Mol Gen Genet (1992) 234:481488 © Springer-Verlag 1992

Molecular cloning of the imidazoleglycerolphosphate dehydratase gene of Trichoderma harzianum by genetic complementation in Saccharomyces cerevis&e using a direct expression vector G.H. Goldman, J. Demolder 1, S. Dewaele 1, A. Herrera-Estreila*, R.A. Geremia, M. Van Montagu, and R. Contreras 1 Laboratorium voor Genetica and 1Laboratorium voor Moleculaire Biologic, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium Received January 23, 1992 / Accepted April 4, 1992

Summary. The Trichoderma harzianum imidazoleglycerolphosphate dehydratase gene (igh) has been isolated by complementation of a Saccharomyces cerevisiae his3 mutant using a direct expression vector. This Escherichia coli-yeast shuttle vector was developed to allow efficient cloning and expression of cDNA libraries. The cDNA is 627 nucleotides long and codes for a protein of 209 amino acids with an apparent molecular mass of 22 466 daltons. The predicted protein sequence showed 63.6%, 58.7 %, and 38.4 % identity respectively to the corresponding enzymes from S. cerevisiae, Pichia pastoris and E. coil Northern analysis showed that the expression of the igh gene in T. harzianum is not inhibited by external histidine and the level of igh m R N A was about threefold higher in cells starved of histidine. Key words: Filamentous fungi - Complementation Yeast - Histidine biosynthetic genes - Shuttle vector

Introduction Parasitism is one of the main antagonistic relationships between microorganisms. When an organism attacks a pathogenic fungus in a biotic system, it can be considered as a mycoparasite (Chet 1990). During the last decade, many data have been accumulated on the efficacy of mycoparasites as efficient biocontrol agents (Baker 1987; Chet 1987). Trichoderma spp. are active as mycoparasites, having been extensively tested in field experiments and shown to be effective as biocontrolling agents against a range of economically important aerial and soil-borne plant pathogens (Chet 1987). Because the sexual stage (Hypocrea) is rare and there is no information about a parasexual cycle in Trichoderma spp., genetic manipulation using transformation and gene cloning * Present address: Centro de Investigaci6n y Estudios Avanzados, Unidad Irapuato, A.P. 629, Irapuato, Mexico Correspondence to : M. Van Montagu

provides the most logical directed approach to dissecting the physiology of these important fungi. Recently, various transformation systems for biocontrol strains of Trichoderma have been described, using as a selective marker a drug-resistant gene of prokaryotic origin (Goldman et al. 1990; Herrera-Estrella et al. 1990). Due to the large number of well-characterized mutants available in Saecharomyces cerevisiae as well as the phylogenetic proximity between these species, cloning metabolic genes of filamentous fungi by complementation of yeast offers many advantages. Such an approach has already been used for the isolation of the alcohol dehydrogenase gene of Aspergillus nidulans (McKnight et al. 1985). With this in mind, we decided to develop an expression system which would allow us to isolate genes of T. harzianum by complementation. In addition, new traits could be introduced into S. cerevisiae. We believe that metabolic genes should be the first target to test in this system since many biosynthetic pathways are genetically well described in yeast. In filamentous fungi, the biosynthetic pathways of most of the amino acids are poorly understood. Although histidine biosynthesis has been elucidated in several prokaryotic and eukaryotic microbes (Berlyn 1967; Burke and Pattee 1972; Carlomagno et al. 1988; Chapman and Nester 1969; Fink 1964; Kloos and Pattee 1965a, b; Legerton and Yanofsky 1985; Struhl 1985), there is very little information available about the genes encoding enzymes involved in this pathway in filamentous fungi. To our knowledge, only one such gene group has been cloned from Neurospora crassa (Legerton and Yanofsky 1985). In prokaryotes such as Salmonella typhimurium and Escherichia coli, the fourth gene of the histidine operon codes for a bifunctional enzyme possessing imidazoleglycerolphosphate dehydratase (EC 4.2.1.19) and histidinol phosphate phosphatase (EC 3.1.3.15) activities. The enzyme catalyses the seventh and ninth steps of histidine biosynthesis (Winkler 1987). In higher unicellular organisms, like N. erassa, the two activities are encoded by separate genes, his4 for the phosphatase and hisl for the dehydratase (Fink 1964). Here, we describe the develop-

482 ment of an E. coli-yeast shuttle vector, which allows efficient cloning and regulated expression of eDNA libraries. The T. harzianum imidazoleglycerolphosphate dehydratase gene (igh) has been isolated by complementation of a S. cerevisiae his3 mutant using this direct expression vector. We suggest that this gene codes only for the dehydratase activity and, thus, propose the existence of another gene, coding for the phosphatase activity. Materials and methods Strains and plasmids. T. harzianum strain IMI206040 was used throughout this work. The yeast strains employed in this work are listed in Table 1. Yeast strains were grown at 30°C in YPD medium (1% yeast extract, 2% Bacto peptone, 2% glucose) or in a defined medium containing 0.67% yeast nitrogen base (YNB) supplemented with 20 mg uracil/1 and the appropriate required amino acids; the carbon source was 2% glucose or 2% galactose (v/v). Bacterial strains were grown at 37 ° C in LB medium (0.5% yeast extract, 1% Bacto peptone, 1% NaC1, adjusted to pH 7.0) supplemented with ampicillin for selection when necessary. E. coli MC1061 was used for the storage and amplification of the yeast-E, coli plasmid. The same strain was also used for the isolation of the eDNA library by electroporation (Bio-Rad) and the frequency of transformation obtained was as described by Bio-Rad. Plasmid pSCGAL 10-SN (see below) was used for the preparation of the eDNA library. The library was stored as plasmid DNA at - 2 0 ° C and retransformed into E. coli cells from which the plasmid DNA was purified (Qiagen) and used for yeast transformation. Preparation o f cell walls. Ten discs of agar (approximately 1 cm z) with Rhizoctonia solani were inoculated per litre

of minimal medium (Del Rey et al. 1979). After 1 week of incubation at 28 ° C, with rotary shaking at 200 rpm, the mycelia were harvested by filtration through filter paper (Whatman no. 1) and washed thoroughly with distilled water. Washed mycelia were frozen in liquid nitrogen and lyophilized overnight. The dried mycelia were ground and the powder was resuspended in distilled water (2g/10ml). This mixture was centrifuged at 30000 x 9 for 20 rain at 4 ° C. The pellet was resuspended in distilled water and sonicated three times for 5 rain each (full amplitude). The suspension was centrifuged at 8000 x 9 for 20 rain at 4 ° C, then the pellet was resuspended in water and centrifuged again at 800 x g for 10min at 4°C (to precipitate coarse particles). The supernatant was centrifuged at 8000 x 9 for 20 rain at Table 1. Yeast strains employedin this

work

4 ° C and the pellet homogenized intermittently in water and centrifuged again until no residual nucleic acids could be detected. The precipitated cell walls were then deep-frozen, lyophilized, and kept as a powder in a sealed container until use. e D N A library. For the construction of the eDNA library, T. harzianum was grown for 24 h in minimal medium

(Geremia et al. 1991) using, as sole carbon source, cell walls (1 mg/ml) of the phytopathogenic fungus R. solani (prepared as described above). Total RNA and poly(A) + RNA were isolated according to an adaptation for the large-scale isolation of RNA from fungal tissues following closely the protocol of Jones et al. (1985) and using the PolyATtract mRNA isolation kit (Promega). The eDNA was prepared by the following protocol, based on the method of Okayama and Berg (1982). The mRNA (5 ~tg in a volume of 5 pl of H20) was heated to 70 ° C in a 1.5 ml centrifuge tube for 10 rain and quenched on ice. In a separate centrifuge tube, the following reagents were mixed: 4 ~1 of reaction buffer (250 mM TRIS-HC1, pH 8.3, 375 mM KC1, 15 mM MgC12), 2 gl of 0.1 M dithiothreitol, 1 gl of a deoxynucleoside triphosphate mix at 10 mM of each (Pharmacia), 2.5 gl of a NotI restriction fragment primer (Fig. 1C), at 2 pmol/~tl, and 0.5 gl of RNase Block II (Stratagene, 1 U/pl). This mixture was added to the mRNA. After 2 rain at 37° C, 5 gl of Superscript reverse transcriptase (BRL, 200 U/~tl) was added and the reaction mixture was incubated at 37 ° C for 1 h. To the reaction mixture was then added 88 gl of diethylpyrocarbonatetreated water, 30 gl of buffer [containing 100 mM TRISHC1, pH 6.9, 450 mM KC1, 23 mM MgC12, 0.75 mM 13-NAD +, 50 mM (NH,,)2SO4], 3 ~tl o f a deoxynucleoside triphosphate mix at 10 mM of each (Pharmacia), 3 ~1 of [~-3aP]dATP (Amersham; 3000 Ci/mmol, 10mCi/ml), 1 ~tl ofE. coli DNA ligase (BRL; 10 U/~tl), 4 ~1 of DNA polymerase I (BRL; 10 U/gl) and 1 pl ofRNase H (BRL; 2 U/gl). After 2 h at 16° C, 20 U of T4 DNA polymerase I (BRL) were added and the mixture was further incubated at 16° C for 5 rain. The reaction was stopped by the addition of 10 tal of 0.5 M of EDTA (pH 8.0). The reaction mixture was extracted with phenol/ chloroform and fractionated over a Sephacryl S-300 spin column (Pharmacia), saturated with buffer (20raM TRIS-HC1, pH 7.5, 10 mM MgC1,_, 10 mM 13-mercaptoethanol). To the eDNA (column effluent approximately 130 gl) were added 1 ~1 of 10 mM rATP, 150 pmol of radioactive phosphorylated SfiI adaptors (5'6a'rGGCCTTrr a'~] , and 15 U of T4 DNA ligase (Pharmacia) CAACCGGA followed by incubation overnight at 12° C. The reaction was phenol extracted, then extracted with diethylether --'

Strain

Relevant characteristics

Source

C13-ABYS-86 83L 84L W3031B/ct W3031B/a

ura3-2leu2 his- pral prbl prcl cpsl a spo11 ura3 can1 cyh2 ade2 his7 hem3 c*spo11 ura3 can1 cyh2 ade2 his7 hem3 a ade2 his3 leu2 trpl ura3 a ade2 his3 leu2 trpl ura3

D. Wolf G. Fink G. Fink P. Slominski P. Slominski

483 and digested with NotI. The radioactive cDNA was fractionated on a Biogel A150m column in 10 mM TRISHC1, 1 mM EDTA, 0.05% SDS buffer. The cDNA fractions were pooled, extracted with phenol-chloroform, ethanol-precipitated with 10 gg of yeast carrier RNA, and resuspended in 100 gl of 10 mM TRIS-HC1, pH 7.5, 1 mM EDTA (TE). The cloning vector, pSCGAL10-SN (described in Fig. 1), was digested with SfiI and NotI and fractionated in agarose gel, then eluted from the gel by centrifugal filtration (Zhu et al. 1985). The cDNA and the vector were titrated in test ligations; usually 1 gg of purified vector was used for the cDNA obtained from 5 gg of poly(A) + RNA. Ligation was performed with 10 U of T4 DNA ligase (Pharmacia) in a volume of 200 rtl overnight at 12° C. The ligation reaction mixtures were phenol-extracted, ethanol-precipitated with 10 gg of yeast carrier RNA, and dissolved in 10 gl of H20.

A

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Results Development of the vector The development of the vector pSCGAL 10-SN is shown schematically in Fig. 1A. The plasmid pUT322 (Gatignol

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Yeast transformations. Yeast transformation was carried out using the lithium acetate method (Ito et al. 1983). Carrier DNA was prepared according to Schiestl and Gietz (1989). Plasmid DNA from yeast cells was isolated as described by Sherman et al. (1986). Approximately 2 ~tg of total DNA was used in each transformation. DNA/RNA manipulations. Restriction enzyme digests and DNA ligations were performed according to the recommendations of the manufacturer. Isolation of plasmid DNA from E. coli, nick translation and Southern blotting were all performed using standard procedures (Maniatis et al. 1982). For Northern analyses, T. harzianum was grown by inoculating 3 × 104 conidiospores per ml of minimal medium (MM), MM plus 10 mM 3-amino-l,2,4-triazole (Sigma, St Louis, Mo.) or MM supplemented with 200 ~tg histidine/ml. The cultures were incubated in a reciprocal shaker at 28 ° C for 30 h, harvested by filtration through Whatman no. 1 filter paper and washed thoroughly with TES buffer (50 mM TRIS-HC1, 50 mM EDTA, 50 mM NaC1, pH 8.0). The culture treated with aminotriazole was subjected to an additional 5 h of growth in the presence of the analog. The mycelium was disrupted and total RNA and poly(A) + RNA extracted (as described above for the cDNA library). Three micrograms of the poly(A) ÷ RNA from each treated culture were then fractionated in a 2.2 M formaldehyde, 1% agarose gel (Maniatis et al. 1982), and then transferred to a Hybond-N membrane (Amersham) in the presence of 20 × SSC (1 × SSC is 0.15 M NaC1, 15 mM sodium citrate). Prehybridization and hybridization were performed according to Maniatis et al. (1982). In all Northern analysis experiments, the RNA concentration was normalized by hybridization with a [3-tubulin gene from T. viride (tub1 ; G.H. Goldman, unpublished results).

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C AGCTTGGCCAAAAAGGCCTAGCGGCCGCCTAGAGGATCCCCGGGCGAGCT (T)6o_0o ACCGGTTTTTCCGGATCGCCGGCGGATCTCCTAGGGGCCCGC Fig. 1. A Scheme illustrating the development of the expression vector pSCGAL10-SN. B The sequence between the XbaI and HindlII sites of the vector pSCGAL10-SN. C NotI restriction fragmentprimer used for the constructionof the cDNA library (see the Materials and methods)

484 et al. 1987) was cut with the restriction enzymes EcoRI and HindIII, and then ligated with an EcoRI-HindIII fragment from the plasmid pSp64-SN (this plasmid will be described in more detail elsewhere), resulting in plasmid pSC-SN. Briefly, between EcoRI and HindIII sites this plasmid contains the SfiI and NotI restriction sites necessary for insertion of the cDNA (as described in the Materials and methods). These latter restriction sites are separated by a piece of stuffer DNA, which facilitates purification of a SfiI-NotI vector. The stuffer D N A is a genomic fragment comprising part of the human fibroblast interferon gene (hlFN-~), and is present as a HincII fragment derived from the plasmid pgHFIF-4 (Degrave et al. 1981) to which synthetic J(hoI linkers (5'CCTCGAGG-3' sequence) had been ligated. The plasmid pSC-SN was cut with HindIII, filled-in with the Klenow fragment of D N A Poll (Po/Ik), and a synthetic XbaI linker, 5'-GACTCTAGAGTC-3', was attached resulting in the plasmid pSC-SN1. This construct was further modified by digesting pSC-SN1 with EcoR1, filling-in the ends with PolIk, and ligation of a synthetic HindIII linker (5'-CACAAGCTTGTG-3') to produce pSC-SN2. The sequence between the J(baI and HindIII sites is shown in Fig. lB. Finally, the plasmid pSC-SN2 was digested with XbaI, treated with T4 polymerase, and further digested with HindIII. The resulting blunt-ended HindIII fragment was isolated and ligated into Sinai + HindIII cleaved pEMBLyex4 (Cesarini and Murray 1987), thus producing pSCGAL10-SN. This vector has the following characteristics: autonomous origins of replication for E. coli and S. cerevisiae, selection markers for E. coli (Ap9 and S. cerevisiae (leu2 and ura3), and allows directional cloning of cDNAs under the control of the regulatable GALIO promoter. This last feature allows controlled expression of the cDNAs by the addition of various concentrations of glucose and galactose. Isolation of the imidazoleglycerolphosphate dehydratase (igh) 9ene The vector pSCGAL10-SN was employed to construct a cDNA library using poly(A) + m R N A from T. harzianum (see the Materials and methods, section cDNA library). The strain C13-ABYS-86 has a high frequency of transformation using the lithium acetate method (Ito et al. 1983), but its his auxotrophy had not been completely characterized. According to the genealogy of the strain, it may be defective in his3 or his7 (D.H. Wolf, Table 2. Sexual crosses carried out Crossing

Formation of diploid

C13-ABYS-86 x 83L C 13-ABYS-86 x 84L C13-ABYS-86 x W3031B/cc C13-ABYS-86 x W3031B/a

(-) (+ ) (-) (-)

Crosses were carried out using the drop-overlay method (Sherman et al. 1986)

personal communication). In addition, the mating type of the strain was also uncharacterized. We decided to characterize this strain by carrying out several sexual crosses using the drop-overlay method (Sherman et al. 1986). Based on these experiments (Table 2), the strain was characterized as carrying a his3 mutation. The igh gene was identified by examining recombinant D N A plasmids for functional complementation of the his3 mutation in strain C13-ABYS-86. Plasmids containing the igh gene were selected in two steps. In the first step, the cDNA library was transformed into this strain using selection for uracil in minimal medium plus glucose. Afterwards, the clones obtained (about 3500 transformants/pg of DNA) were pooled and spread on plates containing minimal medium plus galactose but without histidine. Approximately 30 colonies were obtained and plasmid D N A isolated from 6 of these transformants was used to transform E. coli (the transformant colonies were selected by ampicillin resistance). The plasmids were isolated and subjected to restriction enzyme analysis. All of the transformants showed the presence of pSCGAL10-SN plasmid containing an insert of approximately 1.1 kb. In order to confirm the phenotype conferred by this clone, the plasmid containing the cDNA insert was used to transform ABYSM and complementation of the his3 deficiency was confirmed. Nucleotide sequence of the igh gene The complete sequence of the igh gene of T. harzianum is shown in Fig. 2. The cDNA has an open reading frame of 627 nucleotides. The predicted protein is 209 amino acids long, having a calculated molecular weight of 22 466 and a calculated pI value of 5.81. A comparison of the deduced amino acid sequence of the igh gene of T. harzianum and the sequences of the fused hisB (region coding for IGH activity) of E. coli (Carlomagno et al. 1988), hisB of Azospirillum brasilense (Fani et al. 1989) and the S. cerevisiae his3 peptides (Struhl 1985) is shown in Fig. 3. The following degrees of homology were found: T. harzianum-E, eoli, 38.4% identity and 13.6% similarity; T. harzianum-A, brasilense, 40.1% identity and 18.4% similarity; T. harzianum-S, cerevisiae, 63.6% identity and 12% similarity. The data in Fig. 3 also indicate that in the four proteins there are conserved regions, with identical amino acids in corresponding positions. The major difference between the prokaryotic and eukaryotic proteins is the addition in the latter of a short peptide of 24 residues which is absent in E. coli. This short peptide is also absent in the igh gene (hisB) of A. brasilense. The predicted protein sequence of the igh clone shows 58.7% identity and 13% similarity with the corresponding gene in the yeast Pichia pastoris (M. Logghe, unpublished results). Here, the same short peptide was also found, suggestirig that this portion of the protein serves a specific function in eukaryotic cells. To determine the number of genes for IGH present in the T. harzianum genome, genomic D N A was digested with various restriction enzymes that lacked recognition sites within the cDNA clone, the fragments separated by

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agarose gel electrophoresis and the gel blotted to nylon membrane. The c D N A probe hybridized to only one genomic fragment, indicating that the T. harzianum genome most probably contains only one gene for IGH (data not shown). T. harzianum has seven chromosomes and the gene encoding igh has been mapped to chromosome VI by Southern analysis of chromosomes separated by pulse-field electrophoresis (Geremia et al., in preparation).

GGTCAGGGAG

796 AGTAAAAGAA 866 CGCTATTGCG 936 GCTGGATATC 1006 ACCATTCGCC

Fig. 2. Nucleotide sequence and predicted amino acid sequence of the Trichoderma harzianum igh gene (1.1 kb SfiI-NotI fragment). Conventional one-letter code is used for the amino acids (EMBL accession no. Z11528)

Northern analysis of igh expression in T. harzianum The 1.1 kb SfiI-NotI fragment containing the complete c D N A clone of the igh gene was used as a hybridization probe to investigate the transcription of this gene in T. harzianum. R N A was prepared from T. harzianum cultures grown in minimal medium with either no supplements or containing 200 gg histidine/ml. The probe identified only a single transcript of approximately 1.1 kb

486 E. A. T. S.

coli brasilense harzianum cerevisiae

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E. c o l i A. b r a s i l e n s e T. h a r z i a n u m

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98

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EQATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTE ...**.**.*** .... , * . . . . **..**.***.*

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Fig. 3. Comparison of the amino acid sequence deduced for the T. harzianum. IGtt protein with the corresponding sequence from other IGH proteins: spliced hisB (region coding for IGH activity) of Escherichia eoli (Carlomagno et al. 1988), hisB of Azospirillum brasilense (Favi et al. 1989) and Saeeharomyees eerevisiae his3 gene (Struhl 1985). Conventional one-letter code is used for the amino acids. The asterisks indicate homology between the sequences; similar residues are shown by dots

Discussion

X

X

b

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~'~'~'~'~

Fig. 4. a Northern analysis of ioh expression, b Northern analysis of [3-tubulin (tub1) expression. Lane 1, T. harzianum grown on minimal medium (MM); lane 2, T. harzianum grown on MM supplemented with 200 gg/ml histidine; and lane 3, T. harzianum grown on MM supplemented with 10 mM 3-amino-l,2,4-triazole

(Fig. 4a) which was detected at approximately the same level in the extracts prepared from cultures grown either with or without histidine. F r o m this it may be inferred that the expression of the igh gene in T. harzianum is not repressed by excess histidine. However, we do not know whether expression is constant in other physiological conditions. Aminotriazole is a potent competitive inhibitor of the yeast his3 product (Struhl and Davis 1981). Addition of aminotriazole to the medium results in temporary cessation of growth due to histidine starvation. The levels of many amino acid biosynthetic enzymes are increased when cells resume growth in the presence o f aminotriazole (Wolfner et al. 1975). To determine whether igh gene activity increases under these conditions, aminotriazole was added to minimal medium in which cells were growing. As can be seen in Fig. 4a (lane 3), the transcript accumulated to a level at least three times as high as that seen in untreated cultures, whereas 13-tubulin R N A expression remained constant under all conditions tested (Fig. 4b).

The genetics and biochemistry of histidine biosynthesis have been investigated in a variety of microorganisms. Eukaryotic organisms studied include N. crassa, S. cerevisiae and A. nidulans (Berlyn 1967; Fink 1964; Struhl 1985). The histidine biosynthetic genes are carried on several different chromosomes in all three organisms, but there is one cluster of three genes c o m m o n to each organism. This gene cluster has already been cloned in N. crassa (Legerton and Yanofsky 1985). In prokaryotic organisms, such as E. coli and S. typhimurium, the ten enzymes catalysing the biosynthesis of the amino acid are encoded by nine genes within one single operon. The fourth gene o f this operon (hisB) codes for a bifunctional enzyme possessing imidazoleglycerolphosphate dehydratase and histidinol phosphate phosphatase activities. In all eukaryotic microorganisms studied until now, the activities of these two enzymes are coded by two different genes. In S. cerevisiae, the loci are called his3 for the dehydratase and his2 for the phosphatase (Struhl 1985). In filamentous fungi, for example N. crassa and A. nidulans, these two enzymes are coded by his1 and hisB loci, respectively (Berlyn 1967; Fink 1964). Here, we report the cloning and characterization of the imidazoleglycerolphosphate dehydratase gene from T. harzianum. Two main lines of evidence indicate that the clone described here encodes IGH: (i) it can complement a S. cerevisiae his3 mutant, and (ii) the sequence of the predicted protein encoded by the clone is homologous to the fungal and bacterial I G H sequences over its entire length. In order to clone the igh gene, an expression system was developed which allows efficient cloning and expression of eukaryotic cDNAs in yeast.

487 The strategies used for the development of the vector and further directional cloning of cDNAs were successful not only for the igh gene but also for an alkaline protease gene of T. harzianum (Geremia et al. in preparation) and for a tubulin gene of T. viride (G.H. Goldman, unpublished results). In all examples mentioned, full-length c D N A clones were readily obtained. The regulatable promoter, one of the main characteristics of this system, has also been shown to be functional since the expression of igh and a tubulin gene were controlled by the addition of different concentrations of glucose (data not shown; G.H. Goldman, unpublished results). The hisB gene in both S. typhimurium and E. coli is composed of 1068 nucleotides and encodes a single polypeptide of 39998 daltons (Carlomagno et al. 1988). The most widely accepted model for the two enzymatic activities associated with the hisB gene predicts the existence of two independent domains in the gene, a proximal domain encoding the phosphatase moiety and a distal domain encoding the dehydratase activity. This model is based on many biochemical and genetic lines of evidence (Loper 1961; Brady and Houston 1973; Chumley and Roth 1981). The structural organization of the two enzyme activities in S. cerevisiae supports the twodomain model. In S. cerevisiae, the two activities are encoded by two separate genes, his2 and his3. A similar situation occurs in Streptomyces coelicolor, where the two activities are encoded by two different genes, hisB and hisD ( H o p w o o d et al. 1985). The his2 gene of S. cerevisiae, which codes for the histidinol phosphate phosphatase activity has not yet been cloned but the two yeast enzymes have been purified and the molecular mass of the subunits was reported to be 28000 and 35000 daltons, respectively (Glaser and Houston 1974). These results are not compelling since the his3 gene of S. ce~ revisiae codes for a peptide that has a molecular mass of 23 850 daltons (Struhl 1985) whereas the hisB bacterial gene directs the synthesis o f a single protein with an apparent molecular mass of 40 500 daltons (Carlomagno et al. 1988). The igh gene of T. harzianum has an apparent molecular mass of 22466 daltons which is in agreement with the molecular weight of the peptide encoded by the his3 gene of S. cerevisiae. In addition, the P. pastoris polypeptide shows a similar molecular mass (M. Logghe, unpublished results). The expression of the igh gene, as shown by Northern experiments (Fig. 4a), is not affected by exogenous histidine. This result agrees with transcription studies of the his3 gene region in S. cerevisiae: the transcription pattern of yeast cells growing in glucose minimal medium either in the presence or absence of histidine is identical to that seen when they are grown in rich medium (Struhl and Davis 1981). Expression of trpC, a gene involved in the tryptophan biosynthetic pathway, from Phanerochaete chrysosporium (Schrank et al. 1991), Cochliobolus hetero~ strophus (Turgeon et al. 1986) and Phycomyces blakes~ leeanus (Revuelta and Jayaram 1987) is also constitutive. Nevertheless, this contrasts with the situation in A. nidulans (Yelton et al. 1983) and S. cerevisiae (Prasad et al. 1987) where the transcription o f trpC and trpl, respectively, are induced by starvation.

The activity of a number of histidine biosynthetic enzymes has previously been shown to increase two- to fourfold in response to starvation for histidine produced by exposure of exponential cultures of N. crassa to the histidine analog aminotriazole (Legerton and Yanofsky 1985). The steady-state level of the his3 m R N A of S. cerevisiae is about tenfold higher in cells starved of histidine using the same analog (Struhl and Davis 1981). In both species it is known that starvation for histidine also causes derepression of many other amino acid biosynthetic enzymes. Carsiotis and Jones (1974) have proposed the term cross-pathway regulation for this phenomenon. We have examined the levels of expression of the igh gene of T. harzianum cultures grown in the presence and absence of aminotriazole. At least a threefold increase was observed in cultures subjected to aminotriazole starvation compared to cultures grown in the absence of this analog. These results show that cross-pathway regulation (general amino acid control system) may occur in T. harzianum. Further studies in this direction, using both biochemical and molecular techniques, are needed. Based on the genetic organization of the histidine biosynthetic pathway in N. crassa (Fink 1964), where the igh gene is called hisl, we suggest the same locus name for the igh gene in T. harzianum and in addition propose the existence of another gene coding for histidinol phosphate phosphatase activity.

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