, 2Tulane Univ,, New Orleans, 3Texas A &M Univ, College Station,

4 Michigan St. Univ., East Lansing, 5North Carolina St. Univ., Raleigh, 6Purdue Univ., West Lafayette, 7Univ, of Wisconsin, Madison.

Aflatoxins, secondary metabolites of Aspergillus flavus and A. parasiticus, are furanocoumarins which are toxic and extremely carcinogenic in animal systems. A comprehensive and cooperative research effort has resulted in important breakthroughs in understanding of the molecular regulation of aflatoxin biosynthesis, A "gene cluster" of about 70 kilobases in size has been revealed upon which resides a DNA-binding protein thought to function in aflatoxin pathway regulation. In addition, several aflatoxin pathway genes encoding P450 monooxygenases, dehydrogenases, 0-methyltransferases, polyketide synthase and fatty acid synthase, and some yet uncharacterized open reading frames have been localized on the gene cluster. Recent studies have revealed that there are several additional transcripts beyond the 70 kb cluster; their specific involvement in aflatoxin biosynthesis is being examined. A similar cluster has been characterized from A. nidulans; A. nidulans produces sterigmatocystin (ST) as end product, which is the penultimate precursor of aflatoxin. The genes and the newly identified transcripts involved in aflatoxin and sterigmatocystin biosynthetic gene cluster in A. parasiticus, A. flavus, and in A. nidulans will be discussed.

128. Investigation of the intergenic promoter region of the ferrichrome siderophore biosynthesis Gene Cluster in Ustilago maydis.

Walter Yuan1 and Sally A. Leong1,2. lDepartment of Plant Pathology, University of Wisconsin, Madison, 1630 Linden Drive, Madison, WI.; 2Plant Disease Research Unit, USDA-ARS, Department of Plant Pathology, Univ of Wisconsin, Madison.

Two genes required for ferrichrome siderophore biosynthesis in Ustilago have been cloned and characterized. sidl encodes ornithine-N5oxygenase, while sid2 encodes a ferrichrome peptide synthetase. The two sid genes are divergently transcribed from a 4.5 kb intergenic region. Urbs1, a GATA-family transcription factor, regulates sid gene expression under different iron conditions. Multiple GATA motifs have been identified in the intergenic region by DNA sequence analysis, one pair of GATA sequences in the near center of the intergenic region was shown to be crucial for the iron-mediated regulation of sid1 expression. It is hypothesized that sid1 and sid2 expression may be divergently governed by the same promoter region. A twin reporter system will be exploited to test this hypothesis. LacZ and GUS reporter genes will be translationally fused in opposite orientation with the intergenic region in a self-replicating vector ans site directed mutagenesis will be performed to identify important cis elements. To eliminate copy number efects, the key dual reporter constructs will be further tested at single copy in U. maydis by gene replacement. The extent of the sid gene cluster and its coregulation will be assessed by Northern hybridization analysis of RNA extracted from cells grown in low and high iron media.