The genomic TRP1 gene from basidiomycete Flammulina velutipes was cloned by complementation of yeast Saccharomyces cerevisiae trp1 mutation. Sequencing analysis revealed that the TRP1 gene encoded a single protein consisting of three catalytic functional domains; glutamine amidotransferase, indole-3-glycerol phosphate synthase) and N-(5′-phosphoribosyl) anthranilate isomerase, in order of NH2-glutamine amidotransferase–indole-3-glycerol phosphate synthase–N-(5′-phosphoribosyl) anthranilate isomerase–COOH. The coding sequence of the TRP1 gene was interrupted by a single intron of 48 bases, the position and flanking sequences of which were highly homologous to those of basidiomycete Phanerochaete chrysosporium trpC.
Although the biosynthetic pathway of tryptophan appears to be identical in all organisms studied so far and involves five sequential reactions from chorismic acid , the organization of genes encoding enzymes on the pathway is not always conserved. In Escherichia coli, a single operon contains five genes encoding all enzymes , whereas in the yeast Saccharomyces cerevisiae, five unlinked genes specify the tryptophan biosynthetic pathway . At this point, filamentous fungi exhibit an intermediate position between E. coli and S. cerevisiae, that is, in ascomycete Neurospora crassa, four unlinked genes drive the pathway. One of the four genes, the trp-1, encodes a trifunctional enzyme anthranilate synthase (AS) with domains for glutamine amidotransferase (GAT), indole-3-glycerol phosphate synthase (IGPS) and anthranilate isomerase (PRAI) activities . Although basidiomycetes also include equivalent genes, sequence data are available for only trpC from Phanerochaete chrysosporium.
Transformation systems have been developed in many basidiomycetous fungi, based on auxotrophic [5–8] and drug-resistant markers [9–12]. Heterologous promoters have been used for expression of drug-resistant marker genes, giving insufficient transformation. One of the important points for a transformation system is sufficient expression of a selectable marker gene, which includes recognition of a promoter sequence by transcriptional machinery of host cells. Therefore, endogenous promoters are expected to contribute to an efficient transformation system.
Despite being of economic importance, the edible basidiomycete Flammulina velutipes has no transformation system. In this study, we have cloned and sequenced the TRP1 gene of F. velutipes aiming at its application as a marker gene for a transformation vector.
Materials and methods
Strains and media
F. velutipes dikaryotic strain R15 was obtained from Nagano Vegetable and Ornamental Crops Experiment Station, and was grown at 25°C on peptone–sucrose medium (PSM; 2% sucrose, 0.5% peptone, 0.2% yeast extract, 0.1% KH2PO4, 0.05% MgSO4·7H2O, pH 5.5) to maintain it and to prepare its DNA, and also on complete yeast medium (CYM; 2% glucose, 0.2% yeast extract, 0.2% peptone, 0.046% KH2PO4, 0.1% K2HPO4, 0.05% MgSO4·7H2O) to prepare its RNA. E. coli strains HB101 (F−, supE44, hsdS20 (rB−mB−), ara-14, galK2, lacY1, proA2, rpsL20, xyl-5, mtl-1, recA13, λ−)  and DH10B (F−, mcrA, Δ (mrr-hsdRMS-mcrBC), φ80dlacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, λ−, rpsL, nupG) were used for construction of an F. velutipes genomic library and routine plasmid preparations. S. cerevisiae strain SP1 (MATa, trp1, ura3, his3, ade8, leu2, can1) was the host for complementation experiments.
Vectors and general procedure
Yeast expression vector pAAH5 containing a HindIII restriction site flanked by yeast ADC1 promoter and terminator was used to construct the F. velutipes genomic library. Plasmid vector pUC118  was used for subcloning and sequencing of an F. velutipes gene. General manipulations of DNA, such as restriction enzyme treatment, ligation and dephosphorylation of plasmid were done according to the procedures of Sambrook et al. . Yeast transformation was carried out by the lithium acetate technique .
Construction of the genomic library
Genomic DNA was isolated from lyophilized mycelia by the method of Lichtenstein and Draper  and partially digested with a restriction enzyme HindIII to give a mixture of fragments ranging from 5 to 10 kb, which were electrophoretically fractionated. Then the F. velutipes DNA fragments were inserted into the HindIII site in pAAH5, and recombinant plasmids constructed were used to transform the E. coli HB101 strain, resulting in 3.8×104 independent transformants.
Polymerase chain reaction (PCR)
Two primers were designed, a degenerate primer T323mix from a highly conserved region (sense, at nucleotides 395–413) of amino acid sequences in several fungal AS and a T304A primer (5′-CATCTAGAAGTCGTGGAGGTTGCGG-3′, antisense for nucleotides 1380–1396) from a 5′-truncated F. velutipes TRP1 gene originally cloned in this study, and their binding positions are shown in Fig. 1. The T323mix and the T304A primers were used in the PCR amplification of the F. velutipes TRP1 DNA containing its 5′-region. The reaction sample contained 10 mM Tris–HCl at pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 60 ng of F. velutipes genomic DNA, 0.9 μg of T323mix, and 0.2 μg of T304A. After heating at 94°C for 2 min and addition of 2.5 units of Taq DNA polymerase (Takara Shuzo, Kyoto, Japan), the reaction was cycled 36 times at 94°C for 0.5 min, 60°C for 1 min, and 72°C for 1.3 min.
Nucleotide sequences of the F. velutipes TRP1 gene were determined from both strands of the plasmid clones using DSQ1000L sequencer (Shimadzu, Kyoto, Japan). Nucleotide and amino acid sequences were analyzed using the GENETYX software system (Software Development, Tokyo, Japan).
RNA preparation, Northern analysis and reverse transcription (RT)-PCR
Total RNA was extracted from mycelia according to the standard method using phenol and guanidine thiocyanate. Northern blotting was done with approximately 5 μg of total RNA per sample. A probe of the F. velutipes TRP1 gene was digoxigenin-labeled using DNA Labeling and Detection Kit (Boehringer Mannheim, Mannheim, Germany). Detection of specific RNA was done following the supplier's instructions.
Complementary DNA was created from total RNA using RNA LA PCR™ Kit (AMV) (Takara Shuzo) containing avian myeloblastosis virus (AMV) reverse transcriptase and oligo dT-adaptor primer. Two primers, T5′22S (5′-GGGGTACCGTTCAACTTGAACAATCC-3′, sense for nucleotides −28 to −8) and T3′93A (5′-GGTCTAGACGCTTTACATATTCATCGACG-3′, antisense for nucleotides 2749–2769), whose binding positions are shown in Fig. 1, were used for RT-PCR; and the thermal cycling reaction described above was done, except for annealing at 54°C for 1 min, and extension at 72°C for 3 min.
Primer extension analysis
About 0.5 μg of the fluorescein isothiocyanate-labeled primer (5′-TGCGAATGACTTCGACTTTTGCC-3′, antisense at nucleotides 126–148), whose binding position is shown in Fig. 1, was annealed to 3 μg of total RNA and extended by the AMV reverse transcriptase (Takara Shuzo) at 42°C for 1 h in reaction buffer recommended by the manufacturer. The reaction was terminated by chloroform treatment and resulting molecules of nucleic acids were recovered by ethanol precipitation. The extension products were analyzed by the DSQ1000L sequencer.
Isolation of the F. velutipes TRP1 gene
The genomic library of F. velutipes was used to transform the Trp− strain of S. cerevisiae SP1 to Trp+. Two yeast transformants were obtained on the minimal medium lacking tryptophan, and they had the same recombinant plasmids, designated pFTT1, containing an insert of approximate 7.4 kb, which was then subcloned to pUC118. Partial DNA sequencing analysis of this insert and comparison of its deduced amino acid sequence to those of other fungal protein homologues revealed that the cloned TRP1 gene did not contain an entire sequence.
To obtain upper stream of the 5′-truncated TRP1 gene, a 1.0-kb fragment amplified with T304A and T323mix primers was cloned from F. velutipes genomic DNA being used as a template. Using the 1.0-kb fragment as a hybridization probe, another 4.7-kb DNA fragment was screened from the F. velutipes genomic DNA library, subcloned and sequenced.
Expression of the TRP1 gene was confirmed by dot blot analysis of total RNA extracted from F. velutipes vegetative mycelia grown in CYM (data not shown).
Nucleotide sequence of the F. velutipes TRP1 gene
Sequencing analysis of the clones obtained from the genomic DNA library and the PCR clone showed their similarities to sequences of tryptophan biosynthesis genes previously published [1–4,16–18]. The resulting nucleotide sequence could be translated to an amino acid sequence similar to those deduced from the other fungal genes. Fig. 1 shows the nucleotide sequence containing the TRP1 gene and predicted amino acid sequence of TRP1 polypeptide. Upstream of the presumed translation initiation codon, a sequence similar to the CAAT-box was found at position −279 but not the TATA-box. Primer extension analysis revealed that the transcription initiation site was located at position −204.
Sequence homology in fungal polypeptide
The deduced amino acid sequence of the F. velutipes TRP1 gene (Fig. 1) was compared to the trpC genes of P. chrysosporium, Aspergillus nidulans and Aspergillus niger together with the trp-1 gene of N. crassa and the TRP1 gene of Phycomyces blakesleeanus coding for ASs (Fig. 2). These sequences exhibited remarkable similarity including identical stretches, strongly suggesting that the TRP1 gene in F. velutipes encodes a trifunctional polypeptide consisting of three domains which correspond to the sequences encoding the GAT, IGPS and PRAI activities. Their position was estimated at amino acid residues 1–221, 248–514, and 533–875, respectively, from comparison with the gene homologues in other fungi. These results showed that F. velutipes had AS conserved among filamentous fungi.
Analysis of intron sequence of TRP1 gene
The single presumable intron was found to locate at the position of nucleotides 342–390 (Fig. 1), and its length, position, splice junctions, inside termination codon and flanking sequences were highly homologous to those of P. chrysosporium trpC (Fig. 3). The restriction fragments of genomic- and RT-PCR products using T5′22S and T3′93A primers were compared electrophoretically, indicating that only the fragment containing the presumable intron less migrated (Fig. 4).
The TRP1 gene from F. velutipes in this study exhibited striking similarity to the corresponding genes of other filamentous fungi coding for ASs [1,4,16–18], indicating that F. velutipes had AS conserved among filamentous fungi. So far, only the one gene encoding AS from basidiomycetes is sequenced  other than the F. velutipes TRP1 gene. The intron found in the latter was highly conserved in the former, P. chrysosporium trpC gene , and their introns were specific to basidiomycetes, not to ascomycetes.
Functional analysis of the AS genes from filamentous fungi must also be done in addition to their structural analysis, especially those from basidiomycetes. While sequencing data of the ASs from filamentous fungi show that each of the proteins is the trifunctional enzyme related to tryptophan synthesis, basidiomycete Schizophyllum commune TRP1 gene encoding AS has another function related to mating and fruiting . Moreover the promoter and terminator regions for sufficient expression of the F. velutipes TRP1 gene have not yet been specified in this study. Further investigation is now under way to develop the efficient transformation system of the edible mushroom F. velutipes.
We wish to thank Dr. M. Nakafuku (the University of Tokyo) for his kind supplies of an S. cerevisiae strain and a yeast expression vector, and his valuable discussion and suggestions; to Mr. K. Akahane for his kind supply of F. velutipes R15 strain; and to Dr. S. Eda, Mr. S. Habutsu and Mr. T. Togami for their technical support. This investigation was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and by the Agricultural Chemical Research Foundation.