Note: Descriptions are shown in the official language in which they were submitted.
W O 94/28136PCTrUS94/05795
2 1 63790
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
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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.
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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
21 63790
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).
--10--
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SCHEME 1
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
-12-
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
(1) GENERAL INFORMATION:
~i) APPLICANT: GENENCOR INTERNATIONAL, INC.
(ii) TITLE OF INVENTION: Isolation of Mushroom-Inducing Genes and
Their Use in DNA-Mediated Transformation of Edible
Basidiomycetes
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genencor
(B) STREET: 180 Kimball Way
(C) CITY: South San Francisco
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94080
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Krupen, Karen I.
(B) REGISTRATION NUMBER: 34,647
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 742-7500
(B) TELEFAX: (415) 742-7217
(2) INFORMATION FOR SEQ ID NO:1:
(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
CTGCCATCTT CGCAGCGTTC TCGGCTGCAG CTACCTCCGC AGACCGATCT GAGCGAAGGT 120
CATCGACTCG CTGTGCATCC GCATACGCCG TGTAAATCTT CTCCGCATCC GCCTCGAGCT 180
~ GTGCGAAGCT AGTGACCCTC TGTCCATACT CTTCAAGTGA GCCTACACCC GAGACGAGGA 240
GAAGACAGTG GAGGACGCGA GCGTAGAGAG AGACAAATAT GAGGTCACGG CAGGTTCGGT 300
AGTTTGGGAG AGATGTAAGG GTGATTGGGA GGCGGTGAAG GCGTGCTCGG CGAGCCTCGC 360
CCGCTCCCTC GCTCTCATTC CAAAACCAGA AAAGCCCGGC TGTCGGCCCG TTTGCCGTCC 420
TCCCGATTCA GTCAAAGCCT TGAATCCGTT CGGGCTGTGG CTCGCCTGCG ACTGAACGAG 480
-15-
W O 94~136 2 1 6 3 7 9 0 PCTrUS94/05795
TCTAACCGTA GCCATGGCCC CAGCCCCAGA GCGCGTAGTC GCCATCTCCT GTGAGTACCT 540
TGGACTACCT AAAGACCCTG ATGTGCCTGA GACAGACTCG CCTTAGCCGT CAGCGTTGGG 600
GTAGGCCCTC GCGGAGAGAC AGACATGCTA GCACGGGTGG CCGTGATCGA CTTCACTGGG 660
GCTGTGCTCC TGGACGTGTA TGTCGCGCCC ACCAACCCAG TCCGAGACTA TCGAGAAGCA 720
AAGACTGGCA TCAAGCCGGA GTATCTCTAC TCTTGTAAGC CAACTATTCA lll~llGACG 780
TAl~lC~lCG CTGACCGGCT ATCATGAGCA CGAGCACAGG ATATCCGAGC TGTCTACCAG 840
ACAGTGCGCC AGGTTTTACG CAACAAAGTC GTTGTGGGCC ACAGCATGTG GCTGGACTTC 900
ATGGTCCTTG GTTTGACACA TCCAACAAAG GATACGCGTG ACGTTGCTCT CTACCTTGTA 960
CGAGCCTGCC TTCATACATC TCATGCCTGC CCCGCTTATC ACCCTTGTAG CCTTTTCGAA 1020
ATACGCTCCG CTGCCAACGT ATGATAGGCC TGTGGACCCT CAATTACAGG ~ll~llGGAC 1080
TGCGATGTTC TGCCGCACCC GTTGACCCTT TGGAAAGTGC TCGTGTCGCT CTCAATCTGT 1140
ACCGTTGCTA TGCAGCTCAA TGGGAGGACA CGATATCCTC CCGCTCCTGG CCTTGTGAGC 1200
TACCTCCCCC ATGCTTCCGT GGTTGCTTTA TGTAGCAGGC GTCCTGCATT CATAATTCAA 1260
CCGGACCTAC CGCTCAGTGC TATGGTGGTG ACCAAGGATC A~l~l~lGGT TTAGCAGGAT 1320
GGGACTGAAC TGAAGTGGCG AAAAGGAGGA ATCCATGGGA AGGATGAGGG CGTGATGCAC 1380
AGACTAGCGC GAGGGGGAAT CGCTTGCTCA CTACGGCTCA TCCTCGCGAG CCGTGTACGA 1440
AAACCCGTCC TCCTTTACAC CGTCACGGTA AGCAGAGATT CAGCTCGAAA CATAACTACG 1500
CATATGGCAT GCCTTATACG CTTACGCCTC CCTTCTCACC ATGGCTACTA CTGACTGTGC 1560
TGACCAAGCG CGAGGCTTAC GTACCTCGAG CATGAACAAA TCATTACTTT GTATCAAAAT 1620
ACTGTGCCGG CCCCA 1635
(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:
55 ATGGCCCCAG CCCCAGAGCG CGTAGTCGCC ATCTCCTCCG TCAGCGTTGG GGTAGGCCCT 60
CGCGGAGAGA CAGACATGCT AGCACGGGTG GCCGTGATCG ACTTCACTGG GGCTGTGCTC 120
CTGGACGTGT ATGTCGCGCC CACCAACCCA GTCCGAGACT ATCGAGAAGC AAAGACTGGC 180
ATCAAGCCGG AGTATCTCTA CTCTTCACGA GCACAGGATA TCCGAGCTGT CTACCAGACA 240
GTGCGCCAGG TTTTACGCAA CAAAGTCGTT GTGGGCCACA GCATGTGGCT GGACTTCATG 300
GTCCTTGGTT TGACACATCC AACAAAGGAT ACGCGTGACG TTGCTCTCTA CCTTCCTTTT 360
CGAAATACGC TCCGCTGCCA ACGTATGATA GGCCTGTGGA CCCTCAATTA CAGGCTTCTT 420
GGACTGCGAT GTTCTGCCGC ACCCGTTGAC CCTTTGGAAA GTGCTCGTGT CGCTCTCAAT 480
-16-
WO 94128L~6 2 1 6 3 7 9 0 PCT/US94/05795
CTGTACCGTT GCTATGCAGC TCAATGGGAG GACACGATAT CCTCCCGCTC CTGGCCTTGT 540
GAGCTACCTC CCCCATGCTT CCGTGGTTGC TTTATGTAG 579
(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
1 5 10 15
Gly Val Gly Pro Arg Gly Glu Thr Asp Met Leu Ala Arg Val Ala Val
20 25 30
Ile Asp Phe Thr Gly Ala Val Leu Leu Asp Val Tyr Val Ala Pro Thr
35 40 45
Asn Pro Val Arg Asp Tyr Arg Glu Ala Lys Thr Gly Ile Lys Pro Glu
50 55 60
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
85 90 95
Leu Asp Phe Met Val Leu Gly Leu Thr His Pro Thr Lys Asp Thr Arg
100 105 110
Asp Val Ala Leu Tyr Leu Pro Phe Arg Asn Thr Leu Arg Cys Gln Arg
115 120 125
Met Ile Gly Leu Trp Thr Leu Asn Tyr Arg Leu Leu Gly Leu Arg Cys
130 135 140
Ser Ala Ala Pro Val Asp Pro Leu Glu Ser Ala Arg Val Ala Leu Asn
145 150 155 160
Leu Tyr Arg Cys Tyr Ala Ala Gln Trp Glu Asp Thr Ile Ser Ser Arg
165 170 175
Ser Trp Pro Cys Glu Leu Pro Pro Pro Cys Phe Arg Gly Cys Phe Met
180 185 190
