# Patent

## Title
Use of Galerina marginata genes and proteins for peptide production

## Abstract

The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

## Background
This continuation-in-part application claims benefit of the priority filing date of U.S. patent application Ser. No. 12/268,229 filed on Nov. 10, 2008, now U.S. patent publication No. US-2010-0267019-A1 on Oct. 21, 2010, and of U.S. Provisional Patent Application Ser. No. 61/002,650, filed on Nov. 9, 2007, all of which are herein incorporated by reference.


GOVERNMENT INTERESTS
This invention was made in part with government support under DE-FG02-91ER20021 awarded by the United States Department of Energy. The Government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides including small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.
BACKGROUND
More than 90% of human deaths resulting from mushroom poisoning are due to peptide toxins found in Amanita species of mushrooms, such as A. phalloides, A. bisporigera, A. ocreata, and A. virosa. Animals, especially dogs, are frequent victims of poisoning by Amanita mushrooms. Two dogs died after eating toxin containing mushrooms in Michigan, See Schneider: Mushroom in backyard kills curious puppy, Lansing State Journal, Sep. 30, 2008. Besides species in the genus Amanita, other genera of mushrooms make similar toxins, such as phallotoxins and amatoxins. These other genera include Galerina, Conocybe, and Lepiota. Poisonings due to Galerina species have occurred, see FIG. 31.
High concentrations of peptide toxins are found in the above ground mushroom portion (otherwise known as carpophores or fruiting bodies) of the toxin producing mushroom species. These toxins include two major families of compounds called amatoxins (for example, α-amanitin, FIG. 1A) and phallotoxins (for example, phalloidin, phallacidin, FIG. 1B). Both classes of compounds are bicyclic peptides with a Cys-Trp cross-bridge. In general, amatoxins are 8 amino acids in length while phallotoxins are 7 amino acids in length. Amatoxins are produced by Amanita and some Galerina species of mushrooms. Galerina species in general do not make phallotoxins. Amatoxins survive cooking and remain intact in the intestinal tract where they are absorbed into the body where large doses irreversibly damage the liver and other organs (Enjalbert et al., (2002) J. Toxicol. Clin. Toxicol. 40:715; herein incorporated by reference).
Amatoxins and phallotoxins are used extensively for experimental research. Amatoxins are a family of bicyclic peptides that inhibit RNA polymerase II while phallotoxins bind and stabilize F-actin. However Amanita species do not grow well in the laboratory and harvesting from wild sources limits availability of a natural source of these peptides.
Thus it would be useful to have methods for obtaining large quantities of bicyclic amatoxins in addition to custom designed bicyclic amatoxin and phallotoxin peptides using cultivatable mushrooms.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.
The present invention also relates to a composition comprising a recombinant fungal prolyl oligopeptidase nucleic acid sequence selected from the group consisting of SEQ ID NO: 715 and 717.
The present invention also relates to a composition comprising a Galerina fungus transfected with a recombinant prepropeptide nucleic acid sequence encoding a peptide capable of forming a cyclic peptide. In one embodiment, said prepropeptide nucleic acid sequence is selected from the group consisting of nucleic acid sequences encoding SEQ ID NOs:710 and 713. In one embodiment, said cyclic peptide is a bicyclic peptide. In one embodiment, said bicyclic peptide comprises sequence SEQ ID NO:50.
The present invention also relates to a method of making a peptide from a recombinant prepropeptide sequence, comprising, a) providing, a composition comprising a Galerina fungus and a recombinant prepropeptide nucleic acid sequence further encoding a peptide capable of forming a cyclic peptide, and b) contacting said Galerina fungus with said recombinant prepropeptide nucleic acid sequence under conditions for making said peptide. In one embodiment, said contacting comprises transformation of said Galerina fungus with said recombinant prepropeptide sequence. In one embodiment, said peptide is selected from the group consisting of peptides at least six and up to fifteen amino acids in length. In one embodiment, said peptide is biologically active. In one embodiment, said peptide is a cyclic peptide. In one embodiment, said cyclic peptide is a bicyclic peptide. In one embodiment, said bicyclic peptide comprises sequence SEQ ID NO:50.
The present invention also relates to a method of making a synthetic cyclized peptide, comprising, a) providing, i) a Galerina fungal cell, ii) a recombinant prepropeptide nucleic acid sequence comprising a nucleic acid sequence encoding a peptide capable of forming a cyclic peptide, and b) transforming said Galerina cell with said prepropeptide sequence and c) growing said Galerina fungal cell into a fungus under conditions for expressing said prepropeptide for making a synthetic cyclic peptide. In one embodiment, said recombinant prepropeptide encoding sequence is selected from the group consisting of nucleic acid sequences encoding SEQ ID NOs:710 and 713. In one embodiment, said cyclic peptide is selected from the group consisting of a peptide at least six and up to fifteen amino acids in length. In one embodiment, said cyclic peptide is a bicyclic peptide. In one embodiment, said bicyclic peptide comprises SEQ ID NO:50. In one embodiment, said cyclized peptide is biologically active.
The present invention provides an isolated nucleic acid sequence selected from the group consisting of SEQ ID NOs: 709-714, 715, 717, 723 and fragments thereof.
The present invention provides an isolated amino acid sequence selected from the group consisting of SEQ ID NOs: 704-708, 716, 722, 753 and fragments thereof.
The present invention provides a composition comprising a Galerina fungus transformed with a recombinant propeptide nucleic acid sequence encoding a peptide capable of forming a cyclic peptide.
The present invention provides a composition comprising a Galerina fungus transformed with a recombinant nucleic acid sequence encoding a peptide capable of forming a cyclic peptide. In one embodiment, said peptide is selected from the group consisting of peptides at least six amino acids up to fifteen amino acids in length. In one embodiment, said peptide is a bicyclic peptide. In one embodiment, said bicyclic peptide is an Amanitin peptide.
The present invention provides a composition comprising a Galerina fungal cell and a synthetic propeptide sequence comprising a peptide sequence capable of forming a cyclic peptide. In one embodiment, said synthetic propeptide sequence is SEQ ID NO:249. In one embodiment, said peptide sequence is SEQ ID NO:69. In one embodiment, said Galerina fungal cell is a lysate.
The present invention also relates to compositions and methods comprising genes and peptides associated with cyclic peptide toxins and toxin production in mushrooms. In particular, the present invention relates to using genes and proteins from Amanita species encoding Amanita peptides, specifically relating to amatoxins and phallotoxins. In a preferred embodiment, the present invention also relates to methods for detecting Amanita peptide toxin genes for identifying Amanita peptide-producing mushrooms and for diagnosing suspected cases of mushroom poisoning. Further, the present inventions relate to providing kits for diagnosing and monitoring suspected cases of mushroom poisoning in patients.
The present invention provides an isolated nucleic acid sequence comprising at least one sequence set forth in SEQ ID NOs:1-4, 55-56, 79, 81, 85-86, and 97-98. In one embodiment, the nucleic acid encodes a polypeptide comprising at least one sequence set forth in SEQ ID NOs:50, 113, 118, 121-132, and 135. In one embodiment, the nucleic acid sequence comprises a sequence at least 50% identical to any sequence set forth in SEQ ID NOs: 182, 18-22. In one embodiment, the nucleic acid sequence encodes a peptide set forth in any one of SEQ ID NOs: 136-149 and 80. In one embodiment, the nucleic acid sequence comprises SEQ ID NOs: 86. In one embodiment, the polypeptide is selected from the group consisting of IWGIGCNP (SEQ ID NO: 50) and AWLVDCP (SEQ ID NO: 69). In one embodiment, the invention provides a polypeptide encoded by the nucleic acid sequences SEQ ID NOs: 55-56, 79, 81, and 85-86.
The present invention provides a composition comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises at least one sequence set forth in SEQ ID NOs: 1-4, 55-56, 79, 81, 85-86, and 97-98.
The present invention provides a composition comprising a polypeptide, wherein said polypeptide is encoded by a nucleic acid sequence comprising at least one sequence set forth in SEQ ID NOs: 55-56, 79, 81, and 85-86.
The present invention provides a set of at least two polymerase chain reaction primer sequences, wherein said primers are capable of amplifying a mushroom nucleic acid sequence associated with encoding an Amanita peptide. In one embodiment, the two polymerase chain reaction primer sequences are selected from the group SEQ ID NOs: 1-4, 97-98.
The present invention provides a method of identifying a toxin producing mushroom, comprising, a) providing, i) a sample, ii) a set of at least two polymerase chain reaction primers, wherein said primers are capable of amplifying a mushroom nucleic acid sequence associated with encoding a toxin, and iii) a polymerase chain reaction, b) mixing said sample with said set of polymerase chain reaction primers, c) completing a polymerase chain reaction under conditions capable of amplifying a mushroom nucleic acid sequence associated with encoding a toxin, and d) testing for an amplified toxin associated sequence for identifying a toxin producing mushroom. In one embodiment, the testing comprises detecting the presence or absence of an amplified mushroom nucleic acid sequence. In one embodiment, the sample is selected from the group consisting of a raw sample, a cooked sample, and a digested sample. In one embodiment, the sample comprises a mushroom sample. In one embodiment, the sample is obtained from a subject. The subject may be any mammal, e.g., the subject may be a human. In one embodiment, the set of polymerase chain reaction primer sequences may identify any Amanita peptide. In one embodiment, the set of polymerase chain reaction primer sequences may identify an amanitin peptide. In one embodiment, the set of polymerase chain reaction primer sequences are selected from the group consisting of SEQ ID NOs: 1-4, 97-98.
The present invention provides a diagnostic kit for identifying a poisonous mushroom, providing, comprising, a set of at least two polymerase chain reaction primers, wherein said primers are capable of amplifying a mushroom nucleic acid sequence associated with producing a toxin. In one embodiment, the two polymerase chain reaction primer sequences are selected from the group consisting of SEQ ID NOs: 1-4, 97-98. In one embodiment, the kit further comprises a nucleic acid sequence associated with producing a mushroom toxin, wherein said nucleic acid sequence is capable of being amplified by said polymerase chain reaction primers. In one embodiment, the kit further comprises instructions for amplifying said mushroom nucleic acid sequence. In one embodiment, the kit further comprises instructions for detecting the presence or absence of an amplified mushroom nucleic acid sequence. In one embodiment, the kit further comprises instructions for identifying the species of an amplified mushroom nucleic acid sequence. In one embodiment, the kit further comprises instructions for identifying the presence of a mushroom toxin peptide. In one embodiment, the kit further comprises instructions for identifying the presence of a mushroom toxin nucleic acid sequence.
The present invention provides a polypeptide, wherein said polypeptide is encoded by a sequence derived from a fungal species. In one embodiment, the polypeptide is an isolated polypeptide. In one embodiment, the isolated polypeptide is isolated from a cell. In one embodiment, the cell includes but is not limited to a fungal cell and a bacterial cell. In one embodiment, the isolated polypeptide is a synthetic polypeptide. It is not meant to limit the sequence of the polypeptide. In one embodiment, the polypeptide includes but is not limited to a polypeptide comprising a toxin sequence. In one embodiment, the polypeptide includes but is not limited to a preproprotein. In one embodiment, the polypeptide comprises at least one proprotein sequence set forth in SEQ ID NOs: 23, 26-37, 107-113, 118, 249, 303-306, 308-318. In one embodiment, the polypeptide is an amino acid sequence containing MSDIN upstream of a potential toxin encoding region and downstream conserved sequences. In one embodiment, the polypeptide comprises a toxin amino acid sequence. In one embodiment, the polypeptide comprises IWGIGCNP (SEQ ID NO:50) and AWLVDCP (SEQ ID NO:69). In one embodiment, the polypeptide comprises at least one sequence set forth in SEQ ID NOs: 249, and 318. In one embodiment, the polypeptide is linear. In one embodiment, the polypeptide is cyclic. In one embodiment, the polypeptide comprises at least one sequence set forth in SEQ ID NOs: 23, 26-37, 54, 69, 107-113, 118, 249, 303-306, 308-318. In one embodiment, the polypeptide includes but is not limited to a polypeptide comprising a prolyl oligopeptidase sequence. In one embodiment, the prolyl oligopeptidase sequence comprises at least one sequence set forth in SEQ ID NOs: 236, 237, 250-256, 258-276.
A composition, comprising a polypeptide, wherein said polypeptide is encoded by a sequence derived from a fungal species.
A method, comprising a polypeptide, wherein said polypeptide is encoded by a sequence derived from a fungal species.
The present invention provides an antibody having specificity for a polypeptide comprising a toxin sequence, wherein said a polypeptide is encoded by a nucleotide sequence derived from a fungal species. In one embodiment, the polypeptide includes but is not limited to exemplary Amanita and Galerina spp. peptides, proteins, proproteins and preproproteins. SEQ ID NOs: 50, 110, 113, 118, 121-132, 135, 249, 303-306, and 308-318. In one embodiment, the toxin includes but is not limited to a cyclic toxin, a linear amino acid sequence of a cyclic toxin, a portion of a linear amino acid sequence of a cyclic toxin. In one embodiment, the toxin includes but is not limited to an amatoxin or a phallotoxin. In one embodiment, the toxin includes but is not limited to an amanitin. In one embodiment, the toxin includes but is not limited to alpha, beta, gamma, etc., amanitin, Amanitin, amatoxins, etc. In one embodiment, the toxin includes but is not limited to cyclic forms of SEQ ID NOs: 50, 54, 69, 114, 117 and 135-149. In another embodiment, the invention provides an antibody having specificity for mushroom prolyl oligopeptidase including but not limited to Amanita and Galerina spp. prolyl oligopeptidase.
A composition, comprising an antibody having specificity for a preproprotein comprising a toxin sequence, wherein said preproprotein is encoded by a nucleotide sequence derived from a fungal species.
A method, comprising an antibody having specificity for a preproprotein comprising a toxin sequence, wherein said preproprotein is encoded by a nucleotide sequence derived from a fungal species.
The present invention provides an antibody having specificity for a toxin encoded by a nucleotide sequence derived from a fungal species. In one embodiment, the toxin includes but is not limited to a cyclic toxin, a linear amino acid sequence of a cyclic toxin, a portion of a linear amino acid sequence of a cyclic toxin. In one embodiment, the toxin includes but is not limited to an amanitin and a phallatoxin. In one embodiment, the toxin includes but is not limited to an alpha, beta, gamma, etc., amanitin. In one embodiment, the toxin includes but is not limited to SEQ ID NOs: 50, 54, 69, 114, 117 and 135-149. In one embodiment, the antibody includes but is not limited to a polyclonal antibody and a monoclonal antibody. In one embodiment, the antibody includes but is not limited to a rat, rabbit, mouse, chicken antibody.
A composition, comprising an antibody having specificity for a toxin encoded by a nucleotide sequence derived from a fungal species.
A method, comprising an antibody having specificity for a toxin encoded by a nucleotide sequence derived from a fungal species.
A composition, comprising an antibody having specificity for a prolyl oligopeptidase encoded by a nucleotide sequence derived from a fungal species.
A method, comprising an antibody having specificity for a prolyl oligopeptidase encoded by a nucleotide sequence derived from a fungal species.
The present invention provides an isolated prolyl oligopeptidase protein, wherein said prolyl oligopeptidase protein is encoded by nucleic acid sequence derived from a fungal species. In one embodiment, the prolyl oligopeptidase includes but is not limited to a prolyl oligopeptidase, prolyl oligopeptidase A, prolyl oligopeptidase B, and fragments thereof. In one embodiment, the prolyl oligopeptidase A comprises any one sequence set forth in SEQ ID NOs: 250-252, 254, 258, 261-269, 271-273, 275-276, 330-332, 334-336, 346. In a preferred embodiment, the prolyl oligopeptidase B comprises any one sequence set forth in SEQ ID NOs: 267, 253, 271, 273, 276, 280, 282, 286, 288, 289, 290, 293, 296-297, 332, 343, 345, 346, 336, 337, 339, 343, 302.
A composition, comprising an isolated prolyl oligopeptidase protein, wherein said prolyl oligopeptidase protein is encoded by nucleic acid sequence derived from a fungal species.
A method, comprising an isolated prolyl oligopeptidase protein, wherein said prolyl oligopeptidase protein is encoded by nucleic acid sequence derived from a fungal species.
The present invention provides an antibody having specificity to a prolyl oligopeptidase protein, wherein said prolyl oligopeptidase protein is encoded by a nucleotide sequence derived from a fungal species. In one embodiment, the prolyl oligopeptidase includes but is not limited to a prolyl oligopeptidase, prolyl oligopeptidase A prolyl oligopeptidase B, and fragments thereof. In one embodiment, the prolyl oligopeptidase A comprises any one sequence set forth in SEQ ID NOs: 250-252, 254, 258, 261-269, 271-273, 275-276, 330-332, 334-336, 346. In a preferred embodiment, the prolyl oligopeptidase B comprises any one sequence set forth in SEQ ID NOs: 267, 253, 271, 273, 276, 280, 282, 286, 288, 289, 290, 293, 296-297, 332, 343, 345, 346, 336, 337, 339, 343, 302.
A composition, comprising a mushroom P450 protein.
A method, comprising a mushroom P450 protein.
DEFINITIONS
To facilitate an understanding of the present invention, a number of terms and phrases as used herein are defined below:
The use of the article “a” or “an” is intended to include one or more.
As used herein, terms defined in the singular are intended to include those terms defined in the plural and vice versa.
As used herein, “peptide” refers to compounds containing two or more amino acids linked by the carboxyl group of one amino acid to the amino group of another, i.e. “peptide linkages” to form an amino acid sequence. It is contemplated that peptides may be purified and/or isolated from natural sources or prepared by recombinant or synthetic methods. Amino acid sequences may be encoded by naturally or non-naturally occurring nucleic acid sequences or synthesized by recombinant nucleic acid sequences or artificially synthesized. A peptide may be a linear peptide or a cyclopeptide, i.e. cyclic including bicyclic.
As used herein, “cyclic peptide” or “cyclopeptide” in general refers to a peptide comprising at least one internal bond attaching nonadjacent amino acids of the peptide, such as when the end amino acids of a linear sequence are attached to form a circular peptide. A “bicyclic peptide” may have at least two internal bonds forming a cyclopeptide of the present inventions, such as when the end amino acids of a linear sequence are attached to form a circular peptide in addition to another internal bond attaching two nonadjacent amino acids, for examples, see FIG. 1, amanatoxin and pallotoxins.
As used herein, the term “Amanita peptide” or “Amanita toxin” or “Amanita peptide toxin” refers to any linear or cyclic peptide produced by a mushroom, not restricted to a biologically active toxin. It is not intended that the present invention be limited to a toxin or a peptide produced by an Amanita mushroom and includes similar peptides and toxins produced by other fungi, including but not limited to species of Lepiota, Conocybe, Galerina, and the like. In particular, an Amanita peptide toxin resembles any of the amatoxins and phallotoxins, such as similarity of amino acid sequences, matching toxin motifs as shown herein, encoded between the conserved regions (A and B) of their proproteins, encoded by hypervariable regions of their proproteins (P), and the like. The Amanita peptides include, but are not restricted to, amatoxins such as the amanitins, and phallotoxins such as phalloidin and phallacidin. For example, an exemplary Amanita peptide in one embodiment ranges from 6-15 amino acids in length. In another embodiment an Amanita peptide toxin ranges from 7-11 amino acids in length. In one embodiment, an Amanita peptide is linear. In another embodiment, an Amanita peptide is a bicyclic peptide. It is not meant to limit an Amanita peptide to a naturally produced peptide. In some embodiments, an Amanita peptide has a artificial sequence, in other words a nucleic acid encoding an artificial peptide sequence was not naturally found in a fungus or found encoded by a nucleic acid sequence isolated from a fungus.
As used herein, “biologically active” refers to a peptide that when contacted with a cell, tissue or organ induces a biological activity, such as stimulating a cell to divide, causing a cell to alter its function, i.e. altering T cell function, causing a cell to change expression of genes, etc.
As used herein, a “propeptide” refers to an amino acid sequence containing a smaller peptide representing the amino acid sequence found in mature amatoxins and phallotoxins in addition to new amino acid sequences in the toxin position, for example, a propeptide of GmAMA1, see FIG. 32, comprises an amanitin IWGIGCNP (SEQ ID NO: 50) while exemplary sequences coding for new peptides in the toxin position are shown in Table 10C and 11.
As used herein, a “prepropeptide” refers to an amino acid sequence containing a leader sequence, such as a signal sequence for translation, on the 5′ end prior to the start site, i.e. M, in addition to a smaller peptide representing the amino acid sequence found in mature amatoxins and phallotoxins, for example, LTSHSNSNPRPLLITMSDINATRLPAWLVDCPCVGDDVNRLL (SEQ ID NO. 75) shows an exemplary prepropeptide wherein the propeptide is BOLD and the peptide is underlined.
The terms “peptide,” “polypeptide,” “propeptide,” “propolypeptide,” “prepropeptide,” “prepropolypeptide,” and “protein” in general refer to a primary sequence of amino acids that are joined by covalent “peptide linkages.” Polypeptides may encompass either peptides or proteins. In general, a peptide consists of a few amino acids, and is shorter than a protein. “Amino acid sequence” and like terms, such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
As used herein, the term “synthetic” or “artificial” in relation to a peptide sequence refers to a peptide made either artificially from covalently bonding amino acids, such as by made by a Peptide Synthesizer, (for example, Applied Biosystems) or a peptide derived from an amino acid sequence encoded by a recombinant nucleic acid sequence.
As used herein, the term “toxin” in general refers to any detrimental or harmful effects on a cell or tissue. However for the purpose of the present inventions a “toxin” or “peptide toxin” specifically refers to a peptide sequence found within a propeptide in the position of a known toxin of the present inventions, for examples, see Table 10. Therefore, a peptide found within a propeptide may have a biological activity.
As used herein, the term “toxin” in reference to a poison refers to any substance (for example, alkaloids, cyclopeptides, coumarins, and the like) that is detrimental (i.e., poisonous) to cells and/or organisms, in particular a human organism.
In particularly preferred embodiments of the present inventions, the term “toxin” encompasses toxins, suspected toxins, and pharmaceutically active peptides or biologically active peptides produced by various fungal species, including, but not limited to, a cyclic peptide toxin such as an amanitin, that provides toxic activity towards cells and humans. However, it is not intended that the present invention be limited to any particular fungal toxin or fungal species. Indeed, it is intended that the term encompass fungal toxins produced by any organism. As used herein, a toxin encompasses linear sequences of cyclic pharmaceutically active peptides and linear sequences showing identity to known toxins regardless of whether these sequences are known to be toxic.
As used herein, “amatoxin” generally refers to a family of peptide compounds, related to and including the amanitins. For the purposes of the present inventions, an amatoxin refers to any small peptide, linear and cyclic, comprising an exemplary chemical structure as shown in FIG. 1 or encoded by nucleic acid sequence of the present invention, wherein the nucleic acid sequence and/or proprotein has a higher sequence homology to AMA1 than to an analogous sequence of PHA1.
As used herein, “phallotoxin” generally refers to a family of peptide compounds, related to and including phallacidin and phalloidin. For the purposes of the present inventions, a phallotoxin refers to any small peptide encoded by nucleic acid sequences where the nucleic acid sequence and/or proprotein has a higher sequence homology to PHA1 than to an analogous sequence of AMA1.
As used herein the term “microorganism” refers to microscopic organisms and taxonomically related macroscopic organisms within the categories of algae, bacteria, fungi (including lichens), protozoa, viruses, and subviral agents.
The terms “eukaryotic” and “eukaryote” are used in the broadest sense. It includes, but is not limited to, any organisms containing membrane bound nuclei and membrane bound organelles. Examples of eukaryotes include but are not limited to animals, plants, algae, diatoms, and fungi.
The terms “prokaryote” and “prokaryotic” are used in the broadest sense. It includes, but is not limited to, any organisms without a distinct nucleus. Examples of prokaryotes include but are not limited to bacteria, blue-green algae (cyanobacteria), archaebacteria, actinomycetes and mycoplasma. In some embodiments, a host cell is any microorganism.
As used herein, the term “fungi” is used in reference to eukaryotic organisms such as mushrooms, rusts, molds and yeasts, including dimorphic fungi. “Fungus” or “fungi” also refers to a group of lower organisms lacking chlorophyll and dependent upon other organisms for source of nutrients.
As used herein, “mushroom” refers to the fruiting body of a fungus.
As used herein, “fruiting body” refers to a reproductive structure of a fungus which produces spores, typically comprising the whole reproductive structure of a mushroom including cap, gills and stem, for example, a prominent fruiting body produced by species of Ascomycota and Basidiomycota, examples of fruiting bodies are “mushrooms,” “carpophores,” “toadstools,” “puffballs”, and the like.
As used herein, “fruiting body cell” refers to a cell of a cap or stem which may be isolated or part of the structure.
As used herein, “spore” refers to a microscopic reproductive cell or cells.
As used herein, “mycelium” refers to a mass of fungus hyphae, otherwise known as a vegetative portion of a fungus.
As used herein, “Basidiomycota” in reference to a Phylum or Division refers to a group of fungi whose sexual reproduction involves fruiting bodies comprising basidiospores formed on club-shaped cells known as basidia.
As used herein, “Basidiomycetes” in reference to a class of Phylum Basidiomycota refers to a group of fungi. Basidiomycetes include mushrooms, of which some are rich in cyclopeptides and/or toxins, and includes certain types of yeasts, rust and smut fungi, gilled-mushrooms, puffballs, polypores, jelly fungi, brackets, coral, mushrooms, boletes, puffballs, stinkhorns, etc.
As used herein, “Homobasidiomycetes” in reference to fungi refers to a recent classification of fungi, including Amanita spp., Galerina spp., and all other gilled fungi (commonly known as mushrooms), based upon cladistics rather than morphology.
As used herein, “Heterobasidiomycetes” in reference to fungi refers to those basidiomycete fungi that are not Homobasidiomycetes.
As used herein, “Ascomycota” or “ascomycetes” in reference to members of a fungal Phylum or Division refers to a “sac fungus” group. Of the Ascomycota, a class “Ascomycetes” includes Candida albicans, unicellular yeast, Morchella esculentum, the morel, and Neurospora crassa. Some ascomycetes cause disease, for example, Candida albicans causes thrush and vaginal infections; or produce chemical toxins associated with diseases, for example, Aspergillus flavus produces a contaminant of nuts and stored grain called aflatoxin, that acts both as a toxin and a deadly natural carcinogen.
As used herein, “Amanita” refer to a genus of fungus whose members comprise poisonous mushrooms, e.g., Amanita (A.) bisporigera, A. virosa, A. ocreata, A. suballiacea, and A. tenuifolia which are collectively referred to as “death angels” or “Destroying Angels” and “Amanita phalloides” or “A. phalloides var. alba” or “A. phalloides var. verna” or “A. verna”, referred to as “death cap.” The toxins of these mushrooms frequently cause death through liver and kidney failure in humans. Not all species of this genus are deadly, for example, Amanita muscaria, the fly agaric, induces gastrointestinal distress and/or hallucinations while others do not induce detectable symptoms.
As used herein, nonribosomal peptide synthetase (NRPS) is an enzyme that catalyzes the biosynthesis of a small (20 or fewer amino acids) peptide or depsipeptide, linear or circular, and is composed of one or more domains (modules) typical of this class of enzyme. Each domain is responsible for aminoacyl adenylation of one component amino acid. NRPSs can also contain auxiliary domains catalyzing, e.g., N-methylation and amino acid epimerization (Walton, et al., in Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine, et al., Eds. (Kluwer Academic/Plenum, N.Y., 2004, pp. 127-162; Finking, et al., (2004) Annu Rev Microbiol 58:453-488, all of which are herein incorporated by reference). Examples are gramicidin synthetase, HC-toxin synthetase, cyclosporin synthetase, and enniatin synthetase.
As used herein, “prolyl oligopeptidase” or “POP” refers to a member of a family of enzymes classified and referred to as EC 3.4.21.26-enzymes that are capable of cleaving a peptide sequence, such that hydrolysis of Pro-|-Xaa>>Ala-|-Xaa in oligopeptides, also referred to as any one of “post-proline cleaving enzyme,” “proline-specific endopeptidase,” “post-proline endopeptidase,” “proline endopeptidase,” “endoprolyl peptidase,” “prolyl endopeptidase,” “post-proline cleaving enzyme,” “post-proline endopeptidase,” and “prolyl endopeptidase.” A POPA of the present inventions refers to a mushroom sequence found in the majority of mushrooms. A POPB of the present inventions refers to a sequence which in one embodiment has approximately a 55% amino acid homology to POPA, wherein said POPB sequence is primarily found in Amanita peptideproducing mushroom species.
As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which are any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. Several types of fungi and cultures are available for use as a host cell, such as those described for use in fungal expression systems, described below. Prokaryotes include but are not limited to gram negative or positive bacterial cells. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), an organization that serves as an archive for living cultures and genetic materials (atcc.org). An appropriate host can be determined by one of skill in the art based on the vector nucleic acid sequence and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for expression vector replication and/or expression include, among those listed elsewhere herein, DH5α, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURETM Competent Cells and SOLOPACKTM Gold Cells (Stratagene, La Jolla). Alternatively, bacterial cells such as E. coli LE392 can be used as host cells for phage viruses. In some embodiments, a host cell is used as a recipient for vectors. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. For example, a host cell may be located in a transgenic mushroom. A transformed cell includes the primary subject cell and its progeny.
As used herein, “host fungus cell” refers to any fungal cell, for example, a yeast cell, a mold cell, and a mushroom cell (such as Neurospora crassa, Aspergillus nidulans, Cochliobolus carbonum, Coprinus cinereus, Ustilago maydis, and the like).
As used herein, the term “Fungal expression system” refers to a system using fungi to produce (express) enzymes and other proteins and peptides. Examples of filamentous fungi which are currently used or proposed for use in such processes are Neurospora crassa, Acremonium chrysogenum, Tolypocladium geodes, Mucor circinelloides, Trichoderma reesei, Aspergillus nidulans, Aspergillus niger, Coprinus cinereus, Aspergillus oryzae, etc. Further examples include an expression system for basidiomycete genes (for example, Gola, et al., (2003) J Basic Microbiol. 43(2):104-12; herein incorporated by reference) and fungal expression systems using, for example, a monokaryotic laccase-deficient Pycnoporus cinnabarinus strain BRFM 44 (Banque de Resources Fongiques de Marseille, Marseille, France), and Schizophyllum commune, (for example, Alexandra, et al., (2004) Appl Environ Microbiol. 70(11):6379-638; Lugones, et al., (1999) Mol. Microbiol. 32:681-700; Schuren, et al., (1994) Curr. Genet. 26:179-183; all of which are herein incorporated by reference).
The term “transgene” as used herein refers to a foreign gene, such as a heterologous gene, that is placed into an organism by, for example, introducing the foreign gene into cells or primordial tissue. The term “foreign gene” refers to any nucleic acid (e.g., gene sequence) that is introduced into the genome of a host cell by experimental manipulations and may include gene sequences found in that cell so long as the introduced gene does not reside in the same location as does the naturally-occurring gene.
As used herein, the term “vector” is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term “vehicle” is sometimes used interchangeably with “vector.” A vector “backbone” comprises those parts of the vector which mediate its maintenance and enable its intended use (e.g., the vector backbone may contain sequences necessary for replication, genes imparting drug or antibiotic resistance, a multiple cloning site, and possibly operably linked promoter and/or enhancer elements which enable the expression of a cloned nucleic acid). The cloned nucleic acid (e.g., such as a cDNA coding sequence, or an amplified PCR product) is inserted into the vector backbone using common molecular biology techniques.
A “recombinant vector” indicates that the nucleotide sequence or arrangement of its parts is not a native configuration, and has been manipulated by molecular biological techniques. The term implies that the vector is comprised of segments of DNA that have been artificially joined.
The terms “expression vector” and “expression cassette” refer to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome-binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
As used herein, “recombinant nucleic acid” or “recombinant gene” or “recombinant DNA molecule” or “recombinant nucleic acid sequence” indicates that the nucleotide sequence or arrangement of its parts is not a native configuration, and has been manipulated by molecular biological techniques. The term implies that the DNA molecule is comprised of segments of DNA that have been artificially joined together, for example, a lambda clone of the present inventions. Protocols and reagents to manipulate nucleic acids are common and routine in the art (See e.g, Maniatis et al. (eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, [1982]; Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, NY, [1989]; and Ausubel et al. (eds.), Current Protocols in Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., New York [1994]; all of which are herein incorporated by reference). Similarly, a “recombinant protein” or “recombinant polypeptide” refers to a protein molecule that is expressed from a recombinant DNA molecule. Use of these terms indicates that the primary amino acid sequence, arrangement of its domains or nucleic acid elements which control its expression are not native, and have been manipulated by molecular biology techniques. As indicated above, techniques to manipulate recombinant proteins are also common and routine in the art.
As used herein, “recombinant prepropeptide nucleic acid sequence” refers to a nucleic acid sequence comprising a leader sequence which encodes a propeptide amino acid sequence. Similarly, a “recombinant propeptide nucleic acid sequence” refers to a nucleic acid sequence which encodes a propeptide amino acid sequence. Thus in general, a “recombinant peptide nucleic acid sequence” refers to a nucleic acid sequence which encodes a peptide amino acid sequence, such as a prepropeptide, a propeptide or smaller peptides, for example, peptides capable of forming cyclic peptides.
The terms “exogenous” and “heterologous” are sometimes used interchangeably with “recombinant.” An “exogenous nucleic acid,” “exogenous gene” and “exogenous protein” indicate a nucleic acid, gene or protein, respectively, that has come from a source other than its native source, and has been artificially supplied to the biological system. In contrast, the terms “endogenous protein,” “native protein,” “endogenous gene,” and “native gene” refer to a protein or gene that is native to the biological system, species or chromosome under study. A “native” or “endogenous” polypeptide does not contain amino acid residues encoded by recombinant vector sequences; that is, the native protein contains only those amino acids found in the polypeptide or protein as it occurs in nature. A “native” polypeptide may be produced by recombinant means or may be isolated from a naturally occurring source. Similarly, a “native” or “endogenous” gene is a gene that does not contain nucleic acid elements encoded by sources other than the chromosome on which it is normally found in nature.
As used herein, the term “heterologous gene” refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5′ and 3′ end of the sequences that are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5′ or 3′ to the untranslated sequences present on the mRNA transcript). The 5′ flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3′ flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.
As used herein, the terms “an oligonucleotide having a nucleotide sequence encoding a gene” and “polynucleotide having a nucleotide sequence encoding a gene,” mean a nucleic acid sequence comprising the coding region of a gene or, in other words, the nucleic acid sequence that encodes a gene product. The coding region may be present in a cDNA, genomic DNA, or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
As used herein, the term “regulatory element” refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements include splicing signals, polyadenylation signals, termination signals, etc.
The terms “in operable combination,” “in operable order,” “operably linked” and similar phrases when used in reference to nucleic acid herein are used to refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
A “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence (e.g., a nucleic acid sequence encoding a fusion protein of the present invention) to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” e.g., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR, in connection with the compositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by reference). It is further contemplated that control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment (e.g., comprising nucleic acid encoding a fusion protein of the present invention) in the cell type, organelle, and organism chosen for expression. Those of skill in the art of microbiology and molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989); herein incorporated by reference. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct the desired level of expression of the introduced DNA segment comprising a target protein of the present invention (e.g., high levels of expression that are advantageous in the large-scale production of recombinant proteins and/or peptides). The promoter may be heterologous or endogenous.
Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236: 1237 [1987]; herein incorporated by reference). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells, as well as viruses. Analogous control elements (i.e., promoters and enhancers) are also found in prokaryotes. The selection of a particular promoter and enhancer to be operably linked in a recombinant gene depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional only in a limited subset of cell types (for review, see, Voss et al., Trends Biochem. Sci., 11: 287 [1986] and Maniatis et al., Science 236:1237 [1987]; all of which are herein incorporated by reference).
The term “promoter/enhancer region” is usually used to describe this DNA region, typically but not necessarily 5′ of the site of transcription initiation, sufficient to confer appropriate transcriptional regulation. The word “promoter” alone is sometimes used synonymously with “promoter/enhancer.” A promoter may be constitutively active, or alternatively, conditionally active, where transcription is initiated only under certain physiological conditions or in the presence of certain drugs. The 3′ flanking region may contain additional sequences for regulating transcription, especially the termination of transcription.
The term “introns” or “intervening regions” or “intervening sequences” are segments of a gene which are contained in the primary transcript (i.e., hetero-nuclear RNA, or hnRNA), but are spliced out to yield the processed mRNA form. Introns may contain transcriptional regulatory elements such as enhancers. The mRNA produced from the genomic copy of a gene is translated in the presence of ribosomes to yield the primary amino acid sequence of the polypeptide.
Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
As used herein, the term “promoter/enhancer” denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (i.e., the functions provided by a promoter element and an enhancer element). For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The promoter/enhancer may be “endogenous,” or “exogenous,” or “heterologous.” An “endogenous” promoter/enhancer is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” promoter/enhancer is one placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques such as cloning and recombination) such that transcription of the gene is controlled by the linked promoter/enhancer.
As used herein, the term “subject” refers to both humans and animals.
As used herein, the term “patient” refers to a subject whose care is under the supervision of a physician/veterinarian or who has been admitted to a hospital.
The term “sample” is used in its broadest sense. In one sense it can refer to a mushroom cell or mushroom tissue. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples that may comprise mushroom toxins. Biological samples may be obtained from mushrooms or animals (including humans) and encompass fluids, such as gastrointestinal fluids, solids, tissues, and the like. Environmental samples include environmental material such as mushrooms, hyphae, soil, water, such as cooking water, and the like. These terms encompasses all types of samples obtained from humans and other animals, including but not limited to, body fluids such as digestive system fluid, saliva, stomach contents, intestinal contents, urine, blood, fecal matter, diarrhea, as well as solid tissue, partially and fully digested samples. These terms also refers to swabs and other sampling devices which are commonly used to obtain samples for culture of microorganisms. Biological samples may be food products and ingredients, such as a mushroom sample, a raw sample, a cooked sample, a canned sample, animal, including human, fluid or tissue and waste. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples, as well as samples obtained from food processing instruments, apparatus, equipment, disposable, and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
Whether biological or environmental, a sample suspected of containing a poisonous mushroom cell or mushroom toxin, may (or may not) first be subjected to an enrichment means. By “enrichment means” or “enrichment treatment,” the present invention contemplates (i) conventional techniques for isolating a particular mushroom cell or mushroom toxin or mushroom sequence of interest away from other components by means of liquid, solid, semi-solid based separation technique or any other separation technique, and (ii) novel techniques for isolating particular cells or toxins away from other components. It is not intended that the present invention be limited only to one enrichment step or type of enrichment means. For example, it is within the scope of the present invention, following subjecting a sample to a conventional enrichment means, such as HPLC, to subject the resultant preparation to further purification such that a pure sample or culture of a strain of a species of interest is produced. This pure sample or culture may then be analyzed by the compositions and methods of the present inventions.
Thus, a polynucleotide of the present invention may encode a polypeptide, a polypeptide plus a leader sequence (which may be referred to as a prepolypeptide), a precursor of a polypeptide having one or more prosequences which are not the leader sequences of a prepolypeptide, or a prepropolypeptide, which is a precursor to a propolypeptide, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active forms of the polypeptide.
As used herein, the term “portion” when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
As used herein, the term “target protein” or “protein of interest” when used in reference to a protein or nucleic acid refers to a protein or nucleic acid encoding a protein of interest for which structure or toxicity is to be analyzed and/or altered of the present invention, such as a gene encoding a mushroom toxin or a mushroom peptide. The term “target protein” encompasses both wild-type proteins and those that are derived from wild type proteins (e.g., variants of wild-type proteins or polypeptides, or, chimeric genes constructed with portions of target protein coding regions), and further encompasses fragments of a wild-type protein. Thus, in some embodiments, a “target protein” is a variant or mutant. The present invention is not limited by the type of target protein analyzed.
As used herein, the term “endopeptidase” refers to an enzyme that catalyzes the cleavage of peptide bonds within a polypeptide or protein. Peptidase refers to the fact that it acts on peptide bonds and endopeptidase refers to the fact that these are internal bonds. An exopeptide catalyzes the cleavage of the terminal or penultimate peptide bond, releasing a single amino acid or dipeptide from the peptide chain.
In particular, the terms “target protein gene” or “target protein genes” refer to the full-length target protein sequence, such as a prepropolypeptide. However, it is also intended that the term encompass fragments of the target protein sequences, mutants of the target protein sequences, as well as other domains within the full-length target protein nucleotide sequences. Furthermore, the terms “target protein nucleotide sequence” or “target protein polynucleotide sequence” encompasses DNA, cDNA, and RNA (e.g., mRNA) sequences.
The term “gene of interest” as used herein refers to the gene inserted into the polylinker of an expression vector whose expression in the cell is desired for the purpose of performing further studies on the transfected cell. The gene of interest may encode any protein whose expression is desired in the transfected cell at high levels. The gene of interest is not limited to the examples provided herein; the gene of interest may include cell surface proteins, secreted proteins, ion channels, cytoplasmic proteins, nuclear proteins (e.g., regulatory proteins), mitochondrial proteins, etc.
As used herein, the term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or protein precursor. The polypeptide can be encoded by a full-length coding sequence, or by a portion of the coding sequence, as long as the desired protein activity is retained. Genes can encode a polypeptide or any portion of a polypeptide within the gene's “coding region” or “open reading frame.” The polypeptide produced by the open reading frame of a gene may or may not display functional activity or properties of the full-length polypeptide product (e.g., toxin activity, enzymatic activity, ligand binding, signal transduction, etc.).
In addition to the coding region of the nucleic acid, the term “gene” also encompasses the transcribed nucleotide sequences of the full-length mRNA adjacent to the 5′ and 3′ ends of the coding region. These noncoding regions are variable in size, and sometimes extend for distances up to or exceeding 1 kb on both the 5′ and 3′ ends of the coding region. The sequences that are located 5′ and 3′ of the coding region and are contained on the mRNA are referred to as 5′ and 3′ untranslated regions (5′ UTR and 3′ UTR). Both the 5′ and 3′ UTR may serve regulatory roles, including translation initiation, post-transcriptional cleavage and polyadenylation. The term “gene” encompasses mRNA, cDNA and genomic forms of a gene.
It is contemplated that the genomic form or genomic clone of a gene may contain the sequences of the transcribed mRNA, as well as other non-coding sequences which lie outside of the mRNA. The regulatory regions which lie outside the mRNA transcription unit are sometimes called “5′ or 3′ flanking sequences.” A functional genomic form of a gene must contain regulatory elements necessary for the regulation of transcription.
Nucleic acid molecules (e.g., DNA or RNA) are said to have “5′ ends” and “3′ ends” because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide or polynucleotide is referred to as the “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring and as the“3′ end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5′ and 3′ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5′ of the “downstream” or 3′ elements. This terminology reflects the fact that transcription proceeds in a 5′ to 3′ fashion along the DNA strand. The promoter and enhancer elements that direct transcription of a linked gene are generally located 5′ or upstream of the coding region. However, enhancer elements can exert their effect even when located 3′ of the promoter element or the coding region. Transcription termination and polyadenylation signals are located 3′ or downstream of the coding region.
As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” and similar phrases refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (e.g., protein) chain. The DNA sequence thus codes for the amino acid sequence.
As used herein, the terms “an oligonucleotide having a nucleotide sequence encoding a gene,” “polynucleotide having a nucleotide sequence encoding a gene,” and similar phrases are meant to indicate a nucleic acid sequence comprising the coding region of a gene (i.e., the nucleic acid sequence which encodes a gene product). The coding region may be present in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide, polynucleotide or nucleic acid may be single-stranded (i.e., the sense strand or the antisense strand) or double-stranded.
As used herein, the term “gene expression” refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of the mRNA. Gene expression can be regulated at many stages. “Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decreases mRNA or protein production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called “activators” and “repressors,” respectively.
As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization can be demonstrated using a variety of hybridization assays (Southern blot, Northern Blot, slot blot, phage plaque hybridization, and other techniques). These protocols are common in the art (See e.g., Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, NY, [1989]; Ausubel et al. (eds.), Current Protocols in Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., New York [1994]; all of which are herein incorporated by reference).
Hybridization is the process of one nucleic acid pairing with an antiparallel counterpart which may or may not have 100% complementarity. Two nucleic acids which contain 100% antiparallel complementarity will show strong hybridization. Two antiparallel nucleic acids which contain no antiparallel complementarity (generally considered to be less than 30%) will not hybridize. Two nucleic acids which contain between 31-99% complementarity will show an intermediate level of hybridization. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be “self-hybridized.”
During hybridization of two nucleic acids under high stringency conditions, complementary base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of “weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less. As used herein, two nucleic acids which are able to hybridize under high stringency conditions are considered “substantially homologous.” Whether sequences are “substantially homologous” may be verified using hybridization competition assays. For example, a “substantially homologous” nucleotide sequence is one that at least partially inhibits a completely complementary probe sequence from hybridizing to a target nucleic acid under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be verified by the use of a second target that lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target. When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term “substantially homologous” refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of high stringency.
Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T.sub.m of the formed hybrid, and the G:C ratio within the nucleic acids.
As used herein, the term “stringency” is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acids hybridize. “Low or weak stringency” conditions are reaction conditions which favor the complementary base pairing and annealing of two nucleic acids. “High stringency” conditions are those conditions which are less optimal for complementary base pairing and annealing. The art knows well that numerous variables affect the strength of hybridization, including the length and nature of the probe and target (DNA, RNA, base composition, present in solution or immobilized, the degree of complementary between the nucleic acids, the T.sub.m of the formed hybrid, and the G:C ratio within the nucleic acids). Conditions may be manipulated to define low or high stringency conditions: factors such as the concentration of salts and other components in the hybridization solution (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) as well as temperature of the hybridization and/or wash steps. Conditions of “low” or “high” stringency are specific for the particular hybridization technique used.
As used herein the term “stringency” is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Those skilled in the art will recognize that “stringency” conditions may be altered by varying the parameters just described either individually or in concert. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences (e.g., hybridization under “high stringency” conditions may occur between homologs with about 85-100% identity, preferably about 70-100% identity). With medium stringency conditions, nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (e.g., hybridization under “medium stringency” conditions may occur between homologs with about 50-70% identity). Thus, conditions of “weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less. “High stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 65.degree. C. in a solution consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% sodium dodecyl sulfate (SDS), 5.times.Denhardt's reagent and 100 mu.g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1.times.SSPE, 1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in length is employed.
“Medium stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 55.degree. C. in a solution consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times.Denhardt's reagent and 100 mu.g/ml denatured salmon sperm DNA followed by washing in a solution comprising 1.0.times.SSPE, 1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in length is employed.
“Low stringency conditions” comprise conditions equivalent to binding or hybridization at 42.degree. C. in a solution consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5.times.Denhardt's reagent (50.times.Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)) and 100 mu.g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5.times.SSPE, 0.1% SDS at 42.degree. C. when a probe of about 500 nucleotides in length is employed.
As used herein, the term “T.sub.m” is used in reference to the “melting temperature.” The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated “denatures”) into single strands. The equation for calculating the T.sub.m of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the T.sub.m value may be calculated by the equation: T.sub.m=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references include more sophisticated computations that take structural as well as sequence characteristics into account for the calculation of T.sub.m.
As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5′-A-G-T-3′, is complementary to the sequence 3′-T-C-A-5′. Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in polymerase chain reaction (PCR) amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
As used herein, the terms “antiparallel complementarity” and “complementarity” are synonymous. Complementarity can include the formation of base pairs between any type of nucleotides, including non-natural bases, modified bases, synthetic bases and the like.
The following definitions are the commonly accepted definitions of the terms “identity,” “similarity” and “homology.” Percent identity is a measure of strict amino acid conservation. Percent similarity is a measure of amino acid conservation which incorporates both strictly conserved amino acids, as well as “conservative” amino acid substitutions, where one amino acid is substituted for a different amino acid having similar chemical properties (i.e. a “conservative” substitution). The term “homology” can pertain to either proteins or nucleic acids. Two proteins can be described as “homologous” or “non-homologous,” but the degree of amino acid conservation is quantitated by percent identity and percent similarity. Nucleic acid conservation is measured by the strict conservation of the bases adenine, thymine, guanine and cytosine in the primary nucleotide sequence. When describing nucleic acid conservation, conservation of the nucleic acid primary sequence is sometimes expressed as percent homology. In the same nucleic acid, one region may show a high percentage of nucleotide sequence conservation, while a different region can show no or poor conservation. Nucleotide sequence conservation can not be inferred from an amino acid similarity score. Two proteins may show domains that in one region are homologous, while other regions of the same protein are clearly non-homologous.
Numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term “substantially homologous” refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
A gene may produce multiple RNA species that are generated by differential splicing of the primary RNA transcript. cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon “A” on cDNA 1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; the two splice variants are therefore substantially homologous to such a probe and to each other. When used in reference to a single-stranded nucleic acid sequence, the term “substantially homologous” refers to any probe that can hybridize (i.e., it is the exact or substantially close to the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
The term “amplification” is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art (Dieffenbach and G S Dvekler, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y. [1995]; herein incorporated by reference).
As used herein, the term “polymerase chain reaction” (“PCR”) refers to the methods disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, all of which are incorporated herein by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.”
With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; and/or incorporation of .sup.32P-labeled or biotinylated deoxyribonucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications. Amplified target sequences may be used to obtain segments of DNA (e.g., genes) for the construction of targeting vectors, transgenes, etc. Reverse transcription PCR(RT-PCR) refers to amplification of RNA (preferably mRNA) to generate amplified DNA molecules (i.e. cDNA). RT-PCR may be used to quantitate mRNA levels in a sample, and to detect the presence of a given mRNA in a sample. RT-PCR may be carried out “in situ”, wherein the amplification reaction amplifies mRNA, for example, present in a tissue section.
As used herein, the term “amplifiable nucleic acid” is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “template.” As used herein, the term “template” refers to nucleic acid originating from a sample that is to be used as a substrate for the generation of the amplified nucleic acid.
As used herein, the terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
As used herein, the term “primer” refers to an oligonucleotide, typically but not necessarily produced synthetically, that is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides, an inducing agent such as DNA polymerase, and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
As used herein, the term “amplification reagents” refers to those reagents (e.g., deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
As used herein, the term “sample template” refers to a nucleic acid originating from a sample which is analyzed for the presence of “target,” such as a positive control DNA sequence encoding a mushroom toxin. In contrast, “background template” is used in reference to nucleic acid other than sample template, which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids other than those to be detected may be present as background in a test sample.
As used herein, the term “probe” refers to a polynucleotide sequence (for example an oligonucleotide), whether occurring naturally (e.g., as in a purified restriction digest) or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another nucleic acid sequence of interest, such as a nucleic acid attached to a membrane, for example, a Southern blot or a Northern blot. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that the probe used in the present invention is labeled with any “reporter molecule,” so that it is detectable in a detection system, including, but not limited to enzyme (i.e., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
The terms “reporter molecule” and “label” are used herein interchangeably. In addition to probes, primers and deoxynucleoside triphosphates may contain labels; these labels may comprise, but are not limited to, .sup.32P, .sup.33P, .sup.35S, enzymes, fluorescent molecules (e.g., fluorescent dyes) or biotin.
As used herein, the term “rapid amplification of cDNA ends” or “RACE” refers to methods such as “classical anchored” or “single-sided PCR” or “inverse PCR” or “ligation-anchored PCR” or “RNA ligase-mediated RACE” for amplifying a 5′ or 3′ end of a DNA sequence (Frohman et al., (1988) Proc Natl Acad Sci 85:8998-9002; herein incorporated by reference).
The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state they exist in nature. For example, a given DNA sequence (for example, a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a mushroom toxin includes, by way of example, such nucleic acid in cells ordinarily expressing a mushroom toxin, where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (in other words, the oligonucleotide may be single-stranded), but may contain both the sense and anti-sense strands (in other words, the oligonucleotide may be double-stranded).
As used herein, the term “purified” or “to purify” refers to the removal of contaminants from a sample. For example, recombinant nucleotides are expressed in bacterial host cells and the nucleotides are purified by the removal of host cell nucleotides and proteins; the percent of recombinant nucleotides is thereby increased in the sample.
As used herein, the term “kit” is used in reference to a combination of reagents and other materials. It is contemplated that the kit may include reagents such as PCR primer sets, positive DNA controls, such as a DNA encoding a propolypeptide of the present inventions, diluents and other aqueous solutions, and instructions. The present invention contemplates other reagents useful for the identification and/or determination of the presence of an amplified sequence encoding a mushroom toxin, for example, a colorimetric reaction product.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows exemplary bicyclic structures of (A) amatoxins and (B) phallotoxins. Exemplary amino acids have the L configuration except hydroxyAsp in phallacidin and Thr in phalloidin, which have the D configuration at the alpha carbon.
FIG. 2 shows exemplary fungi of the genus Amanita. A. A. bisporigera (collected in Oakland County, Mich.). B: A. phalloides (Alameda County, Calif.). C: Non-deadly species of Amanita. From left to right: three specimens of A. gemmata, A. muscaria, and two specimens of A. franchetii (Mendocino County, Calif.).
FIG. 3 shows an exemplary hypothetical nonribosomal peptide synthetase showing conserved motifs found in many NRPS proteins that served as the basis for the design of PCR primers (see Table 4).
FIG. 4 shows exemplary amanitin (an amatoxin) cDNA sequences, genomic DNA sequences, prepropolypeptide sequences, and polypeptide sequences coding for peptide toxins, A) shows exemplary cDNA sequences of the .alpha-amanitin gene and predicted amino acid sequence, where 5′ and 3′ ends were determined by Rapid Amplification of cDNA Ends (RACE). * indicates a stop codon. The string of A's at the end are a poly-A tail from the cDNA. The amatoxin peptide sequence is underlined. B) shows an exemplary sequence of genomic DNA covering the amanitin gene based on inverse PCR. The nucleotides encoding the amanitin peptide are underlined.
FIG. 5 shows exemplary phallacidin cDNA, genomic DNA, propolypeptide, and polypeptide sequences encoding phallacidin peptide toxin. A) shows exemplary cDNA sequences and predicted amino acid sequence, where 5′ and 3′ ends were determined by RACE, * indicates the stop codon. The string of A's at the end are the poly-A tail and were found in the cDNA but not the genomic DNA, and B) shows an exemplary genomic nucleic acid coding regions for phallacidin sequence #1, 1893 bp SacI restriction enzyme fragment, and phallacidin sequence #2, 1613 nt PvuI restriction enzyme fragment, where the nucleotides encoding a phallacidin peptide were underlined. These two genomic sequences encoding a phallacidin peptide were obtained by inverse PCR and confirmed by sequencing both strands.
FIG. 6 shows an exemplary alignment of a (A) cDNA nucleotide and (B) predicted amino acid sequences of exemplary coding regions of alpha-amanitin (AMA1) and phallacidin (PHA1) proproteins from A. bisporigera, the mature toxin sequences were underlined, and (C) shows a comparison of nucleic acids between AMA1 and PHA1 proproteins (BLAST results).
FIG. 7 (A-H) shows exemplary fragment genomic DNA sequences from the A. bisporigera genomic survey that contain conserved motifs highly similar to those found in the amanitin and phallacidin genes. Each DNA sequence is followed by the translation of the presumed correct reading frame. Conserved upstream and downstream amino acid sequences with variable known and putative toxin sequences were underlined.
FIG. 8 shows exemplary DNA blots of different species of Amanita. (A) Probed with AMA1 cDNA. (B) Probed with PHA1 cDNA. (C) Probed with a fragment of the .beta.-tubulin gene isolated from A. bisporigera as a control. (D) Ethidium-stained gel showing relative lane loading. Markers are lambda cut with BstEII. Species and provenances: Lane 1, A. aff suballiacea (Ingham County, Mich.); lane 2, A. bisporigera (Ingham County); lane 3, A. phalloides (Alameda County, Calif.); lane 4, A. ocreata (Sonoma County, Calif.); lane 5, A. novinupta (Sonoma County); lane 6, A. franchetii (Mendocino County, Calif.); lane 7, (Sonoma County); lane 8, a second isolate of A. franchetii (Sonoma County); lane 9, A. muscaria (Monterey County, Calif.); lane 10, A. gemmata (Mendocino County); lane 11, A. hemibapha (Mendocino County); lane 12, A. velosa (Napa County, Calif.); lane 13, A. sect. Vaginatae (Mendocino County). Mushrooms represent sect. Phalloideae (#'s 1-4), sect. Validae (#'s 5-8), sect. Amanita (#'s 9-10), sect. Caesarea (#11), sect. Vaginatae (#'s 12-13). Four separate gels were run; the lanes are in the same order on each gel and approximately the same amount of DNA was loaded per lane. A and B are to the same scale, and C and D are to the same scale.
FIG. 9 shows an exemplary schematic of a WebLogo alignment (Crooks et al., 2004, herein incorporated by reference) showing a representation of amino acid frequency within at least 15 predicted Amanita peptide sequences from DNA sequences of Amanita species. The height of the amino acid letter indicates the degree of conservation among the Amanita peptide sequences, some of which are shown in FIG. 7.
FIG. 10 shows an exemplary correlation of toxin genes and expression with toxin producing species of mushrooms in addition to a schematic of types of genes discovered near toxin producing genes in at least one lambda clone from a toxin producing mushroom. A) and B) Southern blot of DNA from species of Amanita that do (A. bisporigera and A. phalloides) or do not (A. gemmata, A. muscaria, A. flavoconia, A. section Vaginatae, and A. hemibapha) make amatoxin (probe used in A) and phallotoxin (probe used in B); C) PCR amplification of the gene for alpha-amanitin. Primers were based on the sequences in FIG. 4. A. gemmata and A. muscaria are species of Amanita that do not make amatoxins (or phallotoxins). A. bisporigera #'s 1-3 are three different specimens of A. bisporigera collected in the wild; and D) Exemplary schematic map of Amanita bisporigera genes predicted in a single lambda clone (13.4 kb) isolated using PHA1 as probe; showing two copies of PHA1 clustered with each other and with three P450 genes, NOTE: P450 genes were predicted using FGENESH and the Coprinus cinereus model; however, Coprinus doesn't have a PHA1 gene.
FIG. 11 shows exemplary sequences found in genomic sequencing of Galerina (G. marginata, Gm) A) Nucleic Acid Sequences (GmAMA1) and B) Amino acid sequences deduced from sequences in A (GmAM1). (.=stop codon)
FIG. 12 shows exemplary Galerina marginata amanitin (GmAM1) preproprotein amino acid sequence alignment between Galerina marginata and Amanita including A) alpha-amanitin toxins and alpha-amanitin/gamma-amanitin from Amanita compared to alpha-amanitin/gamma-amanitin from Galerina marginata and B) a Southern blot of Galerina (G.) marginata (m) (Gm) DNA probed with GmAM1 under high stringency conditions. Alpha and gamma amanitin differ in hydroxylation, which is a post-translational modification not encoded by the DNA nor produced during translation of the proprotein on the ribosome. Therefore, the genetic code for alpha and gamma amanitin are the same. Beta-amanitin, on the other hand, differs from alpha and gamma amanitin by one amino acid, and therefore the gene encoding beta-amanitin must be different from the gene encoding alph and gamma amanitin.
FIG. 13 shows an exemplary RNA blot of the Galerina marginata amanitin gene (GmAMA1). The results show that the gene is expressed in two known amanitin-producing species of Galerina (G. marginata and G. badipes) but not in a species that is a nonproducer of toxin (G. hybrida). Induction of gene expression was triggered by low carbon growth conditions. Lane 1: G. hybrida, high carbon. Lane 2: G. hybrida, low carbon. Lane 3: G. marginata, high carbon. Lane 4: G. marginata, low carbon. Lane 5: G. badipes, high carbon. Lane 6: G. badipes, low carbon. The probe was G. marginata AMA1 gene (GmAMA1) predicted to encode alpha-amanitin (FIG. 4). Each lane was loaded with 15 ug total RNA. Fungi were grown in liquid culture for 30 d on 0.5% glucose (high carbon) then switched to fresh culture of 0.5% glucose or 0.1% glucose (low carbon) for 10 d before harvest. The major band in lanes 3-6 is approximately 300 bp. The high MW signal in lane 1 is spurious.
FIG. 14 shows exemplary Galerina marginata amanitin sequences (GmAMA1). Sequences were found in genomic sequencing of Galerina (G. marginata, Gm) A) Nucleic Acid Sequences (GmAMA1); B) Amino acid sequences deduced from sequences in A (GmAMA1). (.=nonsense codon); and C) Amino acid sequence alignment of two Galerina amanitins GaAMA1 and GaAMA2 sequences.
FIG. 15 shows exemplary BLASTP results using human prolyl oligopeptidase (POP) as query against fungi in GenBank. The results indicate that an ortholog of human POP exists in at least some Homobasidiomycetes (Coprinus) and Heterobasidiomycetes (Ustilago and Cryptococcus) and few other fungal species showing various levels of significant identity and where scores and e-values of the two Aspergillus fungal sequences were considered statistically insignificant.
FIG. 16 shows exemplary genome survey sequences from A. bisporigera that align with human POP (gi:41349456) using TBLASTN. Shown are translations of A. bisporigera DNA sequences and the alignments of the human protein POP (query) with each predicted translation product from A. bisporigera (subject).
FIG. 17 shows A) two exemplary prolyl oligopeptidase (POP)-like A. bisporigera genome sequences POPA and POPB, B) two exemplary cDNA sequences for POPA and POPB, and C) two exemplary amino acid sequences for POPA and POPB.
FIG. 18 shows exemplary Southern blot of different Amanita species probed with (A) POPA or (B) POPB of A. bisporigera. DNA was from the same species of mushroom in lanes of the same order as FIG. 8. Lanes 1-4 are Amanita species in sect. Phalloideae and the others are toxin non-producers. Note the presence of POPA and absence of POPB in sect. Validae (lanes 5-8), the sister group to sect. Phalloideae (lanes 1-4). the weaker hybridization of POPA to the Amanita species outside sect. Phalloideae (lanes 5-13) to lower DNA loading and/or lower sequence identity due to taxonomic divergence. The results show that POPB does not hybridize to any species outside sect. Phalloideae even after prolonged autoradiographic exposure.
FIG. 19 shows exemplary purified POPB protein isolated from Conocybe albipes, also known as C. lactea and C. apala, which produces phallotoxins, separated by standard SDS-PAGE gel electrophoresis and Coomassie Blue dye stained to show the location of protein.
FIG. 20 shows an exemplary experiment demonstrated that POPB of C. albipes processed a synthetic phallacidin propeptide to the mature linear heptapeptide A) HPLC analysis of an enzymatic reaction of a synthetic phallacidin propeptide with a boiled sample of POPB showing no cleavage product at the vertical arrow where a AWLVDCP (SEQ ID NO: 69) should be found and B) cleavage of a synthetic phallacidin precursor by purified Conocybe albipes POPB enzyme (see, FIG. 19) showing a cleavage product matching AWLVDCP (SEQ ID NO: 69) at the vertical arrow. The identity of the cleavage product was confirmed by Mass Spectrometry. The results show that purified POPB cuts a synthetic phallacidin peptide precisely at the flanking Pro residues.
FIG. 21 shows exemplary expression of POPB in E. coli and production of anti-POPB antibodies. Lane 1: Markers; Lane 2: recombinant POPB expressed by E. coli purified from inclusion bodies; Lane 3: Soluble extract of Amanita bisporigera; Lane 4: Immunoblot of POPB inclusion body; Lane 5: Immunoblot of Amanita bisporigera extract with antibody raised against purified POPB; where the crude antiserum (as drawn from rats) was used at 1:5000 dilution and a reaction product was observed with an anti-rat antibody using well known visualization methods, arrows point to the bands corresponding to single band of POPB protein. A) Lanes 1-3: stained with Coomassie Blue. B) Lanes 4-5 antibody binding visualized by enhanced chemiluminescence.
FIG. 22 shows exemplary alignment of concepetual translations of Galerina marginata POP DNA sequences (subject sequences) identified using Amanita bisporigera POPA (A-1 to A-9) and POPB (B-1 to B-8) as query sequences for searching a library of Galerina genomic DNA sequences created by the inventors for their use during the development of the present inventions. The higher scoring hits of two nonidentical contigs were strong evidence that the Galerina genome contains at least two POP genes (named POPA and POPB).
FIG. 23 A-C (a continouse sequence) shows an exemplary sequence found in the genomic schematic sequence of FIG. 10D inserted into a lambda clone; 13,254 bp lambda clone [red/underlined sequences (portions) are two copies of PHA1 encoding phallacidin in B]. The two copies are in opposite orientations, SEQ ID NO: 327.
FIG. 24 A-B, continouse table, shows an exemplary FGENESH 2.5 prediction of potential genes in the lambda clone using the Coprinus cinereus prediction model and sequence.
FIG. 25 shows an exemplary contemplated P450 gene mRNA sequence, A) P450-1 (OP451) and putative encoded amino acid sequences, B) blastp results of Predicted protein(s) P450-1 (OP451) against GenBank sequences, C), BLASTP of OP45-1 against Coprinus cinereus sequences at Broad, D) BLASTP of OP451 against Laccaria bicolor genomic sequences, and E) OP451 as a query sequence for a BLASTP against nr, showing an excellent hit against a Coprinus protein.
FIG. 26 shows an exemplary contemplated P450 mRNA sequence predicted in the lambda clone using FGEHESH and the Coprinus model, A) P450-2 (OP452) and putative encoded amino acid sequences, B) blastp results of predicted protein(s): P450-2 (OP452), C), BLASTP of P450-2 (OP452) against Coprinus at Broad, and D) BLASTP of P450-2 (OP452) against Laccaria genomic sequences.
FIG. 27 shows an exemplary FGENESH predicted mRNA and predicted protein number 3, which has no strong hits in any of the BLAST searches. This region overlaps with PHA1-1, which is on + strand (gene 3 is on − strand).
FIG. 28 shows an exemplary contemplated P450 predicted mRNA sequence, A) P450-3 (OP453) and putative encoded amino acid sequences, B) blastp results of Predicted protein(s): P450-3 (OP453), C), BLASTP of P450-3 (OP453) against Coprinus at Broad, and D) BLASTP of P450-3 (OP453) against Laccaria genomic sequences.
FIG. 29 shows exemplary A) PHA1-2 as described herein (5th identified sequence in the lambda clone shown in FIG. 10D) and B) nucleotide sequence of a predicted mRNA of a 6th predicted gene, and its conceptual translation, of unknown function.
FIG. 30 shows exemplary alignments of a P450 genes 1, 2, 4 corresponding to OP451, OP452 and OP453 to each other (tree) and to genes obtained with a BLAST search (30A1-30A2), exemplary sequences from the entire lambda clone reverse complement (3′-5′) (Sequences 613 and 614 were from pieces of the lambda clone translated in a particular frame to clearly show amino acids of PHA1) (30B1-30B3), and FGENESH of reverse complement showing a different gene 4 (30C), which is gene 3 in the reverse complement, resulting in a new set of exemplary gene identifications (30D) contemplated as P450 genes.
FIG. 31 shows an exemplary Galerina species and the result of detecting a-amanitin in samples of Galerina mushrooms that were implicated in the illness of a person who ate them. A person in Bronx, N.Y., with acute liver failure, had eaten an unknown mushroom from their backyard. This is a result of a sample of the mushrooms collected in this backyard analyzed for amanitin toxin that may have caused her liver injury and associated symptoms.
FIG. 32 shows an exemplary gene structure (introns, exons, and protein coding region) of the two variants of α-amanitin genes in Galerina, and comparison to AMA1 of A. bisporigera A. GmAMA1-1 and B. GmAMA1-2 in Galerina marginata. Exons are indicated by heavy lines and introns by thin lines. The predicted proprotein sequences and their location are indicated in FIG. 32.
FIG. 33 shows exemplary alignments of the predicted amino acid sequences of the proproteins of α-amanitin-encoding genes in G. marginata and A. bisporigera. (A) Alignment of the two copies of the α-amanitin proproteins in G. marginata (GmAMA1-1 and GmAMA1-2), and the consensus. (B) Alignment of AMA1 (encoding α-amanitin) and PHA1 (encoding phallacidin) from A. bisporigera (Ab) and the consensus. A gap was introduced in the sequence of PHA1 because phallacidin has one fewer amino acid than α-amanitin. (C) Consensus between the proproteins of AMA1, the α-amanitin-encoding gene of A. bisporigera, and copy 1 (GmAMA1-1) of the α-amanitin-encoding gene of G. marginata, and the consensus. (D) Consensus among the proproteins of AMA1, PHA1, GmAMA1-1, and GmAMA1-2. (E) Exemplary genomic DNA sequence (SEQ ID NO: 709), transcriptional start for prepropeptide nucleic acid sequence (SEQ ID NO: 710), propeptide amino acid sequence (SEQ ID NO: 711) and predicted amino acid sequence of GmAMA1-1 (SEQ ID NO: 704). (F) Exemplary genomic DNA sequence (SEQ ID NO: 712), transcriptional start for prepropeptide nucleic acid sequence (SEQ ID NO: 713), propeptide sequence 61 amino acids (SEQ ID NO: 690), propeptide nucleic acid sequence for 35 amino acids (SEQ ID NO: 714) and predicted 35 amino acid sequence of GmAMA1-2 (SEQ ID NO: 705).
FIG. 34 shows an exemplary DNA blot of Galerina species. Lane 1, G. marginata; lane 2, G. badipes; lane 3, G. hybrida; lane 4, G. venenata. Panel A: Probed with GmAMA1-1; panel B probed with GmPOPB; panel C, probed with GmPOPA; panel D, gel stained with Ethidium bromide. The results showed that amanitin-producing species of Galerina (namely, G. marginata, G. badipes, and G. venenata) have the GmAMA1 and POPB genes, while POPA was present in all species.
FIG. 35 shows an exemplary reverse-phase HPLC analysis of amatoxins in Galerina marginata strain CBS 339.88 grown on a medium containing low carbon A: α-amanitin standard (arrow). B: extract of G. marginata. Elution was monitored at 305 nm. The mushroom extract has a peak corresponding to the α-amanitin standard (arrow). Identify of this compound to authentic alpha-amanitin was confirmed by mass spectrometry. β-Amanitin elutes just before α-amanitin (Enjalbert et al., 1992, herein incorporated by reference) and appears to be absent in extracts of this G. marginata specimen.
FIG. 36 shows an exemplary RNA blot of Galerina strains under different growth conditions. The probe was GmAMA1-1. Lane 1: G. hybrida grown on high carbon. Lane 2: G. hybrida, low carbon (note absence of hybridization signal in lanes 1 and 2). Lane 3: G. marginata, high carbon. Lane 4: G. marginata, low carbon (see RNAs corresponding to the size of AMA1 at arrows). Lane 5: G. badipes, high carbon, no detectable RNA signal in the region of the arrows. Lane 6: G. badipes, low carbon showing some RNA signal at the arrow. Each lane was loaded with 15 μg total RNA. The major band in lanes 3, 4 and 6 is approximately 300 bp. The higher molecular weight signal in lane 1 does not correspond to a specific signal. Arrows point to the presence of mushroom RNA that hybridized to the GmAMA1-1 probe.
FIG. 37 shows exemplary structures of GmPOPA and GmPOPB genes encoding putative prolyl oligopeptidases from G. marginata. Thick bars indicate exons and thin bars indicate introns. The lines above the gene models indicate the positions of the coding regions.
FIG. 38 shows exemplary sequences of isolated A) GmPOPA cDNA and B) GmPOPB cDNA sequences with predicted encoded polypeptide sequences A) GmPOPA cDNA (SEQ ID NO: 715) and polypeptide (SEQ ID NO: 716) and B) GmPOPB cDNA (SEQ ID NO: 717) and polypeptide (SEQ ID NO: 722).
FIG. 39 shows exemplary growth of large colonies of G. marginata (see arrows) on hygromycin, which indicated resistance to hygromycin due to successful transformation with the hygromycin resistance gene.
FIG. 40 shows exemplary PCR results of amplifying genes using specific primers of the hygromycin resistance transgene (see Experimental section), which indicated which colonies are transformants with the hygromycin transgene as opposed to unwanted selection of natural hygromycin resistant colonies. (A) Arrows indicated the hygromycin resistance gene (transgene) PCR products stained with Ethidium bromide while (B) shows the results of this gel blotted and probed with a copy of the hygromycin transgene in order to confirm the identity of the PCR products (arrow). The large streak crossing several lanes was an artifact.
FIG. 41 shows exemplary contigs that were found in a Galerina genome survey when AbPOPA and AbPOPB sequences were used as queries. TBLASTN (protein against nucleic acid database) was used in order to obtain exemplary amino acid sequences. Note that these 4 contigs were short genomic sequences so none of them covered the entire gene: contig sequence 1 (SEQ ID NO: 723; FIG. 41A-1 to A-2); contig sequence 2 (SEQ ID NO: 732; FIG. 41B-1 to B-2) contig sequence 3 (SEQ ID NO: 741; FIG. 41C-1 to C-2); and contig sequence 4 (SEQ ID NO:750; FIG. 41D). Two Galerina genes (FIG. 38) were subsequently sorted out from the genes represented by the 4 contigs by PCR. Both A. bisporigera and G. marginate were found to have two POP genes. They were similar to each other, so the use of BLAST with either of the Amanita sequences hybridized to these contigs, which corresponded to both of the Galerina POP contigs.



DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.
The present invention also relates to compositions and methods comprising genes and peptides associated with cyclic peptide toxins and toxin production in mushrooms. In particular, the present invention relates to using genes and proteins from Amanita species encoding Amanita peptides, specifically relating to amatoxins and phallotoxins. In a preferred embodiment, the present invention also relates to methods for detecting Amanita peptide toxin genes for identifying Amanita peptide-producing mushrooms and for diagnosing suspected cases of mushroom poisoning. Further, the present inventions relate to providing kits for diagnosing and monitoring suspected cases of mushroom poisoning in patients.
The present inventions further relate to compositions and methods associated with screening a genomic library in combination with 454 pyro-sequencing for obtaining sequences of interest. In particular, the present invention relates to providing and using novel PCR primers for identifying and sequencing Amanita peptide genes, including methods comprising RACE PCR primers and degenerate primers for identifying Amanita mushroom peptides. Specifically, the present inventions relate to identifying and using sequences of interest associated with the production of small peptides, including linear peptides representing cyclic peptides, for example, compositions and methods comprising Amanita amanitin toxin sequences.
The present inventions further relate to compositions and methods associated with conserved genomic regions of the present inventions, in particular those conserved regions located upstream and downstream of small peptide encoding regions of the present inventions. Specifically, degenerate PCR primers based upon these conserved regions are used to identifying Amanita peptide-producing mushrooms.
Unlike genetically based disease susceptibility, every human is susceptible to lethal mushroom toxins due to the direct action of toxins, primarily amatoxins, on ubiquitous cellular organelles. Furthermore, unlike poisonous plants, poisonous mushroom species are ubiquitously found throughout the world. For example, mushrooms in the genus Amanita section Phalloideae are responsible for more than 90% of global (worldwide) fatal mushroom poisonings. Perspectively, there are an estimated 900-1000 species of Amanita wherein the majority do not produce amatoxins (or phallotoxins) of which some are actually safe for humans to eat (FIG. 2C) (Bas, (1969) Persoonia 5:285; Tulloss et al., (2000) Micologico G. Bresadola, 43:13; Wei, et al., (1998) Can J. Bot. 76:1170; all of which are herein incorporated by reference). Thus an accurate pre-ingestion determination of toxic species would prevent accidental poisoning in 100% of cases. However, there are a large number of toxin producing mushrooms commonly misidentified as an edible mushroom, see Tables 1 and 2. Therefore, accurately detecting toxic mushrooms in the wild based upon morphology in order to avoid or identify mushroom poisoning primarily depends upon expert mycological examination of an intact mushroom.
Expert identification opinions are necessary due to the large number of “look-a-like” mushrooms, such as exemplary mushroom in the following Table 1. For example, the Early False Morel Gyromitra esculenta is easily confused with the true Morel Morchella esculenta, and poisonings have occurred after consumption of fresh or cooked Gyromitra. Gyromitra poisonings have also occurred after ingestion of commercially available “morels” contaminated with G. esculenta. The commercial sources for these fungi (which have not yet been successfully cultivated on a large scale) are field collection of wild morels by semi-professionals. Cultivated commercial mushrooms of whatever species are almost never implicated in poisoning outbreaks unless there are associated problems such as improper canning (which lead to bacterial food poisoning).







TABLE 1







Poisonous Mushrooms and their Edible Look-A-likes.*


Mushrooms Containing Amatoxins









Poisonous species
Appearance
Mistaken for





Amanita tenuifolia
pure white
Leucoagaricus naucina (Smoothcap Parasol)


(Slender Death




Angel)




Amanita bisporigera
pure white
Amanita vaginata (Grisette), Leucoagaricus naucina


(Death Angel)

(Smoothcap Parasol), white Agaricus spp. (field




mushrooms), Tricholoma resplendens (Shiny




Cavalier)


Amanita verna
pure white
A. vaginata, L. naucina, white Agaricus spp., T. resplendens


(Fool's Mushroom)




Amanita virosa
pure white
A. vaginata, L. naucina, Agaricus spp., T. resplendens


(Destroying Angel)




Amanita phalloides
pure white
Amanita citrina (False Deathcap), A. vaginata, L. naucina,


(Deathcap)
variety
Agaricus spp., T. resplendens


Buttons of A. bisporigera,.
pure white
Buttons of white forms of Agaricus spp. Puffballs


A. verna,

such as Lycoperdon perlatum, etc.


A. virosa




Amanita phalloides
green =
Russula virescens (Green Brittlegill), Amanita


(Deathcap)
normal cap
calyptrodermia (Hooded Grisette), Amanita fulva



color
(Tawny Grisette), Tricholoma flavovirens (Cavalier




Mushroom), Tricholoma portentosum (Sooty Head)


Amanita phalloides
yellow variety
Amanita caesarea (Caesar's Mushroom)


(Deathcap)




Amanita brunnescens
na
Amanita rubescens (Blusher), Amanita pantherina


(Cleft Foot

(Panthercap)


Deathcap)




Galerina autumnalis
LBM
“Little Brown Mushrooms,” including Gymnopilus


(Autumn Skullcap)

spectabilis (Big Laughing Mushroom) and other




Gymnopilus spp., Armillaria mellea (Honey




Mushroom)


Leucoagaricus
LBM
Lepiota spp., Leucoagaricus spp., Gymnopilus spp.


brunnea (Browning

and other Parasol Mushrooms and LBM's


Parasol)




Lepiota josserandii,
LBM
Lepiota spp., Leucoagaricus spp., Gymnopilus spp.


L. helveola, L. subincarnata

and other Parasol Mushrooms and LBM's





*Na = not available.






Mushrooms that produce mild gastroenteritis are too numerous to list here, where exemplary examples are shown which include members of many of the most abundant genera, including Agaricus, Boletus, Lactarius, Russula, Tricholoma, Coprinus, Pluteus, and others. The Inky Cap Mushroom (Coprinus atrimentarius) is considered both edible and delicious, and only the unwary who consume alcohol after eating this mushroom need be concerned. Some other members of the genus Coprinus (Shaggy Mane, C. comatus; Glistening Inky Cap, C. micaceus, and others) and some of the larger members of the Lepiota genus such as the Parasol Mushroom (Leucocoprinus procera) do not contain coprine and do not cause this effect. The potentially deadly Sorrel Webcap Mushroom (Cortinarius orellanus) is not easily distinguished from nonpoisonous webcaps belonging to the same distinctive genus.







TABLE 2





Mushrooms Producing Severe Gastroenteritis.


Mushrooms Producing Severe Gastroenteritis
















Chlorophyllum molybdites
Leucocoprinus rachodes (Shaggy Parasol),


(Green Gill)
Leucocoprinus procera (Parasol Mushroom)


Entoloma lividum (Gray
Tricholomopsis platyphylla (Broadgill)


Pinkgill)



Tricholoma pardinum
Tricholoma virgatum (Silver Streaks),


(Tigertop Mushroom)
Tricholoma myomyces (Waxygill Cavalier)


Omphalotus olearius (Jack
Cantharellus spp. (Chanterelles)


O'Lantern Mushroom)



Paxillus involutus (Naked
Distinctive, but when eaten raw or


Brimcap)
undercooked, will poison some people





* Bad Bug Book published by the U.S. Food & Drug Administration Center for Food Safety & Applied Nutrition Foodborne Pathogenic Microorganisms and Natural Toxins Handbook, website at cfsan.fda.govt/~mow/table3.html; herein incorporated by reference.






Individual specimens of poisonous mushrooms are characterized by individual variations in toxin content based on mushroom genetics, geographic location, and growing conditions. For example, mushroom intoxications may be more or less serious, depending not on the number of mushrooms consumed, but of the total dose of toxin delivered. In addition, although most cases of poisoning by higher plants occur in children, toxic mushrooms are consumed most often by adults. Adults who consume mushrooms are more likely to recall what was eaten and when, and are able to describe their symptoms more accurately than are children. Occasional accidental mushroom poisonings of children and pets have been reported, but adults are more likely to actively search for and consume wild mushrooms for culinary purposes.
In part because of their smaller body mass, children are usually more seriously affected by normally nonlethal mushroom toxins than are adults and are more likely to suffer very serious consequences from ingestion of relatively smaller doses. Similar to the elder population and debilitated persons who are more likely to become seriously ill from all types of mushroom poisoning, even those types of toxins which are generally considered to be mild.
Recently, in addition to humans, see, FIG. 31, dogs and other animals are becoming frequent victims of poisonous mushrooms. See Schneider: Mushroom in backyard kills curious puppy, Lansing State Journal, Sep. 30, 2008 pg. B.1 (at lansingstatejournal.com.apps/pbcs.dll/article?AID=/20080930/COLUMNISTS09/-809,300 321. Body mass plays a role here in that smaller animals, such as puppies and small dogs, are likely to be more susceptible to smaller amounts of toxins. Thus in some embodiments, PCR primers of the present inventions, including PCR primers made from sequences of the present inventions, are contemplated for use in detecting toxin producing mushrooms in samples obtained from dogs or other animals, such as partially eaten material, samples obtained directly from an animals digestive system, etc. in some embodiments, antibodies of the present inventions are contemplated for use in detecting mushroom toxins in samples obtained from dogs or other animals, such as partially eaten material, samples obtained directly from an animals digestive system, etc.
I. Dangers of Mushroom Poisoning.
Mushroom poisoning in subjects, particularly humans, is caused by the consumption of raw or cooked fruiting bodies of toxin producing mushrooms, also known as toadstools (from the German Todesstuhl, death's stool) to distinguish toxic from nontoxic mushrooms. There is no general rule of thumb for distinguishing edible mushrooms from toxic mushrooms (poisonous toadstools). There are generally no easily recognizable differences between poisonous and nonpoisonous species to individuals who are not experts in mushroom identification (mycologists).
Toxins involved in and responsible for mushroom poisoning are produced naturally by the fungi, with each individual specimen within a toxic species considered equally poisonous. Most mushrooms that cause human poisoning cannot be made nontoxic by cooking, canning, freezing, or any other means of processing. Thus, the only way to completely avoid poisoning is to avoid consumption of the toxic species. Mushroom poisonings are almost always caused by ingestion of wild mushrooms that have been collected by nonspecialists (although specialists have also been poisoned). Most cases occur when toxic species are confused with edible species, and a useful question to ask of the victims or their mushroom-picking benefactors is the identity of the mushroom they thought they were picking. In the absence of a well-preserved specimen, the answer to this question could narrow the possible suspects considerably. Poisoning has also occurred when reliance was placed on some folk method of distinguishing poisonous and safe species. Outbreaks have occurred after ingestion of fresh, raw mushrooms, stir-fried mushrooms, home-canned mushrooms, mushrooms cooked in tomato sauce (which rendered the sauce itself toxic, even when no mushrooms were consumed), and mushrooms that were blanched and frozen at home. Cases of poisoning by home-canned and frozen mushrooms are especially insidious because a single outbreak may easily become a multiple outbreak when the preserved toadstools are carried to another location and consumed at another time.
Poisonings in the United States occur most commonly when hunters of wild mushrooms (especially novices) misidentify and consume a toxic species, when recent immigrants collect and consume a poisonous American species that closely resembles an edible wild mushroom from their native land, or when mushrooms that contain psychoactive compounds are intentionally consumed by persons who desire these effects.
A. Symptoms of Poisoning.
Mushroom poisonings are generally acute and are manifested by a variety of symptoms and prognoses, depending on the amount and species consumed. Because the chemistry of many of the mushroom toxins (especially the less deadly ones) is unknown and positive identification of the mushrooms is often difficult or impossible, mushroom poisonings are generally categorized by their physiological effects. There are four categories of mushroom toxins: protoplasmic poisons (poisons that result in generalized destruction of cells, followed by organ failure); neurotoxins (compounds that cause neurological symptoms such as profuse sweating, coma, convulsions, hallucinations, excitement, depression, spastic colon); gastrointestinal irritants (compounds that produce rapid, transient nausea, vomiting, abdominal cramping, and diarrhea); and disulfuram-like toxins. Mushrooms in this last category are generally nontoxic and produce no symptoms unless alcohol is consumed within 72 hours after eating them, in which case a short-lived acute toxic syndrome is produced.
In one embodiment, the inventors provide herein compositions and methods for providing molecular biology based diagnostic tests for accurately and reproducibly identifying DNA sequences encoding lethal fungal toxins. Thus accurate identification of mushroom toxins may be made from samples of uneaten mushrooms, including raw, cooked, frozen, dried, samples, and patient samples of undigested and partially digested, as in gastric contents, such as from human and dogs.
For comparison, current methods for diagnosing mushroom poisonings are briefly described below.
B. Current Diagnostic Methods.
Symptoms of potentially toxic mushroom poisoning may mimic other types of diseases, such as abnormal conditions or ingestion of other types of toxins which would trigger different and likely less drastric treatments. Exemplary differentials include, Adrenal Insufficiency and Adrenal Crisis, Alcohol and Substance Abuse Evaluation, Anorexia Nervosa, Delirium Tremens, Gastroenteritis, Hepatitis, Methemoglobinemia, Pediatrics, Dehydration, Pediatrics, Gastroenteritis, Salmonella Infection, Toxicity, Anticholinergic, Toxicity, Antihistamine, Disulfuram, Disulfuramlike Toxins, Gyromitra, Mushroom Hallucinogens, Mushroom-Orellanine, Organophosphate, and Carbamate, Theophylline, etc. In addition, an Idiosyncratic reaction mimics toxin poisoning when patients with trehalase deficiency who are unable to break down trehalose, a disaccharide found in mushrooms present with diarrhea after ingestion. Further patients with an immune reaction (Paxillus syndrome) may develop an acquired hypersensitivity-type reaction after repeated ingestions of specific mushrooms. This may result in hemolytic crisis and most commonly involves ingestion of Paxillus involutus. Suillus luteus also has been implicated in a psychosomatic syndrome where some patients were reported to develop anxiety-related symptoms after learning that they ate wild mushrooms. Mushroom-drug interaction-symptoms may occur with ingestion of mushrooms contaminated with bacteria, sprayed with pesticides, or supplemented with drugs such as phencyclidine. Thus, in one embodiment, genes and proteins of the present inventions may find use in identifying the presence or lack of toxin producing mushrooms, i.e. their genes related to toxin production, for example using PCR primers for amplifying genes, peptides related to toxins, for example, using antibodies which recognize toxins, and kits comprising PCR primers or antibodies.
As described above, the protoplasmic poisons are the most likely to be fatal or to cause irreversible organ damage. In the case of poisoning by the deadly species of Amanita and other mushrooms that produce the Amanita peptides, important laboratory indicators of liver (elevated LDH, SGOT, and bilirubin levels) and kidney (elevated uric acid, creatinine, and BUN levels) damage will be present. Unfortunately, in the absence of dietary history, these signs could be mistaken for symptoms of liver or kidney impairment as the result of other causes (e.g., viral hepatitis). It is important that this distinction be made as quickly as possible, because the delayed onset of symptoms will generally mean that the organ has already been damaged. The importance of rapid diagnosis is obvious: victims who are hospitalized and given aggressive support therapy almost immediately after ingestion have a mortality rate of only 10%, whereas those admitted 60 or more hours after ingestion have a 50-90% mortality rate.
1. Intact Mushrooms.
Ideally, once a mushroom poisoning is suspected, identification of suspect toxic mushroom, identical to the one ingested, should be made by a local medical toxicologist (certified through the American Board of Medical Toxicology or the American Board of Emergency Medicine) or at a regional poison control center.
If a pre-digested mushroom sample is available, the following information would be helpful to a mycologist or physician with mushroom poisoning experience for determining the mushroom's identity: Provide any available information, for example, size, shape, and color of the mushroom including a description of the surface and the underside of the cap, the stem, gills, veil, ring, spores and the color and texture of the flesh. It would be helpful to know the location and conditions in which the mushroom grew (e.g., wood, soil). Further, it is suggested that any mushroom samples saved for mycological examination are wrapped in foil or wax paper and stored in a paper bag in a cool dry place, pending transport to the mycologist or other professional. Moreover it is discouraged to store mushroom samples for mycological identification in a plastic bag or container where the mushroom's features may be altered due to moisture condensation and further freezing which is likely to alter or destroy any distinguishing identification features of the mushroom. Alternative methods for identifying mushrooms may be done by referring to the Poisindex or a mycology handbook.
Currently there are several research laboratory tests used for identifying Amanita peptides and toxins, examples of which are briefly described as follows. The Meixner test also known as the “Weiland Test” assay is qualitative assay used to detect amatoxins (eg, alpha-amanitin, beta-amanitin) in the mushroom. It is not recommended for use with stomach contents nor to determine edibility of a mushroom because false-positive and false-negative results have been described. Kuo, M. (2004, November). Meixner test for amatoxins. Retrieved from the MushroomExpert.Com Web site: mushroomexpert.com/meixner; herein incorporated by reference).
Further, an intact or partial undigested mushroom may be analyzed for actual toxic peptides, using chemical methods such as reverse-phase HPLC. In order to rule out other types of food poisoning and to conclude that the mushrooms eaten were the cause of the poisoning, it must be established that everyone who ate the suspect mushrooms became ill and that no one who did not eat the mushrooms became ill. Wild mushrooms eaten raw, cooked, or processed should always be regarded as prime suspects. After ruling out other sources of food poisoning and positively implicating mushrooms as the cause of the illness, further diagnosis is necessary to provide an early indication of the seriousness of the disease and its prognosis.
Therefore, an initial diagnosis is based entirely on symptomology and recent dietary history. Despite the fact that cases of mushroom poisoning may be broken down into a relatively small number of categories based on symptomatology, positive taxonomic identification of the mushroom species consumed remains the only means of unequivocally determining the particular type of poisoning involved, and it is still vitally important to obtain such accurate identification as quickly as possible. Cases involving ingestion of more than one toxic species in which one set of symptoms masks or mimics another set are among many reasons for needing this information.
2. Post-Ingested and Pre-Digested Mushroom Samples.
If the actual mushroom is unavailable, which is frequent in post-ingestion cases with delayed onset of symptoms, the following information may be helpful for determining the mushroom's identity. Save emesis or gastric lavage fluid for microscopic examination for spores. If mushroom fragments are available, they can be stored in a 70% solution of ethyl alcohol, methanol, or formaldehyde and placed in the refrigerator. Otherwise, emesis can be centrifuged and the heavier layer on the bottom can be examined under a microscope for the presence of spores.
Despite the availability of laboratory tests for identifying toxins, diagnosing a mushroom poisoning remains primarily limited to taxonomic identification of the mushroom that was eaten. Accurate post-ingestion analyses for specific toxins when no taxonomic identification is possible is essential for cases of suspected poisoning by toxin containing mushrooms, such as species of Amanita, since prompt and aggressive therapy (including lavage, activated charcoal, and plasmapheresis) can greatly reduce the mortality rate.
Samples of actual mushroom toxins may be recovered from poisonous fungi, cooking water of poisonous fungi, stomach contents with poisonous fungi, serum, and urine from poisoned patients. Procedures for extraction and quantitation of toxins are generally elaborate and time-consuming. In the case of using toxin based diagnostic procedures the patient will in most cases either have recovered or died by the time an analysis is made on the basis of toxin chemistry. However even with toxin chemistry, the exact chemical natures of many toxins, including toxins that produce milder symptoms are unknown. Lethal toxins are identified using chromatographic techniques (TLC, GLC, HPLC) for amanitins, orellanine, muscimol/ibotenic acid, psilocybin, muscarine, and the gyromitrins. Recently, amanitins were determined by commercially available .sup.3H-RIA kits. Amanitin EIA Kit from Alpco Diagnostics of American Laboratory Products Company PO Box 451 Windham, N.H. 03087 Sample Type Urine, Serum, Plasma .alpha.- and .gamma.-amanitin present in human urine, serum and plasma. A polyclonal antibody (Ab) specific for alpha- and gamma-Amanitin Diagnostic Accuracy of Urinary Amanitin in Suspected Mushroom Poisoning: A Pilot Study Butera et al., Clinical Toxicology, Volume 42, Issue 6 Dec. 2004, pages 901-912; herein incorporated by reference).
II. Mushroom Toxins.
A large variety of toxins are produced by mushrooms, including amatoxins, phallotoxins, virotoxins, phallolysins, ibotenic acid/muscimol, alkaloids, cyclopeptides, coumarins, etc. Many of these compounds are active at extremely low concentrations and have a rapid effect including death. Milder toxins such as ibotenic acid and muscimol bind to glutamic acid and GABA receptors, respectively, and thereby interfere with CNS receptors.
Amatoxins, phallotoxins, and virotoxins are found in A. bisporigera, A. ocreata, A. phalloides, A. phalloides var. alba, A. suballiacea, A. tenuifolia, A. virosa, and some other mushrooms. The phallolysins are a recently discovered group of toxins as yet observed only in A. phalloides. Many of the cyclic and noncyclic peptides found in Amanita and other toxin producing genera are toxic to humans and other mammals, ranging from mild symptoms to death.
A. Amanitin Peptide Toxins.
Several mushroom species, including the Death Cap or Destroying Angel (Amanita phalloides, A. virosa), the Fool's Mushroom (A. verna) and several of their relatives, along with the Autumn Skullcap (Galerina marginata, formerly called Galerina autumnalis) and some of its relatives, produce a family of cyclic octapeptides called amanitins. Because of taxonomic revisions, amanatin-producing fungi with different names might actually be the same species. Galerina marginata=G. autumnalis=G. venenata=G. unicolor (G. beinrothii, G. sulciceps, G. fasciculata, G. helvoliceps—may all actually be the same species as G. marginata). Amanitins are lethal toxins A human LD.sub.50 for .alpha.-amanitin is approximately 0.1 mg/kg (see, FIG. 1 for exemplary structures). Such that a fatal dose fatal for at least 50% of people weighing approximately 100-110 kgs (200-220 pounds) and around 100% for people weighing 100 or less pounds is 10-12 mg. For example, one mature destroying angel (A. bisporigera [FIG. 2A], A. virosa, A. suballiacea, and allied species) or death cap (A. phalloides; FIG. 2B) can contain a fatal dose of 10-12 mg of .alpha.-amanitin (Wieland, Peptides of Poisonous Amanita Mushrooms (Springer, N.Y., 1986); herein incorporated by reference). The news gets worse. Toxin producing mushrooms typically demonstrate a higher toxicity than these estimates. An estimated 50% of the amatoxin content of a toxin-producing mushroom is .alpha.-amanitin. Some toxin producing mushrooms can also produce other major amatoxins, such as beta-amanitin and gamma-amanitin resulting in a high death rate from mushroom poisonings.
Amatoxins are a member of a family of related molecules of which at least 9 members are known. Alpha-amanitin is one of the principal amatoxins, comprising approximately 50% of the amatoxin content of some amatoxin-producing mushrooms. Beta-amanitin and gamma-amanitin) are toxic in addition to other types of amatoxins, including but not limited to epsilon-Amanitin, Amanin, Amanin amide, Amanullin, Amanullinic acid, and Proamanullin. Members of this toxin family differ in whether they have asparagine (the position 1 amino acid) or aspartic acid, and in the degree of hydroxylation of the position 3 isoleucine and the tryptophan, and at the Cys-Trp cross-bridge.
Amatoxins can be responsible for fatal human poisonings. After ingestion, amatoxins are taken up by the liver where they begin to cause damage. They are then secreted by the bile into the blood where they are taken up by the liver again, causing a cycle of damage and excretion. In the liver, amatoxins inhibit RNA-polymerase II. The liver is slowly destroyed and is unable to repair itself due to the inactivation of the RNA-polymerase. Thus, the liver slowly dissolves with no hope of repair. Thus, one of the few effective treatments is liver transplantation (Enjalbert et al., (2002) (Treatment of Amatoxin Poisoning: 20-Year Retrospective Analysis, review of poisonings) J. Toxicol. Clin. Toxicol. 40:715; Fabrizio, et al., (2006) Transplant International 19(4):344-345; all of which are herein incorporated by reference).
Poisoning by amanitins is clinically characterized by a long latent period (range 6-48 hours, average 6-15 hours) during which the patient shows few or no symptoms. Symptoms appear at the end of the latent period in the form of sudden, severe seizures of abdominal pain, persistent vomiting and watery diarrhea, extreme thirst, and lack of urine production which lasts for about 24 hours. If this early phase is survived, the patient may appear to recover for a short time, 2-3 days, during which liver damage is ongoing. This second latent period will generally be followed by a rapid and severe loss of strength, prostration, and pain-caused restlessness. During the last stages, hepatic and renal damage becomes clinically evident typically resulting in a coma. Death usually follows a period of comatose condition and occasionally is accompanied by convulsions. If recovery occurs, it generally requires at least a month and is accompanied by enlargement of the liver. Autopsy will usually reveal fatty degeneration and necrosis of the liver and kidneys.
Amatoxins are particularly deadly because they are taken up by cells lining the gut where protein synthesis is immediately inhibited. The toxins are then released into the blood stream and transported to the liver. Once inside the liver cells, amatoxins inhibit RNA-polymerase II, which slows or stops new protein production which begins to cause cellular damage. Bushnell et al., (2002) Proc. Natl. Acad. Sci. USA 99:1218; Kroncke et al., (1986) J. Biol. Chem., 261:12562; Letschert et al., (2006) Toxicol Sci. 91:140; Lindell et al., (1970) Science 170:447; all of which are herein incorporated by reference). The liver secretes excess toxins into bile and into the blood stream where they are taken up by the liver again, causing a cycle of damage and excretion. Thus the liver is slowly destroyed and is unable to repair itself Amanitin toxins are excreted in the urine and evacuated from the body within hours of ingestion. However, if sufficient liver tissue is affected, liver failure will ensure death.
In 50-90% of the cases, death occurs from progressive and irreversible liver, kidney, cardiac, and skeletal muscle damage. The course from ingestion to death may occur in 48 hours (large dose), but effects typically lasts 6 to 8 days in adults and 4 to 6 days in children.
A dose that is likely to kill an average adult human is in the range of 6-7 mg, easily found in the cap of one mature A. phalloides. However, like other fungal toxins, the concentration which is fatal for individuals differs and relates to the concentration in different specimens and environment influences on concentration of toxin produced in one basidiocarp. These examples clearly show that any fungus collected from the field should be properly identified before it is consumed.
B. Phallotoxins.
In addition to bicyclic octapeptide amatoxins, mushrooms naturally produce several bicyclic heptapeptides. In particular, members of Amanita sect. Phalloideae produce bicyclic heptapeptides specifically called phallotoxins (FIG. 1B). Although structurally related to amatoxins, phallotoxins were found to exert a different mode of toxic action in mammalian cells, which was to stabilize F-actin (Enjalbert et al., (2002) J. Toxicol. Clin. Toxicol. 40:715, Lengsfeld et al., (1974) Proc. Natl. Acad. Sci. USA, 71:2803; Bamburg, (1999) Annu. Rev. Cell Dev. Biol. 15:185; all of which are herein incorporated by reference). Phallotoxins were found to destroy liver cells by disturbing the equilibrium of G-actin with F-actin, causing it to shift entirely to F-actin. This leads to numerous exvaginations on the liver cell's membrane which render the cell susceptible to deformity by low-pressure gradients, even those of the portal vein in vivo. This is followed by loss of potassium ions and cytoplasmic enzymes which leads to depletion of ATP and glycogen, causing the final failure of the liver.
Phallotoxins, such as phalloidin and phallacidin, are poisonous when administered parenterally, for example, when administered in a manner other than through the digestive tract, such as by inhalation, intravenous or intramuscular injection. However, because they do not appear to be absorbed by the mammalian digestive tract, they are unlikely to play a primary role in clinical mushroom poisonings.
Biochemically, there are at least seven different naturally occurring phallotoxins: phalloin, phalloidin, phallisin, prophalloin, phallacin, phallacidin, and phallisacin. There are two groups of phallotoxins, neutral and acidic. The neutral phallotoxins, such as phalloidin, contain D-threonine, while the acidic ones contain D-beta-hydroxy-Aspartic acid. Phallacidin (AWLVDCP (SEQ ID NO:69)) also includes Valine whereas phalloidin contains Alanine.
Phallotoxin was once thought to be responsible for the usual symptoms of fatal mushroom poisoning. The compound acts to inhibit F actin in the cell cytoskeleton. It acts immediately, and probably does not move beyond the lining of the gut.
C. Virotoxins.
Although they have the same toxicological effects as and appear to be derived from the phallotoxins, the virotoxins are monocyclic heptapeptides, not bicyclic peptides.
There are at least six virotoxins, viroidin desoxoviroidin, alal-viroidin, alal-desoxoviroidin, viroisin, and desoxoviroisin.
D. Other Types of Mushroom Toxins.
Phallolysins There are at least three phallolysins that are hemolytically active proteins, but, as previously stated, they are heat and acid labile and do not pose a threat to humans.
Ibotenic acid/Muscimol. Ibotenic acid is an Excitatory Amino Acid (EAA) and muscimol is its derivative. These toxins act by mimicking the natural transmitters glutamic acid and aspartic acid on neurons in the central nervous system with specialized receptors for amino acids. These toxins may also cause selective death of neurons sensitive to EAAs. However these are not known to be peptides.
III. Amanita Toxin Peptides in Relation to Other Peptides.
Small, modified, and biologically active peptides synthesized on ribosomes were previously identified from several sources, including bacteria, spiders, snakes, cone snails, and amphibian skin (Escoubas, 2006; Olivera, 2006; Simmaco et al., 1998). Like the Amanita peptide toxins, these peptides are synthesized as precursor proteins and often undergo post-translational modifications, including hydroxylation and epimerization. Circular proteins were discovered in microorganisms, plants and mammals, (for an exemplary review, see, Trabi and Craik, 2002).
Lantibiotics. Lantibiotics, such as nisin, subtilin, and cinnamycin; are produced by species of Lactobacillus, Streptococcus, and other bacteria. They contain 19-38 amino acids. They are characterized by the presence of lanthionine, which is formed biosynthetically by dehydration of an Ala residue followed by intramolecular addition of Cys (Willey and van der Donk, 2007). The lantibiotics are similar to the Amanita peptide toxins in containing a modified, cross-linked Cys residue. However, instead of Ala in the case of lantibiotics, the Cys in the Amanita peptides is cross-linked to a Trp residue. Furthermore, thorough BLAST searching of the genome of Amanita and of all other fungi whose genomes have been sequenced (available in GenBank NR or the DOE Joint Genome Institute) did not identify any orthologs of any of the known lantibiotic dehydratases or cyclases (Willey and van der Donk, 2007).
Cone snail toxins. Cone snail toxins (conotoxins) are 12-40 amino acids. They are linear peptides but are cyclized by multiple disulfide bonds (Bulaj et al., 2003). Like the Amanita peptides, the cone snail toxins exist as gene families, the members of which have hypervariable regions, corresponding to the amino acids present in the mature toxins, and conserved regions found in all members (Olivera, 2006; Woodward et al., 1990, all of which are herein incorporated by reference).
Conotoxins and Amanita peptides differ in many key respects. First, the Amanita peptides are smaller (7-10 amino acids vs. 12-40 for the conotoxins) (Bulaj et al., 2003). Second, the mature conotoxins are at the carboxy termini of the preproproteins and are predicted to be cleaved by a protease that cuts at basic amino acids (Arg or Lys). In contrast, the mature Amanita peptide toxin sequences are internal to the proprotein and are predicted to require two cleavages by one or more prolyl peptidases. Third, the conotoxins are cyclized only by multiple disulfide bonds, whereas the Amanita peptides are cyclized by N-terminus to C-terminus (head-to-tail) peptide bonds and do not have disulfide bonds. Fourth, the conotoxin preproproteins have signal peptides to direct secretion into the venom duct, whereas the Amanita peptides are not secreted (Zhang et al., 2005, herein incorporated by reference) and their proproteins lack predicted signal peptides (FIG. 4).
Amphibian, snake, and spider toxins. Like the conotoxins, these peptides are synthesized on ribosomes as preproproteins, undergo posttranslational modifications, and contain multiple disulfide bonds. None of them are truly cyclic nor and all are much bigger than the Amanita peptide toxins.
Cyclotides. Cyclotides such as kalata are 28-37 amino acids in size (Trabi and Craik, 2002; Craik et al., 2007, all of which are herein incorporated by reference). The precursor structure contains an N-terminal signal peptide followed by a proprotein region and a conserved “N-terminal repeat region” containing a highly conserved domain of .about.20 amino acids, one to three cyclotide domains, and a short C-terminal sequence. An Asn-endopeptidase is responsible for removing the C-terminal peptide from the proprotein and cyclizing the peptide (Saska et al., 2007), but the protease that cuts the N-terminus is apparently not known. The mature cyclotides are true head-to-tail cyclic peptides but, like the conotoxins, also have multiple disulfide bonds.
Bacterial auto-inducing peptides (AIPs). Quorum sensing by certain pathogenic Gram-positive bacteria, such as species of Staphylococcus, involves the secretion and recognition of small (7-9 amino acid) ribosomally-encoded peptides called AIPs (Novicku and Geisinger, 2008). AIPs are posttranslationally cyclized by formation of a thiolactone between the carboxyl group of the C-terminal amino acid and an internal Cys. AIP proproteins are processed at the C-terminus by agrB with simultaneous condensation to form the thiolactone ring (Lyon and Novick, 2004). The inventors determined that there are no proteins related to agrB in the genomes of Amanita, Galerina, or any fungus in GenBank.
Microcin and related molecules. Microcin J25 is a 21-amino acid peptide cyclized between an N-terminal Gly or Cys residue and an internal Glu or Asp residue. It is produced by E. coli; other enterobacteria produce related peptides. Processing of the primary translation product (58 amino acids) involves cleavage of a 37-residue leader peptide and cyclization. Cyclization requires two genes, mcjA and mcjB, which are part of the microcin operon (Duquesne et al., 2007). The maturation reaction requires ATP for amide bond formation. The inventors did not find any orthologs of mcjA or mcjB by BLAST searching of all available fungal genomes, including Amanita bisporigera and Galerina marginata. 
Another example of cycle peptides are thiazolyl peptides, highly rigid trimacrocyclic compounds consisting of varying but large numbers of thiazole rings. The backbone amino acids undergo numerous posttranslational modifications while thiazolyl peptide genes are clustered into operons in bacteria. Derivatives of thiazolyl peptides are sometimes used as antibiotics. Because thiazolyl peptides were synthesized on ribosomes by bacteria such as Streptomyces and Bacillus, the inventors' searched for homologous genes. No homologs of any of the thiazolyl peptide genes were found in the genomes of A. bisporigera, G. marginata, or other fungi in GenBank.
In conclusion, comparison of the Amanita peptide toxins to other known small cyclic peptides indicates that they are unique among microbial natural products in regard to their chemistry, modes of action, and biosynthesis.
A summary of several unique characteristics of Amanita peptide toxins and peptides, linear and cyclic, includes but is not limited to: (1) The Amanita peptide toxins are true head-to-tail cyclic peptides, unlike antibiotics, cone snail toxins, microcins, or AIPs. (2) The tryptathionine moiety (Trp-Cys cross-bridge) is not found in any other natural molecule (May and Perrin, 2007, herein incorporated by reference). (3) The Amanita toxins are the only known ribosomally synthesized cyclic peptides from the Kingdom Mycota (Fungi), the source of many important secondary metabolites that affect human health. (4) The known Amanita peptide toxins have unique modes of action, which contributes to their toxicity and also makes them widely used tools for basic biomedical research. The interaction of alpha-amanitin with pol II is understood in detail (Bushnell et al., 2002, herein incorporated by reference). It is therefore possible that other linear or cyclic ribosomally-synthesized peptides known or predicted to be made by species of Amanita, Galerina, Lepiota, Conocybe, etc. (for example, see, might also have biologically significant modes of action that would make them useful as pharmaceutical agents or research reagents. (5) Amatoxins are not secreted (Zhang et al., 2005, herein incorporated by reference). Consistent with this the proproteins do not have predicted signal peptides. In this regard they differ from conotoxins, lantibiotics, snake and spider venoms, amphibian peptides, or microcins. (6) The Amanita peptide toxins are among the smallest known ribosomally synthesized peptides. Their proproteins (34 and 35 amino acids) are also very small by the standards of typical ribosomally synthesized proteins. (7) No other known peptides are predicted to be processed from their proproteins by a Pro-specific peptidase, and (8) Galerina marginata has advantages over other eukaryotic synthesizers of small peptides. Snakes, amphibians, cone snails, and spiders are difficult to obtain or cultivate and their peptide toxins are made only in small venom ducts.
As described herein the inventors discovered the presence of conserved and hypervariable regions in genes encoding small peptide mushroom toxins After the inventors compared the Amanita peptide toxin genes of the present inventions to known conotoxin genes they discovered that genomic sequences of both organisms are characterized by the presence of conserved and hypervariable regions, however with notable significant differences in the size and structure of the coding regions. Cone snails appear to have the capacity to synthesize a large number of peptides on the same fundamental biosynthetic scaffold (Richter et al., (1990) Proc. Nat. Acad. Sci. USA 87:4836; Woodward et al. (1990), EMBO J. 9:1015; all of which are herein incorporated by reference). However, in contrast to the conotoxins (Olivera, (2006) J. Biol. Chem. 281:31173; herein incorporated by reference), the Amanita peptide toxin genes encode smaller peptides from shorter regions of conserved and hypervariable regions in addition to showing other significant differences, Benjamin, Denis R. 1995. Mushrooms. Poisons And Panaceas. (W.H. Freeman, New York). xxvi+422 pp; herein incorporated by reference).
IV. Contemplated Role of Prolyl Oligopeptidase Family (POP) in Mushroom Peptide Toxin Production.
Prolyl oligopeptidase family (POPs) from other organisms are known to cleave several classes of Pro-containing peptides including mammalian hormones such as vasopressin (Brandt et al., 2007; Cunningham and O'Connor, 1997; Garcia-Horsman et al., 2007; Polgar, 2002; Shan et al., 2005, all of which are incorporated by reference). Changes in human blood serum levels of POP have been associated with depression, mania, schizophrenia, and response to lithium (Williams, 2005, herein incorporated by reference). A POP inhibitor reverses scopolamine-induced amnesia in rats (Brandt et al., 2007, herein incorporated by reference). Mutation of a POP gene in Drosophila melanogaster results in resistance to lithium (Williams et al., 1999, herein incorporated by reference). POPs have been proposed as a treatment for celiac-sprue disease, which is caused by failure to properly digest Pro-rich peptides in gluten (Shan et al., 2002, 2005, all of which are herein incorporated by reference). Despite the demonstration that POP will cleave many small peptides, such as mammalian hormones, apparently the native, endogenous substrates of POPs are not definitively known in any biological system (Brandt et al., 2007, herein incorporated by reference).
The Amanita peptide toxin system is contemplated to represent the first time a native substrate of a POP was identified, as shown during the development of the present inventions (see below and FIG. 20). Specifically, alpha-amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, with an invariant proline residue as the last amino acid in the mature peptide and as the first immediate upstream amino acid in the upstream conserved flanking amino acids. Therefore, a proline-specific peptidase was strongly predicted by the inventors to catalyze cleavage of the proprotein to release the peptide of the mature peptide toxins.
The inventors further identified sequences distantly related to human POP (GenBank accession no. NP002717) (SEQ ID NO:150) in the genome survey sequences of A. bisporigera. Orthologs of human POP (POP-like genes) were also found in every other basidiomycete for which whole genome sequences were available, for example, a POP-like gene was characterized from the mushroom Lyophyllum cinerascens. In contrast, orthologs of human POP are rare or nonexistent in fungi outside of the basidiomycetes. Thus, it appeared that at least one component of the biochemical machinery necessary for the biosynthesis of the Amanita toxins is both widespread in, and restricted to, the basidiomycetes.
V. Genomic Structure of Amanita Peptide Encoding Genes of the Present Inventions.
The inventors discovered the genes encoding the Amanita peptide toxins and the translated peptides relating to Amanita peptide toxins during the development of the present inventions. In particular, the inventors discovered a genomic structure of Amanita peptide toxins, AMA1 and PHA1, relating to amatoxin and phallotoxin toxins. Both types of peptides comprise a conserved stretch (A) of about 9 homologous amino acids, followed by a hypervariable region of 6 to 10 amino acids that are specific for either the two types of toxin peptides, a-amanitin or phallacidin, in addition to longer peptides. These hypervariable regions were followed by an additional conserved stretch (B) of approximately 17 homologous amino acids. The inventors contemplate that the coding sequences of the toxins are part of a larger preproprotein, of approximately 35 amino acids, that is translated and then undergoes post-translational processing to release the active peptide, similar to processing mechanisms of neuropeptides and other small peptide toxins (e.g., conotoxins).
The genome of A. bisporigera contains at least 30 copies of genes coding for the first highly conserved stretch of amino acids (A), followed by a hypervariable region (P), then the second conserved region (B). The primary sequences derived from the cDNA encode peptides AWLVDCP (SEQ ID NO: 69) and IWGIGCNP (SEQ ID NO: 50), which are contemplated to be capable of cyclization into phallacidin and alpha or gamma amanitin, respectively. Neither of these peptides were found after searching the entire GenBank NR database. Therefore, by statistical coincidence they are unlikely to be present in A. bisoporigera; however, experimental results shown herein demonstrate that nucleic acid sequences are present that may encode these peptides.
The Amanita peptide toxins differ from the other known naturally occurring small peptides in several ways. First, the animal peptides are not cyclized by peptide bonds known to be present in Amanita peptide toxins but acquire their essential rigidity by extensive disulfide bonds. Ribosomally synthesized cyclic peptides are known from bacteria, plants, and animals, e.g., the cyclotides and microcin J25 (Craik, (2006) Science 311:1563, Rosengren, et al., (2003), J. Am. Chem. Soc. 125:12464; all of which are herein incorporated by reference), but to the best of the inventor's knowledge all other fungal cyclic peptides are synthesized by nonribosomal peptide synthetases (Walton, et al., (2004) in Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine, J. S. Tkacz, L. Lange, Eds. (Kluwer Academic/Plenum, N.Y., pp. 127-162; Finking, et al., (2004) Annu. Rev. Microbiol. 58:453; all of which are herein incorporated by reference). Second, the Amanita peptide toxins are not secreted, and consistent with this they lack predicted signal peptides in their sequences (FIGS. 4 and 5) (Muraoka, et al., (1999) Appl. Environ. Microbiol. 65:4207, Zhang et al., (2005) FEMS Microbiol. Lett. 252:223; all of which are herein incorporated by reference). Third, whereas the other known ribosomal peptides are processed from their respective proproteins by proteases that recognize basic amino acid residues (Arg or Lys) (Olivera, J. Biol. Chem. 281:31173 (2006), Richter et al., (1990) Proc. Nat. Acad. Sci. USA 87:4836; all of which are herein incorporated by reference), the peptide toxins of Amanita are predicted to be cleaved from their proproteins by a proline-specific protease. As shown herein, the inventors were able to begin confirming their predictions by demonstrating the cleavage of a model phalloidin peptide using an isolated POPB protein, see, FIG. 20.
Sequencing of the genome of A. bisporigera to 20.times. coverage should also yield all of the other members of the Amanita peptide toxin family, which is characterized by “MSDIN” as the first five amino acids of the predicted proproteins. Furthermore, other species of Amanita that make Amanita peptide toxins, such as A. phalloides and A. ocreata, should yield more members of this family. Furthermore, sequencing of additional specimens of these species of Amanita should yield more members. The inventors calculate that there are >30 MSDIN sequences in one isolate of A. bisporigera alone.
Further, the inventors contemplate that genes for Amanita peptide toxin biosynthesis will be clustered within the Amanita genome. As shown herein, an example of genomic organization of PHA1 (for phallacidin) genes in relation to adjacent genes encoding potential enzymes.
VI. Contemplated Role of P450 Homologs in Mushroom Peptide Toxin Production.
Many of the Amanita peptide toxins are hydroxylated at isoleucine, tryptophan, proline, and/or aspartic acid. Hydroxylation of the Amanita peptide toxins might be catalyzed by cytochrome P450 monooxygenases, which are known to catalyze hydroxylation of many other fungal secondary metabolites (e.g., Malonek et al., 2005; Tudzynski et al., 2003). Filamentous fungi differ widely in their numbers of P450's. Whereas some filamentous fungi have >100, the Basidiomycete Ustilago maydis has only .about.17 (drnelson.utmem.edu/CytochromeP450.html). The inventors found three P450 genes clustered with two copies of PHA1 (FIG. 10D and in Example). These are candidates to encode one or more of the enzymes that catalyze hydroxylations of the Amanita peptide toxins.
In terms of identifying new P450 genes contemplated to be involved in Amanita peptide toxin biosynthesis, three candidates in the three P450's were found on a lambda clone clustered with two copies of PHA1 (FIG. 10D). Since secondary metabolites appear to be rare in Basidiomycetes compared to Ascomycetes, the number of P450's in A. bisporigera is probably closer to the Basidiomycete Ustilago (.about.17) than the Ascomycete Fusarium (>100) (http://drnelson.utmem.edu/CytochromeP450.html).
VII. Galerina Mushrooms for Use in the Present Inventions.
Further, the present invention relates to using genes and proteins from Galerina species encoding mushroom peptide toxins, specifically amatoxins. Galerina sequences and Galerina mushrooms are particularly contemplated for use in the present inventions because Galerina, unlike Amanita, is a culturable fungus that produces amanitins in the laboratory. Amatoxins are induced in cultured Galerina, by several methods, for example, Benedict R G, V E Tyler Jr., L R Brady, L J Weber (1966) Fermentative production of amanita toxins by a strain of Galerina marginata. J Bacteriol 91:1380-1381; and preferably using methods described in Muraoka S, T Shinozawa (2000) Effective production of amanitins by two-step cultivation of the basidiomycete, Galerina fasciculata GF-060. J Biosci Bioeng 89:73-76, herein incorporated by reference.
Thus the present inventions further relate to compositions and methods associated with creating and screening genomic libraries from Galerina for sequences of interest. In particular, the present invention relates to providing and using PCR primers for identifying and sequencing Galerina genes, including methods comprising RACE PCR primers. Specifically, the present inventions relate to identifying and using sequences of interest, i.e. sequences encoding proteins associated with the production of small peptides, including cyclic peptides, for example, compositions and methods comprising Galerina POP homologs and amatoxins.
Examples of procedures used to ligate the DNA construct of the invention, the promoter, terminator and other elements, respectively, and to insert them into suitable cloning vehicles containing the information necessary for replication, are well known to persons skilled in the art (see, e.g., Sambrook et al., 1989; herein incorporated by reference).
The polypeptide may be detected using methods known in the art that are specific for the polypeptide. These detection methods may include use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or SDS-PAGE gel blotted onto membranes for immunoblotting. For example, an enzyme assay may be used to determine the activity of the polypeptide. Procedures for determining enzyme activity are known in the art for many enzymes.
A. Peptide Toxin Genes in Galerina Mushrooms.
The inventors' were surprised to discover that sequences of the peptide toxin genes in Galerina marginata is quite different compared to A. bisporigera. See FIGS. 12 and 33A and B for alignments of Galerina and Amanita peptide toxin proteins. For this example, approximately 73 MB of final assembled genomic DNA, as described above, was sequenced by 454 pyrosequencing. 73 MB was estimated to be approximately two times the size of the G. marginata genome based on the average size of known basidiomycete genomes. These sequences were put into a private database and searched using AMA1, PHA1, AbPOPA, and AbPOPB protein sequences The DNA contigs showing predicted protein sequences closely related to AbPOPB and AbPOPA were further analyzed. PCR primers were made to predicted sequences at the two ends of the proteins and used to amplify from genomic and cDNA full length genomic and mRNA copies of the two genes. Four examples of contigs are shown in FIG. 41. The results for GmAMA1 variants are described in this example while the results of screening for POP genes are described in the following example.
Using AMA1 from A. bisporigera as the search query, two orthologs of AMA1 were identified in the partial genome survey sequence of G. marginata and designated as GmAMA1-1 and GmAMA1-2.
PCR primers unique to GmAMA1-1 and GmAMA1-2 were designed. For GmAMA1-1, the unique primers were 5′-CTCCAATCCCCCAACCACAAA-3′ (forward, SEQ ID NO:682) and 5′-GTCGAACACGGCAACAACAG-3′ (reverse, SEQ ID NO:683). For GmAMA1-2, the primers were: 5′-GAAAACCGAATCTCCAATCCTC-3′ (forward, SEQ ID NO:684), and 5′-AGCTCACTCGTTGCCACTAA-3′ (reverse, SEQ ID NO:685). PCR primers for each gene were designed based on the partial sequences and used to amplify full-length copies. The amplicons were cloned into E. coli DH5α and sequenced.
The genomic DNA sequences were used for primer design to obtain full-length cDNAs by Rapid Amplification of cDNA Ends (RACE) using the GeneRacer kit (Invitrogen, Carlsbad, Calif.). A cDNA copy of GmAMA1-1 was obtained using primers 5′-CCAACGACAGGCGGGACACG-3′ (5′-RACE, SEQ ID NO:686) and 5′-GACCTTTTTGCTTTAACATCTACA-3′ (3′-RACE, SEQ ID NO:687), and of GmAMA1-2 with primers 5′-GTCAACAAGTCCAGGAGACATTCAAC-3′ (5′-RACE, SEQ ID NO:688) and 5′-ACCGAATCTCCAATCCTCCAACCA-3′ (3′-RACE, SEQ ID NO:689).
Alignments of genomic and cDNA copies were done using Spidey located at ncbi.nlm.nih gov/spidey/ and Splign ncbi.nlm.nih.gov/sutils/splign/splign.cgi.
GmAMA1-1 contained three introns while GmAMA1-2 contained two introns (FIG. 33). The three introns of GmAMA1-1 were 53, 60, and 60 nt in length in similar locations as the three introns of AMA1. The first intron in both GmAMA1-2 and GmAMA1-2 interrupted the third codon before the stop codon. GmAMA1-1 and GmAMA1-2 differed in at least eight nucleotides out of 108 nucleotides in the coding region (i.e., from the ATG through the TGA stop codon). At least two of these differences resulted in amino acid changes and six changes were silent, i.e no change in amino acid at that location (FIG. 33). There were numerous nucleotide differences between GmAMA1-1 and GmAMA1-2 in the 5′ and 3′ untranscribed regions in addition to having large stretches of close identity. The biggest difference between GmAMA1-1 and GmAMA1-2 was that the latter gene had a 100-bp deletion relative to GmAMA1-1, which spaned the second intron of GmAMA1-1. This deletion was in the 3′ UTR (FIG. 32). This accounted for the presence of only two introns in GmAMA1-2 (FIGS. 32 and 33).
The translational start site of a gene is typically contemplated as the first in-frame ATG, SEQ ID NO:711 after the transcriptional start site, SEQ ID NO:710. When this criterion was applied to GmAMA1-1, a start site was indicated that was analogous to AMA1 of A. bisporigera. This start site resulted in a predicted preproprotein, SPIPQPQT HLTKDLFALTSTMFDTNATRLPIWGIGCNPWTAEHVDQTLASGNDIC, SEQ ID NO: 690, and proprotein, SEQ ID NO: 704. However, when this criteria was applied to GmAMA1-2, there was an in-frame ATG that is 78 nucleotides upstream of the ATG, indicated in FIG. 33, i.e. atgcaagtgaaaaccgaataccaatcctccaaccatcaactcaaccaaagatcttcgcccttgccttaatatctgcc, SEQ ID NO: 690, which would result in a proprotein of 61 amino acids instead of 35 as predicted for AMA1 and GmAMA1-1. Thus two translational start sites were contemplated, one, after the transcriptional start site of SEQ ID NO:713, i.e. SEQ ID NO: 690, that resulted in a 61 amino acid preproprotein, MQVKTESPILQPSTQPKIFALALISAFDTNSTRLPIWGIGCNPWTAEHVDQTLVSG NDIC, SEQ ID NO: 691, and the other, SEQ ID NO:714, in a 35 amino acid proprotein, MFDTNSTRLPIWGIGCNPWTAEHVDQTLVSGNDIC, SEQ ID NO:705. However the inventors' contemplated that the 35 amino acid preproprotein was the target of the Gm POP proteins, for an example showing that prolyl oligopeptidases act on other types of peptides less than 40 amino acids see, Szeltner and Polgar, 2008, herein incorporated by reference).
GmAMA1-1 and GmAMA1-2 were both predicted to encode 35-amino acid proproteins, the same size as the proprotein of AMA1 in A. bisporigera. The toxin-encoding region (IWGIGCNP) (SEQ ID NO: 50) was in the same relative position as it was in AMA1. There were 31 nucleotide differences between GmAMA1-1 and AMA1 in the coding region of 108 nucleotides (ATG through the stop codon). This resulted in a low level of amino acid conservation outside the toxin region and the amino acids immediately upstream of the toxin region (NATRLP, SEQ ID NO:754 (FIG. 33).
The sequenced proproteins were added to a family of genes including and related to AMA1 and PHA1 in A. bisporigera, A. phalloides, and A. ocreata, a group of genes that started with MSDIN. In contrast, when a start codon was contemplated in the same location between GmAMA1-1 and GmAMA1-2 the first five amino acids of the two G. marginata α-amanitin genes were MFDTN, SEQ ID NO: 675. Searching the inventors' G. marginata database with the upstream and downstream regions of GmAMA1-1 and GmAMA1-2 did not reveal any additional related sequences. Conversely, searching with the conserved regions of GmAMA1-1 and GmAMA1-2 did not reveal any related sequences in A. bisporigera beyond the known MSDIN family members described herein.
Distribution of α-Amanitin Genes in the Genus Galerina. 
Within the genus Amanita, AMA1 and PHA1 are known to be present in section Phalloideae, which contains the known amatoxin- and phallotoxin-producing species in this genus. To explore the distribution of the α-amanitin genes in relation to toxin production in Galerina, four species of Galerina were compared by DNA blotting (also known as Southern blotting) and RNA blotting (also known as Northern blotting).
Recent taxonomic revision of this genus indicates that G. marginata and G. venenata are synonyms, whereas G. hybrida and G. badipes are considered as separate species (Enjalbert et al., 2004; Gulden et al., 2001, 2005, all of which are herein incorporated by reference). In Southern blots, a GmAMA1-1 probe [a genomic DNA sequence made with primers (5′-ATGTTCGACACCAACTCCACT-3′, SEQ ID NO:672) and (5′-CGCTACGTAACGGCATGACAGTG-3′, SEQ ID NO:673) hybridized to all three α-amanitin producers (G. marginata, G. badipes, and G. venenata) but not to the toxin nonproducer, G. hybrida (lane 3) (FIG. 34). In contrast to Amanita species, which give multiple hybridizing bands when probed with AMA1 or PHA1, the pattern in Galerina was less complex. Instead of multiple bands, two bands were observed indicating that GmAMA1 is not part of an extended gene family in G. marginata. In order to determine whether there were multiple copies located on the same restriction fragment restriction digests with other enzymes were done; however, these also showed two bands. This pattern of hybridization was consistent with the genome survey sequence that indicated that G. marginata has two sequences closely related to GmAMA1-1. The genome survey sequence and cDNA analysis indicated that both genes encode α-amanitin (FIG. 33), and the inventors' isolate of G. marginata does not make other peptide toxins related to α-amanitin such as beta-amanitin. Because gamma-amanitin has the same amino acid sequence as alpha-amanitin, it is predicted to be encoded by the same gene. The sequenced isolate of G. marginata does not make gamma-amanitin. Further, the genome survey sequence did not contain a DNA sequence that would encode β-amanitin, which differs from α-amanitin by one amino acid (Asp instead of Asn). HPLC analysis of G. marginata CBS 339.88 indicated that it made, at most, a trace of β-amanitin (FIG. 35). The G. marginata sample contained approximately 0.3 mg α-amanitin/g dry weight.
Regulation of GmAMA1 by Low Carbon.
Successful amplification of GmAMA1-1 and GmAMA1-2 by reverse transcriptase PCR with gene-specific probes indicated that both genes are transcribed in culture. Expression was further studied by RNA blotting. Muraoka and Shinozawa (2000, herein incorporated by reference) showed that α-amanitin production in G. fasciculata was upregulated on low glucose medium (carbon starvation). The inventors' found that expression of GmAMA1-1 and/or GmAMA1-2 were also up-regulated by carbon starvation in G. marginata and G. badipes (FIG. 36). Due to their high nucleotide similarity, this experiment did not distinguish between expression of GmAMA1-1 and GmAMA1-2. As expected from the DNA blot results, RNA from the amanitin nonproducer, G. hybrida, gave no signal in either high or low carbon (FIG. 36).
Discovering that G. marginata peptide toxin genes differed from those of A. bisporigera was surprising in several ways. First, the proproteins share little overall amino acid identity except in the toxin region itself (IWGIGCNP) (SEQ ID NO. 50) with the exception of short regions outside of the toxin sequence. For example, whereas the A. bisporigera peptide toxin proproteins start with MSDIN, SEQ ID NO:674, (or with only a single amino acid difference), the two copies of AMA1 in G. marginata started with MFDTN, SEQ ID NO:675. Additionally, the inventors found conservations in the four amino acids after MSDIN, which were also found after MFDTN, and the start of the peptide toxin coding region (IWGIGCNP) (SEQ ID NO: 50) These conserved motif sequences were found as ATRLP, SEQ ID NO:676, or STRLP, SEQ ID NO:677, in the proproteins of both the A. bisporigera peptide toxins and the G. marginata peptide toxins. The complete conservation of the Pro residue immediately upsream of the peptide toxin coding region was believed to be significant because Pro is believed to be required for processing of the proprotein by a prolyl oligopeptidase. The inventors further contemplated that upstream conserved region of amino acids in G. marginata peptide toxin sequences (i.e. N[A/S]TRL, SEQ ID NO:678) is important for recognition of the proproteins by Gm POPB. There was little conservation between the downstream conserved regions of the A. bisiporigera and the G. marginata genes. For example, MFDTNATRLP SEQ ID NO: 679, was unexpectedly found in place of MSDIN.
Second, G. marginata was discovered to contain two nearly identical copies of the α-amanitin gene with at least one variant of each whereas one copy of the α-amanitin gene was found in A. bisporigera. Conversely, A. bisporigera has at least two copies of genes encoding phallacidin (PHA1) while none were found in the sequenced isolate of G. marginata, and phallacidin or other phallotoxins have not been reported from G. marginata. 
Third, the inventors were surprised to find two sequences related to the α-amanitin genes in the genome of G. marginata whereas a large family of related sequences (>30 members), which encode predicted, but chemically unknown, cyclic peptides was discovered in the A. bisporigera genome. These predicted peptides were discovered by translating the A. bisporigera genes contained 7 to 10 amino acids where the majority lacked Trp and Cys predicted to be used to form tryptathionine, which was a characteristic of the amatoxins and phallotoxins of A. bisporigera peptides.
G. marginata and other species of Galerina were known to make α-amanitin (Enjalbert et al., 2004; Muraoka et al., 1999; Muraoka and Shinozawa, 2000, all of which are herein incorporated by reference). However phallotoxins were not found in Galerina species however some species were reported to make β-amanitin. β-amanitin differs from α-amanitin in having Asp in place of Asn. The difference between these two forms of amanitin was predicted to be genetically encoded and not catalyzed by, e.g., a transamidase, because the genome of A. phalloides contains a gene that was predicted to directly encode β-amanitin.
The inventors confirmed that the isolate of G. marginata prepared and used herein did not synthesize β-amanitin and the genome lacks a gene for β-amanitin. In other isolates, traces of β-amanitin from G. marginata grown in culture were detected i.e. Benedict et al. (1966, 1967, all of which are herein incorporated by reference). Further, β-amanitin was not detected in several wild North American specimens of Galerina. Therefore, some species and/or isolates of Galerina do make β-amanitin and others do not, therefore each isolate must be tested. Other forms of amanitin, such as γ-amanitin and ε-amanitin, differ from α-amanitin and β-amanitin in their pattern of hydroxylation. This chemical difference was not found in encoding DNA.
B. Full Length POP Gene Production.
The G. marginata partial genome survey was discovered to contain two orthologs of the POP genes of A. bisporigera. Genomic PCR, reverse transcriptase PCR, and RACE were used, as described herein, to isolate full-length copies of these two genes and determine their intron/exon structures (FIG. 37). GmPOPA had 18 introns, which is the same number found in AbPOPA, while GmPOPB had 17 introns, one fewer than in AbPOPB. The amino acid sequences of the predicted translational products of GmPOPA (738 amino acids) and GmPOPB (730 amino acids) are 57% identical to each other. The GmPOPA protein is 65% identical to AbPOPA and 58% identical to AbPOPB, and GmPOPB is 57% identical to AbPOPA and 75% identical to AbPOPB.
During the development of the present inventions, two orthologs were found in the G. marginata genome sequences corresponding to the two A. bisporigera prolyl oligopeptidases (AbPOPA and AbPOPB) described herein. The G. marginata genes with closest identity to AbPOPA or AbPOPB were designated as GmPOPA and GmPOPB, respectively.
Sequences hybridizing to AbPOPA were found to be present in amatoxin and phallotoxin-producing and non-producing species of Amanita, whereas AbPOPB was found present only in the toxin-producing species. By DNA blotting GmPOPA was present in all four specimens of Galerina, however GmPOPB was not present in the amanitin non-producing species G. hybrida (FIG. 34). The similarity of the hybridization pattern of G. venenata and G. marginata to GmAMA1, GmPOPA, and GmPOPB was consistent with these two isolates belonging to the same species (see, Gulden et al., 2001, herein incorporated by reference). The association of POPB with amanitin production in both A. bisporigera and G. marginata, and the higher amino acid identity of GmPOPA to AbPOPA and of GmPOPB to AmPOPB was consistent with a contemplated role for POPB in amanitin biosynthesis in both species. Other basidiomycetes in GenBank and at the DOE Joint Genome Institute (JGI) have single POP genes, which are contemplated as functional orthologs of POPA.
For isolating and cloning full-length cDNA sequences for GmPOPA and GmPOPB, PCR primers that corresponded to the amino and carboxyl termini of both genes (which were present on different contigs) were designed from the genome survey sequence. The forward primers were 5′-TTTAGGGCAGTGATTTCGTGACA-3′, SEQ ID NO: 692, and 5′-AACAGGGAGGCGATTATTCAAC-3′, SEQ ID NO: 693, and the reverse primers were 5′-GAACAATCGAACCCATGACAAGAA-3′, SEQ ID NO: 694, and 5′-CCCCCATTGATTGTTACCTTGTC-3′, SEQ ID NO: 695. The primer pairs were used in both combinations and successful amplification indicated the correct pairing of 5′ and 3′ primers. The resulting amplicons were cloned into E. coli DH5α and sequenced.
The RACE primers for GmPOPA were 5′-CGGCGTTCCAAGGCGATGATAATA-3′ (5′-RACE), SEQ ID NO: 696, and 5′-CATCTCCATCGACCCCTTTTTCAGC-3′ (3′-RACE), SEQ ID NO: 697, and for GmPOPB 5′-AGTCTGCCGTCCGTGCCTTGG-3′ (5′-RACE), SEQ ID NO: 698, and 5′-CGGTACGACTTCACGGCTCCAGA-3′ (3′-RACE), SEQ ID NO: 699. Sequences generated from the RACE reactions were used to assemble full-length cDNAs of two genes, GmPOPA and GmPOPB (see FIGS. 38A and 38B).
Alignments of genomic and synthetic cDNA copies (see, FIGS. 38A and 38B) were done using Spidey available at National Center for Biotechnology Information (NCBI) at website ncbi.nlm.nih.gov/spidey/ and Splign at ncbi.nlm.nih.gov/sutils/splign/splign.cgi.
GmPOPA and POPB were predicted to encode exemplary polypeptides as shown in FIGS. 38A and 38B.
The inventors' contemplate that POP proteins encoded by the G. marginata POP sequences (known as GmPOP) of the present inventions are capable of enzymatic activity. There are three critical amino acids that constitute the active site in other POP proteins (Szeltner et al., (2008) Current Protein and Peptide Science 9:96-107, herein incorporated by reference). In a crystallized POP protein, the active site residues were Ser554, Asp641, and His680. The location of these active site residues in POPA are: Ser581, Asp665, and His 701. In POPB they are Ser571, Asp661, and His698. Thus the GmPOP genes of the present inventions are contemplated to be capable of encoding POP proteins with these active site amino acids in analogous positions for a protein capable of enzymatic activity.
The inventors showed that isolated prolyl oligpeptidase (POP) proteins of other mushroom species were capable of initial processing of the proproteins of amatoxins and phallotoxins. First, in the extended MSDIN (SEQ ID NO: 674) family of Amanita, discovered by the inventors and now shown to correspond to an MFDTN, SEQ ID NO:675, family of α-amanitin genes of G. marginata, flanking Pro residues are completely conserved. One Pro remains in the mature toxin while the other is removed with the flanking sequence. Second, an enzyme that proteolytically cleaves a synthetic phalloidin proprotein, isolated from the phalloidin-producing fungus Conocybe albipes, was identified during the development of the presence inventions as a POP protein. The same enzyme cleaves at both Pro residues to release the mature linear peptide (AWLATC (SEQ ID NO: 756) in the case of phalloidin). Third, toxin-producing species of Amanita have two POP genes, whereas all other sequenced basidiomycetes have one. One of the Amanita POP genes, AbPOPB, was found during the development of the presence inventions restricted to toxin-producing species, like AMA1 and PHA1 themselves. Fourth, the distribution of AbPOPB and α-amanitin overlap in mushroom tissues was found during the development of the presence inventions, indicating a cytological connection between α-amanitin biosynthesis and accumulation. G. marginata was discovered to have two POP genes, like Amanita but unlike other, toxin non-producing species of mushrooms. GmPOPB is absent from species such as G. hybrida that do not make toxins. Thus, AbPOPB and GmPOPB are believed to be involved in the biosynthesis of the amatoxins and/or phallotoxins in their respective species.
VIII. Recombinant Polypeptide Products of Amanita and Galerina Genes.
A desired end product, i.e., the polypeptide of interest, such as a POP enzyme, may be expressed by a host cell, such as a bacterium, i.e. E. coli, as a heterologous protein or peptide. Thus the polypeptide may be any polypeptide heterologous to the bacterial cell. The term “polypeptide” is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The heterologous polypeptide may also be an engineered variant of a polypeptide. The term “heterologous polypeptide” is defined herein as a polypeptide, which is not native to the host cell. Preferably, the host cell is modified by methods known in the art for the introduction of an appropriate cloning vehicle, i.e., a plasmid or a vector, comprising a DNA fragment encoding the desired polypeptide of interest. The cloning vehicle may be introduced into the host cell either as an autonomously replicating plasmid or integrated into the chromosome. Preferably, the cloning vehicle comprises one or more structural regions operably linked to one or more appropriate regulatory regions.
The structural regions are regions of nucleotide sequences encoding the polypeptide of interest. The regulatory regions include promoter regions comprising transcription and translation control sequences, terminator regions comprising stop signals, and polyadenylation regions. The promoter, i.e., a nucleotide sequence exhibiting a transcriptional activity in the host cell of choice, may be one derived from a gene encoding an extracellular or an intracellular protein, preferably an enzyme, such as an amylase, a glucoamylase, a protease, a lipase, a cellulase, a xylanase, an oxidoreductase, a pectinase, a cutinase, or a glycolytic enzyme.
The resulting polypeptide may be isolated by methods known in the art. For example, the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989, herein incorporated by reference).
IV. Compositions and Methods for Expressing Small Linear Peptides and Cyclic Peptides Using Transformed Galerina Marginata and Lysates.
The inventors grew G. marginata in the laboratory and collected mycelium for use in the following transformation procedure. The inventors show herein the successful transformation of the alpha-amanitin-producing fungus Galerina marginata with a test construct. Thus the inventors' contemplate producing commercial levels of amanatin in addition to novel, non-natural analogs of amanitin. Further, the inventors' contemplate making novel linear and cyclic peptides from synthetic prepropeptides.
The following are exemplary methods for making buffers and reagents for us in the present inventions. Galerina culture methods: Vegetative mycelial stocks were prepared by culturing aseptic fragments of fruiting bodies on HSVA plates. Fungal colonies were transferred and reisolated until pure cultures were obtained. The stocks were subcultured every 6 months. HSV-2C (1 L): 1 g yeast extract, 2 g glucose, 0.1 g NH4Cl, 0.1 g CaSO4.5H2O, 1 mg thiamine.HCl, and 0.1 mg biotin, pH 5.2 (Muraoka and Shinozawa, 2000, herein incorporated by reference). Agar medium (HSVA) for subculture contained 2% agar in HSV. Protoplasting Buffer: In 20 ml of 1.2 M KCl add 500 mg Driselase (Sigma), 1 mg chitinase (Sigma), and 300 mg lysing enzyme from Aspergillus sp. Sigma #L-3768. Stir for 30 min and filter sterilize in a 0.45 um filter. Sorbitol Tris-HCl Ca (STC) buffer: Solution a) 1.2 M sorbitol, 10 mM Tris-HCl (pH8.0), 50 mM CaCl2, autoclaved. Solution b) 30% PEG Solution Mix: 30% (W/V) polyethylene glycol/STC buffer. Filter sterilize in a 0.45 um filter. Regeneration medium (RM): a) HSV-2C (1 L) and b) sucrose 273.5 g/500 ml of water. Autoclave solutions a) and b) separately and combine after autoclaving.
The following is an exemplary Galerina transformation protocol for use in the present inventions. Around 20 pieces of mycelium were used to inoculate 100 ml of HSV-2C broth in a 250 ml Erlenmeyer flask. This inoculate was placed on a shaker at 150 rpm at room temperature for 9-15 days, until cloudy. The culture medium and fungus was used to begin the following steps. The cultures were: 1. Filtered through sterile Miracloth and the collected mycelia was washed thoroughly with sterile water. This fungal mycelium was placed in a sterile 250 ml Erlenmeyer flask. 20 ml Protoplasting Buffer (see recipe below) was added. 2. Digested for 8 hours on a rotary shaker at 26-30 C at 120 rpm. 3. Digestion mix was filtered through a 30 micron Nitex nylon membrane (Tetko Inc. Kansas City, Mo., U.S.A.)) into 1-2 sterile 30 ml Oakridge tubes on ice. Filtered solution was turbulent due to the presence of protoplasts when checked under the microscope. 4. This filtered solution was centrifuged in Oakridge tubes at 4 C at 2000×g for 5 min. 5. Supernatant was carefully poured off and discarded. Protoplast pellet was gently resuspended in approx. 10 ml of STC buffer and resuspended by shaking gently. Solution was spun at 2000×g for 5 min. 6. Repeat step 5 once. 7. Supernatant was discarded and the protoplast pellet was gently resuspended in 1 ml of STC buffer with a wide orifice pipette and transferred to a microcentrifuge tube and spun at room temperature at 4000×g for 6 min. 8. Supernatant was poured off and protoplasts were resuspended in 1 ml of STC in a final volume with concentration of 108-109 protoplast/ml. The tube was placed on ice. 9. The following mixture was combined: 50 μl protoplasts, 50 μl STC buffer, 50 ul 30% PEG solution and 10 ul plasmid or PCR product (1 μg) depending upon the experiment. When plasmids were used they were linearized with a restriction enzyme which cut the DNA in a noncoding region. 10. 2 ml of 30% PEG solution was added and the tubes incubated for 5 min. 11. 4 ml of STC buffer was added and gently mixed by inversion. 12. The mix was added to Regeneration Media (RM) (see below) at 47° C., and mixed by inversion then poured into Petri dishes. Each solution mixture was plated in several plates. 13. Protoplasts were regenerated for up to 20 days until tiny colonies started to appear as viewed by eye. 10 ml of RM amended with 10 ng/ml Hygromycin B was overlayed onto the cultures. 14. Putative transformants were isolated from colonies that grew after the Hygromycin B overlay and eventually emerged on the surface of the overlaid agar. Examples of colonies collected for use in the present inventions are shown by arrows in FIG. 39.
After colonies were collected the presence of the inserted Hygromycin B transgene was tested by PCR. Primers specific to the hygromycin resistance gene used in FIG. 40 were the following: hph_forward 5′-GCGTGGATATGTCCTGCGGG-3′ hph_reverse, SEQ ID NO:700, 5′-CCATACAAGCCAACCACGGC-3′, SEQ ID NO: 701, (Kilaru et al., 2009, Curr Genet 55:543-550, herein incorporated by reference).
The inventor's contemplate that G. marginata can be transformed with synthetic genes, using the G. marginata specific contemplated cut sites, i.e. synthetic sequences comprising nucleotides encoding MDSTN, TRIPL and Prolines in conserved positions. For examples, in one embodiment, a synthetic DNA sequence encoding an amino acid sequence of alpha-amanitin may be expressed. In one embodiment, alpha-amanitin production would be increased, for example, using a high expression promoter, transforming Galerina with multiple copies of the alpha-amanitin gene.
In another contemplated embodiment, a synthetic, novel cyclic peptide is synthesized by transformed Galerina by changing specific bases of synthetic G. marginata alpha-amanitin sequences (including PCR copies of isolated peptide toxin genes and base by base construction of nucleic acid sequences) in order to make other types of peptide toxins and peptides. In one example, replacing the codon AAC (Asn) with GAC (Asp) will encode beta-amanitin instead of alpha-amanitin. Beta-amanitin production in G. marginata would be easily detected by reverse-phase HPLC because the inventor's isolate of G. marginata makes barely detectable levels of beta-amanitin.
The inventors further contemplate changing other amino acids to make non-natural amanitin derivatives, as one example, replacing Gly with Ala by replacing GGT with GCT. Even further, the inventor's contemplate an embodiment for making linear and cyclic peptides of at least six, seven, eight, nine, ten or more amino acids comprising the general formula XWXXXCXP, SEQ ID NO:702, where X is any amino acid. The Pro is retained in these peptides in order for correct processing by POP, and the presence of Trp (W) and Cys (C) will result in the biosynthesis of tryptathionine, a unique hallmark of the Amanita toxin peptides. Expression of synthetic peptides and peptide toxins would be monitored by standard assays including but not limited to PCR generated fragments (as in FIG. 40), and by HPLC methods (as in FIG. 31), and the like. Further, separation of synthetic toxins from endogenous peptide toxin and endogenous small peptides (i.e. peptides produced from genomic DNA originally contained in these Galerina isolates) would be done by standard techniques including but not limited to HPLC methods (as in FIG. 31). Isolated peptides produced by expression of synthetic sequences would be used in assays for assessing biological activity. For example, toxicity of synthetic amanitin toxins would be determined in assays, for one example, to measure inhibition of transcription in eukaryotic cells, such as capability to inhibit RNA Polymerase II. These toxins are contemplated for commercial levels of production.
Even further, the inventors' contemplate making new Galerina isolates that do not produce peptide toxins for use in the present inventions. In one embodiment, the inventors' contemplate knocking out genomic peptide toxin genes for making a new Galerina isolate that does not express peptide toxins. As examples for removing genomic peptide toxin genes in Galerina, i.e. test Galerina (isolates of Galerina used in the following methods) would be subject to homologous integration of transforming DNA that would be used for removing regions of DNA comprising the peptide toxin genes in transformed test Galerina, spontaneous mutants and induced mutants of test Galerina would be made then screened for loss of peptide toxin gene expression and more preferably loss of peptide toxin genes. Another method for eliminating endogenous toxin production is RNAi, which has been used in other basidiomycete fungi (Heneghan et al., Mol Biotechnol. 2007 35(3):283-96, 2007, herein incorporated by reference). Loss of toxin expression in test isolates would be monitored by standard assays including but not limited to genomic sequencing of test Galerina, PCR generated fragments of genomic sequences (as in FIG. 40), PCR generated toxin cDNA (as described herein), and by HPLC methods (as in FIG. 31), and the like. When a test Galerina isolate is shown to lack expression of peptide toxins this isolate would be cultured as a new Galerina laboratory isolate for use in the present inventions.
G. marginata has numerous advantages as an experimental system for use in the present inventions. First, G. marginata is cultured under laboratory conditions, unlike most species of Amanita, which do not grow well in the laboratory (Benedict et al., 1966, 1967; Muraoka and Shinozawa, 2000; Zhang et al., 2005, all of which are herein incorporated by reference). Second, G. marginata produced α-amanitin in culture and production was increased by carbon starvation. Third, genomic sequencing and genetic studies were facilitated by the availability of a peptide toxin-producing monokaryotic strain (isolate) of G. marginata. Fourth, the panoply of peptide toxin genes, estimated greater than 30 members in species of Amanita, was not found in the laboratory isolate of G. marginata, where only two genes were found during the development of the present inventions.
EXPERIMENTAL
The following examples serve to illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosures which follow, the following abbreviations apply: N (normal); M (molar); mM (millimolar); .mu.M (micromolar); mol (moles); mmol (millimoles); .mu.mol (micromoles); nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams); .mu.g (micrograms); ng (nanograms); pg (picograms); L and l (liters); ml (milliliters); .mu.l (microliters); cm (centimeters); mm (millimeters); .mu.m (micrometers); nm (nanometers); U (units); min (minute); s and sec (second); deg (degree); .degree. C. (degrees Centigrade/Celsius).
Example I
Materials and Methods
The following is a description of exemplary materials and methods that were used in subsequent Examples during the development of the present inventions.
A. Exemplary Mushroom Species of the Present Inventions (FIG. 2 and FIG. 31).
The inventors selected the genome of Amanita bisporigera to provide sequences of interest because of reports on consistently high, albeit somewhat variable, levels of amatoxins and phallotoxins within individual fruiting bodies combined with the relative ease of obtaining exemplary wild growing mushrooms by merely identifying and harvesting the mushrooms.
Exemplary Basic Molecular Biology Techniques.
The inventors developed and used the following exemplary materials and methods during the development of the present inventions. During the development of the present inventions the inventors were surprised to successfully clone cDNAs encoding toxin genes from mature mushrooms in addition to mushrooms in the button stage.
Genomic DNA Isolation.
Although the carpophores (fruiting bodies) contain high concentrations of the toxins, like other ectomycorrhizal Basidiomycetes, species of Amanita grow slowly and do not form carpophores in culture (Muraoka et al., (1999) Appl. Environ. Microbiol. 65:4207; Zhang et al., (2005) FEMS Microbiol Lett. 252:223; all of which are herein incorporated by reference). Therefore, A. bisporigera mushrooms, an amatoxin and phallotoxin producing species native to North America, were harvested from the wild. Caps and undamaged stems were cleaned of soil and debris, frozen at −80.degree. C., and lyophilized.
Genomic DNA was extracted from the lyophilized fruiting bodies using cetyl trimethyl ammonium bromide-phenol-chloroform isolation (Hallen, et al., (2003) Mycol. Res. 107:969; herein incorporated by reference). For studies requiring RNA, RNA was extracted using TRIZOL (Invitrogen) (Hallen, et al., (2007) Fung. Genet. Biol., 44:1146; herein incorporated by reference in its entirety). Specifically, DNA for genomic blotting was cut with PstI and electrophoresed in 0.7% agarose.
Probe Labeling, DNA Blotting, and Filter Hybridization.
Standard protocols were followed for these and similar molecular biology procedures (see, Maniatis, et al., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor, N.Y., 1982, herein incorporated by reference) and Singh, et al., (1984) Nucl. Acids Res. 12:5627; herein incorporated by reference). In general, hybridization was done overnight at 65.degree. C. in 4.times.SET (600 mM NaCl, 120 mM Tris-HCl, pH 7.4, 8 mM EDTA), 0.1% sodium pyrophosphate, 0.2% SDS, 10% dextran sulfate, 625 mu.g/ml heparin. Washing: twice in 2.times.SSPE (300 mM NaCl, 20 mM NaH.sub.2PO.sub.4, 2 mM EDTA, pH 7.4), 0.1% SDS at 21.degree. C., then twice in 0.1.times.SSPE and 0.1% SDS at 60.degree. Celcius.
PCR Amplification of Peptide Encoding Genes.
PCR primers for amanitin and phallacidin amplification from A. bigospora were based on fragments within sequences shown in FIGS. 4-6. The primer sequences used are shown in Table 3.







TABLE 3







PCR primers used for making synthetic amanitin (AMA1) and phallacidin


genes (PHA1).









Sequence Name
SEQ ID NO:
SEQUENCE





AMA1, forward
SEQ ID NO: 1
5′ CCATCTGGGGTATCGGTTGC 3′


 


AMA1, reverse
SEQ ID NO: 2
5′ TTGGGATTGTGAGGTTTAGAGGTC 3′


 


PHA1, forward
SEQ ID NO: 3
5′ CGTCAACCGTCTCCTC 3′


 


PHA1, reverse
SEQ ID NO: 4
5′ ACGCATGGGCAGTCTAC 3′









A 551-bp fragment of the A. bisporigera β-tubulin gene was amplified using primers 5′-ACCTCCATCTCGTCCATACCTTCC-3′ (SEQ ID NO: 5) and 5′-TGTTTGCCACGCTGCATACTA-3′ (SEQ ID NO: 6) then used as a control probe on DNA blots. PCR amplification was done using REDTaq ReadyMix DNA polymerase (Sigma) and appropriate reagents under 30 cycles of denaturation (94.degree. C., 30 sec), annealing (55.degree. C., 30 sec), and extension (72.degree.C., 5 min).
Target Genes for Sequencing.
PCR target gene products were purified using Wizard SV Gel and PCR Clean-Up System (Promega) and then cloned into TOPO pCR 4 (Invitrogen) for obtaining sequence information.
B. Exemplary Mushroom Species of the Present Inventions (FIG. 31).
Biological Material.
Four species of Galerina were obtained from Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands, including G. marginata (CBS 339.88), G. badipes (CBS 268.50), G. venenata (CBS 924.72), and G. hybrida (CBS 335.88). G. marginata CBS 339.88 is monokaryotic and was confirmed to make α-amanitin. G. venenata is considered synonymous with G. marginata (Gulden et al., 2001, herein incorporated by reference). The cultures were maintained on potato dextrose agar. For DNA isolation, the isolates were cultured in liquid medium for 15-30 d with rotary shaking at 120 rpm at 23° C. The medium was HSV-2C, which contains (per liter) 1 g yeast extract, 2 g glucose, 0.1 g NH4Cl, 0.1 g CaSO4.5H2O, 1 mg thiamine.HCl, and 0.1 mg biotin, pH 5.2 (Muraoka and Shinozawa, 2000). For induction experiments, the media had the same formulation, except that high carbon (HSV-5C) and low carbon (HSV-1C) media contained 5 g glucose and 1 g glucose, respectively (Muraoka and Shinozawa, 2000, herein incorporated by reference).
Nucleic Acid Isolation and Genome Sequencing.
Lyophilized fungal mycelia were ground in liquid nitrogen with a mortar and pestle. High molecular weight DNA was isolated using Genomic-tip 100/G (Qiagen, Germantown, Md.; catalog #10234) and RNA was extracted with TRIzol (Invitrogen, Carlsbad, Calif.), following the manufacturers' protocols.
Genomic DNA was sequenced by 454 pyrosequencing at the Research Technology Support Facility (RTSF) at Michigan State University. A general library was constructed using standard protocols and sequenced on a 454 GSFLX Titanium Sequencer (Roche manual, 20th ed., herein incorporated by reference). Raw reads were assembled with Newbler and assembled into a searchable database.
Cloning and Gene Characterization.
AMA1 and PHA1 are the designations for the α-amanitin- and phallacidin-encoding genes, respectively, of A. bisporigera; the prefix Ab is used to designate other genes from A. bisporigera. The prefix Gm is used to designate all genes from G. marginata. 
DNA and RNA Blotts.
DNA for Southern blotting was digested with PstI and electrophoresed in 0.7% agarose. Probe labeling, blotting, and filter hybridization followed standard protocols (Scott-Craig et al., 1990, herein incorporated by reference). Hybridizations were performed for 15 hr at 65° C. Roughly 2 μg of DNA were loaded per lane. Probes were made by labeling genomic DNAs of GmAMA1-1, GmPOPA, and GmPOPB with [32P]dCTP.
For the GmAMA1 induction experiment, G. marginata was cultured in HSV-5C media for 30 d and then transferred to HSV-5C or HSV-1C and grown for an additional 10 d. The resulting mycelia were lyophilized and stored at −80° C. prior to RNA extraction. Full-length cDNA was prepared using the GeneRacer RACE kit, following the manufacturer's protocols. Hybridization probes were amplified using a specific 5′ primer (5′-ATGTTCGACACCAACTCCACT-3′, SEQ ID NO:680) and GeneRacer 3′ nested primer (5′-CGCTACGTAACGGCATGACAGTG-3′, SEQ ID NO:681). Probe labeling, RNA gel electrophoresis, and blotting followed standard protocols (Scott-Craig et al., 1990, herein incorporated by reference). Each lane was loaded with 15 μg total RNA.
Amanitin Extraction and Analysis.
G. marginata was cultured in HSV-5C media for 30 d and then transferred to fresh HSV-1C medium for an additional 10 d. After harvest, the mycelium was lyophilized and stored in at −80° C. A portion of dried mycelium (0.2 gm) was ground in liquid nitrogen and mixed with 2 ml methanol:water:0.01 M HCl (5:4:1) (Enjalbert et al., 1992; Hallen et al., 2003, herein incorporated by reference). The suspension was incubated at 22° C. for 30 min and then centrifuged at 10,200×g for 10 min at 4° C. The supernatant was collected and filtered through a 0.22 μl filter. Chromatographic separation was done on a C18 column (Vydac 218TP54) attached to an Agilent Model 1100 HPLC with detection at 230, 290, and 305 nm. Elution solution A was water+0.1% trifluoroacetic acid, and solution B was acetonitrile+0.075% trifluoroacetic acid. The flow rate was 1 ml/min with a gradient from 100% A to 100% B in 30 min. An α-amanitin standard (Sigma A2263) was dissolved in water at a concentration of 100 μg/ml. Loadings were 40 μl unknown or 20 μl standard.
Example II
This example describes exemplary methods for providing a fungal genomic library, specifically an Amanita spp., library.
The inventors initially contemplated the existence of an amatoxin synthetase gene that was a member of the class of enzyme known as nonribosomal peptide synthetases.
However after extensive unsuccessful attempts to obtain amatoxin synthetase genes or gene fragments through PCR-based techniques using isolated genomic DNA, see, Example III, and biochemical methods (such as, ATP-pyrophosphate exchange assay; amino acid feeding studies, etc.), the inventors subsequently initiated a shotgun genome sequencing project for obtaining genes of interest, such as genes associated with cyclized peptide production, toxin production, peptide encoding genes, toxin encoding genes, etc. One genomic library was generated by the Genomics Technology Support Facility at Michigan State University and one was generated by Macrogen, Inc. Each library yielded genomic fragments of approximately 2-kb in length. Random clones were end sequenced by automated dideoxy sequencing.
Approximately 5.7 Mb sequence was generated in approximately 10,000 unidirectional sequencing reads using dideoxy sequencing using an ABI 3730 Genetic Analyzer and an ABI Prism 3700 DNA Analyzer (sequencing performed at the Research Technologies Support Facility at Michigan State University, and by Macrogen, Inc.).
The inventors originally began a public Amanita sequence database; however, after a brief posting of the above-described sequencing results, the inventors removed those sequences from public access (see, Examining amatoxins: The Amanita Genome Project. Hallen, Walton, 159. The utility of the incomplete genome: the Amanita bisporigera genome project. Mar. 15-20, 2005 Asilomar Conference Center, Pacific Grove Calif. Fungal Genetics Newsletter, Volume 52-Supplement XXIII FUNGAL GENETICS CONFERENCE; herein incorporated by reference). Moreover, to the inventors' knowledge, sequences of the present inventions were never publicly available.
The inventors subsequently also completed at least four runs on a Genome Sequencer 20 from 454 Life Sciences (Margulies et al., (2005) Nature 437:376; herein incorporated by reference). This generated approximately 70 MB of sequence data, which is approximately 2×coverage of the genome of A. bisporigera, based on the known size of other Homobasidiomycetes, (Le Quere et al., Fung. Genet. Biol. 36, 234 (2002); Coprinus cinereus Sequencing Project. Broad Institute of MIT and Harvard (broad.mitedu/annotation/genome/coprinus_cinereus/Hom-e.html); all of which are herein incorporated by reference).
The inventors structured and maintained the sequenced DNA in a password-protected, private BLAST-searchable format. The sequences were compared to GenBank's non-redundant database.
BLASTX (translated query against protein database) was used in searching the non-redundant database (NR) at GenBank, and TBLASTX (translated query against translated database) and BLASTN (nucleotide query against nucleotide database) were used in searching the genomes of Coprinopsis cinereus (also known as Coprinus cinereus) and Phanerochaete chrysosporium, the two closest relatives to Amanita bisporigera for which complete genome sequences were available at that time. In some embodiments, BLAST results were examined, catalogued, and automatically annotated.
Example III
This example describes the failure of the inventors to obtain a gene homologous to a fungal nonribosomal peptide synthetases (NRPSs) in Amanita bisporigera, which produces amatoxins, phallotoxins, and other putative Amanita peptide toxins. Details are shown in a poster entitled “Examining amatoxins: The Amanita Genome Project” Hallen Walton 159. The utility of the incomplete genome: the Amanita bisporigera genome project. Mar. 15-20, 2005 Asilomar Conference Center Pacific Grove Calif. Fungal Genetics Newsletter, Volume 52-Supplement XXIII FUNGAL GENETICS CONFERENCE; herein incorporated by reference.
Because known fungal cyclic peptides are biosynthesized by methods comprising nonribosomal peptide synthetases (NRPSs) (Walton, et al., in Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine, et al., Eds. (Kluwer Academic/Plenum, New York, 2004, pp. 127-162; Finking, et al., (2004) Arum Rev Microbiol 58:453-488, all of which are herein incorporated by reference), the inventors initiated an attempt to identify by PCR in the total genomic DNA of Amanita bisporigera sequences encoding an NRPS using PCR primers based on known bacterial and fungal NRPSs and total A. bisporigera DNA as template. The inventors contemplated that any NRPS genes sequences within the Amanita bisporigera genome should have been readily amplified using two or more of PCR primers. Then, from sequencing genomic DNA outward from the PCR products, they should have ultimately identified an NRPS with 8 adenylating domains containing other conserved regions present in all known NRPS-encoding sequences.







TABLE 4







PCR primers used that failed to obtain a NRPS sequence (See FIG. 3).








Forward Primers 5′-3′
Reverse Primers 5′-3′













AIxKAGxA:
GCN ATH TNN AAR GCN GGN
AIxKAGx:
GCN GNN CCN GCY


SEQ ID NO: 7
NCN GC
SEQ ID NO:
TTN NAD ATN GC




8



 


FTSGSTG
TTY ACI TCI GGI TCI ACI GG1
na
na


(JA4F): SEQ ID





NO: 9





 


YTSGSTG1: SEQ
TAY ACN AGY GGN AGY ACN GG
na
na


ID NO: 10





 


YTSGSTG2: SEQ
TAY ACN AGY GGN TCN ACN GG
na
na


ID NO: 11





 


YTSGSTG3: SEQ
TAY ACN TCN GGN TCN ACN GG
na
na


ID NO: 12





 


YTSGSTG4: SEQ
TAY ACN TCN GGN AGY ACN GG
na
na


ID NO: 13





 


SRGKPKG: SEQ
TCT AGA GGN AAR CCN AAR GG2
na
na


ID NO: 14





 


TGKPKG: SEQ
ACN GGN AAR CCN AAR GG4
TGKPKG:
CCY TTN GGY TTN


ID NO: 15

SEQ ID NO:
CCN GT




16



 


YGPTE: SEQ ID
TAY GGN CCN ACN GA4
YGPTE:
TTC NGT NGG NCC


NO: 17

SEQ ID NO:
RTA




18



 


YGPTE2: SEQ ID
TAC GGN CCN ACN GAN
na
na


NO: 19





 


na
na
GELIIGG:
CCN CCN ATN ATN




SEQ ID NO:
AGY TCN CC




20



 


ARGY X: SEQ ID
TBG CNC GNG GNT ACN
ARGY:
GTA NCC NCG NGC


NO: 22

SEQ ID NO:
GAN




21



 


Y K/R TGDL:
TAC ARR ACN GGN GAY CT
YKTGDL:
ARR TCN CCN GTY


SEQ ID NO: 23

SEQ ID NO:
TTR TAT CTA GA2




24



 


YRTGDLV: SEQ
TAY MGI ACI GGI GAY YTI GT
na
na


ID NO: 25





 


Y/F RTGD L/R
TWY GCI ACI GGI GAY YKI GKI
na
na


G/V R(TGD):
CG3




SEQ ID NO: 26





 


ELGEIE: SEQ ID
GAR YTN GSN GAR ATH GA
KDTQVK
GGI ACY TGI TGR


NO: 27

(JA5): SEQ
TCY TT1




ID NO: 28



 


na
na
LLXLGGX
AWI GAR KSI CCI




S (LGG):
CCI RRS IMR AAR




SEQ ID NO:
AA3




29



 


GGDSI A/T: SEQ
GGN GGN GAY TCN ATY RCN
GGDSI A/T
GCN GYD ATN SWR


ID NO: 30

A: SEQ ID
TCN CCN CC




NO: 31



 


na
na
GGHSI A/T
GCN GYR ATN GAR




A: SEQ ID
TGN CCN CC




NO: 544



 


na
na
GDSITA
CGC CGT GAT CGA




Cochliobolus
ATC CCC




victoriae:





SEQ ID NO:





32



 


ISGDW: SEQ ID
CAY CAY NNN ATH WSN GAY
ISGDW:
CCT NCC RTC NSW


NO: 33
GGN TGG
SEQ ID NO:
NAT NNN RTG RTG




34



 


EGHGRE: SEQ
GAR GGN CAY GGN MGN GA
EGHGRE:
TCN CKN CCR TGN


ID NO: 35

SEQ ID NO:
CCY TC




36



 


DAYPCS C.
GAT GCC TAC CCA TGC TCG
DVYPCTP:
GTK CAN GSR WAN


victoriae: SEQ

SEQ ID NO:
ACR TCY TC


ID NO: 37

38



 


PCTPLQ: SEQ ID
CCN TGY ACN CCN YTN CA
PCTPLQ:
TGN ARN GGN GTR


NO: 39

SEQ ID NO:
CAN GG




40



 


na
na
PCTPLQ2:
TGI ARI GGI GTR




SEQ ID NO:
CAI GG




41



 


QEGLMA(JA1):
CAR GAR GGI YTI ATG GC1
QEGLMA:
CGC ATN AGN CCY


SEQ ID NO: 42

SEQ ID NO:
TCC TG




43



 


QEGMLA: SEQ
KAR GGN ATG AWN GC
QEGMLA:
GCN WTC ATN CCY


ID NO: 44

SEQ ID NO:
TMY TG




45





1Primer sequences that the inventors obtained from Dr. Aric Weist


2Primers referenced in Panaccione, (1996) Mycological Research 100: 429-436; herein incorporated by reference.


3Primers referenced in Turgay & Marahiel (1994), Peptide Research 7: 238-241; herein incorporated by reference.


4Primers references in Nikolskaya et al. (1995) Gene 165: 207-211


Abbreviations:


A, adenine;


T, thymine;


G, guanine;


C, cytosine;


I, inosine,


K, G or T;


R, A or G;


M, A or C;


W, A or T;


Y, C or T.


No = not available






In order to find an NRPS in A. bisporigera, the inventors first contemplated that amatoxins were synthesized via a non-ribosomal peptide synthetase (NRPS) as found in other types of fungi (see, example in FIG. 3). Specifically, the inventors further contemplated that an NRPS responsible for biosynthesizing amatoxins would be encoded by a gene of approximately 30 kb in size. Because amatoxins contain eight amino acids, and in NRPS enzymes one domain activates by adenylation one amino acid, the enzyme should be approximately one MDa. Such a protein was predicted to be encoded by a 30-kb gene. The inventors further contemplated random (shotgun) sequencing of the genome and an average read size of 600 by and calculated a >99% probability of hitting a 30 kb target in a 40 Mb genome in 7,000 random, independent sequences.
The inventors generated more than 70 MB of DNA sequence and searched using BLAST and more than 20 known NRPS genes and proteins from prokaryotes and eukaryotes for evidence for an NRPS in the genome of A. bisporigera. However, the inventors did not find evidence for any NRPS-like sequence in A. bisporigera. In contrast, the inventors discovered that the most closely related sequences to NRPSs were orthologs of aminoadipate reductase and acyl-CoA synthase, which, like bacterial and fungal NRPSs, are classified within the aminoacyl-adenylating superfamily (Finking et al., (2004) Annu. Rev. Microbiol. 58:453; herein incorporated by reference).
Approximately 59% of the Amanita bisporigera sequences of the present inventions did not show a hit to the GenBank NR database. This is consistent with results from other fungal genome projects (see, e.g. Schulte, U (2004) Genomics of filamentous fungi. In Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine (JS Tkacz & L Lange, eds.):15-29. Kluwyer Academic/Plenum Publishers, New York; herein incorporated by reference). Little annotation is yet available for fungal genomes, so the proportion of unidentified sequences is high. Three thousand eight sequences that produced no hits to GenBank NR did yield hits to the Phanerochaete chrysosporium and/or Coprinopsis cinereus genomes. The following known genes were identified using BLAST comparisons of the novel Amanita fragments of the present inventions. The inventors found matches contemplated to be Amanita homologs to members of the aminoacyl-adenylating superfamily (Finking et al., (2004) Annu Rev Microbiol 58:453-488; herein incorporated by reference) which includes but is not limited to exemplary sequences of L-aminoadipate-semialdehyde dehydrogenase. In particular, L-aminoadipate-semialdehyde dehydrogenase is related to but is not a non-ribosomal peptide synthetase (NRPS), an enzyme originally contemplated to be responsible for Amanita peptide toxin biosynthesis. The inventors ruled out a NRPS identity of this match after they sequenced the remainder of the clone 16_c01KoreaM13Rrc, then extended the sequence by approximately 700 by using inverse PCR.
Cap64 is a capsule formation protein first identified in the pathogenic basidiomycete Filobasidiella neoformans with a known homolog in the saprophytic basidiomycete Pleurotus ostreatus, of which the later does not form capsules associated with mammalian pathogenicity. The discovery of an AmanitaCap64 homologous sequence was not expected because like Pleurotus, Amanita species are not known to form capsules associated with mammalian pathogenicity.
Laccases, like Cap64, were not expected even though they were previously found to be widespread in saprophytic fungi (Coprinopsis, Melanocarpus, and the white rot fungus Trametes), and in both asco- and basidiomycetes. Their role in an ectomycorrhizal fungus such as Amanita, which is expected to obtain most of its nutrients in the form of photosynthate and would therefore lack the need to degrade plant tissue, is unknown.
Therefore, despite predictions to the contrary, the inventors did not find evidence of an NRPS gene that would likely be involved with synthesizing amatoxins and phallotoxins (Walton et al. (2004) Peptide synthesis without ribosomes. In: Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine. J Tkacz, L Lange, eds, Kluwer Academic, New York, pp. 127-162; herein incorporated by reference). Yet on the other hand, surprisingly, the inventors discovered other types of genes.
Example IV
This example describes exemplary compositions and methods for identifying amatoxin-encoding genes. The inventors initially focused on amatoxins, in particular amanitins, bicyclic octapeptides which are more potent toxins to humans than any of the other mushroom toxins and are directly responsible for the majority of fatal human mushroom poisonings. Specifically, this example describes the discovery of an A. bisporigera gene sequence contemplated to encode alpha amanitin.
An exemplary structure of α-amanitin is cyclic(L-asparaginyl-4-hydroxy-L-prolyl-(R)-4,5-dihydroxy-L-isoleucyl-6-hydroxy-2-mercapto-L-tryptophylglycyl-L-isoleucylglycyl-L-cysteinyl), cyclic (4-8)-sulfide, (R)—S-oxide (ChemIDplus.sup.2), wherein the amino acids have the L configuration and several amino acids are modified by hydroxylation. When simplified to the 20 proteogenic amino acids, the chemical name became cyclic(NPIWGIGC) (SEQ ID NO:46) (ChemIDplus). However because this is a cyclized peptide, the order in which the amino acids are assembled biosynthetically was unknown. Moreover, the structure of .beta.-amanitin, RN: 21150-22-1 was based upon the known chemical structure of .alpha.-amanitin RN: 23109-05-9 and named in a similar manner. .sup.2 chem.sis.nlm.nih.gov/chemidplus/ProxyServlet?objectHandle=DBMaint&actionHandle=default&nextPage=jsp/chemidheavy/ResultScreen.jsp&ROW_NUM=0&TXTSUP ER-LISTID=023109059
Therefore, the inventors searched the DNA sequences from their A. bisporigera genome seeking DNA fragments capable of encoding amino acid sequences of amanitins, such as predicted sequences comprising a predicted sequence of NPIWGIGC (SEQ ID NO:46). Thus the inventors discovered an exemplary sequence encoding .alpha.-amanitin, ECIMO1V02FKY4Z S CCCAACTAAATCCCATTCGAACCTAACTCCAAGACCTCTAAACCTCACAATCC CAATGTCTGACATCAATGCTACCCGTCTCCCCATCTGGGGTATCGGTTGCAAC CCGTGCG, length=113 (SEQ ID NO:48) encoding prepropetide PTKSHSNLTPRPLNLTIPMSDINATRLPIWGIGCNPC (SEQ ID NO:49), propeptide in BOLD, underlined peptide, SEQ ID NO: 50. The inventors' exemplary sequence translated into a IWGIGCNP, SEQ ID NO: 50, which the inventors contemplate would be capable of forming a, cyclo(IWGIGCNP), SEQ ID NO: 50, wherein the inventors further contemplated several posttranslational hydroxylations and a sulfoxide crossbridge between the Trp and the Cys in order to form the bicyclic peptide known as alpha-amanitin. The inventors used the amino acid sequence and the nucleic acid sequences encoding IWGIGCNP (SEQ ID NO: 50) for searching known sequences in GenBank's non-redundant database. There was no evidence of any gene encoding or protein with IWGIGCNP (α- and γ-amanitins) (SEQ ID NO: 50). Therefore, the inventors contemplated that these sequences are unique for A. bisporigera and further these sequence orders were unlikely to be present in an Amanita genome by statistical coincidence.
The inventors also obtained a second and longer sequence comprising nucleotides encoding IWGIGCNP (SEQ ID NO: 50) using inverse PCR (AMA1 forward and reverse primers, see above) and obtained a genomic sequence contig 49252 AATCTCAGCGTTCAGTACCCAACTCCCATTCGAACCTAACTCCAAGACCTCTA AACCTCACAATCCCAATGTCTGACATCAATGCTACCCGTCTCCCCATCTGGGG TATCGGTTGCAACCCGTGCGTCGGTGACGACGTCACTACG, length=146 (SEQ ID NO:52) encoding SQRSVPNSHSNLTPRPLNLTIPMSDINATRLPIWGIGCNPCVGDDVTT (SEQ ID NO:53), propeptide in BOLD, underlined peptide, SEQ ID NO: 50.
Therefore the inventors found nucleotide sequences that encode the amino acid sequence of .alpha.-amanitin with the sequence order of IWGIGCNP (SEQ ID NO. 50), in single letter code, and further identified two larger genomic sequences encoding an IWGIGCNP (SEQ ID NO: 50) amanitin peptide in the genome of A. bisporigera. The inventors contemplated that amanitins would be a cyclic permutation of linear peptides of IWGIGCNP (SEQ ID NO: 50) (α- and γ-amanitins) and IWGIGCDP (SEQ ID NO:54) (β- and ε-amanitins).
Example V
This example demonstrates using amino acid and nucleic acid information of the present inventions, inverse PCR and RACE methods to identify a cDNA and a large genomic fragment that comprises an amanitin gene as indicated in FIG. 4.
The inventors initiated a genomic survey using nucleic acid coding regions encoding the AMA1 gene, as described in the previous Example. SEQ ID NOs: 48, 49, 52, and 53, encoding an AMA1 polypeptide, were used to design AMA1 forward and reverse primers that were used in an inverse PCR reaction to obtain a larger genomic fragment of the AMA1 gene. Specifically, inverse PCR, using circularized PvuI generated genomic fragments as target (template) DNA resulted in the isolation of a 2.5-kb fragment of flanking genomic DNA.
RACE (Rapid Amplification of cDNA Ends) (for example, see, Frohman et al., (1988) Proc Natl Acad Sci 85:8998-9002; herein incorporated by reference), was used to obtain a full-length cDNA copy of AMA1, SEQ ID NO:55, encoding an AMA1 polypeptide, SEQ ID NO:56. When compared to the AMA1 genomic sequence, SEQ ID NO:57, the cDNA indicated that AMA1 contains three introns (53, 59, and 58 nt in length), with canonical GT/AG boundaries. Two of the introns were in the 3′ untranslated region, while the first intron was in the third codon from the end of the coding region (FIG. 4A). The inventors contemplated that translation started at the first ATG downstream of the transcriptional start site thus encoding a proprotein of 35 amino acids (FIG. 4A). The string of A's at the end represents the poly-A tail typical of eukaryotic mRNAs and their corresponding cDNAs (though not encoded within the genomic sequence). The amatoxin prepropeptide and propeptide encoding sequences are shown in relation to the encoded amino acid sequence for an amanitin peptide (underlined), FIG. 4A. The amatoxin prepropeptide and propeptide encoding sequences are shown where the amanitin peptide encoding sequence is underlined, FIG. 4B.







TABLE 5







Examples of RACE primers used herein.











SEQ ID


SEQUENCE Name
SEQUENCE
NO: XX





GeneRacer TM 5′ Primer
5′-GCACGAGGACACUGACAUGGACUGA-3′
SEQ ID




NO: 58


 


GeneRacer TM 5′ Nested
5′-GGACACTGACATGGACTGAAGGAGTA-3′
SEQ ID


Primer

NO: 58


 


GeneRacer TM 3′ Primer
5′-GCTGTCAACGATACGCTACGTAACG-3′
SEQ ID




NO: 60


 


3′ AMA1 RACE initial
5′ CCCATTCGAACCTAACTCCAAGAC 3′
SEQ ID


primer

NO: 61


 


3′ AMA1 RACE primer,
5′ CCTCTAAACCTCACAATCCCAATG 3′
SEQ ID


nested primer

NO: 62


 


5′ AMA1 RACE cDNA,
5′ GCCCAAGCCTGATAACGTCCACAACT 3′
SEQ ID


primer

NO: 63


 


5′ AMA1 RACE cDNA,
5′ TATCGCCCACTACTTCGTGTCATA 3′
SEQ ID


nested primer

NO: 64


 


3′ PHA1, initial primer
5′ GACCTCTGCTCTAAATCACAATG 3′
SEQ ID




NO: 65


 


3′ PHA1, nested primer
5′ ATCAATGCCACCCGTCTTCCTG 3′
SEQ ID




NO: 66


 


5′ PHA1 initial primer
5′ CGGATCATTTACGTGGGTTTTA 3′
SEQ ID




NO: 67


 


5′ nested primer
5′ AACTTGCCTTGACTAGTGGATGAGAC 3′
SEQ ID




NO: 68









Thus an exemplary amino acid sequence of the proprotein of AMA1 is MSDINATRLPIWGIGCNPCIGDDVTTLLTRGEALC, SEQ ID NO: 559, underlined peptide, SEQ ID NO: 50. The inventors further contemplated an exemplary structure of .beta.-amanitin, wherein Asn is replaced by Asp to provide IWGIGCDP, SEQ ID NO:54. Indeed, further investigations described below, did result in the finding of an Amanita PCR product encoding a .beta.-amanitin sequence.
An RNA blot of total RNA extracted from mushrooms of Amanita bisporigera probed with DNA fragment SEQ ID NO: 48 showed an approximately 400 nt band contemplated as an AMA1 mRNA. Minor discrepancies between the genomic and cDNA sequences are likely due to natural variation among the amatoxin genes.
Example VI
This example describes the discovery of an A. bisporigera gene sequence contemplated to encode a phallotoxin, specifically a phallacidin toxin sequence.
An exemplary structure of phallacidin is a cyclic(L-alanyl-2-mercapto-L-tryptophyl-4,5-dihydroxy-L-leucyl-L-valyl-er-ythro-3-hydroxy-D-alpha-aspartyl-L-cysteinyl-cis-4-hydroxy-L-prolyl)cyclic (2-6)-sulfide, RN: 26645-35-2, with predicted amino acid sequences simplified to the 20 proteogenic amino acids comprising cyclo(ATCPAWL), SEQ ID NO:70. Another phallotoxin, phalloidin, RN: 17466-45-4, is a cyclic(L-alanyl-D-threonyl-L-cysteinyl-cis-4-hydroxy-L-prolyl-L-alanyl-2-mercapto-L-tryptophyl-4,5-dihydroxy-L-leucyl), cyclic (3,6)-sulfide, which translates into the sequence cyclo(ATCPAWL), SEQ ID NO:70. Several of the phallacidin and phalloidin amino acids are hydroxylated. The Asp residue (which is replaced by Thr in phalloidin) has the D configuration at the alpha carbon.
A genomic survey of A. bisporigera sequences yielded at least 2 nucleic acid sequences encoding a predicted sequence comprising a linear AWLVDCP, SEQ ID NO:69, which would encode cyclicphallacidin (SEQ ID NO:71), for example, SEQ ID NO:72, ECGK9LO01B8L63 S TGAGGAGACGGTTGACGTCGTCACCGACGCATGGGCAGTCTACAAGCCAAGC AGGAAGACGGGTGGCATTGATGTCAGACATTGTGATTTAGAGTAG, length=97 encoding LLITMSDINATRLPCVGDDVNRLL, SEQ ID NO:73, and SEQ ID NO:74, contig73170, TGAGGAGACGGTTGACGTCGTCACCGACGCATGGGCAGTCTACAAGCCAAGC AGGAAGACGGGTGGCATTGATGTCAGACATTGTGATTTAGAGTAGAGGTCTT GGGTTCGAGTTCGAATGGGAGGTAAG, length 130, encoding a prepropeptide LTSHSNSNPRPLLITMSDINATRLPAWLVDCPCVGDDVNRLL (SEQ ID NO: 75), showing the propeptide in BOLD and underlined peptide SEQ ID NO:69.
Inverse PCR following PvuI and Sad digestion of whole genomic DNA and ligation was used to isolate genomic fragments of 1.6 kb and 1.9 kb, respectively, named phallacidin sequence PHA1#1-1893 bp. SacI, SEQ ID NO:76, and phallacidin-sequence PHA1#2-1613 nt. PvuI, SEQ ID NO:77, collectively named PHA1, comprising phallacidin amino acid sequences. These were two different classes of sequences, identical in the region of phallacidin, SEQ ID NO:78, but diverged approximately 135 nt upstream. These two sequences showed that A. bisporigera genome has at least two copies of the PHA1 gene, both of which encode a phallacidin toxin sequence, FIG. 5. Furthermore, a cDNA for PHA1, SEQ ID NO:44, was isolated by 5′ and 3′ RACE (FIG. 5) using methods similar to those used in Example IV in combination with PHA1 RACE primers listed above. Nucleotide sequences of a cDNA for PHA1 are shown in FIG. 5A. When the genomic sequence (FIG. 5, #2) was compared to a cDNA sequence, the inventors found three introns (50-69 nt). Two of the introns were in the 3′ untranslated region, while the first intron was in the third codon from the end of the coding region. Carats marked within the sequence indicate the positions of introns. The c DNA sequence, SEQ ID NO:79, is predicted to encode an amino acid sequence as a proprotein of PHA1 that is 34 amino acids in length, SEQ ID NO: 80, translating into MSDINATRLPAWLVDCPCVGDDVNRLLTRSLC (SEQ NO: 350) (phallacidin sequence, SEQ ID NO: 69 in BOLD), whose coding sequence was underlined in FIG. 5A. Because two different phallacidin genomic sequences were obtained, the inventors contemplate that A. bisporigera has at least two copies of PHA1. Further, the inventors concluded that these two PHA1 sequences represent natural variants of the phallacidin gene because both are present in the same isolate of A. bisporigera. The inventors further contemplate that these two PHA1 genes arose as a gene duplication event.
Example VII
This example describes methods and results from exemplary comparisons of AMA1 and PHA1 for obtaining exemplary consensus sequences.
Based on the cDNA sequence, the inventors chose the first ATG sequence downstream of the transcriptional start site as the translational start site of the proprotein polypeptides and the first in-frame stop codon as the translational stop. AMA1 and PHA1 nucleic acid and predicted amino acid sequences were compared by alignment of each set of two target sequences using a BLAST engine for local alignment through the NCBI website, (world wide web.ncbi.nlm.nih.gov/blast/b12 seq/wblast2.cgi).
Alignment of the predicted proproteins, amanitin to phallacidin sequences, is shown in FIG. 6A. Proproteins of amanitin and phallacidin were 35 and 34 amino acids in length, respectively. Sequences corresponding to amanitin and phallacidin are underlined, and for clarity are separated by spaces from the upstream and downstream amino acid sequences.
When the inventors compared the sequences of genomic and cDNA copies of AMA1 and PHA1, the inventors observed that both comprise 3 introns (approximately 57, 70, and 51 nt in length), in approximately the same positions. Furthermore, AMA1 and PHA1 gene sequences and their translation products were found to be similar in overall size and sequence, except strikingly in the region encoding the peptide toxins themselves (FIG. 6 and Table 6).
Within amino acid encoding regions (the proproteins), nucleic acid sequence regions upstream of IWGIGCNP (amatoxin) (SEQ ID NO: 50) and AWLVDCP (phallotoxin) (SEQ ID NO: 69) comprise 28 of 30 identical nt (93%), while regions downstream of IWGIGCNP (SEQ ID NO. 50) and AWLVDCP (SEQ ID NO 69) comprise 41 of 50 identical nt (82%). However, these findings were in contrast to the amatoxin and phallotoxin-encoding regions themselves (IWGIGCNP and AWLVDCP) (SEQ ID NOs: 50 and 69, respectively) where merely 12 of 24 nt were identical (50%). Thus the inventors designated these proprotein areas of .alpha.-amanitin and phallacidin as being composed of three domains, one conserved upstream region (A), one conserved downstream region (B), and a hypervariable peptide region (P) encoding amatoxin and phallotoxin. In other words, proprotein sequences of the present inventions consist of an upstream conserved region (A), a downstream conserved region (B) in relation to a variable region (P), such that the variable Amanita cyclic peptide toxin region is flanked by two conserved regions, (FIG. 6B). Because amatoxins contain 8 amino acids and phallotoxins contain 7 amino acids, the inventors inserted a 3-nucleotide gap ( - - - ) in the cDNA sequence and a one-amino acid space (-) in the proprotein sequence in order to emphasize the alignment of the conserved sequences downstream of the amatoxin and phallotoxin-encoding regions (FIG. 7A).







TABLE 6







Exemplary comparisons between AMA1 and PHA1 using BLASTN.











Comparison and Identity


SEQ ID NO:
Sequence
No. aa/No. aa (percent identity)





AMA1 A,
atg tct gac atc aat gct



SEQ ID NO:
acc cgt ctt ccc (30aa)



182




 


PHA1 A,
atg tct gac atc aat gcc
AMA1A v. PHA1 A


SEQ ID NO:
acc cgt ctt ccc (30aa)
29/30 (96%),


82




 


AMA1 B,
tgc atc ggt gac gac gtc



SEQ ID NO:
act aca ctc ctc act cgt



19
ggc gag gcc ctt tgt (51aa)



 


PHA1 B,
tgc gtc ggt gac gat gtc
AMA1 B v. PHA1 B


SEQ ID NO:
aac cgt ctc ctc act cgt
41/50 (82%)


83
ggc gag agc ctt tgg (48aa)



 


AMA1 toxin,
atc tgg ggt atc ggt tgc aac ccg



SEQ ID NO:
(24aa)



85




 


PHA1 toxin,
gct tgg ctt gta gat tgc --- cca
AMA1 toxin v. PHA1 toxin


SEQ ID NO:
(21aa)
12/24


86

(50%)

















TABLE 7A








Exemplary BLAST searches for AMA1 and PHA1 using BLAST.











SEQ


Comparison



ID 
Query

and Identity
percent


NO.
SEQ
Hit
No. aa/No. aa
identity





572
Alpha-
Rhodococcus sp. gb|CP000431.1|
28/32
 87%



Amanitin
CGGGTACAACACGTGCATCGGTGACGCCGTC






A




 


579

Zebrafish DNA sequence emb|CR385042.30|
28/33
 84%




CGACACTACCCTCACCACTCGTGCCCTTAGT






TA




 


522
Phallacidin
Agrobacterium tumefaciens gb|AE009415.1|
31/35
 88%




TCTGTGACGATGTCATCCAGTCTC-






TCACTCGTA




 


580

CP000479.1 Mycobacterium avium 104
28/33
 84%




CGTCGGTGACGATGTACACCGTCGCCACGCT






CG




 


521

AC112739.5 Rattus norvegicus 7 BAC CH230-
26/30
 86%




108Al2






TGTCAACCGTCTCCTCTGTCGTTTCCTTTG




 


578

XM_382946.1 Gibberella zeae PH-1 chromosome 1
25/28
 89%




conserved hypothetical protein (FG02770.1) partial






mRNA






CGTCGGTGACGATGTCCTCCGTCTCTTC




 


523

AM444890.2 Vitis vinifera contig
22/23
 95%




TTGTAGACTGCCCATGCGTCTGT




 


541

gb|AAQY01001277.1| Phytophthora sojae strain
21/21
100%




P6497 CGGTGACGATGTCAACCGTCT




 


540

gb|AAQR01490933.1| Otolemur garnettii
21/21
100%




cont1.490932






TGTCTGACATCAATGCCACCC
















TABLE 7B







Exemplary BLAST searches for AMA1 and PHA1 using BLASTN.














Comparison



SEQ ID


and Identity
percent


NO:
Query SEQ
Hit
No. aa/No. aa
identity





524
Amanitin A
ATGTCTGACATCAATGCTACCCGT
30/30
100%




CTCCCC




 


563

ref|XM_001182437.1| PREDICTED:
19/20
 95%




Strongylocentrotus purpuratus similar to






ESP-1 (LOC574923), purple sea urchin






TGTCTGACATCAATGGTACC




 


530

dbj|AK173931.1| Ciona intestinalis
18/18
100%




cDNA, ATGTCTGACATCAATGCT




 


564

ref|XM_001365250.1| Monodelphis
17/17
100%




domestica similar to transducin beta-3-






subunit mRNA short-tailed opossums,






GTCTGACATCAATGCTA




 


568

ref|XM_814507.1| Trypanosoma cruzi
16/16
100%




strain CL Brener kinesin






AATGCTACCCGTCTCC




 


565

ref|XM_652576.1| Aspergillus nidulans
16/16
100%




FGSC A4 hypothetical protein






(AN0064.2 TGTCTGACATCAATGC




 


537

emb|BX842594.1| Neurospora crassa
16/16
100%




DNA linkage group II BAC clone B18P7






TGTCTGACATCAATGC




 


532

dbj|AP007162.1| Aspergillus oryzae
16/16
100%




RIB40 genomic DNA, SC102






CTGACATCAATGCTAC




 


 82
Phallacidin A
ATGTCTGACATCAATGCCACCCGT
30/30
100%




CTTCCC




 


567

ref|XM_753671.1| Corn smut is of maize
20/21
 95%




caused by the pathogenic plant fungus






Ustilago maydis






CATCAATGCCACCCGCCTTCC




 


542

gb|AC122231.2| Mus musculus BAC
19/19
100%




clone RP23-






135M3ATGTCTGACATCAATGCCA




 


536

emb|AL031736.16| Human DNA
19/19
100%




sequence from clone RP4-






738P11ATGTCTGACATCAATGCCA




 


562

ref|NM_202010.2| Arabidopsis thaliana
18/18
100%




FUS5 (FUSCA 5); MAP kinase kinase






(FUS5) CAATGCCACCCGTCTTCC




 


566

ref|XM_652576.1| Aspergillus nidulans
18/18
100%




FGSC A4 hypothetical protein






(AN0064.2),






TGTCTGACATCAATGCCA




 


533

dbj|AP008214.1| Oryza sativa (japonica
18/18
100%




cultivar-group) genomic






TCTGACATCAATGCCACC




 


543

gb|EF469872.1| Helianthus annuus RFLP
17/17
100%




probe ZVG13 mRNA sequence






AATGCCACCCGTCTTCC




 


538

emb|CR619305.1| B cells (Ramos cell
17/17
100%




line) GTCTGACATCAATGCCA




 


538

emb|CR595196.1| T cells (Jurkat cell
17/17
100%




line) GTCTGACATCAATGCCA




 


538

emb|CR592893.1| Neuroblastoma of
17/17
100%




Homo sapiens (human)






GTCTGACATCAATGCCA




 


531

dbj|AK173931.1| Ciona intestinalis or
17/17
100%




Sea squirt. ATGTCTGACATCAATGC




 


525
Amanitin B
TGCATCGGTGACGACGTCACTACT
45
100%




CTCCTCACTCGTGCCCTTTGT




 


573

Strongylocentrotus purpuratus
19/19
100%




CATCGGTGACGACGTCACT




 


548

Ostreococcus lucimarinus unicellular
18/18
100%




coccoid green alga






GCATCGGTGACGACGTCA




 


529

Chaetomium globosum dematiaceous
18/18
100%




filamentous fungus infectious in humns






CTCCTCACTCGTGCCCTT




 


546

Human DNA sequence from clone
18/18
100%




XXyac-60D10






TCACTACTCTCCTCACTC




 


561

Rattus norvegicus LEA_4 domain
17/17
100%




containing protein






ACGTCACTACTCTCCTC




 


526

Atlantic Salmon
17/17
100%




CTCCTCACTCGTGCCCT




 


527

Burkholderia cenocepacia Gram-
17/17
100%




negative bacteria Pathogen






ATCGGTGACGACGTCAC




 


547

Ornithorhynchus anatinus Platypus
17/17
100%




ACGTCACTACTCTCCTC




 


 82
Phallacidin B
TGCGTCGGTGACGATGTCAACCGT
45
100%




CTCCTCACTCGTAGCCTTTGG




 


528

Chaetomium globosum CBS 148.51
24/26
 92%




GGTGACGATGACAACCGCCTCCTC






AC




 


545

Gibberella zeae
23/25
 92%




CGTCGGTGACGATGTCCTCCGTCTC




 


571

Rhizobium leguminosarum bv. viciae
20/21
 95%




chromosome






CGTCGGTGACGAGGTCAACCG




 


574

Tetraodon nigroviridis
19/19
100%




GATGTCAACCGTCTCCTCA









The conserved amino acid regions encoded by conserved domains A and B and consensus region B were used as query sequences for BLAST searching the GenBank public NR database. These sequences per se were not found within the database, however somewhat similar sequences were discovered, with exemplary sequences shown below.








TABLE 8








Exemplary homology comparisons using Consensus MSDINATRLP (SEQ ID NO: 88),



XWXXXCXP (SEQ ID NO: 135), and CVGDDVXXLLTRALC (SEQ ID NO: 581) as query sequences using BLASTP



(MSDINATRLPXWXXXCXPCVGDDVXXLLTRALC, SEQ ID NO: 87).










SEQ ID 

Identitiy



NO.
SEQUENCE
No. aa/matching No. aa
GenBank sequence hit





88
AMA1
7/10 (70%),
gb|EDN21666.1| predicted protein



Conserved A

[Botryotinia fuckeliana B05.10]



MSDINATRLP




 




7/8 (87%),
gb|EAT86097.1| hypothetical protein





SNOG_06266 [Phaeosphaeria





nodorum SN15]


 




7/9 (77%),
gb|EAK82279.1| hypothetical protein





UM01662.1 [Ustilago maydis 521]


 




6/9 (66%),
gb|EAU90435.1| predicted protein





[Coprinopsis cinerea okayama7#130]


 


582

MREINSTRLP 7/10 (70%)
predicted protein [Botryotinia





fuckeliana B05.10]. Pathogenic fungus





(aka Botrytis cinerea) that causes gray





mold rot in plants


 


583

MSNIAAPRLP 7/10 (70%)
gb|ABD10583.1| Endopeptidase Clp





[Frankia sp. CcI3]


 


584

MSDIAWEIPDNATR 8/13 (61%)
hypothetical protein CC1G_09232





[Coprinopsis cinerea okayama7#130]


 


585

SDVNAPRLP 7/9 (77%)
hypothetical protein UM01662.1





[Ustilago maydis 521]


 


586

SDI-ATRLP 8/9 (88%)
non-ribosomal peptide synthetase





[Saccharopolyspora erythraea





NRRL 2338]


 


89
AMA1
8/11 (72%)
gb|ABF87913.1| ATP-binding protein,



Conserved

ClpX family [Myxococcus xanthus DK



Region B

1622]



CIGDDVTTLL





TRGEALC




 




8/10 (80%)
emb|CAG61741.1| unnamed protein





product [Candida glabrata CBS 138]


 




10/16 (62%)
gb|EAK84527.1| hypothetical protein





UM03624.1 [Ustilago maydis 521]


 




11/16 (68%)
gb|EAU39589.1| conserved





hypothetical protein [Aspergillus





terreus NIH2624]


 




8/8 (100%)
dbj|BAE56937.1| unnamed protein





product [Aspergillus oryzae]


 


90
PHA1
14/21 (66%)
gb|AAZ10451.1| hypothetical protein



Conserved

Tb927.3.4180 [Trypanosoma brucei]



Region B





CVGDDVNRL





LTRGESLC




 




11/18 (61%)
gb|EAQ84320.1| hypothetical protein





CHGG_10724 [Chaetomium globosum





CBS 148.51]


 




9/11 (81%)
gb|ABE92653.1| Peptidase, cysteine





peptidase active site; Aromatic-ring





hydroxylase [Medicago truncatula]


 




9/14 (64%)
gb|EDN63642.1| conserved protein





[Saccharomyces cerevisiae YJM789]


 


91, 569
Consensus B
9/14 (64%)
ref|XP_760134.1| hypothetical protein



CXGDDVXXL
GDDVAALLSRRVLC
UM03987.1 [Ustilago maydis 521]



LTRXLC
SEQ ID NO: 569




SEQ ID NO: 91




 


570

8/12 (66%)
ref|ZP_00591779.1| ClpX, ATPase




GDDVETILTRLL
regulatory subunit [Prosthecochloris





aestuarii DSM 271]green sulfur





bacterium









Example VIII
This example describes materials and methods for determining whether the amatoxin and phallotoxin-encoding nucleic acids are specific for Amanita mushroom species that produce amatoxins and phallotoxins.
Many secondary metabolites such as mushroom peptide toxins are limited in their taxonomic distribution; for example, most species of Amanita do not make amatoxins or phallotoxins. Thus the inventors contemplated whether the lack of amatoxin and phallotoxin production among other species of Amanita was due to absence of the encoding genes or due to the absence of productive translation of the genes. The inventors tested for the presence of amatoxins such as alpha-amanitin and phallotoxins such as phallacidin and in the same mushrooms tested for the presence of DNA encoding alpha amanitin (AMA1) and phallacidin (PHA1). The inventors tested for the presence of AMA1 and PHA1 in the genomes of known amatoxin and phallotoxin-producing mushroom species and non-producing mushroom species in order to associate the AMA1 and PHA1 sequences with amatoxin and phallotoxin production.
Preparation and Isolation of Amanita Genomic Sequences.
DNA was extracted from a variety of species of Amanita that were either known as amatoxin and phallotoxin-producers (A. bisporigera, A. ocreata, A. aff. suballiacea and A. phalloides) or were known to not produce amatoxins (A. novinupta, A. franchetti, A. porphyria, A. velosa, A. gemmata, A. muscaria, A. flavoconia, A. section Vaginatae, and A. hemibapha). DNA was extracted from lyophilized fruiting bodies using cetyl trimethyl ammonium bromide-phenol-chloroform isolation (Hallen, (2003) Mycol. Res. 107:969; herein incorporated by reference). Following the usual preparation methods, sequences were separated by gel electrophoresis and then transferred to blotting media for subsequent probe hybridization.
Southern blots of DNA were probed with AMA1 and PHA1 as described. As shown in FIG. 8, Panel A was probed with an amanitin gene AMA1 (nt 1710-2175 as numbered in FIG. 5) while Panel B was probed with a phallacidin gene PHA1 (nt 635-1115 in phallacidin #2, see, FIG. 6). For references on amatoxin and phallotoxin production in relation to Amanita taxonomy, see website http://pluto.njcc.com/.about.ret/amanita/mainaman.html; Hallen (2002) Studies in amatoxin-producing genera of fungi: phylogenetics and toxin distribution. Ph.D. dissertation, East Lansing, Mich.: Michigan State University. 192 pp.; and Arora D (1986) Mushrooms Demystified, Second Edition. Ten Speed Press, Berkeley; (Bas, Persoonia 5, 285 (1969); Tulloss et al., Boll Gruppo Micologico G Bresadola, 43, 13 (2000); WeiB et al., Can J. Bot. 76, 1170 (1998); all of which are herein incorporated by reference).
The results showed that AMA1 and PHA1 sequences hybridized to DNA from known amatoxin and phallotoxin-producing species but did not hybridize to the species known to not produce these compounds. The inventors concluded that these genes were present in amatoxin and phallotoxin-producing species and absent in non-producers, thus providing additional evidence that the genes described herein encode amatoxins and phallotoxins.
Extraction and Analysis of Amatoxins and Phallotoxins.
Variability in toxin content is known even within species of Amanita that normally produce amatoxins and phallotoxins (Beutler, et al., (1981) J. Nat. Prod. 44:422 and Tyler, et al., (1966) J. Pharm. Sci. 55:590; all of which are herein incorporated by reference in its entirety). Therefore in order to confirm that the presence of AMA1 and PHA1-encoding sequences correlates with actual production of amatoxins and phallotoxins, the inventors tested the same mushrooms that were used for extraction of DNA and Southern blotting (FIG. 8) for the presence of amatoxins and phallotoxins. Thus amatoxins and phallotoxins were extracted from these mushrooms, then analyzed by established HPLC methods (Hallen, et al., Mycol. Res. 107:969 (2003), Enjalbert, (1992) J. Chromatogr. 598:227; all of which are herein incorporated by reference in its entirety). Standards of α-amanitin, β-amanitin, phalloidin, and phallacidin were purchased from Sigma.
Each of the tested mushrooms that contain amatoxins and phallotoxins, but none of the nonproducers, hybridizes to AMA1 and PHA1. This is consistent with AMA1 and PHA1 as being responsible for alpha-amanitin and phallacidin biosynthesis and provides a molecular explanation for why Amanita species outside of sect. Phalloideae are not deadly poisonous. Some of the species of Amanita that do not make amatoxins or phallotoxins are edible, but others make toxic compounds chemically unrelated to the Amanita cyclic peptide toxins.
Example IX
This Example demonstrates PCR amplification of an α-amanitin gene in mushroom species known to produce α-amanitin while failing to amplify DNA from species that do not produce alpha-amanatin (FIG. 10C).
PCR amplification of the gene for α-amanitin. Primers were based on the sequences in FIGS. 4, 5 and 6. The primer sequences used were: forward primer: 5′-AGCATCTGCCCGCACCTTACG-3′, SEQ ID NO:92; Reverse primer: 5′ ACTGCCTTGTATCACCGTTATG-3′, SEQ ID NO:93. PCR mixtures and running conditions were REDTaq ReadyMix DNA polymerase (Sigma), 30 cycles of denaturation (94.degree. C., 30 sec), annealing (55.degree. C., 30 sec), and extension (72.degree. C., 5 min).
A. gemmata and A. muscaria are species of Amanita that do not make amatoxins (or phallotoxins) and did not yield a PCR product using these primers (FIG. 10C). “A. b.” No.'s1-3 indicate three different isolates of A. bisporigera, all of which produced alpha-amanitin, and all of which yielded PCR products, indicating the presence of the gene for alpha-amanitin (FIG. 10).
Example X
This Example shows the development of conserved regions upstream and downstream of Amanita peptide encoding regions.
The unexpected complex hybridizaton patterns shown in FIG. 8 led the inventors to contemplate that AMA1 and PHA1 are members of gene families such that additional short peptides related to AMA1 and PHA1 should be encoded by genes in A. bisporigera. 
The conserved upstream and downstream amino acid sequences of AMA1 and PHA1 were used as queries using BLASTP to search for additional related sequences in the A. bisporigera genome sequence database. The inventors thereby found at least 12 new related DNA sequences that could encode proproteins as long or longer than the proproteins of AMA1 and PHA1 and another 10-15 partial sequences (missing the upstream or the downstream conserved sequences) see exemplary sequences, including partial sequences in FIG. 7). These new sequences comprise an upstream conserved sequence MSDINTARLP (SEQ ID NO: 575) MSDIN (SEQ ID NO: 5×7), R, and P were invariant yielding an exemplary consensus sequence MSDINXXRXP, SEQ ID NO: 94), and a downstream conserved sequence CVGDDV (SEQ ID NO: 534), wherein the first D is invariant, for a consensus sequence CVGDXV, SEQ ID NO: 95, and a consensus sequence CVGDDVXXXDXX, SEQ ID NO: 96. The regions capable of comprising interesting peptides are those in the same positions relative to the upstream and downstream conserved regions in AMA1 and PHA1, namely, starting immediately downstream of the first invariant Pro residue and ending just after a second invariant Pro residue. These regions between these two absolutely conserved Pro residues are much more variable (“hypervariable”) in predicted amino acid sequence compared to the upstream and downstream conserved sequences. The “hypervariable regions” between the two invariant Pro residues are predicted to contain from seven to ten amino acids. Among the described putative new hypervariable regions (FIGS. 7 and 9) all twenty proteinogenic amino acids are represented in at least one. These new hypervariable sequences might represent previously unknown linear and cyclic peptides made by A. bisporigera. 
Example XI
This example describes methods and results of using conserved regions of AMA1 and PHA1 for obtaining additional regions encoding potentially biologically active linear or cyclic peptides from A. bisporigera, A. phalloides, and other species of Amanita. In particular, a DNA sequence encoding amino acid sequences was found that was highly similar to .alpha.-amanitin and comprising the amino acid sequence found in .beta.-amanitin, and a DNA that was highly similar to phallacidin and comprising the amino acid sequence found in phalloidin.
During the course of developing the present inventions, the inventors discovered regions of conserved sequence whose use resulted in the discovery of additional sequences contemplated to encode proproteins related to amatoxin and phallotoxin proproteins, which could encode novel small linear or cyclic peptides. Degenerate primers were designed against the conserved sequences of AMA1 and PHA1. DNA extracted from A. phalloides and A. ocreata was used as template. This also shows that the AMA1 and PHA1 genes and related genes are conserved in other species of amatoxin and phallotoxin-producing Amanita species, and that PCR primers designed against one species (A. bisporigera) function to identify amatoxin and phallotoxin genes in other species of Amanita. 
New degenerate PCR primer sequences that the inventors developed and used on genomic DNA as a template were 5′-ATGTCNGAYATYAAYGCNACNCG (forward), SEQ ID NO: 97, and 5′-AAGGSYCTCGCCACGAGTGAGGAGWSKRKTGAC (reverse), SEQ ID NO: 98, W indicates A or T, S indicates C or G, K indicates G or T, R indicates A or G, and Y indicates T or C. The resulting PCR products (approximately 100 nt) were cloned and sequenced. Exemplary sequences of three amplicons are:







number 1:


ATGTCTGATATTAATGCAACGCGTCTTCCCTTCAATATTCTGCCATTCA


 


TGCTTCCCCCGTGCGTCAGTGACGATGTCAATATACTCCTCACTCGTGG


 


CGAG, SEQ ID NO: 99,


 


translation: MSDINATRLPFNILPFMLPPCVSDDVNILLTRGE,


 


SEQ ID NO: 100, [predicted to encode a unique


 


linear and cyclic peptide, underlined,


 


SEQ ID NO: 114];


 


number 2:


ATGTCAGATATCAATGCGACGCGTCTTCCCATATGGGGAATAGGTTGCG


 


ACCCGTGCATCGGTGACGACGTCACCATACTCCTCACTCGTGGCGAG


 


translation, SEQ ID NO: 101, MSDINATRLPIWGIGCDPCI


 


GDDVTILLTRGE, SEQ ID NO: 102, [predicted to encode


 


beta-amanitin SEQ ID NO: 54];


 


number 3:


ATGTCGGATATTAATGCTACACGTCTTCCAATTATTGGGATCTTACTTC


 


CCCCGTGCATCGGTGACGATGTCACCCTACTCCTCACTCGTGGCGAG,


 


SEQ ID NO: 103, [translation:


 


MSDINATRLPIIGILLPPCIGDDVTLLLTRGE, SEQ ID NO: 104,


 


[predicted to encode a unique linear or cyclic


 


peptide, underlined SEQ ID NO: 117];


and


 


number 4:


ATGTCAGACA TTAACGCGAC CCGTCTTCCCGCCTGGCTCGCCACCTG


 


CCC GTGCGCCGGTGACGACGTCA ACCCTCTCCT CACTCGTGGC  


 


GAG, SEQ ID NO: 105, translation:


 


MSDINATRLPAWLATCPCAGDDVNPLLTRGE, SEQ ID NO: 106,


 


[predicted to encode phalloidin, underlined


 


(SEQ ID NO: 136].













TABLE 9







Exemplary comparisons of Amanita peptide sequences.










Identity
Percent


Preprotprotein nucleic acid
No. na/matching No. na
Identity





Alpha-Amanitin vs. new peptide
35/41
85%


1 SEQ ID NO: 114


Alpha-Amanitin vs. new peptide
79/91
86%


2, beta-Amanitin


Alpha-Amanitin vs. new peptide 3
36/41
87%


SEQ ID NO: 117


Phallacidin vs. new peptide 1
34/40
85%


SEQ ID NO: 114


Phallacidin vs. new peptide 2,
33/40
82%


beta-Amanitin


Phallacidin vs. new peptide 3
35/40
87%


SEQ ID NO: 117









The inventors then initiated a BLASTN and TBLASTN search of the Amanita bisporigera genome DNA sequences using conserved region A for identifying homologous sequences. The inventors discovered numerous nucleic acid sequences encoding MSDINVTRLP SEQ ID NO:88 or versions thereof, followed by variable short regions that were in turn followed by regions homologous to regions B of AMA1 and PHA1, see, FIG. 9, and the Table below. The inventors contemplated that these sequences encode additional proproteins and biologically active linear or cyclic peptides, such as toxins or enzyme inhibitors.







TABLE 10A







Exemplary comparisons to AMA1 and PHA1.









Name
Proprotein
Identity





[amanitin]
MSDINATRLP IWGIGCNP CVGDDVTTLLTRGE
100%


peptide
SEQ ID NO: 107



 


[phallacidin],
MSDINATRLP AWLVDCP CVGDDVNRLLTRGE
25/32 (78.1%)



SEQ ID NO: 108



 


[consensus],
MSDINATRLP XWXXXCXP CVGDDVXXLLTRGE




SEQ ID NO: 109



 


new potential
MSDINATRLP FNILPFMLPP CVSDDVNILLTRGE
AMA1 23/34


peptide 1
SEQ ID NO: 110
(67%)




PHA1 22/34 (64%)


 


new potential
MSDINATRLP IWGIGCDP CIGDDVTILLTRGE
AMA1 29/32


peptide 2
SEQ ID NO: 111
(90%)




PHAl 24/32 (75%)


 


new potential
MSDINATRLP IIGILLPP CIGDDVTILLTRGE
AMA1 26/32


peptide 3
SEQ ID NO: 112
(81%)




PHA1 22/32 (68%)


 


new potential
MSDINATRLP AWLATCPC AGDDVNPLLTRGE
AMA1 26/32


peptide 4
SEQ ID NO: 113
(81%)




PHA1 22/32 (68%)
















TABLE 10B





Exemplary comparisons using Amanita peptide sequences as query


sequences in GenBank (BLASTP).


















Alpha-amanitin
IWGIGCNP (8)
6/8 (75%)
gb|AAZ19981.1| conserved


(AMA1)
(SEQ ID NO: 50)
IWGIGCVL
hypothetical protein




(SEQ ID NO:
[Psychrobacter arcticus 273-




655) 6/8 (75%)
4] gb|EAU82808.1|





hypothetical protein





CC1G_J1325 [Coprinopsis





cinerea okayama7#130]


 


Alpha-amanitin
IWGIGCNP (8)
5/8 (40.0%)
AWLVDCP (PHA1)


(AMA1)
(SEQ ID NO: 50)

(SEQ ID NO: 69)


 


phallacidin
AWLVDCP (7)
AWLVDC
gb|EAV54171.1| sigma54


(PHA1)
(SEQ ID NO: 69)
(SEQ ID NO:
specific transcriptional




656)
regulator, Fis family




6/7 (85.5%)
[Burkholderia ambifaria




AWVVDCP
MC40-6]




(SEQ ID NO:
gb|AAG04585.1|AE004550_1




657) 6/7 (85.5%)
probable transcriptional





regulator [Pseudomonas





aeruginosa PAO1]





gb|EAL84365.1| conserved





hypothetical protein





[Aspergillus fumigatus





Af293]


 


Peptide 1 SEQ
FNILPFMLPP
2/10 (20%)
AMA1 PHA1


ID NO: 114
(10)
2/10 (20%)
ref|ZP_01047917.1|




8/10 (80%)
hypothetical protein





NB311A_09386 [Nitrobacter





sp. Nb-311A]


 


beta-amanitin
IWGIGCDP (8)
7/8 (87%)
AMA1


SEQ ID NO: 54

5/8 (40.0%)
PHA1




7/8 (87%)
ref|YP_265415.1|





hypothetical protein





Psyc_2134 [Psychrobacter





arcticus 273-4]


 


Peptide 3
IIGILLPP (8)
4/8 (50%)
AMA1


SEQ ID NO:

1/8 (12.5%)
PHA1


117

7/8 (87%)
gb|ABR79950.1| hypothetical




IIGILLP
protein [Klebsiella pneumoniae




7/7 (100%)
subsp. pneumoniae MGH





78578]





ref|YP_001292803.1





hypothetical protein





[Haemophilus influenzae





PittGG]





ref|XP_001139896.1|





PREDICTED: prolyl 4-





hydroxylase, alpha I subunit





isoform 2 [Pan troglodytes]
















TABLE 10C







Exemplary sequences related to AMA1 and PHA1. Predicted amino acid


sequences encoded by genomic survey sequences of A. bisporigera (FIG. 7). Spaces


were sometimes inserted before and after the peptide/toxin regions (underlined),


when the peptide/toxin region had fewer than 10 predicted amino acids, in order to


emphasize the conservation of the upstream and downstream sequences. *indicates


stop codon. These are genomic survey sequences. Based on the cDNA sequences of


AMA1 and PHA1, an intron is contemplated near the C-terminus of the indicated


proproteins.








SEQ ID NO:
Exemplary Amanita peptides





SEQ ID NO: 118
MSDINATRLP HPFPLGLQP  CAGDVDNLTLTKGEG


 


SEQ ID NO: 111
MSDINATRLP IWGIGCDP   CIGDDVTILLTRGE


 


SEQ ID NO: 113
MSDINATRLP AWLATCP    CAGDDVNPLLTRGE


 


SEQ ID NO: 121
MSDINVTRLP GFVPILFP   CVGDDVNTALT


 


SEQ ID NO: 122
MSDINTARLP FYQFPDFKYP CVGDDIEMVLARGER*


 


SEQ ID NO: 123
MSDINTARLP FFQPPEFRPP CVGDDIEMVLTRG*


 


SEQ ID NO: 124
MSDINTARLP LFLPPVRMPP CVGDDIEMVLTRGER*


 


SEQ ID NO: 125
MSDINTARLP LFLPPVRLPP CVGDDIEMVLTR


 


SEQ ED NO: 126
MSDINTARLP YVVFMSFIPP CVNDDIQVVLTRGEE*


 


SEQ ID NO: 127
MSDINTARLP CIGFLGIP   SVGDDIEMVLRH


 


SEQ ID NO: 128
MSDINTARLP LSSPMLLP   CVGDDILMV


 


SEQ ID NO: 129
MSDINAIRAP ILMLAILP   CVGDDIEVLRRGEG*


 


SEQ ID NO: 130
MSDINGTRLP IPGLIPLGIP CVSDDVNPTLTRGER*


 


SEQ ID NO: 131
MSDINATRLP GAYPPVPMP  CVGDADNFTLTRGEK*


 


SEQ ID NO: 132
MSDINATRLP GMEPPSPMP  CVGDADNFTLTRGN


 


SEQ ID NO: 118
MSDINATRLP HPFPLGLQP  CAGDVDNLTLTKGEG*









In particular, the inventors analyzed three sequences encoding short peptides and potential toxins including comparing sequence homology to α-amanitin and phallacidin.







TABLE 11A







Exemplary Amanita Peptides.










Peptide sequence
SEQ ID Number.






IWGIGCNP
SEQ ID NO: 50


 



AWLVDCP
SEQ ID NO: 69


 



XWXXXCXP
SEQ ID NO: 135


 



FNILPFMLPP
SEQ ID NO: 114


 



IWGIGCDP
SEQ ID NO: 54


 



IIGILLPP
SEQ ID NO: 117


 



AWLATCP
SEQ ID NO: 136


 



GFVPILFP
SEQ ID NO: 137


 



FYQFPDFKYP
SEQ ID NO: 138


 



FFQPPEFRPP
SEQ ID NO: 139


 



LFLPPVRMPP
SEQ ID NO: 140


 



LFLPPVRLPP
SEQ ID NO: 141


 



YVVFMSFIPP
SEQ ID NO: 142


 



CIGFLGIP
SEQ ID NO: 143


 



LSSPMLLP
SEQ ID NO: 144


 



ILMLAILP
SEQ ID NO: 145


 



IPGLIPLGIP
SEQ ID NO: 146


 



GAYPPVPMP
SEQ ID NO: 147


 



GMEPPSPMP
SEQ ID NO: 148


 



HPFPLGLQP
SEQ ID NO: 149









Example XII
This example shows the complex hybridization patterns of Example VIII, FIG. 8, that indicated that AMA1 and PHA1 are members of gene families.
Using the conserved upstream and downstream amino acid sequences of AMA1 and PHA1 as queries, the inventors found at least 15 new related sequences (Table 11B and another 10-15 partial sequences in the genome survey sequence of A. bisporigera. Each of them had an upstream conserved consensus sequence MSDINATRLP (MSD, N, R, and P are invariant), and a downstream conserved consensus CVGDDXXXXLTRGE (D is invariant). The putative peptide toxin regions, which start immediately downstream of an invariant Pro residue and end just after an invariant Pro residue, are more variable compared to the upstream and downstream sequences. The hypervariable regions contain seven to ten amino acids, while all of the twenty proteinogenic amino acids are represented at least once (FIGS. 7 and 9). With specific 5′ PCR primers and oligo-dT, the inventors demonstrated that at least two of the sequences starting with “MSDIN” or closely similar sequence (FIG. 7) are expressed at the mRNA level.







TABLE 11B







AMA1 and PHA1 related sequences.








Fifteen additional AMA1 and PHA1 related sequences



found in a genome survey of A. bisporigera using



conserved upstream and downstream amino acid



sequences of AMA1 and PHA1 as queries.
SEQ ID NO: XX





MSDINATRLPIWGIGCN--PCVGDDVTILLTRGE
SEQ ID NO: 303


 


MSDINATRLPAWLVDC---PCVGDDVNRLLTRGE
SEQ ID NO: 304


 


MSDINATRLPIWGIGCD--PCIGDDVTILLTRGE
SEQ ID NO: 305


 


MSDINATRLPIIGILLP--PCIGDDVTLLLTRGE
SEQ ID NO: 306


 


MSDINATRLPFNILPFMLPPCVSDDVNILLTRGE
SEQ ID NO: 110


 


MSDINTARLPFYQFPDFKYPCVGDDIEMVLARGE
SEQ ID NO: 308


 


MSDINTARLPFFQPPEFRPPCVGDDIEMVLTRGE
SEQ ID NO: 309


 


MSDVNDTRLPFNFFRFPY-PCIGDDSGSVLRLGE
SEQ ID NO: 310


 


MSDINTARLPLFLPPVRMPPCVGDDIEMVLTRGE
SEQ ID NO: 311


 


MSDINTARLPYVVFMSFIPPCVNDDIQVVLTRGE
SEQ ID NO: 312


 


MSDINAIRAPILMLAIL--PCVGDDIEVLRRGEG
SEQ ID NO: 313


 


MSDINGTRLPIPGLIPLGIPCVSDDVNPTLTRGE
SEQ ID NO: 314


 


MSDINATRLPGAYPPVPM-PCVGDADNFTLTRGE
SEQ ID NO: 315


 


MSDINATRLPHPFPLGLQ-PVAGDVDNLTLTKGE
SEQ ID NO: 316


 


MSDINATRLPAWLATC---PCAGDDVNPLLTRGE
SEQ ID NO: 317









Fifteen sequences listed in Table 11B were used for constructing a WebLogo graphic (Crooks et al., 2004, herein incorporated by reference) showing the relative conservation by letter size representing amino acids, such that highly conserved amino acids are represented by large letters (for example, MSDIN; positions 1-5, and P; positions 10 and 20) while less conserved amino acids have smaller letters (for example A/T, G/S; positions 6 and 23, respectively) and low areas of conserved amino acids have small letters (for example, in regions 11-18). These results showed upstream MSDINATRLP (SEQ ID NO: 88) (MSD, N, R, and P are invariant, consensus was MSDXNXXRXP) and downstream conserved consensus CVGDDXXXXLTRGE (SEQ ID NO: 239) (D is invariant). FIG. 9. Because WebLogo requires that all sequences have the same length, therefore the spaces were replaced with one, two, or three X's within the toxin region before the second conserved Pro residue for toxin peptides of nine, eight, or seven amino acids, respectively.
Example XIII
This example shows exemplary sequences for amanitin produced by G. marginata mushrooms. 
Galerina marginata (a synonym for G. autumnalis) produces amatoxins but not phallotoxins (Benedict et al., 1966). This fungus is contemplated as a potentially valuable experimental system for elucidating the biosynthesis and regulation of amatoxin biosynthesis because, unlike Amanita, it is saprophytic and grows and produces amatoxins in culture (Muraoka and Shinozawa, 2000). Galerina spp. are relatively small and rare, but they nonetheless sometimes cause mushroom poisonings (e.g., Kaneko et al, 2001, herein incorporated by reference, and FIG. 31).
Therefore, the inventors sequenced about 40 MB of G. marginata and identified two genomic sequences that could encode alpha-amanitin (GmAMA1) (FIGS. 11 and 12). Comparison of the DNA and amino acid sequences of AMA1 and GmAMA1 (FIG. 12A) indicated that amatoxins are also made on ribosomes in Galerina and probably processed similarly. DNA probed with GmAM1 under high stringency conditions showed at least 2 sequences, a Southern blot of G. autumnalis FIG. 12B. Lanes 1-4 are samples of total genomic DNA cut with PstI, HindIII, EcoRV, and BamHI. The blot shows that there are two copies of GmAMA1. This corresponds to the two copies of GmAM1. One was identified by 454 sequencing and the other by inverse PCR (see herein). However, the upstream and downstream sequences are much less well conserved when compared to the Amanita alpha amanitin sequence. The four amino acids immediately upstream of the toxin region (TRLP) are conserved in Amanita and Galerina (FIG. 11). This might be an indication that these amino acids are important for processing of the proproteins by prolyl oligopeptidase (see below).
An RNA blot of the Galerina marginata amanitin gene (GmAMA1) showed that the gene is expressed in two known amanitin-producing species of Galerina (G. marginata and G. badipes) and not in a nonproducer (G. hybrida), and that the gene is induced by low carbon. Lane 1: G. hybrida, high carbon. Lane 2: G. hybrida, low carbon. Lane 3: G. marginata, high carbon. Lane 4: G. marginata, low carbon. Lane 5: G. badipes, high carbon. Lane 6: G. badipes, low carbon. Each lane was loaded with 15 ug total RNA. The agarose gel was blotted to nitrocellulose by standard methods and probed with the G. marginata AMA1 gene (GmAMA1) predicted to encode alpha-amanitin. Fungi were grown in liquid culture for 30 d on 0.5% glucose (high carbon) then switched to fresh culture of 0.5% glucose or 0.1% glucose (low carbon) for 10 d before harvest. The major band in lanes 3-6 is .about.300 bp. The high MW signal in lane 1 is spurious.
Therefore, by RNA blotting, the inventors found that GmAMA1 is expressed in culture and is induced by carbon starvation, as has been reported for the toxin itself (Muraoka and Shinozawa, 2000, herein incorporated by reference) (FIG. 13).
Genomic DNA Isolation.
Galerina marginata, an amatoxin producing species of circumboreal distribution, was harvested from the wild. Caps and undamaged stems were cleaned of soil and debris, frozen at −80.degree. C., and lyophilized.
Genomic DNA was extracted from the lyophilized fruiting bodies using cetyl trimethyl ammonium bromide-phenol-chloroform isolation (Hallen, et al., (2003) Mycol. Res. 107:969; herein incorporated by reference). For studies requiring RNA, RNA was extracted using TRIZOL (Invitrogen) (Hallen, et al., (2007) Fung. Genet. Biol., 44:1146; herein incorporated by reference in its entirety). The inventors used a Genome Sequencer FLX from 454 Life Sciences (Margulies, et al., (2005) Nature 437:376; herein incorporated by reference) for generating sequences from Galerina species genomic DNA. There was no subcloning necessary. The inventors structured and maintained the sequenced DNA in a password-protected, private BLAST-searchable format.
Therefore, the inventors searched the DNA sequences from their Galerina marginata genome seeking DNA fragments capable of encoding amino acid sequences of amanitins, such as predicted sequences comprising a known predicted sequence of IWGIGCNP (SEQ ID NO: 50) Thus the inventors discovered an exemplary DNA sequence encoding either or both .alpha.-amanitin and/or .gamma.-amanitin (these two forms of amanitin have the same amino acid sequence because they differ only in hydroxylation, which is a posttranslational modification). The sequences were compared (BLAST) to Amanita sequences previously discovered by the inventor and disclosed in a Provisional U.S. Patent Application Ser. No. 61/002,650 (FIG. 12A and FIG. 14). Therefore the inventors found nucleotide sequences that encode the amino acid sequence of .alpha.-amanitin or or .gamma.-amanitin with the sequence order of IWGIGCNP (SEQ ID NO: 50), in single letter code, in the genome of G. marginata. The inventors contemplate that IWGIGCNP (SEQ ID NO: 50) would form a cyclic .alpha.-amanitin and/or .gamma.-amanitin, which is also known to be present in G. marginata. 
Specifically, PCR primers were designed based on the full-length (248 bp) Genome Sequencer 454 FLX read encoding IWGIGCNP (SEQ ID NO: 50) and were used successfully to amplify the predicted amanitin coding region from G. marginata genomic DNA for use as probes in Southern and Northern blots. Primers were also designed for inverse PCR, in order to isolate and sequence DNA upstream and downstream of the amanitin-encoding region. Primers are as follows: A) Gal 454 start F: CCA GTG AAA ACC GAG TCT CCA; SEQ ID NO: 319, B) Gal before MFD F: CAA AGA TCT TCG CCC TTG CCT; SEQ ID NO: 320; C) Gal CDS MFD F: ATG TTC GAC ACC AAC TCC ACT, SEQ ID NO: 321; D) Gal end 454 R: ACA CAT TCA ACA AAT ACT AAC; SEQ ID NO: 322; E) Gal inverse->: GCT GAA CAC GTC GAT CAA ACT; SEQ ID NO: 323; F) Gal inverse<-: TCC ATG GGT TGC AGC CAA TAC; SEQ ID NO: 324. Primer combinations A:D, B:D, and C:D amplify unique PCR products from G. marginata of sizes 244, 201 and 169 bp, respectively; when cloned and sequenced, these PCR products are perfect matches to the Genome Technologies 454 FLX sequence. FIG. 14. Unlike GmAMA1, GmAMA2 (MFD2) was obtained by inverse PCR on genomic DNA of Galerina using primers GCT GAA CAC GTC GAT CAA ACT; SEQ ID NO: 323 and TCC ATG GGT TGC AGC CAA TAC; SEQ ID NO: 324. This yielded one PCR product (MFD2). Thus the inventors showed that Galerina has at least two genes encoding for amanitin.
Example XIV
This Example describes identifying potential prolyl oligopeptidase (POP)—like genes in fungal species.
The inventors discovered during the development of the present inventions, that both sequences of the present inventions and the structurally resolved Amanita cyclic peptides (amatoxins and phallotoxins) contained conserved Prolines. In particular, the inventors found in each predicted peptide sequence a Proline was located downstream of a N-terminal conserved region where proline (Pro) was the last amino acid of the sequence, while the last amino acid in the peptide toxin region itself was always a conserved Pro (for examples, FIGS. 5, 7). Thus the inventors contemplated that during processing of the propeptides of AMA1 and PHA1 to smaller peptides representing the amino acids found in the final mature amatoxins and phallotoxins, there would be a role for a proline-specific peptidase, for example a prolyl oligopeptidase enzyme, which is a peptidase or protease that cuts peptide bonds specifically after Pro residues. It was contemplated that such an enzyme also processes the other proproteins related to AMA1 and PHA1, resulting in the release of a small (7-10 amino acid) peptide that could be subsequently modified by, e.g., cyclization, hydroxylation, epimerization, and other posttranslational modifications.
Based on the conservation of a Pro residue immediately upstream of the peptide toxin region, and of a Pro as the last amino acid in the toxin region of all Amanita peptide toxin family members the inventors contemplated that an enzyme that recognizes and cleaves peptides at the carboxy side of Pro residues catalyzes the first post-translational step in Amanita toxin biosynthesis. Further, Based on the properties of the known proline-specific peptidases (Cunningham, et al., (1997) Biochim Biophys Acta 1343:160, Polgar, (2002) Cell. Mol. Life Sci. 59:349; all of which are herein incorporated by reference), the inventors contemplated that a member of the prolyl oligopeptidase family (POP) (EC 3.4.21.26) family was the most likely to be involved in the processing of the proproteins encoded by AMA1 and PHA1.
POPs are known to be widespread in animals, plants, and bacteria. However, none of the other known Pro-recognizing proteases specifically cleave at internal Pro residues of small peptides (Cunningham and O'Connor, 1997; Gass and Khosla, 2007).
Thus, the inventors used a human POP sequence (GenBank NP—002717, SEQ ID NO: 150) as a query sequence to search GenBank and known fungal genomes in order to identify a candidate fungal POP (see Table 12 below). A TBLASTN search was conducted using human POP (GenBank NP—002717) as query. BLASTP (default parameters) identified no orthologs of human POP with a score >53 and E value <e-06 in any fungus outside the Basidiomycetes, except perhaps Phaeosphaeria nodorum (SNOG.sub. —11288; score=166; E value=3e-40) (FIG. 15).
Orthologs of human POP are were present in other Basidiomycetes including Coprinopsis cinereus (GenBank CC1G.sub. —09936), Ustilago maydis (UM05288), Cryptococcus neoformans (XP.sub. —567311 and XP.sub. —567292), Laccaria bicolor (Lacbi1|303722) hypertext transfer protocol site:genomejgi-psforg/Lacbi1/Lacbi1.home.html), Phanerochaete chrysosporium (Phchr1|1293) hypertext transfer protocol site:genomejgi-psf.org/Phchr1/Phchr1.home.html), and Sporobolomyces roseus (Sporo1|33368) hypertext transfer protocol site:genome.jgi-psf.org/Sporo1/Sporo1.home.html). A POP enzyme has been previously purified from the mushroom Lyophyllum cinerascens (Yoshimoto, et al., (1988) J. Biochem. 104:622; herein incorporated by reference). Surprisingly, POP orthologs (POP-like genes and proteins) are rare or nonexistent in fungi outside of the Basidiomycetes, a possible exception being one in the Ascomycete Phaeosphaeria (Septoria) nodorum (SNOG—11288). However, this single potential Ascomycete POP-like gene is much less similar to human POP than any of the POP-like genes found in Basidiomycetes.







TABLE 12







Exemplary results using human prolyl oligopeptidase (POP;


(GenBank NP_002717, SEQ ID NO: 150) as a query sequence


for fungal sequences (BLAST of GenBank unless otherwise noted).









Fungal sequences




related to human




POP found in public




databanks
Sequence Reference No.
SEQ ID NO: XX





human prolyl
(GenBank NP_002717)
SEQ ID NO: 150


oligopeptidase (POP).




Coprinopsis
(GenBank CC1G_09936)
SEQ ID NO: 151


(Coprinus) cinereus




Ustilago maydis
(GenBank UM05288)
SEQ ID NO: 152


Cryptococcus
(GenBank XP_567311)
SEQ ID NO: 153


neoformans




Cryptococcus
(GenBank XP_567292)
SEQ ID NO: 154


neoformans




Laccaria bicolor*
(The DOE Joint Genome
SEQ ID NO: 155



Institute (JGI) Lacbi1|303722)



Phanerochaete
(The DOE Joint Genome
SEQ ID NO: 348


chrysosporium*
Institute (JGI) Phchr1|1293)



Puccinia graminis
PGTG_14822.2
na


Sporobolomyces
(The DOE Joint Genome
SEQ ID NO: 349


roseus*
Institute (JGI) 1|33368;




Sporo1|33368)



mushroom Lyophyllum
Yoshimoto, et al., (1988)
na


cinerascens
J. Biochem. 104: 622; herein




incorporated by reference



Ascomycete
(GenBank SNOG_11288)
SEQ ID NO: 158


Phaeosphaeria




(Septoria) nodorum









Based upon these discoveries the inventors contemplated that a POP-like protease was rare or nonexistent in the Ascomycota yet found widespread within the Basidiomycota.
Example XV
This example describes the identification and isolation of an Amanita bisporigera orthologous to human prolyl oligopeptidase (POP). The inventors used the sequence for human POP (GenBank NP.sub. —002717) for screening their A. bisporigera genomic DNA sequence database.
Genome survey sequences were identified in the A. bisporigera genome (subject) by TBLASTN using human POP (GenBank accession no. NP002717, SEQ ID NO:150) as a query sequence (FIG. 16 and Table 13).







TABLE 13







Exemplary homology results using human prolyl


oligopeptidase (POP) as a query sequence


 (BLAST of A. bisporigera genome).









Sequences related




to human POP




found in the




Amanita genome




of the present

SEQ ID


inventions
SEQUENCE
NO:





ECGK9LO02JKSHR R
TTGAGAGCACACAAGTCTGGTATG
SEQ ID



AGAGCAAAGACGGAACGAAAGTTC
NO: 159



CAATGTTCATCGTTCGTCACAAAT




CAACGAAATTTGACGGAACGGCGC




CGGCGATTCAAAACGG



 


ECGK9LO02JKSHR R
ESTQVWYESKDGTKVPMFIVRHKS
SEQ ID



TKFDGTAPA
NO: 160


 


contig26093
CGTATATCGAACTGCCAAGGTCAA
SEQ ID



GGGTTTAAATCCGAACGATTTCGA
NO: 161



GGCTCGACAGGTGACTAGTTGGTT




TTATATTGCATGAAAAGTGCGTCT




CATGCGGTCTAGGTGTGGTATGAC




AGCTACGACGGAACAAAGATTCCA




ATGTTCATCGTCCGTCACAAGAAT




ACCAAATTTAATGGGACGGCGCCA




GCTATACAATATGG



 


contig26093
VWYDSYDGTKIPMFIVRHKNTKFN
SEQ ID



GTAPAIQY
NO: 162


 


ECIMO1V02I2IO5 S
CGACAAACAAGTAACACCTACGCG
SEQ ID



CGAAAAACTCGCGATCTCCGGCGG
NO: 163



CAGCAACGGCGGACTCCTCGTCGG




CGCAAGCCGATTGACCCAGCGCCC




CGACCTCTTCG



 


ECIMO1V02I2IO5 S
EKLAISGGSNGGLLVGASRLTQR
SEQ ID



PDLF
NO: 164


 


ECIMO1V01CKHE5 R
ATCCTCGGATGGCACAGCCTCGCT
SEQ ID



CTCCATGTATGATTTCTCACACTG
NO: 165



TGGCAAATACTTCGCATATGGTAT




TTCTCTTTCCGTATGTAATTTT



 


ECIMO1V01CKHE5 R
SSDGTASLSMYDFSHCGKYFAYGI
SEQ ID



SLS
NO: 166


 


EEISCGG02IHTSV R
GGGATAATTAATTGCAGCGAGTTA
SEQ ID



TGACAACGGAAAAACCCACCTCTT
NO: 167



CTCAGTAGATTTTCCTCCGCCATG




CCCCGCTTTCTTGTCTACACGTAG




CAGAAGTGGA



 


EEISCGG02IHTSV R
PLLLRVDKKAGHGGGKSTEK
SEQ ID




NO: 168


 


ECIMO1V02H2WNR S
DGTKVPMFIVRHKSTK
SEQ ID




NO: 169









After identifying homologous fragments, the inventors used PCR to amplify two Amanita prolyl oligopeptidase (POP)-like genes, with primers shown in Tables 14A and 14B. The full genomic sequences of prolyl oligopeptidas-likeA (POPA), SEQ ID NO: 170 and prolyl oligopeptidas-likeB (POPB), SEQ ID NO: 171 are shown in FIG. 17. Based on 5′ and 3′ RACE, using primers shown in Tables 14A and 14B, cDNA clones were obtained and sequenced, SEQ ID NOs: 234 and 235. Comparison of full length genomic and cDNA sequences (FIG. 17A) indicated that POPA and POPB each have 19 introns. The cDNA sequences of POPA and POPB are shown (FIG. 14B). The amino acid sequences of POPA and POPB are shown in (FIG. 17C), SEQ ID NOs: 236 and 237.







TABLE 14A







PCR primers used to amplify prolyl oligopeptidase-likeA (POPA)


genomic sequences and for 5′ and 3′ RACE to identify full-length


cDNA clones of POPA.









Primer
Sequence
SEQ ID NO:





PopA genomic
5′ GAAACGAGAGGCGAAGTCAAGGTG 3′
SEQ ID NO:


forward primer

172


 


PopA genomic
5′ AAGTGGATGACGATTATGCGGCAG 3′
SEQ ID NO:


reverse primer

173


 


PopA gene-
5′ GATTGGGTATTTGGCGCAGAAGTCACG 3′
SEQ ID NO:


specific primer

174


for 3′ RACE




(used with




GeneRacer 3′




primer)




 


PopA gene-
5′ ATGTCTCGCCGAACTCGCCGCCTCCTC 3′
SEQ ID NO:


specific primer

175


for 5′ RACE




(used with




GeneRacer 5′




primer)
















TABLE 14B







PCR primers used to amplify prolyl oligopeptidase-like B (POPB)


genomic sequences and for 5′ and 3′ RACE to identify full-length


cDNA clones of POPB.









Primer
Sequence
SEQ ID NO:





PopB genomic
5′ TCAAATGAAGTAGACGAATGGAC 3′
SEQ ID NO:


forward primer

176


 


PopB genomic
5′ CACACGGATGAGCAATGGATGAG 3′
SEQ ID NO:


reverse primer

177


 


PopB gene-
5′ AAAGTTCCAATGTTCATCGTTCCTCA 3′
SEQ ID NO:


specific primer

178


for 3′ RACE




(used with




GeneRacer 3′




primer)




 


PopB gene-
5′ TGGGACTAAAGAATGGATCGGCTGTAAT 3′
SEQ ID NO:


specific primer

179


for 5′ RACE




(used with




GeneRacer 5′




Primer)









The finding of a second POP gene was unexpected. Furthermore, the inventors found at least two POP genes in A. bisporigera, while the majority of other mushrooms whose genomes were examined by BLAST had only one POP (i.e., Coprinus cinerea, Laccaria bicolor, Phanerochaete chrysosporium, and Agaricus bisporus). Based on genome survey sequences, Galerina species are contemplated to contain genes for the two types of POPs (see above). By Southern blotting, POPA is present in all Amanita species (FIG. 18A). POPB, on the other hand, is present only in peptide toxin-producing species, corresponding to the discovery of genes encoding its putative substrates, AMA1 and PHA1 (FIG. 18B). In these experiments, the Southern blot of different Amanita species probed with (A) POPA or (B) POPB of A. bisporigera. Lanes 1-4 are Amanita species in sect. Phalloideae and the others are peptide toxin non-producers. Note the presence of POPA and absence of POPB in sect. Validae (lanes 5-8), the sister group (i.e., the section most closely related) to sect. Phalloideae (lanes 1-4). We attribute the weaker hybridization of POPA to the Amanita species outside sect. Phalloideae (lanes 5-13) to lower DNA loading and/or lower sequence identity due to taxonomic divergence.
POPB fragments were not observed to hybridize to any species tested outside of sect. Phalloideae even after prolonged autoradiographic exposure. Therefore, the inventors contemplated that while POPA appears to be present in the genomes of peptide toxin producing and peptide nontoxin producing mushrooms, the presence of POPB appears to be limited to peptide toxin producing mushroom species and thus identifies an amanitin-toxin producing mushroom from a nontoxin (at least for amanitin) producing mushroom.
Example XVI
This example describes the expression and isolation of prolyl oligopeptidase (POP) of the present inventions.
The inventors first tried to express POP genes from A. bisporigera in a heterologous system, which has been successful with porcine and bacterial POPs (Szeltner et al., 2000; Shan et al., 2005). Exhaustive attempts were made to express these fungal proteins in E. coli or Pichia pastoris in a soluble, active form but were unsuccessful. However the inventors were able to use the inclusion bodies to raise antibodies; see below.
Therefore, the inventors purified POP from the mushroom Conocbye lactea (also known as C. albipes or C. apala). Conocbye lactea was chosen as a source of POP because (1) it produces phalloidin, one of the phallotoxins; (2) it grows abundantly in the lawns of Michigan State University while Amanita mushrooms themselves are less common and more restricted in their fruiting season. Proteins isolated from Conocybe were assayed for POP activity with a standard colorimetric substrate (Z-Gly-Pro-pNA) and was inhibited by a specific POP inhibitor, Z-Pro-Prolinal.
The inventors synthesized model peptides, ATRLPIWGIGCNPCVGDD (SEQ ID NO:318), MSDINATRLPAWLATCPCAGDD, and ATRLPAWLVDCPCVGDD (SEQ ID NO:249), i.e., the mature toxin peptides flanked by five amino acids on each end. Based on other successful synthetic POP substrates (e.g., Shan et al., 2005; Szeltner et al., 2000), these were contemplated as test mimics of the proproteins. The peptides IWGIGCNP (SEQ ID NO:50), AWLATCP (SEQ ID NO:136), and AWLVDCP (SEQ ID NO:69) were also synthesized as standards.
Extracts of Conocybe mushrooms catalyze the cleavage of a model phalloidin peptide to the mature heptamer. The responsible enzyme was purified. Specifically, Conocybe mushrooms were freeze-dried, ground in buffer, and the extracts concentrated by ammonium sulfate precipitation. After desalting, the proteins were fractionated by anion exchange high-performance liquid chromatography (or high pressure liquid chromatography, HPLC). FIG. 19.
Fractions containing peptides were assayed using Z-Gly-Pro-pNA and the model phallacidin substrate. Reaction products were separated by reverse phase HPLC (FIG. 20). In some experiments the HPLC eluant was analyzed by MS, while in other cases the peaks of UV absorption were collected and analyzed by MS in the inventors' lab and the central LC/MS facility, in particular for long HPLC run times. The Michigan State University Proteomics and Mass Spectrometry facilities are equipped with several suitable mass spectrometers, including a Waters Quattro Premier XE LC MS/MS (for simultaneous separation and identification), vMALDI MS/MS, and a Shimadzu MALDI TOF MS/MS (for analysis of collected HPLC fractions). PepSeq within the MassLynx program was used to determine peptide sequences. The peptides eluting from HPLC were monitored at 280 nm.
The inventors purified the enzyme responsible for cleaving synthetic model compounds to the linear, mature forms to a single band on an SDS-PAGE gel. Sequencing of this protein showed high sequence similarity to POPA and POPB from A. bisporigera and POP proteins from other organisms including pig and human. After incubation of the test propeptide and the isolated POPB, the inventors consistently observed the production of a mature seven-amino acid product (FIG. 20B), whose identity was confirmed by the high resolution mass of the parent compound and the deduced amino acid sequence derived from MS/MS fragmentation. The inventors also detected one of the two possible intermediate products (i.e., MSDINATRLPAWLATCP (SEQ ID NO: 755)) transiently, but not a compound of the right mass to be the cyclized product. Thus, the same enzyme cuts the phalloidin precursor at both Pro residues, and cuts first at the second (C-terminal) Pro. The cleavage activity was sensitive to boiling of the mushroom extract (FIG. 20A) indciating that the reaction is catalyzed by a labile protein, and was inhibited by Z-Pro-Prolinal, a specific POP inhibitor, which is further evidence that a POP catalyzes this reaction. The same fractions showed activity against the colorimetric generic POP substrate Z-Gly-Pro-pNA and against the synthetic peptide. Confirmation of reaction product structures was accomplished by MS/MS.
The results showed that purified POP cuts a synthetic phalloidin peptide precisely at the expected flanking Pro residues. The purified POP also cut a synthetic amanitin precursor and a synthetic phallacidin precursor.
Further contemplated products (shown in Table 15) for alpha-amanitin; phalloidin precursors where natural or synthetic propeptide sequences will be the substrates for Conocybe POPB protein.







TABLE 15







Peptides and their corresponding molecular mass


for use in the present inventions.










SEQ ID
Peptide

Mr (molecular


NO:
No.
AMA1 peptides
mass)













549
1
TRLPIWGIGCNPCIGD
1714.99




(substrate)



 


549
2
TRLPIWGIGCNPCIGD
1712.99




(substrate, Cys





oxidized to





disulfide)



 


551
3
TRLPIWGIGCNP (cut at
1326.55




C side)



 


552
4
IWGIGCNPCIGD (cut at
1247.42




N side)



 


552
5
IWGIGCNPCIGD (cut at
1245.42




N side, oxidized)



 


50
6
IWGIGCNP (final
858.98




product, cut both





sides)



 


51
7
IWGIGCNP (cyclized)
840.97









Thus, the inventors found production of the mature heptapeptide of phalloidin by extracts of Conocybe, i.e. isolated POPB extracts (FIG. 20). Thus purified POPs from Amanita and Galerina are contemplated to release peptides 3, 4, and/or 6 from an amanitin precursor (prepropeptide or portion thereof).
Amanita species in sect. Phalloideae, and Galerina, have two predicted POP genes (FIG. 17).
Example XVII
In this Example, POPA and POPB of A. bisporigera were expressed in inclusion bodies, purified and used to provide rat anti-POPA and POPB antibodies for use in the present inventions.
E. coli were engineered for expressing POPA and POPB (in separate bacterium). Expression of recombinant POP was done by the procedures outlined in the pET handbook (Novagen). Briefly, a pET vector engineered to comprise a POP coding sequence of the present inventions was transformed into Escherichia coli AD494 cells, and cultures were grown according to the manufacturer's instructions in Luria-Bertani medium and then induced with isopropyl-D-thiogalactoside (final concentration of 1 mM) for 3 h. Pelleted cells were lysed with a French press (16,000 p.s.i.) and recentrifuged, and the pellet was extracted with B-Per II reagent (Pierce, Rockford, Ill.). The resulting purified inclusion bodies were solubilized and refolded using the Protein Refolding Kit (Novagen) according to the manufacturer's instructions.
The inventors raised antibodies against POPA and POPB of A. bisporigera (POPB shown in FIG. 21A) showing immunoreactivity to a band of the same molecular weight as POPB (arrows) (FIG. 21B). The inventors observed that anti-POPB antibodies did not cross-react with POPA. Cross-reactivity between POPB and POPA was not contemplated to be a concern because POPA and POPB are merely 55% identical at the amino acid level, and the immunoblot showed a single band (FIG. 21; Lane 1: Markers. Lane 2: POPB purified from inclusion bodies. Lane 3: Total soluble extract of Amanita bisporigera. Lanes 1-3 were stained with Coomassie blue. Lane 4: immunoblot of POPB inclusion body. Lane 5: immunoblot of Amanita bisporigera extract. Crude antiserum was used at 1:5000 dilutions.
Example XVIII
In this example, exemplary Galerina POP sequences identified using Amanita bisporigera POPA and POPB were used as query sequences for searching a library of Galerina sequences created by the inventors for their use during the development of the present inventions, and additional mushroom libraries. These Galerina sequences were obtained by the inventors from 454 sequencing (45 Mb total), see above. Not every sequence with identity to these genes are shown, merely what are considered the best examples.
Galerina marginata POP sequences were identified using Amanita bisporigera POPA (FIG. 22A) and POPB (FIG. 22B) as query sequences. The specific regions of identity and corresponding sequences are listed. The higher scoring hits (areas of identity) were strong evidence that the Galerina genome contains at least two POP genes. The inventors contemplate using these fragments for isolating full-length sequences for use in the present inventions.
Example XIX
Genes for fungal secondary metabolites are typically clustered (Walton, 2000; Keller et al., 2005). Examples include aflatoxin, penicillin, HC-toxin, fumonisin, sirodesmin, and gibberellins (Ahn et al., 2002; Gardiner et al., 2004; Tudzynski and Holter, 1998). From Basidiomycetes, an example of clustering are the genes for ferrichrome (Welzel et al., 2005).
To test clustering of Amanita toxin genes, the inventors constructed a partial lambda genomic library of A. bisporigera (insert size .about.15 kb) and screened it with PHA1. One exemplary lambda clone was found to contain two copies of PHA1 and three putative cytochrome P450 genes (FIG. 10D). (Based on inverse PCR results, the inventors also discovered two copies of PHA1 in A. bisporigera on a single lambda clone. Thus, at least two Amanita peptide toxin genes are clustered in the genome of A. bisporigera. Furthermore, because Amanita peptide toxins undergo three to five hydroxylations (FIG. 1), which reactions are often catalyzed by P450's in fungi and other organisms (e.g., Malonek et al., 2005; Tudzynski et al., 2003), one or all of these three P450 genes also has a plausible role in the biosynthesis of the Amanita peptide toxins. Therefore, on both theoretical and experimental grounds the inventors contemplated finding additional Amanita peptide toxin biosynthetic genes by examining regions of DNA adjacent to the known Amanita peptide toxin genes.
In this example, a software program and system, FGENESH, Salamov and Solovyev, Genome Res. 2000. 10:516-522, at softberry.com, //linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=-gfind. was used to identify and predict novel sequences adjacent to PHA genes of a 13,254 bp lambda clone (SEQ ID NO:327). This software predicts genes (by which we mean predicting where the gene starts and stops and where intron and exons are) when the gene is pasted in as genomic sequence. In recent rice genome sequencing projects, this software was cited “the most successful (gene finding) program (Yu et al. (2002) Science 296:79) and was used to produce 87% of all high-evidence predicted genes (Goff et al. (2002) Science 296:79).
However, gene prediction is an inexact science, so the FGENESH software is “trained” with known gene structures from different organisms. That is, different organisms' have different (and poorly understood) rules for gene structure. Gene structure in humans isn't the same as plants, etc. To get the best prediction, an organism on which the software has been trained that is taxonomically closest to the source of the DNA was used. Therefore, the inventors used a known Coprinus (Coprinopsis) cinerea model for their Amanita genes.
Using this type of analysis as shown in FIGS. 24-30, the inventors found in an adjacent piece of genomic DNA, two PHA1 genes (one by FGENESH) and 3 P450's, P450-1 (OP451), P450-2 (OP452) and P450-3 (OP453). For comparison, an estimated number of P450 genes in other organisms are provided as follows: Human 50, Arabidopsis 273, Phanerochaete 149, Fusarium 110, Ustilago 17, while there are 282 families of fungal P450's. For each contemplated gene, a BLASTp search was made in the inventors' mushroom libraries and publically available libraries including NCBI GENBANK and Coprinus cinereus genome annotations (Broad contigs) at llgenome.semo.edu/cgi-bin/gbrowse/cc/?reset=1, Genomic sequence data from the Broad Institute (broad.miteduannotation/genome/coprinus_cinereus/Home.html, herein incorporated by reference in it's entirety). The predictions may not find every sequence, however the inventors at this time show that the lambda clone analyzed herein contains at least three P450 genes, genes 1, 2, and 4, at least one PHA gene, gene 5, and at least one unidentified gene that is not PHA1-2, Gene 6, Gene 6 has no significant match to any protein in NCBI GenBank. In addition to the genes listed in the Figures, a PHA1-2 was found (where the software analysis showed a start, stop, and introns correctly) but FGENESH did not predict PHA1-1, which, however, is clearly present by manual annotation.
This example shows that two copies of PHA1 are clustered with each other and with three P450 genes. A map of predicted genes in this lambda clone (13.4 kb), isolated using PHA1 as probe is shown in FIG. 10D.
Example XX
This example shows identification of exemplary variants of two α-amanitin genes identified in laboratory isolates of Galerina marginata. 
The inventors' were surprised to discover that sequences of the peptide toxin genes in Galerina marginata are quite different compared to A. bisporigera. See FIGS. 12 and 33A and B for alignments of Galerina and Amanita peptide toxin proteins. For this example, approximately 73 MB of final assembled genomic DNA, as described above, was sequenced by 454 pyrosequencing. 73 MB was estimated to be approximately two times the size of the G. marginata genome based on the average size of known basidiomycete genomes. These sequences were put into a private database and searched using AMA1, PHA1, AbPOPA, and AbPOPB protein sequences The DNA contigs showing predicted protein sequences closely related to AbPOPB and AbPOPA were further analyzed. PCR primers were made to predicted sequences at the two ends of the proteins and used to amplify from genomic and cDNA full length genomic and mRNA copies of the two genes. Four examples of contigs are shown in FIG. 41. The results for GmAMA1 variants are described in this example while the results of screening for POP genes are described in the following example.
Using AMA1 from A. bisporigera as the search query, two orthologs of AMA1 were identified in the partial genome survey sequence of G. marginata and designated as GmAMA1-1 and GmAMA1-2.
PCR primers unique to GmAMA1-1 and GmAMA1-2 were designed. For GmAMA1-1, the unique primers were 5′-CTCCAATCCCCCAACCACAAA-3′ (forward, SEQ ID NO:682) and 5′-GTCGAACACGGCAACAACAG-3′ (reverse, SEQ ID NO:683). For GmAMA1-2, the primers were: 5′-GAAAACCGAATCTCCAATCCTC-3′ (forward, SEQ ID NO:684), and 5′-AGCTCACTCGTTGCCACTAA-3′ (reverse, SEQ ID NO:685). PCR primers for each gene were designed based on the partial sequences and used to amplify full-length copies. The amplicons were cloned into E. coli DH5α and sequenced.
The genomic DNA sequences were used for primer design to obtain full-length cDNAs by Rapid Amplification of cDNA Ends (RACE) using the GeneRacer kit (Invitrogen, Carlsbad, Calif.). A cDNA copy of GmAMA1-1 was obtained using primers 5′-CCAACGACAGGCGGGACACG-3′ (5′-RACE, SEQ ID NO:686) and 5′-GACCTTTTTGCTTTAACATCTACA-3′ (3′-RACE, SEQ ID NO:687), and of GmAMA1-2 with primers 5′-GTCAACAAGTCCAGGAGACATTCAAC-3′ (5′-RACE, SEQ ID NO:688) and 5′-ACCGAATCTCCAATCCTCCAACCA-3′ (3′-RACE, SEQ ID NO:689).
Alignments of genomic and cDNA copies were done using Spidey located at (ncbi.nlm.nih.gov/spidey/) and Splign (ncbi.nlm.nih.gov/sutils/splign/splign.cgi).
GmAMA1-1 contains three introns while GmAMA1-2 contains two introns (FIG. 33). The three introns of GmAMA1-1 are 53, 60, and 60 nt in length in similar locations as the three introns of AMA1. The first intron in both GmAMA1-2 and GmAMA1-2 interrupts the third codon before the stop codon. GmAMA1-1 and GmAMA1-2 differ in at least eight nucleotides out of 108 nucleotides in the coding region (i.e., from the ATG through the TGA stop codon). At least two of these differences result in amino acid changes and six changes are silent, i.e no change in amino acid at that location (FIG. 33). There are numerous nucleotide differences between GmAMA1-1 and GmAMA1-2 in the 5′ and 3′ untranscribed regions in addition to having large stretches of close identity. The biggest difference between GmAMA1-1 and GmAMA1-2 is that the latter gene has a 100-bp deletion relative to GmAMA1-1, which spans the second intron of GmAMA1-1. This deletion is in the 3′ UTR (FIG. 32). This accounts for the presence of only two introns in GmAMA1-2 (FIGS. 32 and 33).
The translational start site of a gene is typically contemplated as the first in-frame ATG after the transcriptional start site. When this criterion was applied to GmAMA1-1, a start site was indicated that was analogous to AMA1 of A. bisporigera. However, when this criteria was applied to GmAMA1-2, there was an in-frame ATG that is 78 nucleotides upstream of the ATG indicated in FIG. 33, which would result in a proprotein of 61 amino acids instead of 35 as predicted for AMA1 and GmAMA1-1. Thus two start sites are contemplated, one that results in a 61 amino acid preproprotein, SEQ ID NO:690, and the other in a 35 amino acid proprotein, SEQ ID NO:691. However the inventors' contemplate that the 35 amino acid preproprotein is the target of the Gm POP proteins, for an example showing that prolyl oligopeptidases act on other types of peptides less than 40 amino acids see, Szeltner and Polgar, 2008, herein incorporated by reference).
GmAMA1-1 and GmAMA1-2 were both predicted to encode 35-amino acid proproteins, the same size as the proprotein of AMA1 in A. bisporigera. The toxin-encoding region (IWGIGCNP) (SEQ ID NO: 50) was in the same relative position as it was in AMA1. There were 31 nucleotide differences between GmAMA1-1 and AMA1 in the coding region of 108 nucleotides (ATG through the stop codon). This results in a low level of amino acid conservation outside the toxin region and the amino acids immediately upstream of the toxin region (NATRLP, SEQ ID NO:754 (FIG. 33).
The sequenced proproteins were added by the inventors to form a group of a family of genes including and related to AMA1 and PHA1 in A. bisporigera, A. phalloides, and A. ocreata start with MSDIN. In contrast, when a start codon is contemplated in the same location between GmAMA1-1 and GmAMA1-2 the first five amino acids of the two G. marginata α-amanitin genes are MFDTN, SEQ ID NO: 675. Searching of the G. marginata database with the upstream and downstream regions of GmAMA1-1 and GmAMA1-2 did not reveal any additional related sequences. Conversely, searching with the conserved regions of GmAMA1-1 and GmAMA1-2 did not reveal any related sequences in A. bisporigera beyond the known MSDIN family members described herein.
Example XXI
This example shows identification of two exemplary full-length genes encoding orthologs of Prolyl oligopeptidase genes, i.e. POPA and POPB proteins, isolated from G. marginata. 
During the development of the present inventions, using a G. marginata partial genome survey, the inventors' discovered two orthologs of the POP genes of A. bisporigera. These two orthologs corresponded to the two A. bisporigera prolyl oligopeptidases (AbPOPA and AbPOPB) described herein. The G. marginata genes with closest identity to AbPOPA or AbPOPB were designated as GmPOPA and GmPOPB, respectively. Genomic PCR, reverse transcriptase PCR, and RACE were used, as described herein, to isolate full-length copies of these two genes and determine their intron/exon structures (FIG. 37). GmPOPA had 18 introns, which is the same number found in AbPOPA, while GmPOPB had 17 introns, one fewer than in AbPOPB. The amino acid sequences of the predicted translational products of GmPOPA (738 amino acids) and GmPOPB (730 amino acids) are 57% identical to each other. The GmPOPA protein is 65% identical to AbPOPA and 58% identical to AbPOPB, and GmPOPB is 57% identical to AbPOPA and 75% identical to AbPOPB.
Sequences hybridizing to AbPOPA were found to be present in amatoxin and phallotoxin-producing and non-producing species of Amanita, whereas AbPOPB was found present only in the toxin-producing species. By DNA blotting GmPOPA was present in all four specimens of Galerina, however GmPOPB was not present in the amanitin non-producing species G. hybrida (FIG. 34). The similarity of the hybridization pattern of G. venenata and G. marginata to GmAMA1, GmPOPA, and GmPOPB was consistent with these two isolates belonging to the same species (see, Gulden et al., 2001, herein incorporated by reference). The association of POPB with amanitin production in both A. bisporigera and G. marginata, and the higher amino acid identity of GmPOPA to AbPOPA and of GmPOPB to AmPOPB was consistent with a contemplated role for POPB in amanitin biosynthesis in both species. Other basidiomycetes in GenBank and at the DOE Joint Genome Institute (JGI) have single POP genes, which are contemplated as functional orthologs of POPA.
For isolating and cloning full-length cDNA sequences for GmPOPA (SEQ ID NO: 715) and GmPOPB (SEQ ID NO: 717), PCR primers that corresponded to the amino and carboxyl termini of both genes (which were present on different contigs) were designed from the genome survey sequence. The forward primers were 5′-TTTAGGGCAGTGATTTCGTGACA-3′, SEQ ID NO: 692, and 5′-AACAGGGAGGCGATTATTCAAC-3′, SEQ ID NO: 693, and the reverse primers were 5′-GAACAATCGAACCCATGACAAGAA-3′, SEQ ID NO: 694, and 5′-CCCCCATTGATTGTTACCTTGTC-3′, SEQ ID NO: 695. The primer pairs were used in both combinations and successful amplification indicated the correct pairing of 5′ and 3′ primers. The resulting amplicons were cloned into E. coli DH5α and sequenced.
The RACE primers for GmPOPA were 5′-CGGCGTTCCAAGGCGATGATAATA-3′ (5′-RACE), SEQ ID NO: 696, and 5′-CATCTCCATCGACCCCTTTTTCAGC-3′ (3′-RACE), SEQ ID NO: 697, and for GmPOPB 5′-AGTCTGCCGTCCGTGCCTTGG-3′ (5′-RACE), SEQ ID NO: 698, and 5′-CGGTACGACTTCACGGCTCCAGA-3′ (3′-RACE), SEQ ID NO: 699. Sequences generated from the RACE reactions were used to assemble full-length cDNAs of two genes, GmPOPA and GmPOPB (see FIGS. 38A and 38B).
Alignments of genomic and synthetic cDNA copies (see, FIGS. 38A and 38B) were done using Spidey available at National Center for Biotechnology Information (NCBI) at websites ncbi.nlm.nih.gov/spidey/ and Splign ncbi.nlm.nih gov/sutils/splign/splign.cgi.
GmPOPA and POPB were predicted to encode exemplary polypeptides as shown in FIG. 38A (SEQ ID NO: 716) and 38B (SEQ ID NO: 722), respectively.
Example XXII
This example shows an exemplary successful transformation of G. marginata. 
The inventors grew G. marginata in the laboratory and collected mycelium for use in the following transformation procedure. The inventors show herein the successful transformation of the alpha-amanitin-producing fungus Galerina marginata with a test construct. Thus the inventors' contemplate producing commercial levels of amanatin in addition to novel, non-natural analogs of amanitin. Further, the inventors' contemplate making novel linear and cyclic peptides from synthetic prepropeptides.
The following are exemplary methods for making buffers and reagents for us in the present inventions. Galerina culture methods: Vegetative mycelial stocks were prepared by culturing aseptic fragments of fruiting bodies on HSVA plates. Fungal colonies were transferred and reisolated until pure cultures were obtained. The stocks were subcultured every 6 months. HSV-2C (1 L): 1 g yeast extract, 2 g glucose, 0.1 g NH4Cl, 0.1 g CaSO4.5H2O, 1 mg thiamine.HCl, and 0.1 mg biotin, pH 5.2 (Muraoka and Shinozawa, 2000, herein incorporated by reference). Agar medium (HSVA) for subculture contained 2% agar in HSV. Protoplasting Buffer: In 20 ml of 1.2 M KCl add 500 mg Driselase (Sigma), 1 mg chitinase (Sigma), and 300 mg lysing enzyme from Aspergillus sp. Sigma #L-3768. Stir for 30 min and filter sterilize in a 0.45 um filter. Sorbitol Tris-HCl Ca (STC) buffer: Solution a) 1.2 M sorbitol, 10 mM Tris-HCl (pH8.0), 50 mM CaCl2, autoclaved. Solution b) 30% PEG Solution Mix: 30% (W/V) polyethylene glycol/STC buffer. Filter sterilize in a 0.45 um filter. Regeneration medium (RM): a) HSV-2C (1 L) and b) sucrose 273.5 g/500 ml of water. Autoclave solutions a) and b) separately and combine after autoclaving.
The following is an exemplary Galerina transformation protocol for use in the present inventions. Around 20 pieces of mycelium were used to inoculate 100 ml of HSV-2C broth in a 250 ml Erlenmeyer flask. This inoculate was placed on a shaker at 150 rpm at room temperature for 9-15 days, until cloudy. The culture medium and fungus was used to begin the following steps. The cultures were: 1. Filtered through sterile Miracloth and the collected mycelia was washed thoroughly with sterile water. This fungal mycelium was placed in a sterile 250 ml Erlenmeyer flask. 20 ml Protoplasting Buffer (see recipe below) was added. 2. Digested for 8 hours on a rotary shaker at 26-30 C at 120 rpm. 3. Digestion mix was filtered through a 30 micron Nitex nylon membrane (Tetko Inc Kansas City, Mo., U.S.A.)) into 1-2 sterile 30 ml Oakridge tubes on ice. Filtered solution was turbulent due to the presence of protoplasts when checked under the microscope. 4. This filtered solution was centrifuged in Oakridge tubes at 4 C at 2000×g for 5 min. 5. Supernatant was carefully poured off and discarded. Protoplast pellet was gently resuspended in approx. 10 ml of STC buffer and resuspended by shaking gently. Solution was spun at 2000×g for 5 min. 6. Repeat step 5 once. 7. Supernatant was discarded and the protoplast pellet was gently resuspended in 1 ml of STC buffer with a wide orifice pipette and transferred to a microcentrifuge tube and spun at room temperature at 4000×g for 6 min. 8. Supernatant was poured off and protoplasts were resuspended in 1 ml of STC in a final volume with concentration of 108-109 protoplast/ml. The tube was placed on ice. 9. The following mixture was combined: 50 μl protoplasts, 500 STC buffer, 50 ul 30% PEG solution and 10 ul plasmid or PCR product (1 μg) depending upon the experiment. When plasmids were used they were linearized with a restriction enzyme which cut the DNA in a noncoding region. 10. 2 ml of 30% PEG solution was added and the tubes incubated for 5 min. 11. 4 ml of STC buffer was added and gently mixed by inversion. 12. The mix was added to Regeneration Media (RM) (see below) at 47° C., and mixed by inversion then poured into Petri dishes. Each solution mixture was plated in several plates. 13. Protoplasts were regenerated for up to 20 days until tiny colonies started to appear as viewed by eye. 10 ml of RM amended with 10 μg/ml Hygromycin B was overlayed onto the cultures. 14. Putative transformants were isolated from colonies that grew after the Hygromycin B overlay and eventually emerged on the surface of the overlaid agar. Examples of colonies collected for use in the present inventions are shown by arrows in FIG. 39.
After colonies were collected the presence of the inserted Hygromycin B transgene was tested by PCR. Primers specific to the hygromycin resistance gene used in FIG. 40 were the following: hph_forward 5′-GCGTGGATATGTCCTGCGGG-3′ hph_reverse, SEQ ID NO:700, 5′-CCATACAAGCCAACCACGGC-3′, SEQ ID NO: 701, (Kilaru et al., 2009, Curr Genet 55:543-550, herein incorporated by reference).
The inventor's contemplate that G. marginata can be transformed with synthetic genes, using the G. marginata specific contemplated cut sites, i.e. synthetic sequences comprising nucleotides encoding MDSTN, TRIPL and Prolines in conserved positions. For examples, in one embodiment, a synthetic DNA sequence encoding an amino acid sequence of alpha-amanitin may be expressed. In one embodiment, alpha-amanitin production would be increased, for example, using a high expression promoter, transforming Galerina with multiple copies of the alpha-amanitin gene.
In another contemplated embodiment, a synthetic, novel cyclic peptide is synthesized by transformed Galerina by changing specific bases of synthetic G. marginata alpha-amanitin sequences (including PCR copies of isolated peptide toxin genes and base by base construction of nucleic acid sequences) in order to make other types of peptide toxins and peptides. In one example, replacing the codon AAC (Asn) with GAC (Asp) will encode beta-amanitin instead of alpha-amanitin. Beta-amanitin production in G. marginata would be easily detected by reverse-phase HPLC because the inventor's isolate of G. marginata makes barely detectable levels of beta-amanitin.
The inventors further contemplate changing other amino acids to make non-natural amanitin derivatives, as one example, replacing Gly with Ala by replacing GGT with GCT. Even further, the inventor's contemplate an embodiment for making linear and cyclic peptides of at least six, seven, eight, nine, ten or more amino acids comprising the general formula XWXXXCXP, SEQ ID NO:702, where X is any amino acid. The Pro is retained in these peptides in order for correct processing by POP, and the presence of Trp (W) and Cys (C) will result in the biosynthesis of tryptathionine, a unique hallmark of the Amanita toxin peptides. Expression of synthetic peptides and peptide toxins would be monitored by standard assays including but not limited to PCR generated fragments (as in FIG. 40), and by HPLC methods (as in FIG. 31), and the like. Further, separation of synthetic toxins from endogenous peptide toxin and endogenous small peptides (i.e. peptides produced from genomic DNA originally contained in these Galerina isolates) would be done by standard techniques including but not limited to HPLC methods (as in FIG. 31). Isolated peptides produced by expression of synthetic sequences would be used in assays for assessing biological activity. For example, toxicity of synthetic amanitin toxins would be determined in assays, for one example, to measure inhibition of transcription in eukaryotic cells, such as capability to inhibit RNA Polymerase II. These toxins are contemplated for commercial levels of production.
Even further, the inventors' contemplate making new Galerina isolates that do not produce peptide toxins for use in the present inventions. In one embodiment, the inventors' contemplate knocking out genomic peptide toxin genes for making a new Galerina isolate that does not express peptide toxins. As examples for removing genomic peptide toxin genes in Galerina, i.e. test Galerina (isolates of Galerina used in the following methods) would be subject to homologous integration of transforming DNA that would be used for removing regions of DNA comprising the peptide toxin genes in transformed test Galerina, spontaneous mutants and induced mutants of test Galerina would be made then screened for loss of peptide toxin gene expression and more preferably loss of peptide toxin genes. Another method for eliminating endogenous toxin production is RNAi, which has been used in other basidiomycete fungi (Heneghan et al., Mol Biotechnol. 2007 35(3):283-96, 2007, herein incorporated by reference). Loss of toxin expression in test isolates would be monitored by standard assays including but not limited to genomic sequencing of test Galerina, PCR generated fragments of genomic sequences (as in FIG. 40), PCR generated toxin cDNA (as described herein), and by HPLC methods (as in FIG. 31), and the like. When a test Galerina isolate is shown to lack expression of peptide toxins this isolate would be cultured as a new Galerina laboratory isolate for use in the present inventions.
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in mycology, molecular biology, biochemistry, chemistry, botany, and medicine, or related fields are intended to be within the scope of the following claims.

## Claims

1. A fungus cell transfected with a recombinant prepropeptide nucleic acid encoding a proline-containing peptide operably linked to a promoter, wherein the fungus cell comprises a fungal prolyl oligopeptidase nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 236, 237, 348, 716, and 722.

2. The cell of claim 1, wherein said prepropeptide nucleic acid comprises a sequence selected from the group consisting of nucleic acid sequences encoding SEQ ID NOs:710 and 713.

3. The cell of claim 1, wherein said cyclic peptide is a bicyclic peptide.

4. The cell of claim 3, wherein said bicyclic peptide comprises sequence SEQ ID NO:50.

5. A method of making a peptide from a recombinant prepropeptide sequence, comprising,
a) providing, a fungus cell comprising a nucleic acid encoding a fungal prolyl oligopeptidase with an amino acid sequence selected from the group consisting of SEQ ID NO: 236, 237, 348, 716, and 722 and a recombinant prepropeptide nucleic acid encoding a proline-containing prepropeptide, and
b) growing said fungus cell to make said peptide.

6. The method of claim 5, wherein said peptide is at least six and up to fifteen amino acids in length.

7. The method of claim 5, wherein said peptide is biologically active.

8. The method of claim 5, wherein said peptide is a cyclic peptide.

9. The method of claim 5, wherein said cyclic peptide is a bicyclic peptide.

10. The method of claim 9, wherein said bicyclic peptide comprises sequence SEQ ID NO:50 (IWGIGCNP).

11. A method of making a synthetic cyclized peptide, comprising,
a) providing,
i) a fungal cell comprising a fungal prolyl oligopeptidase with an amino acid sequence selected from the group consisting of SEQ ID NOs: 236, 237, 348, 716, and 722,
ii) a recombinant prepropeptide nucleic acid comprising a nucleic acid sequence encoding a proline-containing prepropeptide, and

b) transforming said cell with said prepropeptide nucleic acid and
c) growing said fungal cell under conditions for expressing said prepropeptide and thereby making the synthetic cyclized peptide.

12. The method of claim 11, wherein said recombinant prepropeptide nucleic acid is selected from the group consisting of nucleic acid sequences encoding SEQ ID NOs:710 and 713.

13. The method of claim 11, wherein said cyclized peptide is selected from the group consisting of a peptide at least six and up to fifteen amino acids in length.

14. The method of claim 11, wherein said cyclized peptide is a bicyclic peptide.

15. The method of claim 14, wherein said bicyclic peptide comprises SEQ ID NO:50.

16. The method of claim 11, wherein said cyclic peptide is biologically active.