Recombinant materials and methods for production of limonene hydroxylases

cDNA encoding (-)-limonene-6-hydroxylase from spearmint and (-)-limonene-3-hydroxylase from peppermint have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Accordingly, isolated DNA sequences are provided which code for the expression of (-)-limonene-6-hydroxylase from spearmint (SEQ ID No:1, from Mentha spicata) and (-)-limonene-3-hydroxylase from peppermint (SEQ ID No:3 and SEQ ID No:5, from Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for limonene hydroxylase or for a base sequence sufficiently complementary to at least a portion of the limonene hydroxylase DNA or RNA to enable hybridization therewith (e.g., antisense limonene hydroxylase RNA or fragments of complementary limonene hydroxylase DNA which are useful as polymerase chain reaction primers or as probes for limonene hydroxylase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding limonene hydroxylase. Thus, systems and methods are provided for the recombinant expression of limonene hydroxylase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant limonene hydroxylase (or of the primary enzyme products, trans-carveol or trans-isopiperitenol, respectively) for subsequent use, to obtain expression or enhanced expression of limonene hydroxylase in plants to attain enhanced production of trans-carveol or trans-isopiperitenol as a predator or pathogen defense mechanism, or may be otherwise employed for the regulation or expression of limonene hydroxylase or the production of trans-carveol or trans-isopiperitenol.

FIELD OF THE INVENTION
 The present invention relates to nucleic acid sequences which code for
 cytochrome P450 limonene hydroxylases, such as (-)-limonene-6-hydroxylase
 from Mentha spicata and (-)-limonene-3-hydroxylase from Mentha piperita,
 and to vectors containing the sequences, host cells containing the
 sequences and methods of producing recombinant limonene hydroxylases and
 their mutants.
 BACKGROUND OF THE INVENTION
 Several hundred naturally occurring, monoterpenes are known, and
 essentially all are biosynthesized from geranyl pyrophosphate, the
 ubiquitous C.sub.10 intermediate of the isoprenoid pathway (Croteau and
 Cane, Methods of Enzymology 110:383-405 [1985]; Croteau, Chem. Rev.
 87:929-954 [1987]). Monoterpene synthases, often referred to as
 "cyclases," catalyze the reactions by which geranyl pyrophosphate is
 cyclized to the various monoterpene carbon skeletons. Many of the
 resulting carbon skeletons undergo subsequent oxygenation by cytochrome
 P450 hydroxylases to give rise to large families of derivatives. Research
 on biosynthesis has been stimulated by the commercial significance of the
 essential oils (Guenther, The Essential Oils, Vols. III-VI (reprinted) R.
 E. Krieger, Huntington, N.Y. [1972]) and aromatic resins (Zinkel and
 Russell, Naval Stores: Production, Chemistry, Utilization, Pulp Chemicals
 Association, New York [1989]) and by the ecological roles of these
 terpenoid secretions, especially in plant defense (Gershenzon and Croteau,
 in "Herbivores: Their Interactions with Secondary Plant Metabolites," Vol.
 I, 2nd Ed. (Rosenthal and Berenbaum, eds.) Academic Press, San Diego,
 Calif., pp. 165-219 [1991]; Harborne, in "Ecological Chemistry and
 Biochemistry of Plant Terpenoids," (Harborne and Tomas-Barberan eds.)
 Clarendon Press, Oxford, Mass., pp. 399-426 [1991]).
 The reactions catalyzed by the cytochrome P450-(-)-limonene hydroxylases
 determine the oxidation pattern of the monoterpenes derived from limonene
 (see FIGS. 1A-1C). These reactions are completely regiospecific and are
 highly selective for (-)-limonene as substrate. The primary products of
 limonene hydroxylation (trans-carveol and trans-isopiperitenol) are
 important essential oil components and serve as precursors of numerous
 other monoterpenes of flavor or aroma significance (see FIGS. 1A-1C).
 One of the major classes of plant monoterpenes is the monocyclic p-menthane
 (1 -methyl-4-isopropylcyclohexane) type, found in abundance in members of
 the mint (Mentha) family. The biosynthesis of p-menthane monoterpenes in
 Mentha species, including the characteristic components of the essential
 oil of peppermint (i.e., (-)-menthol) and the essential oil of spearmint
 (i.e., (-)-carvone), proceeds from geranyl pyrophosphate via the cyclic
 olefin (-)-limonene and is followed by a series of enzymatic redox
 reactions that are initiated by cytochrome P450 limonene hydroxylases
 (e.g., limonene-3-hydroxylase in peppermint and limonene-6-hydroxylase in
 spearmint and related species; Karp et al., Arch. Biochem. Biophys.
 276:219-226 [1990]; Gershenzon et al., Rec. Adv. Phytochem. 28:193-229
 [1994]; Lupien et al., Drug Metab. Drug Interact. 12:245-260 [1995]). The
 products of limonene hydroxylation and their subsequent metabolites (as
 shown in FIGS. 1A-1C) also serve ecological roles in plant defense
 mechanisms against herbivores and pathogens, and may act as signals in
 other plant-insect relationships (e.g., as attractants for pollinators and
 seed dispersers).
 A detailed understanding of the control of monoterpene biosynthesis and of
 the reaction mechanisms, enzymes and the relevant cDNA clones as tools for
 evaluating patterns of developmental and environmental regulation, for
 examining active site structure-function relationships and for the
 generation of transgenic organisms bearing such genes are disclosed in
 part in parent U.S. related application Ser. No. 08/582,802 filed Jan. 4,
 1996 as a continuation of application Ser. No. 08/145,941 filed Oct. 28,
 1993, the disclosures of which are incorporated herein by this reference,
 which disclose the isolation and sequencing of cDNAs encoding
 (-)4S-limonene synthase, the enzyme responsible for cyclizing geranyl
 pyrophosphate to obtain (-)-limonene. To date, however, no information has
 been available in the art regarding the protein and nucleotide sequences
 relating to the enzymes through which (-)-limonene is hydroxylated (by the
 action of (-)-limonene-6-hydroxylase to form trans-carveol or by the
 action of (-)-limonene-3-hydroxylase to form trans-isopiperitenol as shown
 in FIG. 1).
 SUMMARY OF THE INVENTION
 In accordance with the foregoing, cDNAs encoding (-)-limonene hydroxylase,
 particularly (-)-limonene-6-hydroxylase from spearmint and
 (-)-limonene-3-hydroxylase from peppermint, have been isolated and
 sequenced, and the corresponding amino acid sequences have been deduced.
 Accordingly, the present invention relates to isolated nucleic acid
 sequences which code for the expression of limonene hydroxylase, such as
 the sequence designated SEQ ID No:1 which encodes
 (-)-limonene-6-hydroxylase (SEQ ID No:2) from spearmint (Mentha spicata),
 or the sequence designated SEQ ID No:3 which encodes
 (-)-limonene-3-hydroxylase (SEQ ID No:4) from peppermint (Mentha
 piperita), or the sequence designated SEQ ID No:5 which encodes another
 (-)-limonene-3-hydroxylase (SEQ ID No:6) from peppermint (Mentha
 piperita). In other aspects, the present invention is directed to
 replicable recombinant cloning vehicles comprising a nucleic acid
 sequence, e.g., a DNA sequence, which codes for limonene hydroxylases or
 for a base sequence sufficiently complementary to at least a portion of
 the limonene hydroxylase DNA or RNA to enable hybridization therewith
 (e.g., antisense limonene hydroxylase RNA or fragments of complementary
 limonene hydroxylase DNA which are useful as polymerase chain reaction
 primers or as probes for limonene hydroxylases or related genes). In yet
 other aspects of the invention, modified host cells are provided that have
 been transformed, transfected, infected and/or injected with a recombinant
 cloning vehicle and/or DNA sequence of the invention. Thus, the present
 invention provides for the recombinant expression of limonene
 hydroxylases, and the inventive concepts may be used to facilitate the
 production, isolation and purification of significant quantities of
 recombinant limonene hydroxylase (or of the primary enzyme products,
 trans-carveol in the case of (-)-limonene-6-hydroxylase or
 trans-isopiperitenol in the case of (-)-limonene-3-hydroxylase) for
 subsequent use, to obtain expression, or enhanced expression, or altered
 expression, of limonene hydroxylase in plants to attain enhanced
 trans-carveol or trans-isopiperitenol production as a predator or pathogen
 defense mechanism, attractant or environmental signal, or may be otherwise
 employed in an environment where the regulation or expression of limonene
 hydroxylase is desired for the production of limonene hydroxylase or the
 enzyme products, trans-carveol or trans-isopiperitenol, or their
 derivatives.
 In another aspect, the present invention is directed to isolated nucleic
 acid molecules that hybridize under stringent hybridization conditions to
 a fragment (having a length of at least 15 bases) of any one of the
 nucleic acid molecules of the present invention encoding a
 limonene-3-hydroxylase or limonene-6-hydroxylase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
 As used herein, the terms "amino acid" and "amino acids" refer to all
 naturally occurring L-.alpha.-amino acids or their residues. The amino
 acids are identified by either the single-letter or three-letter
 designations:

Asp D aspartic acid
 Thr T threonine
 Ser S serine
 Glu E glutamic acid
 Pro P proline
 Gly G glycine
 Ala A alanine
 Cys C cysteine
 Val V valine
 Met M methionine
 Ile I isoleucine
 Leu L leucine
 Tyr Y tyrosine
 Phe F phenylalanine
 His H histidine
 Lys K lysine
 Arg R arginine
 Trp W tryptophan
 Gln Q glutamine
 Asn N asparagine
 As used herein, the term "nucleotide" means a monomeric unit of DNA or RNA
 containing a sugar moiety (pentose), a phosphate and a nitrogenous
 heterocyclic base. The base is linked to the sugar moiety via the
 glycosidic carbon (1' carbon of pentose) and that combination of base and
 sugar is called a nueleoside. The base characterizes the nucleotide with
 the four bases of DNA being adenine ("A"), guanine ("G"), cytosine ("C"),
 thymine ("T") and inosine ("I"). The four RNA bases are A,G,C and uracil
 ("U"). The nucleotide sequences described herein comprise a line array of
 nucleotides connected by phosphodiester bonds between the 3' and 5'
 carbons of adjacent pentoses.
 "Oligonucleotide" refers to short length single or double stranded
 sequences of deoxyribonucleotides linked via phosphodiester bonds. The
 oligonucleotides are chemically synthesized by known methods and purified
 on polyacrylamide gels.
 The term "limonene hydroxylase" is used herein to mean an enzyme capable of
 catalyzing the hydroxylation of limonene to its hydroxylated products,
 such as trans-carveol in the case of (-)-limonene-6-hydroxylase or
 trans-isopiperitenol in the case of (-)-limonene-3-hydroxylase, as
 described herein.
 The terms "alteration", "amino acid sequence alteration", "variant" and
 "amino acid sequence variant" refer to limonene hydroxylase molecules with
 some differences in their amino acid sequences as compared to native
 limonene hydroxylase. Ordinarily, the variants will possess at least about
 70% homology with native limonene hydroxylase, and preferably, they will
 be at least about 80% homologous with native limonene hydroxylase. The
 amino acid sequence variants of limonene hydroxylase falling within this
 invention possess substitutions, deletions, and/or insertions at certain
 positions. Sequence variants of limonene hydroxylase may be used to attain
 desired enhanced or reduced enzymatic activity, modified regiochemistry or
 stereochemistry, or altered substrate utilization or product distribution
 such as enhanced production of other products obtained from alternative
 substrates, such as those shown in FIG. 2.
 Substitutional limonene hydroxylase variants are those that have at least
 one amino acid residue in the native limonene hydroxylase sequence removed
 and a different amino acid inserted in its place at the same position. The
 substitutions may be single, where only one amino acid in the molecule has
 been substituted, or they may be multiple, where two or more amino acids
 have been substituted in the same molecule. Substantial changes in the
 activity of the limonene hydroxylase molecule may be obtained by
 substituting an amino acid with a side chain that is significantly
 different in charge and/or structure from that of the native amino acid.
 This type of substitution would be expected to affect the structure of the
 polypeptide backbone and/or the charge or hydrophobicity of the molecule
 in the area of the substitution.
 Moderate changes in the activity of the limonene hydroxylase molecule would
 be expected by substituting an amino acid with a side chain that is
 similar in charge and/or structure to that of the native molecule. This
 type of substitution, referred to as a conservative substitution, would
 not be expected to substantially alter either the structure of the
 polypeptide backbone or the charge or hydrophobicity of the molecule in
 the area of the substitution.
 Insertional limonene hydroxylase variants are those with one or more amino
 acids inserted immediately adjacent to an amino acid at a particular
 position in the native limonene hydroxylase molecule. Immediately adjacent
 to an amino acid means connected to either the (.alpha.-carboxy or
 .alpha.-amino functional group of the amino acid. The insertion may be one
 or more amino acids. Ordinarily, the insertion will consist of one or two
 conservative amino acids. Amino acids similar in charge and/or structure
 to the amino acids adjacent to the site of insertion are defined as
 conservative. Alternatively, this invention includes insertion of an amino
 acid with a charge and/or structure that is substantially different from
 the amino acids adjacent to the site of insertion.
 Deletional variants are those where one or more amino acids in the native
 limonene hydroxylase molecule have been removed. Ordinarily, deletional
 variants will have one or two amino acids deleted in a particular region
 of the limonene hydroxylase molecule.
 The terms "biological activity", "biologically active", "activity" and
 "active" refer to the ability of the limonene hydroxylase molecule to
 convert (-)-limonene to carveol and isopiperitenol and co-products as
 measured in an enzyme activity assay, such as the assay described in
 Example 7 below. Amino acid sequence variants of limonene hydroxylase may
 have desirable altered biological activity including, for example, altered
 reaction kinetics, substrate utilization product distribution or other
 characteristics such as regiochemistry and stereochemistry.
 The terms "DNA sequence encoding", "DNA encoding" and "nucleic acid
 encoding" 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 translated polypeptide
 chain. The DNA sequence thus codes for the amino acid sequence.
 The term "essential oil plant," or "essential oil plants," refers to a
 group of plant species that produce high levels of monoterpenoid and/or
 sesquiterpenoid and/or diterpenoid oils, and/or high levels of
 monoterpenoid and/or sesquiterpenoid and/or diterpenoid resins. The
 foregoing oils and/or resins account for greater than about 0.005% of the
 fresh weight of an essential oil plant that produces them. The essential
 oils and/or resins are more fully described, for example, in E. Guenther,
 The Essential Oils, Vols. I-VI, R. E. Krieger Publishing Co., Huntington
 N.Y, 1975, incorporated herein by reference. The essential oil plants
 include, but are not limited to:
 Lamiaceae, including, but not limited to, the following species: Ocimum
 (basil), Lavandula (Lavender), Origanum (oregano), Mentha (mint), Salvia
 (sage), Rosmecinus (rosemary), Thymus (thyme), Satureja and Monarda.
 Umbelliferae, including, but not limited to, the following species: Carum
 (caraway), Anethum (dill), feniculum (fennel) and Daucus (carrot).
 Asteraceae (Compositae), including, but not limited to, the following
 species: Artemisia (tarragon, sage brush), Tanacetum (tansy).
 Rutaceae (e.g., citrus plants); Rosaceae (e.g., roses); Myrtaceae (e.g.,
 eucalyptus, Melaleuca); the Gramineae (e.g. Cymbopogon (citronella));
 Geranaceae (Geranium) and certain conifers including Abies (e.g., Canadian
 balsam), Cedrus (cedar) and Thuja and Juniperus.
 The range of essential oil plants is more fully set forth in E. Guenther,
 The Essential Oils, Vols. I-VI, R. E. Krieger Publishing Co., Huntington
 N.Y, 1975, which is incorporated herein by reference.
 The term "percent identity" means the percentage of amino acids or
 nucleotides that occupy the same relative position when two amino acid
 sequences, or two nucleic acid sequences, are aligned side by side.
 The term "percent similarity" is a statistical measure of the degree of
 relatedness of two compared protein sequences. The percent similarity is
 calculated by the GAP program from GCG Version 9.0 (1996) which utilizes
 the algorithm of Needleman S. B. and Wunsch C. D., J. Mol. Biol. 48:
 443-453 (1970), incorporated herein by reference.
 The terms "replicable expression vector" and "expression vector" refer to a
 piece of DNA, usually double-stranded, which may have inserted into it a
 piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is
 DNA not naturally found in the host. The vector is used to transport the
 foreign or heterologous DNA into a suitable host cell. Once in the host
 cell, the vector can replicate independently of or coincidental with the
 host chromosomal DNA, and several copies of the vector and its inserted
 (foreign) DNA may be generated. In addition, the vector contains the
 necessary elements that permit translating the foreign DNA into a
 polypeptide. Many molecules of the polypeptide encoded by the foreign DNA
 can thus be rapidly synthesized.
 The terms "transformed host cell" and "transformed" refer to the
 introduction of DNA into a cell. The cell is termed a "host cell", and it
 may be a prokaryotic or a eukaryotic cell. Typical prokaryotic host cells
 include various strains of E. coli. Typical eukaryotic host cells are
 plant cells, such as maize cells, yeast cells, insect cells or animal
 cells. The introduced DNA is usually in the form of a vector containing an
 inserted piece of DNA. The introduced DNA sequence may be from the same
 species as the host cell or a different species from the host cell, or it
 may be a hybrid DNA sequence, containing some foreign and some homologous
 DNA.
 In accordance with the present invention, cDNA encoding limonene
 hydroxylase was isolated and sequenced in the following manner.
 (-)-Limonene hydroxylase is located exclusively in the glandular trichome
 secretory cells and catalyzes the hydroxylation of (-)-limonene in these
 essential oil species. Known methods for selectively isolating secretory
 cell clusters from these epidermal oil glands and for extracting these
 structures were employed to obtain sufficient amounts of light membranes
 (microsomes). The light membranes were solubilized and the resulting
 protein subjected to hydrophobic interaction chromatography which served
 to purify a spectrally characterized (Omura et al., J. Biol. Chem.
 239:2379-2385 [1964]) cytochrome P450 enzyme from spearmint secretory
 glands. This approach, however, does not differentiate between
 enzymatically distinct cytochrome P450 species. Amino acid sequence
 information derived from the purified protein was employed in a molecular
 approach to the isolation of gland specific cDNA clones encoding such
 cytochromes. Following isolation and sequencing of the cytochrome P450
 cDNA (SEQ ID No:1) from spearmint, functional expression was required to
 confirm the catalytic identity of the enzyme encoded. A
 Spodoptera-Baculovirus expression system, combined with the in situ
 bioassay (feeding (-)-limonene substrate during recombinant protein
 expression), successfully confirmed that the target clone
 (limonene-6-hydroxylase) had been isolated. Sequence information from the
 full length spearmint limonene hydroxylase cDNA (SEQ ID No:1) was utilized
 to construct a selective probe for the isolation of the related
 (-)-limonene-3-hydroxylase gene (SEQ ID No:3) from peppermint secretory
 glands. Functional expression in the Spodoptera-Baculovirus expression
 system, by in situ bioassay, also confirmed the peppermint
 limonene-3-hydroxylase clone (SEQ ID No:3), which was fully sequenced.
 Sequence comparison showed the two regiospecific hydroxylases from
 spearmint and peppermint to be very similar, as expected, since spearmint
 (M. spicata) is a tetraploid and parent of peppermint (M. piperita=Mentha
 aquatica.times.spicata), a hexaploid (Harley and Brighton, Bot. J. Linn.
 Soc. 74:71-96 [1977]). In vitro studies confirmed the recombinant enzymes
 to resemble their native counterparts. An additional
 limonene-3-hydroxylase cDNA clone (SEQ ID No:5) was isolated from a
 peppermint cDNA library.
 The isolation of the limonene hydroxylase cDNAs (SEQ ID No:1; SEQ ID No:3;
 SEQ ID No:5) permits the development of an efficient expression system for
 these functional enzymes with which detailed mechanistic structural
 studies can be undertaken. The limonene hydroxylase cDNAs (SEQ ID No:1;
 SEQ ID No:3; SEQ ID No:5) also provide useful tools for isolating other
 monoterpene hydroxylase genes and for examining the developmental
 regulation of monoterpene biosynthesis.
 Although the limonene hydroxylase cDNA set forth in SEQ ID No:1 directs the
 enzyme to plastids, substitution of the targeting sequence (SEQ ID No:1,
 nucleotides 20 to 146) with other transport sequences well known in the
 art (see, e.g., Keegstra et al., supra; von Heijne et al., supra) may be
 employed to direct the limonene hydroxylase to other cellular or
 extracellular locations.
 In addition to the native (-)-limonene-6-hydroxylase amino acid sequence of
 SEQ ID No:2, encoded by the DNA sequence of SEQ ID No:1, and the native
 (-)-limonene-3-hydroxylase amino acid sequences of SEQ ID No:4 and SEQ ID
 No:6, encoded by the DNA sequences set forth in SEQ ID No:3 and SEQ ID
 No:5, respectively, sequence variants produced by deletions,
 substitutions, mutations and/or insertions are intended to be within the
 scope of the invention except insofar as limited by the prior art. The
 limonene hydroxylase amino acid sequence variants of this invention may be
 constructed by mutating the DNA sequence that encodes wild-type limonene
 hydroxylase, such as by using techniques commonly referred to as
 site-directed mutagenesis. Various polymerase chain reaction (PCR) methods
 now well known in the field, such as a two primer system like the
 Transformer Site-Directed Mutagenesis kit from Clontech, may be employed
 for this purpose.
 Following denaturation of the target plasmid in this system, two primers
 are simultaneously annealed to the plasmid; one of these primers contains
 the desired site-directed mutation, the other contains a mutation at
 another point in the plasmid resulting in elimination of a restriction
 site. Second strand synthesis is then carried out, tightly linking these
 two mutations, and the resulting plasmids are transformed into a mutS
 strain of E. coli. Plasmid DNA is isolated from the transformed bacteria,
 restricted with the relevant restriction enzyme (thereby linearizing the
 unmutated plasmids), and then retransformed into E. coli. This system
 allows for generation of mutations directly in an expression plasmid,
 without the necessity of subcloning or generation of single-stranded
 phagemids. The tight linkage of the two mutations and the subsequent
 linearization of unmutated plasmids results in high mutation efficiency
 and allows minimal screening. Following synthesis of the initial
 restriction site primer, this method requires the use of only one new
 primer type per mutation site. Rather than prepare each positional mutant
 separately, a set of "designed degenerate" oligonucleotide primers can be
 synthesized in order to introduce all of the desired mutations at a given
 site simultaneously. Transformants can be screened by sequencing the
 plasmid DNA through the mutagenized region to identify and sort mutant
 clones. Each mutant DNA can then be restricted and analyzed by
 electrophoresis on Mutation Detection Enhancement gel (J. T. Baker) to
 confirm that no other alterations in the sequence have occurred (by band
 shift comparison to the unmutagenized control).
 In the case of the hydrophobic cleft of the hydroxylases, a number of
 residues may be mutagenized in this region. Directed mutagenesis can also
 be used to create cassettes for saturation mutagenesis. Once a hydrophobic
 segment of the active site is identified, oligonucleotide-directed
 mutagenesis can be used to create unique restriction sites flanking that
 region to allow for the removal of the cassette and the subsequent
 replacement with synthetic cassettes containing any number of mutations
 within. This approach can be carried out with any plasmid, without need
 for subcloning or generation of single-stranded phagemids.
 The verified mutant duplexes in the pET (or other) overexpression vector
 can be employed to transform E. coli such as strain E. coli
 BL21(DE3)pLysS, for high level production of the mutant protein, and
 purification by metal ion affinity chromatography and thrombin
 proteolysis. The method of FAB-MS mapping can be employed to rapidly check
 the fidelity of mutant expression. This technique provides for sequencing
 segments throughout the whole protein and provides the necessary
 confidence in the sequence assignment. In a mapping experiment of this
 type, protein is digested with a protease (the choice will depend on the
 specific region to be modified since this segment is of prime interest and
 the remaining map should be identical to the map of unmutagenized
 protein). The set of cleavage fragments is fractionated by microbore HPLC
 (reversed phase or ion exchange, again depending on the specific region to
 be modified) to provide several peptides in each fraction, and the
 molecular weights of the peptides are determined by FAB-MS. The masses are
 then compared to the molecular weights of peptides expected from the
 digestion of the predicted sequence, and the correctness of the sequence
 quickly ascertained. Since this mutagenesis approach to protein
 modification is directed, sequencing of the altered peptide should not be
 necessary if the MS agrees with prediction. If necessary to verify a
 changed residue, CAD-tandem MS/MS can be employed to sequence the peptides
 of the mixture in question, or the target peptide purified for subtractive
 Edman degradation or carboxypeptidase Y digestion depending on the
 location of the modification.
 In the design of a particular site directed mutagenesis, it is generally
 desirable to first make a non-conservative substitution (e.g., Ala for
 Cys, His or Glu) and determine if activity is greatly impaired as a
 consequence. The properties of the mutagenized protein are then examined
 with particular attention to the kinetic parameters of K.sub.m and
 k.sub.cat as sensitive indicators of altered function, from which changes
 in binding and/or catalysis per se may be deduced by comparison to the
 native cyclase. If the residue is by this means demonstrated to be
 important by activity impairment, or knockout, then conservative
 substitutions can be made, such as Asp for Glu to alter side chain length,
 Ser for Cys, or Arg for His. For hydrophobic segments, it is largely size
 that we will alter, although aromatics can also be substituted for alkyl
 side chains. Changes in the normal product distribution can indicate which
 step(s) of the reaction sequence have been altered by the mutation.
 Modification of the hydrophobic pocket can be employed to change binding
 conformations for substrates and result in altered regiochemistry and/or
 stereochemistry.
 Other site directed mutagenesis techniques may also be employed with the
 nucleotide sequences of the invention. For example, restriction
 endonuclease digestion of DNA followed by ligation may be used to generate
 limonene hydroxylase deletion variants, as described in section 15.3 of
 Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold
 Spring Harbor Laboratory Press, New York, N.Y. [1989]). A similar strategy
 may be used to construct insertion variants, as described in section 15.3
 of Sambrook et al., supra.
 Oligonucleotide-directed mutagenesis may also be employed for preparing
 substitution variants of this invention. It may also be used to
 conveniently prepare the deletion and insertion variants of this
 invention. This technique is well known in the art as described by Adelman
 et al. (DNA 2:183 [1983]). Generally, oligonucleotides of at least 25
 nucleotides in length are used to insert, delete or substitute two or more
 nucleotides in the limonene hydroxylase molecule. An optimal
 oligonucleotide will have 12 to 15 perfectly matched nucleotides on either
 side of the nucleotides coding for the mutation. To mutagenize the
 wild-type limonene hydroxylase, the oligonucleotide is annealed to the
 single-stranded DNA template molecule under suitable hybridization
 conditions. A DNA polymerizing enzyme, usually the Klenow fragment of E.
 coli DNA polymerase I, is then added. This enzyme uses the oligonucleotide
 as a primer to complete the synthesis of the mutation-bearing strand of
 DNA. Thus, a heteroduplex molecule is formed such that one strand of DNA
 encodes the wild-type limonene hydroxylase inserted in the vector, and the
 second strand of DNA encodes the mutated form of limonene hydroxylase
 inserted into the same vector. This heteroduplex molecule is then
 transformed into a suitable host cell.
 Mutants with more than one amino acid substituted may be generated in one
 of several ways. If the amino acids are located close together in the
 polypeptide chain, they may be mutated simultaneously using one
 oligonucleotide that codes for all of the desired amino acid
 substitutions. If however, the amino acids are located some distance from
 each other (separated by more than ten amino acids, for example) it is
 more difficult to generate a single oligonucleotide that encodes all of
 the desired changes. Instead, one of two alternative methods may be
 employed. In the first method, a separate oligonucleotide is generated for
 each amino acid to be substituted. The oligonucleotides are then annealed
 to the single-stranded template DNA simultaneously, and the second strand
 of DNA that is synthesized from the template will encode all of the
 desired amino acid substitutions. An alternative method involves two or
 more rounds of mutagenesis to produce the desired mutant. The first round
 is as described for the single mutants: wild-type limonene hydroxylase DNA
 is used for the template, an oligonucleotide encoding the first desired
 amino acid substitution(s) is annealed to this template, and the
 heteroduplex DNA molecule is then generated. The second round of
 mutagenesis utilizes the mutated DNA produced in the first round of
 mutagenesis as the template. Thus, this template already contains one or
 more mutations. The oligonucleotide encoding the additional desired amino
 acid substitution(s) is then annealed to this template, and the resulting
 strand of DNA now encodes mutations from both the first and second rounds
 of mutagenesis. This resultant DNA can be used as a template in a third
 round of mutagenesis, and so on.
 The genes encoding the (-)-limonene hydroxylase enzymes may be incorporated
 into any organism (intact plant, animal, microbe or cell culture, etc.)
 that produces limonene (either as a native property or via transgenic
 manipulation of one or more of the enzymes involved in limonene synthesis,
 such as limonene synthase) to affect the conversion of limonene to carveol
 or isopiperitenol (and their subsequent metabolites, depending on the
 organism) to produce or modify flavor and aroma properties, to improve
 defense capability, or to alter other ecological interactions mediated by
 these metabolites or for the production of the metabolites themselves. The
 expressed hydroxylases may also be used outside of living cells as a
 reagent to catalyze the corresponding oxidations of limonene in vitro.
 Since (+)-limonene also serves as a substrate for these hydroxylases
 (albeit less efficiently, see FIG. 2), the methods and recombinant enzymes
 of the present invention are useful for the production of all
 stereoisomeric products derived by either C3- or C6-hydroxlyation of (+)-
 or (-)-limonene or related compounds.
 Eukaryotic expression systems are commonly employed for cytochrome P450
 expression since they carry out any required posttranslational
 modifications, direct the enzyme to the proper membrane location, and
 possess a compatible reductase to deliver electrons to the cytochrome. A
 representative eucaryotic expression system for this purpose uses the
 recombinant baculovirus, Autographa californica nuclear polyhedrosis virus
 (AcNPV; M. D. Summers and G. E. Smith, A Manual of Methods for Baculovirus
 Vectors and Insect Cell Culture Procedures [1986]; Luckow et al.,
 Bio-technology 6:47-55 [1987]) for expression of the limonene hydroxylases
 of the invention. Infection of insect cells (such as cells of the species
 Spodoptera frugiperda) with the recombinant baculoviruses allows for the
 production of large amounts of the limonene hydroxylase protein. In
 addition, the baculovirus system has other important advantages for the
 production of recombinant limonene hydroxylase. For example, baculoviruses
 do not infect humans and can therefore be safely handled in large
 quantities. In the baculovirus system, a DNA construct is prepared
 including a DNA segment encoding limonene hydroxylase and a vector. The
 vector may comprise the polyhedron gene promoter region of a baculovirus,
 the baculovirus flanking sequences necessary for proper cross-over during
 recombination (the flanking sequences comprise about 200-300 base pairs
 adjacent to the promoter sequence) and a bacterial origin of replication
 which permits the construct to replicate in bacteria. The vector is
 constructed so that (i) the DNA segment is placed adjacent (or operably
 linked or "downstream" or "under the control of") to the polyhedron gene
 promoter and (ii) the promoter/limonene hydroxylase combination is flanked
 on both sides by 200-300 base pairs of baculovirus DNA (the flanking
 sequences).
 To produce the limonene hydroxylase DNA construct, a cDNA clone encoding
 the full length limonene hydroxylase is obtained using methods such as
 those described herein. The DNA construct is contacted in a host cell with
 baculovirus DNA of an appropriate baculovirus (that is, of the same
 species of baculovirus as the promoter encoded in the construct) under
 conditions such that recombination is effected. The resulting recombinant
 baculoviruses encode the full limonene hydroxylase. For example, an insect
 host cell can be cotransfected or transfected separately with the DNA
 construct and a functional baculovirus. Resulting recombinant
 baculoviruses can then be isolated and used to infect cells to effect
 production of the limonene hydroxylase. Host insect cells include, for
 example, Spodoptera frugiperda cells, that are capable of producing a
 baculovirus-expressed limonene hydroxylase. Insect host cells infected
 with a recombinant baculovirus of the present invention are then cultured
 under conditions allowing expression of the baculovirus-encoded limonene
 hydroxylase. Limonene hydroxylase thus produced is then extracted from the
 cells using methods known in the art. For a detailed description of the
 use of the baculovirus/Spodoptera expression system, see Examples 5 and 6,
 infra.
 Other eukaryotic microbes such as yeasts may also be used to practice this
 invention. The baker's yeast Saccharomyces cerevisiae, is a commonly used
 yeast, although several other strains are available. The plasmid YRp7
 (Stinchcomb et al., Nature 282:39 [1979]; Kingsman et al., Gene 7:141
 [1979]; Tschemper et al., Gene 10:157 [1980]) is commonly used as an
 expression vector in Saccharomyces. This plasmid contains the trp1 gene
 that provides a selection marker for a mutant strain of yeast lacking the
 ability to grow in tryptophan, such as strains ATCC No. 44,076 and PEP4-1
 (Jones, Genetics 85:12 [1977]). The presence of the trp1 lesion as a
 characteristic of the yeast host cell genome then provides an effective
 environment for detecting transformation by growth in the absence of
 tryptophan. Yeast host cells are generally transformed using the
 polyethylene glycol method, as described by Hinnen (Proc. Natl. Acad. Sci.
 USA 75:1929 [1978]).
 Suitable promoting sequences in yeast vectors include the promoters for
 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073
 [1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.
 7:149 [1968]; Holland et al., Biochemistry 17:4900 [1978]), such as
 enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
 decarboxylase, phosphofructokinase glucose-6-phosphate isomerase,
 3-phosphoglycerate mutase, pyruvate kinase, triose-phosphate isomerase,
 phosphoglucose isomerase, and glucokinase. In the construction of suitable
 expression plasmids, the termination sequences associated with these genes
 are also ligated into the expression vector 3' of the sequence desired to
 be expressed to provide polyadenylation of the mRNA and termination. Other
 promoters that have the additional advantage of transcription controlled
 by growth conditions are the promoter region for alcohol dehydrogenase 2,
 isocytochrome C, acid phosphatase, degradative enzymes associated with
 nitrogen metabolism, and the aforementioned glyceraldehyde-3 -phosphate
 dehydrogenase, and enzymes responsible for maltose and galactose
 utilization. Any plasmid vector containing yeast-compatible promoter,
 origin of replication and termination sequences is suitable.
 Cell cultures derived from multicellular organisms and multicellular
 organisms, such as plants, may be used as hosts to practice this
 invention. For example, transgenic plants can be obtained such as by
 transferring plasmids that encode limonene hydroxylase and a selectable
 marker gene, e.g., the kan gene encoding resistance to kanamycin, into
 Agrobacterium tumifaciens containing a helper Ti plasmid as described in
 Hoeckema et al., Nature 303:179-181 [1983] and culturing the Agrobacterium
 cells with leaf slices of the plant to be transformed as described by An
 et al., Plant Physiology 81:301-305 [1986]. Transformation of cultured
 plant host cells is normally accomplished through Agrobacterium
 tumifaciens, as described above. Cultures of mammalian host cells and
 other host cells that do not have rigid cell membrane barriers are usually
 transformed using the calcium phosphate method as originally described by
 Graham and Van der Eb (Virology 52:546 [1978]) and modified as described
 in sections 16.32-16.37 of Sambrook et al., supra. However, other methods
 for introducing DNA into cells such as Polybrene (Kawai and Nishizawa,
 Mol. Cell. Biol. 4:1172 [1984]), protoplast fusion (Schaffner, Proc. Natl.
 Acad. Sci. USA 77:2163 [1980]), electroporation (Neumann et al., EMBO J.
 1:841 [1982]), and direct microinjection into nuclei (Capecchi, Cell
 22:479 [1980]) may also be used. Transformed plant calli may be selected
 through the selectable marker by growing the cells on a medium containing
 e.g., kanamycin, and appropriate amounts of phytohormone such as
 naphthalene acetic acid and benzyladenine for callus and shoot induction.
 The plant cells may then be regenerated and the resulting plants
 transferred to soil using techniques well known to those skilled in the
 art.
 In addition, a gene regulating limonene hydroxylase production can be
 incorporated into the plant along with a necessary promoter which is
 inducible. In the practice of this embodiment of the invention, a promoter
 that only responds to a specific external or internal stimulus is fused to
 the target cDNA. Thus the gene will not be transcribed except in response
 to the specific stimulus. As long as the gene is not being transcribed,
 its gene product is not produced (nor is the corresponding hydroxylation
 product of limonene).
 An illustrative example of a responsive promoter system that can be used in
 the practice of this invention is the glutathione-S-transferase (GST)
 system in maize. GSTs are a family of enzymes that can detoxify a number
 of hydrophobic electrophilic compounds that often are used as pre-emergent
 herbicides (Weigand et al., Plant Molecular Biology 7:235-243 [1986]).
 Studies have shown that the GSTs are directly involved in causing this
 enhanced herbicide tolerance. This action is primarily mediated through a
 specific 1.1 kb mRNA transcription product. In short, maize has a
 naturally occurring quiescent gene already present that can respond to
 external stimuli and that can be induced to produce a gene product. This
 gene has previously been identified and cloned. Thus, in one embodiment of
 this invention, the promoter is removed from the GST responsive gene and
 attached to a limonene hydroxylase gene that previously has had its native
 promoter removed. This engineered gene is the combination of a promoter
 that responds to an external chemical stimulus and a gene responsible for
 successful production of limonene hydroxylase.
 In addition to the methods described above, several methods are known in
 the art for transferring cloned DNA into a wide variety of plant species,
 including gymnosperms, angiosperms, monocots and dicots (see, e.g., Glick
 and Thompson, eds., Methods in Plant Molecular Biology, CRC Press, Boca
 Raton, Fla. [1993], incorporated by reference herein). Representative
 examples include electroporation-facilitated DNA uptake by protoplasts in
 which an electrical pulse transiently permeabilizes cell membranes,
 permitting the uptake of a variety of biological molecules, including
 recombinant DNA (Rhodes et al., Science, 240(4849):204-207 [1988]);
 treatment of protoplasts with polyethylene glycol (Lyznik et al., Plant
 Molecular Biology, 13:151-161 [1989]); and bombardment of cells with
 DNA-laden microprojectiles which are propelled by explosive force or
 compressed gas to penetrate the cell wall (Klein et al., Plant Physiol.
 91:440-444 [1989] and Boynton et al., Science, 240(4858):1534-1538
 [1988]). Transformation of gymnosperm species can be achieved, for
 example, by employing the methods set forth in Han et al, Plant Science,
 95:187-196 (1994), incorporated by reference herein. A method that has
 been applied to Rye plants (Secale cereale) is to directly inject plasmid
 DNA, including a selectable marker gene, into developing floral tillers
 (de la Pena et al., Nature 325:274-276 (1987)). Further, plant viruses can
 be used as vectors to transfer genes to plant cells. Examples of plant
 viruses that can be used as vectors to transform plants include the
 Cauliflower Mosaic Virus (Brisson et al., Nature 310: 511-514 (1984)).
 Additionally, plant transformation strategies and techniques are reviewed
 in Birch, R. G., Ann Rev Plant Phys Plant Mol Biol, 48:297 (1997);
 Forester et al., Exp. Agric., 33:15-33 (1997). The aforementioned
 publications disclosing plant transformation techniques are incorporated
 herein by reference, and minor variations make these technologies
 applicable to a broad range of plant species.
 Each of these techniques has advantages and disadvantages. In each of the
 techniques, DNA from a plasmid is genetically engineered such that it
 contains not only the gene of interest, but also selectable and screenable
 marker genes. A selectable marker gene is used to select only those cells
 that have integrated copies of the plasmid (the construction is such that
 the gene of interest and the selectable and screenable genes are
 transferred as a unit). The screenable gene provides another check for the
 successful culturing of only those cells carrying the genes of interest. A
 commonly used selectable marker gene is neomycin phosphotransferase II
 (NPT II). This gene conveys resistance to kanamycin, a compound that can
 be added directly to the growth media on which the cells grow. Plant cells
 are normally susceptible to kanamycin and, as a result, die. The presence
 of the NPT II gene overcomes the effects of the kanamycin and each cell
 with this gene remains viable. Another selectable marker gene which can be
 employed in the practice of this invention is the gene which confers
 resistance to the herbicide glufosinate (Basta). A screenable gene
 commonly used is the .beta.-glucuronidase gene (GUS). The presence of this
 gene is characterized using a histochemical reaction in which a sample of
 putatively transformed cells is treated with a GUS assay solution. After
 an appropriate incubation, the cells containing the GUS gene turn blue.
 Another screenable gene is a transcriptional activator for anthocyanin
 biosynthesis, as described in the copending application of Bowen et al.,
 U.S. patent application Ser. No. 387,739, filed Aug. 1, 1989. This gene
 causes the synthesis of the pigment anthocyanin. Cells transformed with a
 plasmid containing this gene turn red. Preferably, the plasmid will
 contain both selectable and screenable marker genes.
 The plasmid containing one or more of these genes is introduced into either
 plant protoplasts or callus cells by any of the previously mentioned
 techniques. If the marker gene is a selectable gene, only those cells that
 have incorporated the DNA package survive under selection with the
 appropriate phytotoxic agent. Once the appropriate cells are identified
 and propagated, plants are regenerated. Progeny from the transformed
 plants must be tested to insure that the DNA package has been successfully
 integrated into the plant genome.
 Mammalian host cells may also be used in the practice of the invention.
 Examples of suitable mammalian cell lines include monkey kidney CVI line
 transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line
 293S (Graham et al., J. Gen. Virol. 36:59 [1977]); baby hamster kidney
 cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (Urlab and Chasin,
 Proc. Natl. Acad. Sci USA 77:4216 [1980]); mouse sertoli cells (TM4,
 Mather, Biol. Reprod. 23:243 [1980]); monkey kidney cells (CVI-76, ATCC
 CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human
 cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,
 ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung
 cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
 mammary tumor cells (MMT 060562, ATCC CCL 51); rat hepatoma cells (HTC,
 MI.54, Baumann et al., J. Cell Biol. 85:1 [1980]); and TRI cells (Mather
 et al., Annals N.Y. Acad. Sci. 383:44 [1982]). Expression vectors for
 these cells ordinarily include (if necessary) DNA sequences for an origin
 of replication, a promoter located in front of the gene to be expressed, a
 ribosome binding site, an RNA splice site, a polyadenylation site, and a
 transcription terminator site.
 Promoters used in mammalian expression vectors are often of viral origin.
 These viral promoters are commonly derived from polyoma virus,
 Adenovirus2, and most frequently Simian Virus 40 (SV40). The SV40 virus
 contains two promoters that are termed the early and late promoters. These
 promoters are particularly useful because they are both easily obtained
 from the virus as one DNA fragment that also contains the viral origin of
 replication (Fiers et al., Nature 273:113 [1978]). Smaller or larger SV40
 DNA fragments may also used, provided they contain the approximately
 250-bp sequence extending from the HindIII site toward the BglI site
 located in the viral origin of replication.
 Alternatively, promoters that are naturally associated with the foreign
 gene (homologous promoters) may be used provided that they are compatible
 with the host cell line selected for transformation.
 An origin of replication may be obtained from an exogenous source, such as
 SV40 or other virus (e.g., Polyoma, Adeno, VSV, BPV) and inserted into the
 cloning vector. Alternatively, the origin of replication may be provided
 by the host cell chromosomal replication mechanism. If the vector
 containing the foreign gene is integrated into the host cell chromosome,
 the latter is often sufficient.
 Satisfactory amounts of limonene hydroxylase are produced by transformed
 cell cultures. However, the use of a secondary DNA coding sequence can
 enhance production levels. The secondary coding sequence typically
 comprises the enzyme dihydrofolate reductase (DHFR). The wild-type form of
 DHFR is normally inhibited by the chemical methotrexate (MTX). The level
 of DHFR expression in a cell will vary depending on the amount of MTX
 added to the cultured host cells. An additional feature of DHFR that makes
 it particularly useful as a secondary sequence is that it can be used as a
 selection marker to identify transformed cells. Two forms of DHFR are
 available for use as secondary sequences, wild-type DHFR and MTX-resistant
 DHFR. The type of DHFR used in a particular host cell depends on whether
 the host cell is DHFR deficient (such that it either produces very low
 levels of DHFR endogenously, or it does not produce functional DHFR at
 all). DHFR-deficient cell lines such as the CHO cell line described by
 Urlaub and Chasin, supra, are transformed with wild-type DHFR coding
 sequences. After transformation, these DHFR-deficient cell lines express
 functional DHFR and are capable of growing in a culture medium lacking the
 nutrients hypoxanthine, glycine and thymidine. Nontransformed cells will
 not survive in this medium.
 The MTX-resistant form of DHFR can be used as a means of selecting for
 transformed host cells in those host cells that endogenously produce
 normal amounts of functional DHFR that is MTX sensitive. The CHO-Kl cell
 line (ATCC No. CL 61) possesses these characteristics, and is thus a
 useful cell line for this purpose. The addition of MTX to the cell culture
 medium will permit only those cells transformed with the DNA encoding the
 MTX-resistant DHFR to grow. The nontransformed cells will be unable to
 survive in this medium.
 Prokaryotes may also be used as host cells for the initial cloning steps of
 this invention. They are particularly useful for rapid production of large
 amounts of DNA, for production of single-stranded DNA templates used for
 site-directed mutagenesis, for screening many mutants simultaneously, and
 for DNA sequencing of the mutants generated. Suitable prokaryotic host
 cells include E. coil K12 strain 294 (ATCC No. 31,446), E. coli strain
 W3110 (ATCC No. 27,325) E. Coli X1776 (ATCC No. 31,537), and E coli B;
 however many other strains of E. coli, such as HBO101, JM101, NM522,
 NM538, NM539, and many other species and genera of prokaryotes including
 bacilli such as Bacillus subtilis, other enterobacteriaceae such as
 Salmonella typhimurium or Serratia marcesans, and various Pseudomonas
 species may all be used as hosts. Prokaryotic host cells or other host
 cells with rigid cell walls are preferably transformed using the calcium
 chloride method as described in section 1.82 of Sambrook et al., supra.
 Alternatively, electroporation may be used for transformation of these
 cells.
 As a representative example, cDNA sequences encoding limonene hydroxylase
 may be transferred to the (His).sub.6. Tag pET vector commercially
 available (from Novagen) for overexpression in E Coli as heterologous
 host. This pET expression plasmid has several advantages in high level
 heterologous expression systems. The desired cDNA insert is ligated in
 frame to plasmid vector sequences encoding six histidines followed by a
 highly specific protease recognition site (thrombin) that are joined to
 the amino terminus codon of the target protein. The histidine "block" of
 the expressed fusion protein promotes very tight binding to immobilized
 metal ions and permits rapid purification of the recombinant protein by
 immobilized metal ion affinity chromatography. The histidine leader
 sequence is then cleaved at the specific proteolysis site by treatment of
 the purified protein within thrombin, and the limonene hydroxylase again
 purified by immobilized metal ion affinity chromatography, this time using
 a shallower imidazole gradient to elute the recombinant hydroxylase while
 leaving the histidine block still adsorbed. This
 overexpression-purification system has high capacity, excellent resolving
 power and is fast, and the chance of a contaminating E. coli protein
 exhibiting similar binding behavior (before and after thrombin
 proteolysis) is extremely small.
 As will be apparent to those skilled in the art, any plasmid vectors
 containing replicon and control sequences that are derived from species
 compatible with the host cell may also be used in the practice of the
 invention. The vector usually has a replication site, marker genes that
 provide phenotypic selection in transformed cells, one or more promoters,
 and a polylinker region containing several restriction sites for insertion
 of foreign DNA. Plasmids typically used for transformation of E. coli
 include pBR322, pUC18, pUC19, pUCI18, pUC119, and Bluescript M13, all of
 which are described in sections 1.12-1.20 of Sambrook et al., supra.
 However, many other suitable vectors are available as well. These vectors
 contain genes coding for ampicillin and/or tetracycline resistance which
 enables cells transformed with these vectors to grow in the presence of
 these antibiotics.
 The promoters most commonly used in prokaryotic vectors include the
 .beta.-lactamase (penicillinase) and lactose promoter systems (Chang et
 al. Nature 375:615 [1978]; Itakura et al., Science 198:1056 [1977];
 Goeddel et al., Nature 281:544 [1979]) and a tryptophan (trp) promoter
 system (Goeddel et al., Nucl. Acids Res. 8:4057 [1980]; EPO Appl. Publ.
 No. 36,776), and the alkaline phosphatase systems. While these are the
 most commonly used, other microbial promoters have been utilized, and
 details concerning their nucleotide sequences have been published,
 enabling a skilled worker to ligate them functionally into plasmid vectors
 (see Siebenlist et al., Cell 20:269 [1980]).
 Many eukaryotic proteins normally secreted from the cell contain an
 endogenous secretion signal sequence as part of the amino acid sequence.
 Thus, proteins normally found in the cytoplasm can be targeted for
 secretion by linking a signal sequence to the protein. This is readily
 accomplished by ligating DNA encoding a signal sequence to the 5' end of
 the DNA encoding the protein and then expressing this fusion protein in an
 appropriate host cell. The DNA encoding the signal sequence may be
 obtained as a restriction fragment from any gene encoding a protein with a
 signal sequence. Thus, prokaryotic, yeast, and eukaryotic signal sequences
 may be used herein, depending on the type of host cell utilized to
 practice the invention. The DNA and amino acid sequence encoding the
 signal sequence portion of several eukaryotic genes including, for
 example, human growth hormone, proinsulin, and proalbumin are known (see
 Stryer, Biochemistry W. H. Freeman and Company, New York, N.Y., p. 769
 [1988]), and can be used as signal sequences in appropriate eukaryotic
 host cells. Yeast signal sequences, as for example acid phosphatase (Arima
 et al., Nuc. Acids Res. 11:1657 [1983]), alpha-factor, alkaline
 phosphatase and invertase may be used to direct secretion from yeast host
 cells. Prokaryotic signal sequences from genes encoding, for example, LamB
 or OmpF (Wong et al., Gene 68:193 [1988]), MalE, PhoA, or beta-lactamase,
 as well as other genes, may be used to target proteins from prokaryotic
 cells into the culture medium.
 As described above, the limonene-6-hydroxylase amino terminal membrane
 insertion sequence resides at SEQ ID No:2, residues 1 through 42, and in
 the embodiment shown in SEQ ID No:2 directs the enzyme to endoplasmic
 reticulum membranes. Alternative trafficking sequences from plants,
 animals and microbes can be employed in the practice of the invention to
 direct the gene product to the cytoplasm, plastids, mitochondria or other
 cellular components, or to target the protein for export to the medium.
 These considerations apply to the overexpression of
 (-)-limonene-6-hydroxylase or (-)-limonene-3-hydroxylase, and to direction
 of expression within cells or intact organisms to permit gene product
 function in any desired location.
 The construction of suitable vectors containing DNA encoding replication
 sequences, regulatory sequences, phenotypic selection genes and the
 limonene hydroxylase DNA of interest are prepared using standard
 recombinant DNA procedures. Isolated plasmids and DNA fragments are
 cleaved, tailored, and ligated together in a specific order to generate
 the desired vectors, as is well known in the art (see, for example,
 Maniatis, supra, and Sambrook et al., supra).
 As discussed above, limonene hydroxylase variants are preferably produced
 by means of mutation(s) that are generated using the method of
 site-specific mutagenesis. This method requires the synthesis and use of
 specific oligonucleotides that encode both the sequence of the desired
 mutation and a sufficient number of adjacent nucleotides to allow the
 oligonucleotide to stably hybridize to the DNA template.
 The foregoing may be more fully understood in connection with the following
 representative examples, in which "Plasmids" are designated by a lower
 case p followed by an alphanumeric designation. The starting plasmids used
 in this invention are either commercially available, publicly available on
 an unrestricted basis, or can be constructed from such available plasmids
 using published procedures. In addition, other equivalent plasmids are
 known in the art and will be apparent to the ordinary artisan.
 "Digestion", "cutting" or "cleaving" of DNA refers to catalytic cleavage of
 the DNA with an enzyme that acts only at particular locations in the DNA.
 These enzymes are called restriction endonucleases, and the site along the
 DNA sequence where each enzyme cleaves is called a restriction site. The
 restriction enzymes used in this invention are commercially available and
 are used according to the instructions supplied by the manufacturers. (See
 also sections 1.60-1.61 and sections 3.38-3.39 of Sambrook et al., supra.)
 "Recovery" or "isolation" of a given fragment of DNA from a restriction
 digest means separation of the resulting DNA fragment on a polyacrylamide
 or an agarose gel by electrophoresis, identification of the fragment of
 interest by comparison of its mobility versus that of marker DNA fragments
 of known molecular weight, removal of the gel section containing the
 desired fragment, and separation of the gel from DNA. This procedure is
 known generally. For example, see Lawn et al. (Nucleic Acids Res.
 9:6103-6114 [1982]), and Goeddel et al. (Nucleic Acids Res., supra).
 The following examples merely illustrate the best mode now contemplated for
 practicing the invention, but should not be construed to limit the
 invention. All literature citations herein are expressly incorporated by
 reference.
 EXAMPLES
 Example 1
 Plant Material and Limonene-6-Hydroxylase Isolation
 Plant materials--Spearmint (Mentha spicata) plants were propagated from
 rhizomes or stem cuttings in peat moss:pumice:sand (58:35:10, v/v/v) and
 were grown in a greenhouse with supplemental lighting (16 h, 21,000 lux
 minimum) and a 30.degree./15.degree. C. (day/night) temperature cycle.
 Plants were watered as needed and fertilized daily with a complete
 fertilizer (N:P:K, 20:20:20) plus iron chelate and micronutrients. Apical
 buds of vegetative stems (3-7 weeks old) were used for the preparation of
 glandular trichome cells for enzyme extraction and for nucleic acid
 isolation. (-)-4S-Limonene (97%) and other monoterpene standards were part
 of the lab collection or were purchased from Sigma or Aldrich and were
 purified by standard chromatographic methods.
 Limonene-6-hydroxylase isolation--Limonene-6-hydroxylase was extracted from
 a purified preparation of glandular trichome secretory cell clusters
 isolated from spearmint (Mentha spicata). To obtain these clusters, plant
 material was soaked in ice-cold, distilled water for 1 h and gently
 abraded in a cell disrupter of our own design (Colby et al., J. Biol.
 Chem. 268:23016-23024 [1993]). Batches of 45-60 g of spearmint apical
 tissue were abraded in the 600 ml polycarbonate cell disruption chamber
 with 140 ml of glass beads (500 .mu.m diameter, Bio-Spec Products), 35 g
 Amberlite XAD-4 resin and .about.300 ml of extraction buffer consisting of
 (25 mM MOPSO, 0.5 mM sodium phosphate (pH 7.4), 200 mM sorbitol, 10 mM
 sucrose. 10 mM sodium-metabisulfite, 10 mM ascorbate, 1% (w/v)
 polyvinylpyrrolidone (M.sub.r 40,000), 0.6% methyl cellulose, and 1 mM
 DTT). Removal of glandular trichome secretory cells was accomplished by
 three 1 min pulses of operation with the rotor speed controlled by a
 rheostat set at 85-95 V. This procedure was carried out at 4.degree. C.,
 and after each pulse the chamber was allowed to cool for 1 min. The
 isolated secretory cell clusters were separated from the glass beads,
 XAD-4 resin and residual plant material by sieving through a series of
 nylon meshes. The secretory cell clusters (approximately 60 .mu.m in
 diameter) readily passed through meshes of 350 and 105 .mu.m and were
 collected on a mesh of 20 .mu.m. After filtration, cell clusters were
 washed to remove chloroplasts and other contaminates, and suspended in 50
 ml of cell disruption (sonication) buffer (100 mM sodium phosphate (pH
 7.4), 250 mM sucrose, 1 mM DTT, 1 mM PMSF, 1 mM sodium EDTA, and 5 .mu.M
 flavins (FAD and FMN)). Suspensions (50 ml) of isolated secretory cell
 clusters (.about.1.6.times.10.sup.6 cells/ml) were disrupted by sonication
 in the presence of 25% (v/v) XAD-4 resin and 0.5-0.9 g of
 Polyvinylpolypyrrolidone (added based on the level of phenolics observed
 during tissue harvesting) with the probe (Braun-Sonic 2000) at maximum
 power; five times for 15 see with 1 min cooling periods between each 15
 sec burst. After sonication, protein was extracted by gentle stirring at
 4.degree. C. for 20 min. The resulting extract was filtered through, and
 washed on, a 20 .mu.m nylon mesh on a Buchner funnel under vacuum to
 remove XAD-4 beads, PVPP, and cell debris. The resulting filtrate
 (.about.80 ml) was homogenized in a chilled Tenbroek glass homogenizer and
 brought to 100 ml with sonication buffer. The sonicate was then
 centrifuged at 18,000.times.g to remove cellular debris and the resulting
 supernatant was centrifuged at 195,000.times.g to yield the glandular
 microsomal fraction. Microsomal pellets prepared from gland sonicates
 (originating from 110 g of spearmint apical tissue) were resuspended and
 homogenized in 6 ml of solubilization buffer (25 mM Tris (pH 7.4), 30%
 glycerol, 1 mM DTT, 1 mM EDTA, 20 mM octylglucoside) and incubated on ice
 at 4.degree. C. overnight (under N.sub.2). Insoluble material was removed
 by centrifugation at (195,000.times.g) for 90 min at 4.degree. C. to
 provide the soluble supernatant used as the enzyme source for further
 purification.
 Example 2
 (-)-Limonene-6-Hydroxylase Purification
 The solubilized protein fraction from Example 1 containing the
 (-)-limonene-6-hydroxylase was subjected to two rounds of hydrophobic
 interaction chromatography on methyl-agarose (Sigma Lot #97F9710, Aug. 6,
 1992), followed by further purification by SDS-PAGE (Laemmli, Nature
 227:680-685 [1970]). Hydrophobic interaction chromatography was performed
 at room temperature. Samples were kept on ice before loading and as
 fractions were collected. Typically, 3 to 6 nmol of solubilized cytochrome
 P450 measured by the method of Omura and Sato (Omura et al., J. Biol.
 Chem. 239:2379-2385 [1964]) were loaded onto a 3 ml methyl-agarose column
 (C-1), that was prepared and equilibrated with solubilization buffer. The
 flow-through of the first C-1 column (12 ml) was collected and loaded onto
 a second C-1 column (equilibrated as before). Following the removal of
 contaminants achieved on the first C-1 column, the cytochrome P450 bound
 to the second column and was selectively eluted with solubilization buffer
 plus substrate (2 .mu.l/ml (-)-limonene mixed to an emulsion in buffer).
 Although this procedure proved useful for purification of the
 (-)-limonene-6-hydroxylase and for obtaining amino acid micro-sequence
 data from the pure enzyme, it was not reproducible with additional lots of
 methyl-agarose from Sigma and recovery yields varied greatly between
 individual protein preparations. To establish this example, it was
 therefore necessary to develop an alternative, reproducible protein
 purification strategy which is described for the first time in the
 following paragraph.
 Alternative protein purification method--Microsomal pellets prepared from
 gland sonicates originating from 200-250 g of spearmint leaves (16-20)
 were resuspended in 5 ml of 25 mM HEPES buffer (pH 7.2), containing 20%
 glycerol, 25 mM KCl, 10 mM MgCl.sub.2, 5 mM DTT, 0.2 mM PMSF, 50 .mu.M
 BHT, and 10 mg/liter leupeptin using a glass Tenbroeck homogenizer. An
 equal volume of the same buffer containing 1% Emulgen 911 was added slowly
 dropwise while stirring on ice, and the stirring continued for 1 h. The
 suspension was then centrifuged for 90 min at 195,000.times.g. The
 resulting solubilized microsomes were used as the source of (-)-limonene
 hydroxylase for further purification, which consisted of a polyethylene
 glycol, (PEG) precipitation step followed by anion-exchange chromatography
 on DEAE Sepharose and chromatography on ceramic hydroxylapatite (the
 latter serves a dual function as a final purification step and a detergent
 removal step which is required to reconstitute (-)-limonene-6-hydroxylase
 catalytic activity in homogeneous protein preparations).
 A 60% suspension of polyethylene glycol (M.sub.r 3,350) in HEPES buffer
 (above) with out detergent was added slowly dropwise to the solubilized
 microsomes while stirring on ice to give a final PEG concentration of 30%;
 stirring was continued for 30 min. The suspension was then centrifuged at
 140,000.times.g for 60 min and the supernatant discarded. The resultant
 0-30% PEG pellet was then resuspended in 5 ml of buffer containing 25 mM
 Tris-Cl (pH 7.0), 20% glycerol, 1 mM DTT and 50 .mu.M BHT using a glass
 homogenizer. To this suspension was slowly added (dropwise) an equal
 volume of the same buffer containing 0.2% Emulgen 911 followed by stirring
 on ice for an additional 30 min. The suspension was then clarified by
 centrifugation at 140,000.times.g for 30 min.
 The clarified PEG suspension was applied to a 3.5.times.1.75 cm column of
 DEAE Sepharose (Sigma or Pharmacia) equilibrated and washed with buffer
 (25 mM Tris-Cl (pH 7.0) containing 20% glycerol, 1 mM DTT, 50 .mu.M BHT,
 and 0.1% Emulgen 911), at a rate of 1.75 ml/min. The remaining bound
 protein was eluted stepwise (75 ml/step) with the same buffer containing
 50, 125, 250, and 1000 mM KCl. DEAE anion-exchange chromatography
 performed in this manner yields 45-60% of the microsomal P-450 measured by
 the method of Omura and Sato (Omura, supra) as an essentially homogeneous
 57 kD protein (with a 21% P-450 yield relative to the glandular sonicate).
 Cytochrome P-450 containing fractions from the anion-exchange column were
 concentrated by Amicon YM-30 ultrafiltration (Amicon) and bound to ceramic
 hydroxylapatite (Sigma). Emulgen 911 was removed by washing the matrix
 with 5 mM potassium, 40 .mu.m (Bio-Rad Laboratories) phosphate buffer (pH
 7.4) containing 20% glycerol, 1 mM DTT, and 10 mM CHAPS. The matrix was
 further washed with the same phosphate buffer containing no detergent,
 after which the (-)-limonene-6-hydroxylase is eluted from hydroxylapatite
 with 240 mM potassium phosphate buffer containing 20% glycerol and 1 mM
 DTT.
 Purified cytochrome P-450-containing fractions were combined and
 concentrated by TCA precipitation in preparation for SDS-PAGE. This
 protocol was shown to provide pure samples suitable for amino acid
 sequence analysis. TCA was added to protein samples at 8% (v/v), and the
 mixture was vigorously vortexed and incubated on ice for 40 min.
 Precipitated protein was pelleted by centrifugation for 15 min at
 10,000.times.g at 4.degree. C. The pellets were washed twice with ice cold
 acetone and vacuum desiccated to remove traces of organic solvent. The
 resulting pellets were resuspended in 75 .mu.l of 1.times.Laemmli loading
 buffer (Laemmli, supra), frozen at -80.degree. C. overnight and then
 heated for 15 min at 55.degree. C. prior to SDS-PAGE.
 Example 3
 Amino Acid Analysis and Protein Sequencing
 For obtaining N-terminal amino acid sequence data, the gels were
 electroblotted to polyvinyldifluoride membranes (Immobilon-P.sup.SQ,
 Millipore) in 25 mM Tris, 192 mM glycine (pH 8.3) containing 20% (v/v)
 methanol (Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350-4354 [1979]).
 Membranes were stained in 0.1% Coomassie Brilliant Blue R-250 in
 (methanol:acetic acid:water (50:10:40, v/v/v)) and destained with
 methanol:acetic acid:water (50:5:45). The resolved bands containing
 cytochrome P450 at .about.57 kDa ((-)-limonene-6-hydroxylase) were
 excised, washed by vortexing in distilled water, and the membrane
 fragments containing the target proteins were subjected to sequence
 analysis via edman degradation on an Applied Biosystems 470 sequenator (at
 The Washington State University Laboratory for Bioanalysis and
 Biotechnology, Pullman, Wash.).
 In order to obtain internal amino acid sequence information, protein
 samples were subjected to SDS-PAGE as described above. In this case,
 however, the gels were not directly electroblotted but were visualized by
 staining with 0.2% Coomassie Brilliant Blue R-250 in methanol:acetic
 acid:water (30:10:60, v/v/v) and destained with methanol:acetic acid:water
 (5:8:93, v/v/v) to avoid gel shrinkage. The gel band at 57 kDa was
 excised, washed with distilled water, and equilibrated in SDS-sample
 buffer (Laemmli, supra) for 5 min at room temperature. In a second
 SDS-PAGE step, the gels were polymerized with an extra large stacking gel
 and pre-electrophoresed as described above. The equilibrated gel slices
 from above were inserted into the sample well of the second SDS-10%
 polyacrylamide vertical slab gel (16 cm.times.18 cm.times.1.0 mm) which
 was previously filled with SDS-running buffer (Laemmli, supra). V-8
 protease (2 .mu.g) from Sigma was added to SDS sample buffer with 20%
 (v/v) glycerol and loaded using a Hamilton syringe into the sample well
 surrounding the gel slice. The samples were electrophoresed at 90 V
 (.about.2/3 of the way into the stacking gel). The power was turned off
 for 30 min in order to allow proteolytic cleavage. Electrophoresis was
 then continued at 90 V until the Bromophenol Blue dye front had entered
 the resolving gel. At this time, cooling was maintained at 20.degree. C.
 and electrophoresis was continued at 20 mA constant current for .about.3
 h. Following electrophoresis, the gel was electroblotted, the resulting
 membrane was coomassie stained, and the resolved peptide bands were
 prepared for microsequence analysis as described above. This method of
 proteolytic cleavage routinely yielded three peptide fragments whose
 combined molecular weights equaled approximately 57 kDa.
 Peptides were sequenced via Edman degradation on an Applied Biosystems 470
 sequenator at the Washington State University Laboratory for Bioanalysis
 and Biotechnology, Pullman, Wash.
 These methods yielded 20-25 residues of amino acid sequence data from each
 of the three V-8 derived peptides, as well as from the N-terminus of
 uncleaved (native) protein. The sequence of the second largest proteolytic
 peptide, V-8.2 (SEQ ID No:7) was identical to that of the corresponding
 region of the uncleaved protein (amino acid residues 1-21 of the sequence
 set forth in SEQ ID No:2) representing the N-terminus of the native
 enzyme. The V-8.3 (SEQ ID No:8) sequenced fragment could be most easily
 aligned with the C-terminal region of an avocado P450 (Bozak et al., Proc.
 Natl. Acad Sci. USA 87:3904-3908 [1990]) suggesting its origin from the
 same C-terminal region on the (-)-limonene hydroxylase. The V-8.3 fragment
 (SEQ ID No:8) corresponds to amino acids 375-398 of the amino acid
 sequence set forth in SEQ ID No:2. The third peptide fragment, V-8.1, (SEQ
 ID No:9) was assumed to be located somewhere between V-8.2 (SEQ ID No:7)
 and V-8.3 (SEQ ID No:8). [The avocado P450 was not a useful probe for
 limonene hydroxylases as it was not sufficiently similar]. The V-8.1
 fragment (SEQ ID No:9) corresponds to amino acids 176-200 of the amino
 acid sequence set forth in SEQ ID No:2.
 Example 4
 PCR-based Probe Generation
 Degeneracy considerations prevented the direct use for library screening of
 the amino acid sequence data generated from the purified
 (-)-limonene-6-hydroxylase from spearmint. PCR methods were employed to
 amplify the nucleotide sequences corresponding to the amino acid data. Six
 short, degenerate PCR primers were designed to prime the termini of each
 encoded peptide fragment. These primers are shown in the following Table
 1:
 TABLE 1
 PCR Primers
 Primer
 Name Primer Sequence (5' to 3') SEQ ID No.
 1.AC GTI ACI AAA ATG AC 10
 TG G T
 1.AG GTI ACI AAA ATG AG 11
 TG G T
 1.B GC CTC IGA ICC CTG ATC CTT 12
 T CT T G T
 1.C G TGT GTC GTC GTG TGC AGG GCG GCG TTC G 13
 2.AA ATG GAG CTI GAC CTI CTI A 14
 A T G T T G T G
 A A A
 2.AT ATG GAG CTI GAC CTI CTI T 15
 A T G T T G T G
 A A A
 2.B TC IAT ATA IGT IGC IAC 16
 G
 3.A ATG GAG GTI AAC GGI TAC AC 17
 A T T
 3.B TTT TTT TTT TTT TTT TTT A 18
 T
 C
 3.C CC GAT IGC GAT IAC GTT IAT AAA AAT ICT IGT CTT IGC IGG 19
 T T A G G G T
 A A T
 I = Inosine
 Primer 1.AC (SEQ ID No:10) was designed to prime the 5' end of the
 proteolytic peptide fragment V-8.1 (SEQ ID No:9) in the forward
 orientation. This primer was combined with primer 1.AG (SEQ ID No:11)
 during PCR to create the oligonucleotide mixture called primer 1.A which
 was successfully employed to amplify the 75 bp nucleotide sequence
 encoding the V-8.1 peptide fragment (SEQ ID No:9).
 Primer 1.AG (SEQ ID No:11) was designed for the same purpose as primer 1.AC
 (SEQ ID No:10). Primers 1.AC (SEQ ID No:10) and 1.AG (SEQ ID No:11) were
 synthesized separately and combined to create the primer 1.A in order to
 reduce the population degeneracy level in the primer pool.
 Primer 1.C (SEQ ID No:13) primes the central region of the V-8.1 peptide
 fragment (SEQ ID No:9). This primer is a non-degenerate primer oriented in
 the forward direction and was successfully employed when combined with the
 primer 3.C (SEQ ID No:19) to amplify the nucleotide sequence spanning the
 V-8.1 (SEQ ID No:9) and V-8.3 (SEQ ID No:8) proteolytic peptide fragments.
 The amplified nucleotide sequence was utilized as a cDNA hybridization
 probe and named LH-1 (SEQ ID No:20).
 Primer 2.AA (SEQ ID No:14) was designed to prime the amino-terminus of the
 nucleotide sequence based on the 5' end of the V-8.2 peptide fragment (SEQ
 ID No:7). This primer is oriented in the forward direction and was
 combined with the primer 2.AT (SEQ ID No:15) during PCR to achieve a lower
 degeneracy level in the primer pool.
 Primer 2.AT (SEQ ID No:15) was designed for the same purpose and at the
 same location as the primer 2.AA (SEQ ID No:14).
 Primer 2.B (SEQ ID No:16) was designed to prime the 3' end of the V-8.2
 peptide fragment (SEQ ID No:9) in the reverse orientation.
 Primer 3.A (SEQ ID No:17) designed to prime the 5' end of the V-8.3 peptide
 (SEQ ID No:8) fragment in the forward direction.
 Primer 3.B (SEQ ID No:18) primes the poly(A) tail on cDNA molecules. This
 primer was designed in the reverse orientation to amplify nucleotide
 fragments when combined with any of the other forward primers.
 Primer 3.C (SEQ ID No:19) was designed to prime the 3' end of the V-8.3
 peptide fragment (SEQ ID No:8) in the reverse orientation.
 Additional primers were designed to amplify regions spanning the three
 peptide fragments.
 The PCR primers were employed in all possible combinations with a range of
 amplification conditions using spearmint gland cDNA as template. Analysis
 of PCR products by gel electrophoresis indicated that one primer set (1.A
 (the mixture of SEQ ID No:10 plus SEQ ID No:11) and 1.B (SEQ ID No:12))
 had amplified the appropriate sized DNA fragment corresponding to the
 V-8.1 peptide (SEQ ID No:9). This 75 bp fragment was cloned into pT7Blue
 (Novagen), sequenced (by the chain termination method using Sequenase
 Version 2.0, United States Biochemical Corp.), and shown to code for the
 V-8.1 peptide (SEQ ID No:9). A non-degenerate forward primer (1.C) (SEQ ID
 No:13) was then designed from the internal coding sequence of V-8.1 (SEQ
 ID No:9) which, when combined with the degenerate reverse primer 3.C (SEQ
 ID No:19) designed to the V-8.3 peptide (SEQ ID No:8), permitted the
 amplification of a specific 700 bp DNA fragment (SEQ ID No:20). This
 fragment was cloned in to pT7Blue and sequenced as above, confirming that
 it coded for the sequence which spanned the V-8.1 (SEQ ID No:9) and V-8.3
 (SEQ ID No:8) peptides. This fragment (LH-1, SEQ ID No:20) was then
 labeled with [.alpha.-.sup.32 P-dATP] via the random hexamer reaction
 (Tabor et al., in Current Protocols in Molecular Biology. Sections
 3.5.9-3.5.10, John Wiley and Sons Inc. New York [1991]) and was used as a
 hybridization probe to screen the spearmint oil gland cDNA library.
 Example 5
 Plasmid Formation and Screening
 cDNA Library Construction--Spearmint (Mentha spicata) and peppermint
 (Mentha piperita) oil gland specific cDNA libraries were constructed. As
 published (Gershenzon et al., Anal. Biochem. 200:130-138 [1992]), the
 glandular trichome secretory cell isolation procedure does not protect RNA
 from degrading during a long water imbibition prior to surface abrasion.
 To protect RNA from degradation, published RNA purification protocols
 require either immediate freezing of tissue in liquid nitrogen or
 immersion in either strong organic solvents or chaotropic salts (see prior
 RNA isolation methods submitted with limonene synthase patent). These
 protocols have proven themselves to be incompatible with gland cluster
 isolation. Additionally, most tissues do not have the high levels of RNA
 degrading phenolics found in mint secretory glands. Therefore, a
 reproducible procedure was developed that protects the RNA from
 degradation during leaf imbibition and subsequent gland isolation and
 extraction. Additions of the low molecular weight RNase inhibitor,
 aurintricarboxylic acid (ATCA) (Gonzales et al., Biochemistry 19:4299-4303
 [1980]) and the low molecular weight polyphenyloxidase inhibitor, thiourea
 (Van Driessche et al., Anal. Biochem. 141:184-188 [1984]), to the water
 used during imbibition were tested. These additions were shown not to
 adversely effect water imbibition and gland isolation, yet to greatly
 improve the yield and quality of subsequent RNA isolation. Optimum
 concentrations for ATCA and thiourea were found to be 5 mM and 1 mM,
 respectively. These modifications allowed gland clusters to be isolated
 that consistently contained undegraded RNA. RNA extraction and
 purification using the improved method of Logemann et al. (Logemann et
 al., Anal. Biochem. 163:16-20 [1987]) was compromised by phenolics
 released during initial disruption of the purified gland cells. The
 inclusion of insoluble polyvinylpolypyrrolidone (PVPP) (Lewinsohn et al.,
 Plant Mol. Biol. Rep. 12(1):20-25 [1994]) to the RNA extraction buffer of
 Logemann et al., sufficiently sequestered phenolics and eliminated
 degradation. These modifications to the gland cell cluster isolation and
 RNA purification protocols consistently yield intact RNA that is useful
 for further manipulation. Poly (A)+ RNA was isolated on oligo
 (dT)-cellulose (Pharmacia Biotech, Inc.), and 5 .mu.g of the resulting
 purified mRNA was utilized to construct a .lambda.ZAP cDNA library for
 each Mentha species according to the manufacturer's instructions
 (Stratagene).
 Spearmint gland cDNA Library Screening--The 700 bp nucleotide probe (LH-1,
 SEQ ID No:20) generated by the PCR strategy of Example 4 was employed to
 screen replicate filter lifts of 1.times.10.sup.5 primary plaques grown in
 E. coli XL1-Blue MRF' using Stratagene protocols. Hybridization according
 to the DuPont-New England Nuclear protocol was for 24 h at 65.degree. C.
 in 25 ml of hybridization solution consisting of 5.times.SSPE
 (1.times.SSPE=150 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA),
 5.times.Denhardts, 1% SDS and 100 .mu.g/ml denatured sheared salmon sperm
 DNA. Blots were washed twice for 10 min with 2.times.SSPE at room
 temperature, twice with 2.times.SSPE containing 2% SDS for 45 min at
 65.degree. C., and, finally, twice with 0.1.times.SSPE for 15 min at room
 temperature.
 Of the plaques affording positive signals, 35 were purified through two
 additional cycles of hybridization. Thirty pure clones were in vivo
 excised as Bluescript SK (-) phagemids and their insert sizes were
 determined by PCR using T3 and T7 promoter primers. The largest 6 clones
 (.about.1.6 kb) were partially sequenced using T3 and T7 promoter primers.
 Three of these cDNA clones, 8A, 11A and 22C, were completely sequenced
 using nested deletion subclones generated with the Exo III/MungBean
 Nuclease Deletion Kit (Stratagene) as per manufacturer's instructions;
 additional sequencing primers, shown in the following Table 2 were also
 employed.
 TABLE 2
 Sequencing Primers
 SEQ ID
 Designation Sequence No.
 22CR3 CACGACATCTTCGACACCTCCTCC 21
 22CF1 GCAACCTACATCGTATCCCTCC ** 22
 NTREV1 GGCTCGGAGGTAGGTTTTGTTGGG 23
 NTREV2 GATTAGGAGGGATACGATGTAGGTTGC 24
 11A4.25R6 CTGGGCTCAGCAGCTCTGTCAA 25
 4.25R5 GGGCTCAGCAGCTCTCTC 26
 4.25R3 CTTCACCAACTCCGCCAACG ** 27
 11A4.25R2 GCTCTTCTTCTCCCTATGC 28
 11A4.25R TAGCTCTTGCACCTCGCTC 29
 11A.1F4 TTCGGGAGTGTGCTCAAGGACCAGG 30
 11A1F3 GTTGGTGAAGGAGTTCGCTG 31
 11A.1F2 CTTACAACGATCACTGG 32
 S12.2PF1 GACATCGTCGACGTTCTTTTCAGG 33
 S12.2PF2 CTACCACTTCGACTGGAAATTGC 34
 S12.2PF3 CTGAGATCGGTGTTAAAGGAGAC 35
 S12.2PR1 GCCACCTCTATAAGACACTCCTC 36
 S12-2PR2 GATCTCAACATTTGCCAGC 37
 S12BF GAAACCATGGAGCTCGACC 38
 P17.1F2 CGACGACATCATCTTCAGC 39
 P17F1 AGTACGGTCCAGTGGTGCACGTGC 40
 P17.1.2F3 GAGGAGCTGGTGGAGCTGGTGAAG 41
 P17.1.2F5 CGAGATCATGCAGAGAAGAATGC 42
 P17R1 ATGGGACCTCAACATTTGGCAAC 43
 P17.1R2 ATGTTCTTGGCCTTATTCG 44
 P17.1.2R4 CAGAGCAAGTTGAGGAGCTTGGAGG 45
 P17.1.2F4 CCATCACCACCAACGCCATCAAAGC 46
 P17.1.2R6 GTACTGCTTCGCCACGCTGG 47
 BLUT3 CGCGCAATTAACCCTCACTAAAGGG 48
 11A4.10F GCTGAATGGGCAATGG 49
 11A.1F-A CACCTCCACTTCCTGTGG 50
 P17.1.2R5 GCTGAAGAGCTCGGAGACGCAGATC 51
 **These primers were used as PCR primers to construct the cDNA
 hybridization probe LH-2 (SEQ ID No:52) in addition to being used as
 sequencing primers.
 DNA fragments were assembled, and the sequence was analyzed using Seq AID
 II version 3.8 (a public domain program provided by Rhodes, D. D., and
 Roufa, D. J., Kansas State University) and the Genetics Computer Group
 Packet (The Genetics Computer Group, Program Manual for the Wisconsin
 Packet, Version 8, Genetics Computer Group, Madison, Wis. [1994]).
 Following alignment of the cDNA sequences with the peptide sequences
 obtained, it was determined that all three of these cDNA clones were
 truncated at the N-terminus; clone 22C was also truncated at the
 C-terminus and clone 8A was shuffled. Therefore, a second nucleotide probe
 (LH-2, SEQ ID No:52) was generated by PCR using a new forward primer
 (22CF, SEQ ID No:53), homologous to the 20 most N-terminal bases of clone
 22C and a new reverse primer 4.25R3, SEQ ID No:54 (priming a region 500 bp
 downstream on clone 22C). The resulting DNA fragment (probe LH-2, SEQ ID
 No:52) was employed to re-screen the spearmint gland library as above. The
 second screen yielded 30 purified clones, which were in vivo excised and
 partially sequenced (Dye Deoxy Terminator Cycle Sequencing. Applied
 Biosystems). A single full-length clone, designated pSM12.2 (SEQ ID No:1),
 was isolated (1762 bp in length) and found to encode the entire protein
 (SEQ ID No:2) by comparison to the original amino acid sequence data. The
 limonene-6-hydroxylase protein set forth in SEQ ID No:2 includes a
 membrane insertion sequence at amino acids 1-42; a halt-transfer signal at
 amino acids 38-42, and a heme-binding region at amino acids 423-448.
 Isolation of peppermint cytochrome P450 cDNA clones--One hundred thousand
 primary (peppermint gland cDNA) plaques were grown and screened by
 hybridization with probe LH-2 (SEQ ID No:52) employing the same methods,
 as described above, used to isolate the spearmint cDNA clone pSM12.2 (SEQ
 ID No:1). Of the 25 plaques that were purified, ten were in vivo excised
 and partially sequenced with T3 and T7 promoter primers. Sequence
 alignment indicated that seven of these were representatives of the same
 gene (one of which, pPM17, was a full length clone and was completely
 sequenced). The nucleotide sequences for both cloned inserts, pSM12.2
 ((-)-limonene-6-hydroxylase), and pPM17 ((-)-limonene-3-hydroxylase) are
 shown in SEQ ID No:1 and SEQ ID No:3, respectively.
 Baculovirus Constructs--Site directed mutagenesis PCR was employed to
 subclone the (-)-limonene-6-hydroxylase cDNA (pSM12.2, SEQ ID No:1) into
 the baculovirus transfer vector pBlueBac3 (Invitrogen). PCR primers (see
 Table 3, below) were designed to add restriction sites (NcoI) at the 5'
 translation initiation codon extending to a second primer at a position 20
 bp downstream of the translation termination codon, thus creating a
 HindIII site. The resulting fragment was digested, gel purified, ligated
 into NcoI-HindIII restricted pBlueBac3, and transformed into E. coli
 DH5.alpha. cells, thus creating the baculovirus transfer vector pBac12.2.
 TABLE 3
 PCR Primers used to construct the
 baculovirus transfer vectors pSM12.2 and pPM17.35:
 Designation Sequence SEQ ID No.
 P17START ATGGAGCTTCAGATTTCG 55
 p17RSTOP GCACTCTTTATTCAAAGG AGC 56
 S12BF GAAACCATGGAGCTCGACC 57
 S12BR TATGCTAAGCTTCTTAGTGG 58
 BAC4PCR-F TTTACTGTTTTCGTAACAGTTTTG 59
 BAC4PCR-R CAACAACGCACAGAATCTAGC 60
 BAC3PCR-F TTTACTGTTTTCGTAACAGTTTTG 61
 BAC3PCR-R CAACAACGCACAGAATCTAGC 62
 The (-)-limonene-3-hydroxylase cDNA (pPM17, SEQ ID No:3) was cloned into
 the baculovirus transfer vector pBlueBac4 (Invitrogen) by PCR using the
 thermal stable, high fidelity, blunting polymerase Pfu I (Stratagene) with
 PCR primers pE17Start (at the translation initiation ATG) and pE17Stop
 (extending 21 bp downstream of the translation termination codon) into the
 3' untranslated region. The resulting blunt-ended fragment was ligated
 into Nhe I digested pBlueBac4 (Invitrogen), that had been filled in via
 Klenow enzyme (Boehringer Mannheim), and was transformed into E. coli
 DH5.alpha., thus yielding the baculovirus transfer vector pBac17.35. Both
 transfer vectors were completely resequenced to verify cloning junctions;
 no errors were introduced by polymerase reactions.
 Recombinant baculovirus was constructed as described by Summers and Smith
 (Summers et al, A Manual of Methods for Baculovirus Vectors and Insect
 Cell Culture Procedures, Bulletin No. 1555, Texas Agricultural Experiment
 Station, College Station, Tex. [1988]). Briefly, CsCl banded transfer
 vector was cotransfected into Spodoptera frugiperda (Sf9) cells with
 purified, linearized AcMNPV DNA by the method of cationic liposome
 mediated transfection (Invitrogen) as per the manufacturer's instructions.
 Recombinant virus was identified by the formation of blue (occlusion
 negative) plaques using established plaque assay procedures (Summers et
 al., supra; O'Reilly et al., Baculovirus Expression Vectors, A Laboratory
 Manual, Oxford: Oxford University Press, pp. 45-50, 109-166 [1994]; Smith
 et al., Lancet 339:1375-1377 [1992]). Putative recombinant viruses were
 monitored for purity by PCR analysis and gel electrophoresis.
 Example 6
 cDNA Expression
 Sf9 Cell Culture and Recombinant Protein Expression--Spodoptera frugiperda
 (Sf9) cells were maintained as monolayers or in suspension (85-90 RPM)
 culture at 27.degree. C. in Grace's media (Gibco BRL supplemented with 600
 mg/L L-glutamine, 4 g/L yeastolate, 3.3 g/L, lactoalbumin hydrolyste, 10%
 (v/v) fetal bovine serum, 0.1% pluronic F-68, and 10 .mu.g gentamicin/ml).
 For the generation of high titer viral stocks, suspension cultures of log
 phase cells (1.1 to 1.6.times.10.sup.6 cells/ml) were infected at a
 multiplicity of infection (MOI) equal to .about.0.1 PFU/cell, and then
 allowed to grow until near complete cell lysis had occurred. Cell debris
 was pelleted by centrifugation and the media stored at 4.degree. C. For
 expression, log phase suspension cultures of Sf9 cells were supplemented
 with 3 .mu.g hemin chloride/ml (Sigma) in 75 mM sodium phosphate and 0.1 N
 NaOH (pH 7.6) and infected with recombinant baculovirus at an MOI of
 between 5 and 10 PFU/cell. The addition of hemin to the culture media was
 required to compensate for the low heme synthetic capability of the insect
 cells. Cells were harvested at various time intervals (between 24 and 96
 hours post infection) by centrifugation (800.times.g, 10 min), then washed
 with PBS, and resuspended in 75 mM sodium phosphate buffer (pH 7.4)
 containing 30% glycerol, 1 mM DTT, and 1 mM EDTA.
 Example 7
 Limonene Hydroxylase Analysis
 Product analysis and other analytical methods--An in situ bioassay was
 developed to evaluate functional expression of (-)-limonene hydroxylase
 activity. Expression cultures were incubated in the presence of .about.300
 .mu.M (-)-(4S)-limonene, which was added to the culture medium immediately
 following infection. At zero and various time intervals, 50-100 ml culture
 samples were removed and cells were harvested by centrifugation, washed,
 and resuspended in 3-6 ml of sodium phosphate buffer as described above.
 Resuspended cell suspensions were chilled on ice and extracted twice with
 3 ml portions of ice cold ether after the addition of 25 nmol camphor as
 internal standard. The extract was decolorized with activated charcoal,
 backwashed with water, and the organic phase containing the products was
 passed through a short column of anhydrous MgSO.sub.4 and activated
 silica. The purified extracts were then concentrated to .about.500 .mu.l
 under N.sub.2 and analyzed by capillary GLC (Hewlett-Packard 5890). GLC
 was performed on 0.25 mm i.d.times.30 m of fused silica capillary columns
 coated with superox FA or AT-1000 using "on column" injection and flame
 ionization detection with H.sub.2 as carrier gas at 13.5 psi (programmed
 from 45.degree. C. (5 min) to 220.degree. C. at 10.degree. C. per min).
 The identities of the products, (-)-trans-carveol from C-6 hydroxylation
 and (-)-trans-isopiperitenol from C-3 hydroxlyation, were confirmed by
 coincidence of retention times with the corresponding authentic standard.
 Peak quantitation was by electronic integration based on the internal
 standard.
 Functional expression of the (-)-limonene-6-hydroxylase (pSM12.2)(SEQ ID
 No:1), from spearmint and the (-)-limonene-3-hydroxylase from peppermint
 (pPM17)(SEQ ID No:3), using the in situ bioassay thus confirmed the
 identity of the clones. GLC and GLC-MS analysis of Sf9 expression cultures
 infected with Baculovirus clones pBac12.2 and pBac17.35 verified the
 production of between 15 and 35 nmol of the expected oxygenated
 monoterpene product ((-)-trans-carveol from the spearmint clone and
 (-)-trans-isopiperitenol from the peppermint clone) per 50 ml of
 expression culture. Non-infected Sf9 control cultures grown under
 expression conditions and fed limonene substrate, control cultures
 infected with recombinant baculovirus but not fed limonene, and Sf9 cells
 alone evidenced no detectable carveol or isopiperitenol production, as
 expected. Cell free extracts of the transfected cells yielded a typical
 CO-difference spectrum (Omura et al., J. Biol. Chem. 239:2379-2385 [1964])
 and afforded a positive Western blot (using antibody directed against the
 native spearmint 6-hydroxylase) thus demonstrating the recombinant enzymes
 to resemble their native counterparts, which have been previously isolated
 and characterized (but not previously purified) from the respective mint
 species (Karp et al., Arch. Biochem. Biophys. 276:219-226 [1990]), and
 confirming that the isolated genes are those controlling the oxidation
 pattern of limonene in monoterpene metabolism (Gershenzon et al., Rec.
 Adv. Phytochem. 28:193-229 [1994]).
 Example 8
 Cloning and Characterization of an Additional cDNA Molecule Encoding
 Limonene-3-Hydroxylase from Peppermint
 An additional cDNA molecule called PM2 (SEQ ID No:5), encoding a
 naturally-occurring limonene-3-hydroxylase variant (SEQ ID No:6), was
 cloned, concurrently with limonene-3-hydroxylase cDNA PM17 (SEQ ID No:3),
 from a peppermint cDNA library using the LH-2 fragment (SEQ ID No:52) as a
 probe. The PM2 cDNA (SEQ ID No:5) exhibits 93% identity with the nucleic
 acid sequence set forth in SEQ ID No:3 which also encodes a
 limonene-3-hydroxylase from peppermint (SEQ ID No:4). The
 limonene-3-hydroxylase (SEQ ID No:6), encoded by the cDNA having the
 nucleic acid sequence set forth in SEQ ID No:5 exhibits 97% similarity
 with the limonene-3-hydroxylase (SEQ ID No:4) encoded by the cDNA having
 the nucleic acid sequence set forth in SEQ ID No:3.
 Example 9
 Physical Properties of Isolated, Recombinant Limonene-3-Hydroxylase and
 Limonene-6-Hydroxylase Proteins of the Present Invention
 Preferred limonene-3-hydroxylase and limonene-6-hydroxylase proteins of the
 present invention are members of the cytochrome P450 oxygenase superfamily
 (subfamily Cyp71D) described in Nelson et al., DNA Cell Biol. 12:1-51
 (1993) and Nelson et al., Pharmacogenetics 6: 1-42 (1996), and require
 oxygen and NADPH or NADH for biological activity. Preferred
 limonene-3-hydroxylase and limonene-6-hydroxylase proteins of the present
 invention exhibit pH optima in the range of pH 7.0 to pH 8.0, pI values of
 pH 7.0+/-1.5, and are about 56 kDa to about 60 kDa in size. Preferred
 limonene-3-hydroxylase and limonene-6-hydroxylase proteins of the present
 invention are inhibited by substituted azoles and carbon monoxide (with
 reversal of inhibition by the latter by blue light having a wavelength of
 450 nm), bind tightly to their substrates (K.sub.m &lt;50 .mu.M) and exhibit
 relatively low turnover (K.sub.cat &lt;100). The preferred substrate of
 preferred limonene-3-hydroxylase and limonene-6-hydroxylase proteins of
 the present invention is (-)-limonene, but these enzymes can also utilize
 (+)-limonene and the dihydrolimonenes. Preferred limonene-3-hydroxylases
 of the present invention can form both cis and trans C3 hydroxylation
 products, while preferred limonene-6-hydroxylases of the present invention
 can form both cis and trans C6 hydroxylation products.
 Example 10
 Hybridization of Fragment of Spearmint Limonene-6-Hydroxylase cDNA (SEQ ID
 No:52) to Other Nucleic Acid Sequences of the Present Invention
 Utilizing Northern blot analysis, the spearmint limonene-6-hydroxylase cDNA
 fragment set forth in SEQ ID No:52 (fragment LH-2), or its complementary
 sequence, is capable of hybridizing to other nucleic acids of the present
 invention under the following conditions: hybridization in 3.times.SSC at
 65.degree. C. for 16 hours. Once hybridized under the foregoing
 hybridization conditions, the spearmint limonene-6-hydroxylase cDNA
 fragment set forth in SEQ ID No:52, or its complementary sequence, is
 capable of remaining hybridized to other nucleic acids of the present
 invention under the following wash conditions: two washes in 2.times.SSC
 at room temperature (20.degree. C. to 25.degree. C.) for 20 minutes per
 wash, followed by one wash in 0.5.times.SSC at 50.degree. C. for 30
 minutes.
 The ability of the nucleic acid molecules of the present invention to
 hybridize, and to remain hybridized, to the nucleic acid sequence set
 forth in SEQ ID No:52, or to the complementary sequence of the nucleic
 acid sequence set forth in SEQ ID No:52, can be determined utilizing the
 technique of hybridizing radiolabelled nucleic acid probes to nucleic
 acids immobilized on nitrocellulose filters or nylon membranes as set
 forth, for example, at pages 9.52 to 9.55 of Molecular Cloning, A
 Laboratory Manual (2nd edition), J. Sambrook, E. F. Fritsch and T.
 Maniatis eds, the cited pages of which are incorporated herein by
 reference.
 Utilizing the foregoing hybridization and wash conditions, the nucleic acid
 sequence set forth in SEQ ID No:52, and its antisense complement, were
 radiolabelled and used to probe a Northern blot, bearing RNA samples from
 several essential oil plant species, in the following manner. The method
 of Lewinsohn et al. (Lewinsohn et al., Plant. Mol. Biol. Rep. 21: 20-25
 (1994)) was utilized to isolate RNA from young leaves of the following
 essential oil plant species: Mentha arvensis, Mentha pulegium, Mentha
 gentilis (gracilis), Nepeta cataria (unknown chemotype), Perilla
 frutescens (unknown chemotype), Tanacetum vulgare (local chemotype),
 Pelargonium graveolens (unknown variety) and Carum carvi. 20 .mu.g of
 total RNA from each of the foregoing species were separated on a 1.5%
 agarose gel containing 6% formaldehyde.
 The gel containing the separated RNA samples was blotted onto Hybond
 N.sup.+ Nylon membrane (Amersham) and was prehybridized for one hour at
 42.degree. C. The nucleic acid sequence set forth in SEQ ID No:52 was used
 as a template for generating a .sup.32 P-labelled hybridization probe. The
 hybridization and wash conditions were as described in the present
 Example. Autoradiography revealed that the hybridization probe recognized
 a single RNA band of 1.6-1.8 kb in each of the samples.
 Example 11
 Additional Representative Nucleic Acid Molecules Encoding Either
 Limonene-3-Hydroxylase or Limonene-6-Hydroxylase
 In addition to the nucleic acid sequence set forth in SEQ ID No:1, examples
 of representative nucleic acid sequences of the present invention that
 encode a limonene-6-hydroxylase and which hybridize to the complementary
 sequence of the nucleic acid sequence disclosed in SEQ ID No:1 under the
 hybridization conditions set forth in Example 10 (and which remain
 hybridized under the wash conditions set forth in Example 10) are set
 forth in SEQ ID No:63 (encoding the limonene-6-hydroxylase variant set
 forth in SEQ ID No:64); and SEQ ID No:65 (encoding the
 limonene-6-hydroxylase variant set forth in SEQ ID No:66).
 In addition to the nucleic acid sequences set forth in SEQ ID No:3 and SEQ
 ID No:5, examples of representative nucleic acid sequences of the present
 invention that encode a limonene-3-hydroxylase and which hybridize to the
 complementary sequence of the nucleic acid sequence disclosed in SEQ ID
 No:3 under the hybridization conditions set forth in Example 10 (and which
 remain hybridized under the wash conditions set forth in Example 10) are
 set forth in SEQ ID No:67 (encoding the limonene-3-hydroxylase variant set
 forth in SEQ ID No:68) and SEQ ID No:69 (encoding the
 limonene-3-hydroxylase variant set forth in SEQ ID No:70).
 The nucleic acid sequences set forth in SEQ ID No:63, SEQ ID No:65, SEQ ID
 No:67 and SEQ ID No:69 were generated using a computer. The protein
 encoded by the nucleic acid set forth in SEQ ID No:63 is identical to the
 protein encoded by the nucleic acid set forth in SEQ ID No:1, except that
 the protein encoded by the nucleic acid set forth in SEQ ID No:63 includes
 a proline residue at position 228 of the protein sequence set forth in SEQ
 ID No:64, whereas the protein encoded by the nucleic acid set forth in SEQ
 ID No:1 has a leucine residue at position 228 of the protein sequence set
 forth in SEQ ID No:2.
 The protein encoded by the nucleic acid set forth in SEQ ID No:65 is
 identical to the protein encoded by the nucleic acid set forth in SEQ ID
 No:1, except that the protein encoded by the nucleic acid set forth in SEQ
 ID No:65 includes a glutamic acid residue at position 117 of the protein
 sequence set forth in SEQ ID No:66, whereas the protein encoded by the
 nucleic acid set forth in SEQ ID No:1 has an aspartic acid residue at
 position 117 of the protein sequence set forth in SEQ ID No:2.
 The protein encoded by the nucleic acid set forth in SEQ ID No:67 is
 identical to the protein encoded by the nucleic acid set forth in SEQ ID
 No:3, except that the protein encoded by the nucleic acid set forth in SEQ
 ID No:67 includes a lysine residue at position 289 of the protein sequence
 set forth in SEQ ID No:68, whereas the protein encoded by the nucleic acid
 set forth in SEQ ID No:3 has an asparagine residue at position 289 of the
 protein sequence set forth in SEQ ID No:4.
 The protein encoded by the nucleic acid set forth in SEQ ID No:69 is
 identical to the protein encoded by the nucleic acid set forth in SEQ ID
 No:3, except that the protein encoded by the nucleic acid set forth in SEQ
 ID No:69 includes an aspartic acid residue at position 254 of the protein
 sequence set forth in SEQ ID No:70, whereas the protein encoded by the
 nucleic acid set forth in SEQ ID No:3 has a glutamic acid residue at
 position 254 of the protein sequence set forth in SEQ ID No:4.
 Example 12
 Hybridization Under Stringent Hybridization Conditions
 The present invention provides isolated nucleic acid molecules that
 hybridize under stringent hybridization conditions to a fragment (having a
 length of at least 15 bases) of any one of the nucleic acid molecules set
 forth in SEQ ID No:1, SEQ ID No:3, SEQ ID No:5, SEQ ID No:63, SEQ ID
 No:65, SEQ ID No:67 and SEQ ID No:69. Hybridization under stringent
 hybridization conditions is achieved as follows. Nitrocellulose membranes
 are washed three to five times in 3.times.SSC with 0.1% SDS at room
 temperature and prehybridized for one hour at 37.degree. C. in
 6.times.SSC, 5.times.Denhart's solution, 0.05% sodium pyrophosphate, 0.5%
 SDS and 100 .mu.g/ml boiled herring sperm DNA. High stringency
 hybridization is conducted in 6.times.SSC, 1.times.Denhart's solution, 0.1
 mg/ml yeast tRNA, 0.05% sodium pyrophosphate at 45.degree. C. for twenty
 four to forty eight hours. The filters are washed three to five times for
 ten minutes per wash in 6.times.SSC, 0.5% sodium pyrophosphate at room
 temperature, and once for 30 minutes in 6.times.SSC, 0.05% sodium
 phosphate at 55.degree. C. Low stringency hybridization is identical to
 high stringency hybridization, except that the hybridization temperature
 is 35.degree. C. and the last wash is conducted at 50.degree. C. instead
 of 55.degree. C.
 Presently preferred fragments useful for hybridizing to isolated nucleic
 acid molecules of the present invention include: nucleic acid residues
 1274-1288 of SEQ ID No:1; nucleic acid residues 1301-1315 of SEQ ID No:1
 and nucleic acid residues 1385-1399 of SEQ ID No:1.
 The abbreviation "SSC" refers to a buffer used in nucleic acid
 hybridization solutions. One liter of the 20.times. (twenty times
 concentrate) stock SSC buffer solution (pH 7.0) contains 175.3 g sodium
 chloride and 88.2 g sodium citrate.
 A 50.times.stock solution of Denhart's solution contains 5 g Ficoll (Type
 400, Pharmacia), 5 g of polyvinylpyrrolidone, 5 g of bovine serum albumin
 (Fraction V, Sigma) and water to 500 ml.
 While the preferred embodiments of the invention have been illustrated and
 described, it will be appreciated that various changes can be made therein
 without departing from the spirit and scope of the invention. For example,
 sequence variations from those described and claimed herein as deletions,
 substitutions, mutations, insertions and the like are intended to be
 within the scope of the claims except insofar as limited by the prior art.