CA2163790A1 - Isolation of mushroom-inducing genes and their use in dna-mediated transformation of edible basidiomycetes - Google Patents

Abstract

The present invention is directed to a process for inducing the development of fruiting bodies in host cells, preferably in edible Basidiomycete host cells. The present invention is further directed to the isolation and identification of a mushroon-inducing gene from the Basidiomycete Schizophyllum commune called Frt1 and methods of transforming appropriate host cells with the Frt1 gene or portions or modifications thereof. Another aspect to the present invention relates to comparable Frt1 genes in edible species that are known to be relates to S.commune.

Description

W O 94/28136PCTrUS94/05795 ISO~ATION OF NUSHROON-Ih~u~lwG GENES
A-wD THEIR USE IN DNA-MFnT~Fn TRAw~O~TION OF
EDIB~E ~-eTnIONYCETES

Field of the Invention:
The present invention relates to a process for inducing the development of mushrooms, also called fruiting bodies, in host cells. Furthermore, this invention relates to a method of transforming a Basidiomycete strain with DNA encoding for all or part of a mushroom-inducing gene. In addition, the present invention relates to identification and isolation of a novel fruiting gene from the Basidiomycete Schizophyllum commune.

State of the Art:
Schizophyllum commune is a wood-rotting Basidiomycete related to various species of edible mushrooms including Agaricus bisporus, Agaricus bitorquis, Pleurotus species, Flammulina velutipes, Lentinus edodes and Volvariella volvacea. S. commune has long served as a model system for the study of genes regulating mating and fruiting in fungi. While edible, its mushrooms are relatively puny and chewy and, therefore, of little or no commercial value for gastronomic purposes. However, the species is ideal for genetic studies, not only because it is haploid throughout most of its life cycle, but also because it can be made to complete this cycle on chemically defined media within a relatively short period of time. The mating and fruiting of S. commune has been extensively reviewed, see for example Raper, C.A. 1983 in Secondary Metabolism and Differentiation in Funqi, pp. 195-238, (edited by J. Bennett and A. Ciegler) Marcel Dekker, New York; and Raper, C.A., 1988 in Genetics of Pathoqenic Funqi, Vol. 6, 30 pp. 511-522 (edited by G.S. Sidham) Advances in Plant Patholoqy (edited by D.S. Ingrams and P.H. Williams) Marcel Dekker, New York; and Stankis, et al, 1990, in Seminars in Developmental Biology, Vol. 1, pp. 195-206 (edited by C.A. Raper and D. I. Johnson) W.B. Saunders Co., London.

As in most species of edible mushrooms, fruiting in S. commune is normally under the control of the mating-type genes (Raper, J.R., 1966, Genetics of Sexuality in Hiqher Funqi, pp. 283, Ronald Press, New York). Fruiting is known to involve a number of other genes as well as environmental factors (Raper, J.R. and Krongelb G.S., 1958, Mycologia 59:707-740). A number of genes that are specifically transcribed in the differentiated tissue of fruiting bodies in S. commune have been cloned and are being characterized W 0 94/28136 2 1 6 3 7 9 0 PCTrUS94/05795 (Mulder, G.H. and Wessels, J.G.H., 1986, Experimental Mycology 10:214-227;
Wessels, J.G.H. (1992) Mycological Res. 96(8):609-620). These genes, called Sc genes, are thought to encode protein products essential to the differentiation of fruiting tissue.

A gene called Frtl that induces fruiting body (mushroom) development has been isolated from a strain of the Basidiomycete S. commune (Horton, J.S.
and Raper, C.A., 1991, Genetics 129:702-716). Sequences similar to Frtl have been identified in other strains.
5. commune is a heterothallic Basidiomycete species in which mushrooms normally develop only after activation of the mating-type genes by the coupling of two homokaryons of different mating types to form a dikaryon.
The cloned Frtl gene is capable of inducing mushroom development in certain unmated homokaryons when integrated into the genome by DNA-mediated transformation, overriding the normal requirement of a compatible mating interaction for fruiting in this fungus.

It is believed that the Frtl gene is involved in the regulation of other genes in the developmental pathway of fruiting. Thus, if this group of genes is conserved in related species of edible Basidiomycetes, it may be possible to enhance the mushroom production of such other related strains by either transforming the Frtl gene or a modification thereof, or transforming a related gene into a more commercially valuable edible Basidiomycete. In light of the various applications of the Frtl gene, the present invention is directed to the complete characterization of Frtl isolated from S. commune, i.e., the DNA fragment and sequence encoding Frtl, and a process for transforming edible Basidiomycetes with the Frtl gene to enhance mushroom production.
Summary of the Invention:
The present invention relates to a novel gene isolated from S. commune, such gene having mushroom-inducing activity, expression vectors comprising the DNA of such gene or modifications thereof, and the protein product of such gene, produced either by S. commune or E. coli or any other organism.

In addition, the present invention relates to a method for DNA-mediated transformation of an appropriate host organism, i.e., a Basidiomycete, preferably a commercially valuable and edible species, with a vector comprising a DNA fragment encoding a mushroom-inducing gene or modifications thereof.

Accordingly, one embodiment of the present invention relates to a process for inducing or enhancing the development of fruiting bodies in host cells, the process comprising the identification of a DNA sequence encoding a fruiting gene from edible Basidiomycetes, optionally modifying such DNA

W O 94/28136 2 1 6 3 7 9 0 PCTrUS94/05795 sequence, and integrating the modified or unmodified DNA sequence into an appropriate host cell via DNA-mediated transformation to induce or enhance the development of fruiting bodies in the presence or absence of an activated mating-type gene. In a preferred embodiment, the mushroom-inducing gene is isolated from S. commune, and more preferably the Frtlgene having the sequence set forth in SEQ ID NO:l or a modification thereof.

A further embodiment of the present invention comprises utilizing a DNA
sequence, preferably the Frtl gene or a modification thereof, to isolate related or comparable genes in more commercially valuable and edible species that are known to be related to S. commune.

In yet a further embodiment, the present invention relates to a process for transforming one or more Basidiomycete species comprising the process of treating the Basidiomycete cells or protoplasts with recombinant DNA under conditions permitting at least some of the Basidiomycete cells to take up the recombinant DNA and form transformants, and isolating the Basidiomycete transformants. Useful Basidiomycete species for such transformation include but are not limited to Agaricus bisporus, Agaricus bitorquis, Pleurotus species, Flammulina velutipes, Lentinus edodes and Volvariella volvacea.

In addition, the present invention relates to a process for transforming the above-listed Basidiomycete species such that the transformants express the fruiting gene to elicit or enhance fruiting. A particular aspect of this embodiment is that the Basidiomycete cells or protoplasts are transformed with recombinant DNA linked to a homologous or heterologous promoter, which is optionally linked to an inducible promoter under conditions permitting at least some of the Basidiomycete cells to take up the recombinant DNA and form transformants.

The present invention further relates to transformed Basidiomycete cells produced by the above processes containing the DNA fragment having mushroom-inducing activity.

In yet a further embodiment, the present invention relates to a recombinant DNA construct which comprises a selectable marker gene and all or part of a fruiting gene or a modification thereof. Preferably, the above DNA
construct comprises a promoter which is either homologous or heterologous to the mushroom-inducing gene and, optionally, is an inducible promoter.

A further embodiment of the present invention relates to the protein product of the fruiting gene or antibodies raised against the protein product of the fruiting gene, preferably Frtl, expressed in E. coli, a Basidiomycete or any other suitable expression system. In particular the W O 94/28136 2 1 6 3 7 9 0 PCT~US94/05795 protein product of the present invention is Frtl, having the amino acid sequence shown in Figure 3 or a modification of such sequence.

Various other objects and advantages of the present invention will become obvious from the drawings and the following description of the invention.

The entire contents of all references cited above are incorporated herein by reference.

Brief Description of the Drawinqs:
SEQ ID NO:l depicts the genomic DNA sequence of the Frtl gene including bracketed segments representing the intron sequences.

SEQ ID NO:2 depicts the cDNA sequence of the Frtl gene. This sequence begins and terminates at the translation start and stop sites respectively.

SEQ ID NO:3 is the predicted amino acid sequence of the Frtl protein product, derived from translation of the Frtl cDNA shown in SEQ ID NO 2.
The "P-loop" region is delineated by brackets.

Detailed Description of the Invention:
As noted above, the present invention generally relates to a process for inducing mushroom or mushroom-development in host cells and the isolation and characterization of a novel gene and protein translated therefrom that induces such development in a species of Basidiomycete. Prior to discussing the invention in further detail, the following terms will be defined.

The term "alleles," "alternate alleles" or "allelic variations~ refers to a series of possible alternative forms of a given gene differing in DNA
sequence and usually affecting the functioning of a single RNA or protein product.

The term "amino acid sequence" refers to the linear order of amino acids in a peptide or protein.

The term "antibody" refers to a protein produced in mammals in response to a foreign substance (antigen), e.g., a protein that is capable of coupling specifically with that antigen.

The term "Basidiomycete" refers to a class of fungi bearing specialized cells known as basidia upon which are born Basidiospores containing nuclei which are the products of meiosis.
The term "cDNA" refers to complementary DNA which is the DNA complement of W O 94l28136 PCTrUS94/05795 an RNA sequence. The RNA sequence, termed "messenger RNA," is derived by transcription of the DNA sequence constituting the gene, and the transcript, in turn, can be translated into a protein product.

A "genetic clone" refers to an amplified sequence of DNA using the method of in vitro recombination to insert the sequence into a vector molecule (construct), thus, producing a recombinant plasmid which can be replicated in a suitable host cell, e.g., the bacterium E. coli .

A "genomic clone bank" refers to a collection of genetic clones containing a vector and inserts which together comprise the entire genome of an organism.

A "consensus sequence" refers to a particular nucleotide sequence which occurs, with some minor variations, in genes of known similar function or origin.

A '~cosmid" is a plasmid vector which contains cohesive ends of a bacteriophage and one or more selectable markers. Cosmid clones generally include 30-40 kb of genomic DNA derived from the organism of interest.

"Constructs" refer to recombinant DNA molecules constructed in vitro.

The term "derivative" is intended to include derivatives of SEQ ID NO:3 shown by the addition of one or more amino acid residues to either or both the C- and N-terminus of the sequence, substitution of one or more amino acid residues at one or more sites in the sequence, deletion of one or more amino acid residues at either or both ends of the sequence, or deletions from within or insertion of one or more amino acid residues at one or more sites within the sequence, such that a sequence identity of at least 30%
with SEQ ID NO:3 is retained and containing mushroom-inducing activity.

The term "dikaryon" refers to a colony of fungal cells each cell having two genetically different nuclei (usually two types of haploid nuclei of opposite mating types in a Basidiomycete fungus).

"DNA" is deoxyribonucleic acid, the molecule of inheritance in organisms comprising a duplexed sequence of nucleotides having deoxyribose as their sugar.
The term "encode" refers to that portion of a gene which determines the amino acid sequence of its protein product.
.

The term "expression vector" refers to a vector containing a promoter sequence which facilitates the efficient transcription of an inserted gene and, therefore, results in the production of a high concentration of the W O 94/28136 2 1 6 3 7 9 0 PCTrUS94/05795 inserted gene's expressed protein product within the host cell.

"Fruiting body" relates to the sexually reproducing organ in fungi. In Basidiomycetes it is commonly known as a mushroom, which is edible in some species.

The term "gene" refers to the unit of hereditary function; a DNA sequence which encodes a functional protein.

The term "genome" refers to the entire complement of genetic material in a cell.

The term "genotype" refers to the genetic constitution of an organism as revealed by genetic or molecular genetic analyses. It consists of the specific allelic composition of a gene or genes in a cell or strain.

A "haploid" cell contains only one complete set of chromosomes which contain one set of genes, as compared to the usual condition in most cells of higher organisms which contain two sets of identical chromosomes and are called "diploid" cells.

"Heterothallic Basidiomycete species" are species which fruit and complete the sexual phase of the life cycle only by the mating of two compatible homokaryons.
The term "homokaryon" refers to a colony of fungal cells having genetically identical nuclei.

"Homologous genes" refer to genes with strong similarities (ca. 80% or greater) to one another in their DNA sequence.

"Homologous promoter" and "heterologous promoter" refer to the natural promoter of a given gene and a promoter that belongs to a different gene, respectively. A heterologous promoter may be substituted for a homologous promoter by isolating a gene, removing its natural promoter and recombining the isolated heterologous promoter upstream of that gene. This is an example of genetic engineering.

The term "host cells" relates to cells of an organism or strain that are used as recipients in DNA-mediated transformation.

The term "hybridization" relates to the formation of stable duplexes between two complementary polynucleotide strands (either RNA or DNA) from different sources.
"In vitro mutagenesis" is a technique in which a cloned gene is wo 94~28136 2 1 6 3 7 9 0 PCTAUS94/05795 specifically altered (mutated) in vitro, i.e., outside the organism.
Usually the mutated gene is then tested for altered activity by transformation into the appropriate recipient cells.

The term "inducible promoter" refers to a promoter that functions in response to a specific chemical or physical agent, e.g., galactose instead of glucose as a nutritive carbon source.

The term "intronic sequence" refers to a DNA sequence intervening within the coding region (exons) of a gene. Intronic sequences (introns) are spliced out of the gene after transcription.

The term "kb" is the abbreviation for kilobase pair (one thousand base pairs of nucleic acid, DNA or RNA).
"Mating-type genes" are genes determining sexual compatibility, hence fertility, i.e., genes governing the capability of an individual fungal colony to produce fruiting bodies. When the mating-type genes are activated, either by pairing of opposite types or by mutation to constitutive function, fruiting may occur.

The term "modifications" of the DNA sequence is intended to include nucleotide substitutions, deletions, or insertions which give rise to another form of FRT1 protein containing mushroom-inducing activity.
An ~oligonucleotide" is a short chain of nucleic acid molecules.

An "open reading frame" refers to a reading frame of DNA sequence uninterrupted by stop codons. Generally, it is a sequence that encodes the product of the gene.

The term "P-loop" refers to a sequence of six amino acids, Glycine-x-Glycine-x-x-Glycine, where x is any amino acid. This region is thought to act as a flexible hinge in proteins which undergo a conformational change.
The term "phenotype" refers to the observable or detectable outward characteristics of an organism determined by its genotype as influenced by the environment.

The term Nplasmid" refers to an extrachromosomal element capable of independent replication when introduced into cells of an organism, e.g., bacterium or yeast. Plasmids can be engineered to carry genes of interest and are often used to amplify the DNA encoding those genes.

"Poly(A)+RNA" refers to RNA that has a sequence of adenylate residues at its 3' end.

W O 94/28136 PCTrUS94/05795 The term "polymerase chain reaction (PCR)" is an enzymatic method for in vitro amplification of specific DNA fragments. PCR is based on the use of two oligonucleotides to prime DNA polymerase-catalyzed synthesis from opposite strands across a region spanned by the priming sites.

"Primers" are short oligonucleotides to which DNA polymerase is able to add nucleotides to a free 3' hydroxyl group.

A "promoter" is the part of a gene to which RNA polymerase binds prior to the initiation of transcription. The promoter is usually found just upstream from the coding region of the gene.

The term "protoplast" refers to the cell of a fungus or plant which has had its wall removed, usually by the action of lytic enzymes. Protoplasts are used to facilitate DNA-mediated transformation and can regenerate into a network of cells with normal morphology.

The term 'irecombinant DNA" refers to DNA molecules in which sequences which are not naturally contiguous have been placed next to each other by manipulations outside the cell, i.e., in vi tro.

A "restriction enzyme" is an endonuclease which recognizes a specific sequence of bases within double-stranded DNA.
"RNA" is ribonucleic acid, the molecule resulting from transcription of DNA. RNA is single stranded nucleic acid similar to DNA but having ribose sugar rather than deoxyribose sugar in its backbone, and uracil rather than thymine as one of its bases.
"Southern hybridization" is a technique in which DNA fragments are first separated in an agarose gel, denatured, transferred to a solid support and hybridized with a 32P-labeled, single-stranded DNA probe.

The term "spawn" refers to fungal cells (usually of a Basidiomycete fungus) growing on a substrate and capable of producing fruiting bodies (mushrooms) when used as inoculum to a bed of substrate for mass cultivation of mushrooms. Spawn is sold for purposes of commercial mushroom production.

A "selectable marker" is a gene incorporated into a vector, which is able to complement a deficiency in that gene's function in the recipient (host) cell used in DNA-mediated transformation. For example, a host cell which is incapable of synthesizing tryptophan due to a mutation in an essential gene, e.g., Trpl in the tryptophan synthesis pathway, will gain the competence for synthesizing this amino acid if it is transformed by the integration of the wild-type Trpl gene and this transformant can be W O 94/28136 2 1 6 3 7 9 0 PCTrUS94/0579~ selected on a so-called selective medium which does not include tryptophan.
Tryptophan-competent transformants can then be examined for other phenotypic effects which may be due to the presence of another gene of interest, e.g., the Frtl gene, that is incorporated within the same transforming plasmid.

"DNA-mediated transformation" relates to a mechanism of gene transfer which involves the uptake of purified DNA into host cells to generate a transformant. A transformant is usually identified first by a change in phenotype, e.g., by a change from tryptophan-requiring to tryptophan-competence and, possibly, from non-fruiting to fruiting, if the transforming DNA is a plasmid containing the Trpl and Frtl genes and the host cell contains neither of these genes.

The term "transcription" refers to the formation of a complementary RNAmolecule (transcript) upon a DNA template. The transcript, also called messenger RNA, is single-stranded and incorporates a complement of that portion of the gene that encodes the product of the gene. The messenger RNA serves as a template for the assembly of a sequence of amino acids constituting the product of the gene.

A "vector" or "construct" refers to a DNA molecule derived from a plasmid or bacteriophage into which fragments of DNA may be inserted or cloned.

The isolation and characterization of genomic clones containing the Frtl gene have been described by Horton, J.S. and Raper, C.A. (1991) Genetics 129:707-716, and are described below in the Examples, as is the sequencing of the Frtl gene, which has not been previously described.

Both classical and molecular genetic studies of Frtl have established the following principles concerning its activity within living cells of Schizophyllum commune: 1~ Cloned Frtl integrates stably when introduced into the genome of recipient cells via DNA-mediated transformation and appears to be trans-acting; 2) the integration of cloned Frtl has the effect of not only inducing de novo the formation of homokaryotic fruiting bodies in certain strains, but also of enhancing the formation of dikaryotic fruiting bodies after the mating of homokaryons transformed for Frtl; and 3) functional equivalency of presumed alternate alleles for the Frtl gene has been demonstrated by showing that alternate alleles from other strains can operate in a fruiting dikaryon in which Frtl has been deleted from one of the comoponent homokaryotic genomes. The alternate allele from the wild-type mate complements the deficiency of the mutant mate in which Frtl was deleted.

The present invention encompasses the concept that the Frtl alleles mayregulate the expression of other genes in the fruiting pathway. Potential W O 94l28136 PCTtUS94tO5795 candidates for genes regulated by the Frtl gene may be found among the Sc genes which are transcribed preferentially at the time of fruiting (Mulder and Wessels 1986, Wessels 1991). A primary target might be a homologue of the fruiting specific Sc7 gene that was located 2 kb away from the Frtl gene by DNA hybridization experiments. Frtl itself may be a target for activity of the mating-type genes.

By understanding the relationships among genes regulating mushroom development in Schizophyllum commune, it is contemplated that one skilled in the art, using S. commune as a model system, may apply such an understanding to comparable genes in edible species.

Accordingly, one aspect of the present invention relates to a method for isolating mushroom-inducing genes from the genomes of other Basidiomycetes.
It is possible to design effective strategies for identifying, isolating and characterizing comparable genes in edible Basidiomycetes, (Raper, C.A.
and Horton, J.S., 1992, in Genetics and Breeding of Edible Mushrooms, pp.
285-296, edited by S.T. Chang, P.G. Miles and J.A. Buswell, Gordon and Breach Inc., Philadelphia.) A preliminary survey of the genomes of other species for the presence of Frtl-like sequences may be accomplished by DNA hybridization analyses according to the methods of Southern, 1975, J. Molecular Biol., Vol.
98:503-517, in which the Frtl gene or portion thereof may be used as a probe against restriction enzyme digests of genomic DNAs of a variety of other Basidiomycetes. Any positive results would identify species for further investigations, but negative results would not be definitive. The DNA sequences must be very similar (approximately 80% or more) for detection by this method.
A more reliable approach would employ the Polymerase Chain Reaction, known as PCR (Saiki, R.L., l990, in PCR Protocols: A Guide to Methods and Applications, pp. 13-20, edited by M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White, Academic Press, San Diego), to amplify those parts of a gene that have been shown to be conserved and important for function. For Frtl, an optimal choice of the sequence to be amplified would be based upon comparative sequence analyses with other alleles existing in S. commune and in vi tro mutagenesis experiments. Oligonucleotide primers corresponding to important consensus sequences could then be designed for use in Polymerase Chain Reactions to amplify related sequences from the genomic DNA of other Basidiomycetes (see Scheme 1).

W O 94/28136 PCTrUS94/05795 Donne tRrconsensus se~uences In S ta i vnc ~.
Oeslgn ol~gonucleotld-¦ ~ Rmpll-~l potentl~l prlmers ¦ mus~room lnduclng ~ se~uence(s) by PCR
Isol~te genomlc D~R trom edlble B~sidlomycete Ueritu bg Sout~-rn ~ brldl2~tlon wlt~l tRr-spetlrle P~obe Use ~mplltled se~uentes ~s pro~s to ~lect genomlc elones Test tor musnroom-lnauclng ~ctlulty by tr~nstorm~tlon ~oletul~r ~n~ sls ~nd manlpul~tlon Genetlc engineering ot edlble ~sldlomycetes Scheme 1. Proposed strategy for isolation of mushroom-inducing genes from edible Basidiomycetes.

wo 94~28136 2 1 6 3 7 9 0 PCTnJS94/05795 The amplified sequences may be checked for correspondence to the desired sequence since the PCR procedure sometimes results in the amplification of artifactual sequences. The desired sequence may be identified by sequencing the cloned PCR product and comparing this sequence to that of the Frtl gene. A PCR product may represent only a portion of the coding sequence of the putative mushroom-inducing gene and may, therefore, not be useful for testing in vivo activity via transformation. Hence, the amplified sequence may be used as a probe to screen a genomic clone bank for clones containing the corresponding Frt gene. Selected clones may be tested for function in transformation experiments and subsequently subcloned using Southern hybridization analyses to identify the region for similarity to the Frtl gene.

After demonstrating a functional role for a Frt gene isolated from an edible Basidiomycete, the Frt gene may be subjected to molecular analyses to characterize and manipulate the sequence. As with the Frtl gene of S.
commune, the cloned sequence linked to its own promoter may enhance the normal fruiting process in transformants for this isolated gene and, thus, improve crop production. Transformation with constructs in which the gene is linked to a stronger promoter may result in an overexpression of the Frt gene and greater mushroom yield. Constructs incorporating an inducible promotor may be useful in producing synchronous flushes of mushrooms, a definite advantage in harvesting the crop.

Those skilled in the art will understand the value of applying known molecular genetic techniques to other species of Basidiomycetes. The related Basidiomycete Coprinus cinereus could be used in initial experiments to test this approach. Although not commercially valuable as an edible species, C. cinereus is an excellent candidate for such tests because all the necessary techniques of molecular genetics, including DNA-mediated transformation, have been developed for this species (Pukkila, P.
and Casselton, L., 1991, in ~More Gene Manipulations in Fungi", pp. 126-150, Bennett, J. and Lasure L., Eds., Academic Press, New York) Frtl of 5.
commune or a Frt gene isolated from C. cinereus may be tested for function in vivo in transformation experiments and subsequently analyzed using the techniques of in vitro mutagenesis.

Certain aspects of the inventions are described in greater detail in the non-limiting Examples that follow.
EXAMPLES
Example 1 - Isolating the Frtl Clone Frtl was selected from a cosmid clone bank of Schizophyllum commune genomic DNA. The vector used in construction of this clone bank contained the ~rpl gene of S. commune as a selectable marker. The random inserts of DNA
averaged 35 kb in length. Transformation was carried out according to a wo 94J28136 2 1 6 3 7 9 0 PCTrUS94/05795 protocol devised by Specht et al (1988) Experimental Mycoloqy 12:357-366 and modified by Horton, J.S. and Raper, C.A. (1991) Current Genetics 19:77-80 and Horton, J.S. and Raper, C.A., Genetics 129:707-716. Recipient cells in the transformation experiments were homokaryons, wild-type for the mating-type genes but mutated for Trpl, hence tryptophan-requiring.
Transformants were identified by their ability to grow on selective medium in the absence of tryptophan. While screening the clone bank for developmental genes, one cosmid was identified which was capable of inducing the formation of fruiting bodies in the homokaryotic recipient.
This clone was subsequently subcloned to produce a 1.4 kb active fragment.
(See Horton, J.S. and Raper, C.A., supra).

Example 2 - Sequencing the Frtl Gene DNA sequencing of the region surrounding and inclusive of the Frtl gene was accomplished by the following methods. A set of nested deletions overlapping the Frtl gene was generated by using the Exonuclease III
digestion procedure of Henikoff, S. (1984) Gene 28:351-359, utilizing the plasmid vector pGEM7Zf(+) (available from Promega Biotech). Double stranded plasmid DNA was isolated by the method of Saunders, S.E. and J.F.
20 Burke (1990) Nucleic Acids Research 18:4948, and sequenced according to the procedure of Zhang et al (1988) Nucleic Acids Research 16:1220, using the Sequenase Version 2.0 Sequencing Kit (commercially available from US
Biochemical). Sequencing reactions were run on 6% polyacrylamide gels which were dried and autoradiographed using standard methods (Ausubel et al, 1989, Current Protocols in Molecular Biology, John Wiley and Sons, New York). Both strands were sequenced over the entire 1635 nucleotide region.
Oligonucleotide primers were designed and used to close any gaps in the sequence.

The cDNA sequencing of the Frtl coding region was done in the followingmanner. Total cDNA was synthesized from poly(A)+RNA isolated from Schizophyllum commune strain H9-1, the strain from which Frtl was isolated.
Frtl cDNA was generated by using the Polymerase Chain Reaction (PCR) to amplify cDNA between primers A15 and A16 (nucleotides 464-1386 of the genomic sequence), using total cDNA as the template. The product was separated by gel-electrophoresis, purified from the gel, then reamplified, repurified and cloned into the pGEM7 vector. Double-stranded sequencing was performed as described for the genomic DNA. Assembly of the cDNA and genomic sequence, and identification of intronic sequences, and the FRTl open reading frame were done with the aid of the Mac Vector Program (IBI), run on a Macintosh IIcx computer.

The genomic DNA and cDNA sequence of Frtl is provided in SEQ ID NO:1 and SEQ ID NO:2, respectively. The predicted amino acid sequence of the protein produced from the cDNA sequence of Frtl is provided in SEQ ID NO:3.
There are three introns in the genomic sequence provided in SEQ ID NO:1.

W 0 94/28136 2 1 6 3 7 9 0 PCTtUS94tO5795 The first intron sequence extends from nucleotide 531 to nucleotide 586.
The second intron sequence extends from nucleotide 755 to nucleotide 808.
The third intron sequence extends from nucleotide 958 to nucleotide 1010.
Furthermore, SEQ ID NO:1 contains a start codon at nucleotides 494 through 496 and a stop codon at nucleotide 1233 through 1235.

Example 3 - Characterization of the Frtl Gene and Predicted Protein Product A transcript for the Frtl gene is present during mushroom development.
This was determined by using the cloned gene as a probe against polyadenylated RNA isolated from fruiting cultures. A 600 nucleotide transcript was thus identified. The methods used followed standard procedures as described in Ausubel et al, 1989.

Analysis of the Frtl sequence has revealed that the predicted protein is small (22 kD), potentially phosphorylated at three threonine residues and has a HP-loop" motif found in many nucleotide-binding proteins. In vitro mutagenesis of the Frtl genomic sequence at the site of 19Gly (second glycine of the "P-loop") in which either a valine or a proline would be substituted for this amino acid, resulted in the abolishment of the mushroom-inducing activity of the cloned DNA. The predicted amino acid sequence does not appear to have any significant similarity to any other proteins in the databases searched.

Sequence divergence of Frtl between different strains was implicated by the results of DNA hybridization experiments in which it was shown that Frtl hybridizes faintly to similar sequences in the genomes of strains other than the one from which it was isolated. Divergence was evidenced also by strain-specific polymorphisms with respect to location of restriction enzyme sites within the hybridizing sequences. These hybridizing sequences in other strains are referred to as alleles of Frtl.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.

W 0 94/28136 2 1 6 3 7 q O PCTrUS94/05795 SEQUENCE LISTING

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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1635 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
55 CATATCACCC ~ AGA AAGCGTTGTC GGAGCTTGAT ACGGACACCG GCCTCCGCAG 60 ~ GTGCGAAGCT AGTGACCCTC TGTCCATACT CTTCAAGTGA GCCTACACCC GAGACGAGGA 240 W O 94~136 2 1 6 3 7 9 0 PCTrUS94/05795 AAGACTGGCA TCAAGCCGGA GTATCTCTAC TCTTGTAAGC CAACTATTCA lll~llGACG 780 TAl~lC~lCG CTGACCGGCT ATCATGAGCA CGAGCACAGG ATATCCGAGC TGTCTACCAG 840 ATACGCTCCG CTGCCAACGT ATGATAGGCC TGTGGACCCT CAATTACAGG ~ll~llGGAC 1080 CCGGACCTAC CGCTCAGTGC TATGGTGGTG ACCAAGGATC A~l~l~lGGT TTAGCAGGAT 1320 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 579 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

WO 94128L~6 2 1 6 3 7 9 0 PCT/US94/05795 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 192 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Ala Pro Ala Pro Glu Arg Val Val Ala Ile Ser Ser Val Ser Val Gly Val Gly Pro Arg Gly Glu Thr Asp Met Leu Ala Arg Val Ala Val Ile Asp Phe Thr Gly Ala Val Leu Leu Asp Val Tyr Val Ala Pro Thr Asn Pro Val Arg Asp Tyr Arg Glu Ala Lys Thr Gly Ile Lys Pro Glu Tyr Leu Tyr Ser Ser Arg Ala Gln Asp Ile Arg Ala Val Tyr Gln Thr Val Arg Gln Val Leu Arg Asn Lys Val Val Val Gly His Ser Met Trp Leu Asp Phe Met Val Leu Gly Leu Thr His Pro Thr Lys Asp Thr Arg Asp Val Ala Leu Tyr Leu Pro Phe Arg Asn Thr Leu Arg Cys Gln Arg Met Ile Gly Leu Trp Thr Leu Asn Tyr Arg Leu Leu Gly Leu Arg Cys Ser Ala Ala Pro Val Asp Pro Leu Glu Ser Ala Arg Val Ala Leu Asn Leu Tyr Arg Cys Tyr Ala Ala Gln Trp Glu Asp Thr Ile Ser Ser Arg Ser Trp Pro Cys Glu Leu Pro Pro Pro Cys Phe Arg Gly Cys Phe Met

Claims (23)

WHAT IS CLAIMED IS:

1. A DNA fragment isolated from Schizophyllum commune having mushroom-inducing activity and having the sequence shown in SEQ ID NO:1 or modifications thereof.

2. A protein having the amino acid sequence shown in SEQ ID NO:3 or a derivative thereof having mushroom-inducing activity.

3. An expression vector comprising the sequence of claim 1.

4. A host cell transformed with the expression vector of claim 4.

5. A process for inducing the development of fruiting bodies in a non-fruiting host cell strain, the process comprising the steps of:
a) identifying a DNA sequence encoding a mushroom-inducing gene from edible Basidiomycete; and b) integrating said DNA sequence or a modification thereof into a non-fruiting host cell.

6. The process of claim 5 wherein the fruiting bodies are induced in the absence of activity of a mating-type gene.

7. A process for enhancing the development of fruiting bodies in a host cell strain, the process comprising the steps of:
a) identifying a DNA sequence encoding a fruiting gene from an edible Basidiomycete; and b) integrating said DNA sequence or a modification thereof into a host cell during a phase of the host cell life cycle wherein the mating-type gene is activated.

8. The process of claim 5 wherein the DNA sequence encodes for a mushroom-inducing gene from Schizophyllum commune or a modification thereof.

9. The process of claim 7 wherein the DNA sequence encodes for a mushroom-inducing gene from Schizophyllum commune or a modification thereof.

10. A process for identifying a DNA sequence encoding a mushroom-inducing gene, the process comprising utilizing the DNA sequence or portion thereof of the sequence shown in SEQ ID NO:1 to isolate related sequences from endogenous to edible fungi.

11. A process for transforming a Basidiomycete species of commercial value, the process comprising the steps of:

a) treating the Basidiomycete cells or protoplasts with recombinant DNA under conditions permitting at least some of the Basidiomycete cells to take up the recombinant DNA and form transformants therewith; and b) obtaining Basidiomycete transformants.

12. The process of claim 11 wherein the recombinant DNA is linked to a homologous or heterologous promoter.

13. The process of claim 12 wherein the promoter is an inducible promoter.

14. A process of claim 12 wherein the Basidiomycete species are selected from the group consisting of Agaricus bisporus, Agaricus bitorquis, Flammulina velutipes, Lentinus edodes and Volvariella volvacea.

15. A transformed Basidiomycete cell containing DNA having mushroom-inducing activity.

16. A recombinant DNA construct which contains a selectable marker gene and all or part of the DNA fragment shown in SEQ ID NO:1.

17. A recombinant DNA construct which contains a selectable marker gene and all or part of a DNA sequence identified by a process of claim 10 .

18. A plasmid which contains the recombinant DNA construct of claim 16.

19. A plasmid which contains the recombinant DNA construct of claim 17.

20. A fruiting body made by the process of claim 5.

21. A fruiting body made by the process of claim 7.

22. A fruiting body made by the process of claim 11.

23. A DNA sequence encoding FRT1 protein from Schizophyllum commune comprising the cDNA sequence shown in SEQ ID NO:2 or a modification thereof encoding FRT? protein containing mushroom-inducing activity.