Cytochrome P450s and Uses Thereof Joe Chappell University of Kentucky, [email protected]

Cytochrome P450s and Uses Thereof Joe Chappell University of Kentucky, Chappell@Uky.Edu

University of Kentucky UKnowledge

Plant and Soil Sciences Faculty Patents Plant and Soil Sciences

5-21-2013 Cytochrome P450s and Uses Thereof Joe Chappell University of Kentucky, [email protected]

Lyle F. Ralston Right click to open a feedback form in a new tab to let us know how this document benefits oy u.

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Recommended Citation Chappell, Joe and Ralston, Lyle F., "Cytochrome P450s and Uses Thereof" (2013). Plant and Soil Sciences Faculty Patents. 12. https://uknowledge.uky.edu/pss_patents/12

This Patent is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Plant and Soil Sciences Faculty Patents by an authorized administrator of UKnowledge. For more information, please contact [email protected]. USOO8445231B2

(12) United States Patent (10) Patent N0.2 US 8,445,231 B2 Chappell et al. (45) Date of Patent: May 21, 2013

(54) CYTOCHROME P4505 AND USES THEREOF 7,442,785 B2 10/2008 Chappelletal...... 536/236 7,622,614 B2 11/2009 Julien etal...... 568/327 . - . 8,106,260 B2 1/2012 Chappelletal. .. .. 800/298 (75) Inventors ioslelgllghlai’pené?exmiwl?’ 8,192,950 B2 6/2012 Chappelletal. .. 435/41 ye - as 0“, ester 6 a ( ) 2003/0166255 A1 9/2003 Chappell .. 435/2523

2004/ 0078840 A1 4/ 2004 Chappell et . .. .. 800/278 (73) Assignee: University of Kentucky Research 2006/0218661 A1 9/2006 Chappellet a1. .. .. 800/278 Foundation, Lexington, KY (Us) 2007/0231861 A1 10/2007 Millis et a1. . 435/69.1 2007/0238157 A1 10/2007 Millis et a1. 435/166 * - . - - - - 2007/0238159 A1 10/2007 Millis et a1. 435/25233 ( ) Nonce' SUbJeCt.t° any (3531mm? the garmgftgl; 2007/0238160 A1 10/2007 Millis et a1. 435/25233 Patent 15 men 6 or a Juste un er 2007/0254354 A1 11/2007 Millis et a1. 435/25233 U.S.C. 154(b) by 0 days. 2008/0178354 A1 7/2008 Chappelletal. 800/298 2008/0233622 A1 9/2008 Julien et a1. .. 435/148 (21) Appl.No.: 13/199,349 2010/0035329 A1 2/2010 Millisetal. 435/2542 2010/0120110 A1 5/2010 Chappell .. 435/166 - . 2010/0151519 A1 6/2010 Julien et a1. . 435/69.1 (22) Flled' Aug' 26’ 2011 2010/0151555 A1 6/2010 Julien et a1. .. 435/193 _ _ _ 2010/0216186 A1 8/2010 Chappelletal. .. . 435/69.1 (65) Prlor Publlcatlon Data 2011/0081703 A1 4/2011 Chappelletal...... 435/193 US 2011/0318797 A1 Dec. 29, 2011 FOREIGN PATENT DOCUMENTS JP 2000-511404 9/2000 Related US. Application Data WO 96/36697 11/1996 _ _ _ _ W0 97/38571 10/1997 (63) Contlnuatlon of apphcatlon No. 12/ 182,000, ?led on WO 97/33703 10/1997 Jul. 29, 2008, now Pat. No. 8,263,362, which is a W0 WO 97/37664 10/1997 continuationMar. 8, 2002, ofnow application Pat. No. 7,405,057.No. 10/097,559, ?led on WO 2010/019696 20010

(60) Provisional application No. 60/274,421, ?led on Mar. OTHER PUBLICATIONS 9, 2001, provisional application No. 60/275,597, ?led on Mar. 13’ 2001. Akiyoshi-Shibata et a1., “Further oxidation of hydroxycalcidiol by calcidiol 24 -hydroxylase. A study with the mature enzyme expressed in Escherichia coli,” Eur. J. Biochem. 224:335-343 (1994). (51) Int“ Cl“ An et al., “Organ-speci?c and developmental regulation of the C1 2N 1/00 (2006-01) nopaline synthase promoter in transgenic tobacco plants,” Plant C12N1/06 (2006.01) Physiol. 88:547-552 (1988). C1 2N 5/00 (200601) An et al., “Functional analysis of the 3‘ control region of the potato C1 2N 5/07 (201001) wound-inducible proteinase inhibitor II gene,” Plant Cell 1:115-122 (1989). (52) U‘s‘ Cl“ Back et a1., “Expression of a plant sesquiterpene cyclase gene in USPC ~~~~~ -- 435/69-1; 435/468; 435648; 435/252-1; Escherichia coli,” Arch. Biochem. Biophys. 315:527-532 (1994). 435/252.2; 435/155; 435/419 Back, K. and J. Chappell, “Cloning and bacterial expression of a (58) Field of Classi?cation Search sesquiterpene cyclase from Hyoscyamus muticus and its molecular None comparison to related terpene cyclases,” J. Biol. Chem. 27017375 See application ?le for complete search history. 7381 (1995) Back, K. and J. Chappell, “Identifying functional domains within (56) References Cited terpene cyclases using a domain-swapping strategy,” Proc. Natl. Acad. Sci. USA. 93:6841-6845 (1996). Back et al., “Cloning and bacterial expression of sesquiterpene U'S' PATENT DOCUMENTS cyclase, a key branch point enzyme for the synthesis of 5,589,619 A 12/1996 Chappell et a1...... 800/205 sesquiterpenoid phytoalexin capsidiol in UV-challenged leaves of 5,672,487 A 9/1997 SChweden et al~ ~~~~~~~~~ ~~ 435/69~1 Capsicum annuum,” Plant Cell. Physiol. 39:899-904 (1998). 5,741,674 A 4/1998 Schweden et al...... 435/69.1 5,766,911 A 6/1998 Koike et a1. 435/193 (Continued) 5,824,774 A 10/1998 Chappell et al. 530/350 _ _ _ _ 5,981,843 A 11/1999 Chappell et a1. 800/301 Prlmary Exam/"er * Medlna A Ibrahlm 2 gloteaunet ?l~l ~~ 5433672233? (74) Attorney, Agent, orFirm * McKenna Long &Aldridge , , appe et a . .. . . ~ - 6,100,451 A 8/2000 Chappell et a1. 800/298 LLP’ Stephame seldman 6,117,649 A 9/2000 Bellamine et al. .. 435/25 (57) ABSTRACT 2321323 3} 32221 285:: 2:1; ;; ;;; 83/92 The feamFes cytochrome 6,368,837 B1 4/2002 Gatenby et a1, , u 435/146 t1des and nuclelc a01d molecules, as well as expresslon vec 6,468,772 B1 10/2002 Chappell et a1. 435/183 tors and transgenic plants containing these molecules. In 6,495,354 B2 12/2002 Chappell et a1. 435/183 addition, the invention features uses of such molecules in 6,531,3036,559,297 B1B2 3/20035/2003 MillisChappell et a1.et al. 5433655135? methods of increasing the level of resistance against a disease 6,569,656 B2 5/2003 Chappell et al. 435/18'3 caused by a plant pathogen in a transgenic plant, in methods 6,645,762 B2 11/2003 Chappell et a1. 43 5 /3 2 5 for producing altered compounds, for example, hydroxylated 6,689,593 B2 2/2004 Millis et al. 435/155 compounds, and in methods of producing isoprenoid com 6,890,752 B2 5/2005 Chappell et a1. 435/325 pounds_ 7,186,891 B1 3/2007 Chappellet al. .. 800/298 7,405,057 B2 7/2008 Chappell et al...... 435/69.1 16 Claims, 11 Drawing Sheets US 8,445,231 B2 Page 2

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Zool. 272(1):25-33 (1995). 11 pages. Wu et al., “Expression cloning and characterization of human 17 Of?ce Action, issued Apr. 14, 2010, in connection With correspond beta-hydroxysteroid dehydrogenase type 2, a microsomal enzyme ing U.S. Appl. No. 12/182,000, 16 pages. possessing 20 alpha-hydroxysteroid dehydrogenase activity,” J. Biol. Examination Report, issued Aug. 2, 2010, in connection With corre Chem. 268(17): 12964-12969 (1993). sponding Canadian Patent Application No. 2,440,278, 3 pages. Appeal Decision, issued Oct. 21, 2011, in connection With corre Intent to Grant, issued Dec. 29, 2010, in connection With correspond sponding Japanese Patent Application No. 2002-571814, 17 pages. ing European Patent Application No. 02709797.l, 5 pages. Response to Examination Report, submitted Feb. 2, 2012, in connec Of?ce Action, issued Jan. 6, 2011, in connection With corresponding tion With corresponding Canadian Patent Application No. 2,440,278, U.S. Appl. No. 12/182,000, 10 pages. 36 pages. Decision to Grant, issued May 19, 2011, in connection With corre Examination Report, issued Nov. 20, 2012, in connection With cor sponding European Patent Application No. 02709797.l, 1 page. responding Canadian Patent Application No. 2,440,278, 7 pages. US. Patent May 21, 2013 Sheet 1 0f 11 US 8,445,231 B2

FIG. 1

EAS / \.

famesyl diphosphateOPP ’. 5-ep1-arlstolochene_

+NADPH +02 1-de3xycapsidiol S-Eeoxycapsidioi (s-txyumxy-s-epi-ansmlochene) (1-hydroxy-5-epi-arisioiochena)

psidiol US. Patent May 21, 2013 Sheet 2 0f 11 US 8,445,231 B2

FIG. 2

100

Enzymeactivity(%ofmaximum) 50

0369121518212427 Time after elicitation (h) US. Patent May 21, 2013 Sheet 3 or 11 US 8,445,231 B2

FIG. 3A

125

100 Enzymeactivity(%ofmaximum) 75 50

0 r 1 r r O 25 50 75 1 DO inhibitor concentration (pM) FIG. 38

125

100

75 Enzymeactivity(%maximum)of 50

0 i I l 0 25 50 75 100 inhibitor concentration (11M) US. Patent May 21, 2013 Sheet 4 0f 11 US 8,445,231 B2

FIG‘ 4A

(Hem:

H6“ 48 US. Patent May 21, 2013 Sheet 5 or 11 US 8,445,231 B2

swim; aiiaitantreated a 24 025 w a __ 12

CYPY1 i3

CYP73A

CYPEZE

CYPQZA

Leading ‘ comm! US. Patent May 21, 2013 Sheet 6 0f 11 US 8,445,231 B2

FIG. 6A

0.006 I

-0.006

Absorbance .0 O a N I

450 500 Wavelength (nm)

----- ,‘

-0.006 Absorbance 0.012 -0.018

-0.024 I 400 450 500 Wavelength (nm) US. Patent May 21, 2013 Sheet 7 0f 11 US 8,445,231 B2

FIG. 7A i i z; a 710 A+ T: E 3 2 i E i 71DA 1%

l l I I l l l I | I FIG. 7B empty A+ empty A

FIG. 7C

71D D+ 71D D .M3 . j ' Wva~ l 1 I ! l l ‘ l l i I FIG. 7D empty D+ empty D

Time (min) US. Patent May 21, 2013 Sheet 8 or 11 US 8,445,231 B2

?gure8A US. Patent May 21, 2013 Sheet 9 or 11 US 8,445,231 B2 US. Patent May 21, 2013 Sheet 10 0f 11 US 8,445,231 B2

&%@@wwa @@@a?@ %@@@@E Q@@@@@

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i f@@@% US. Patent May 21, 2013 Sheet 11 0f 11 US 8,445,231 B2

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1.) E P S V S Y US 8,445,231 B2 1 2 CYTOCHROME P450S AND USES THEREOF fore concluded that the 3-hydroxylase, responsible for hydroxylation of 5-epi-aristolochene at C3 to generate RELATED APPLICATIONS 1-deoxycapsidiol, was pathogen/elicitor inducible, while the 1-hydroxylase, responsible for hydroxylating 1-deoxycap This application is a continuation of US. patent applica sidiol at the C1 to generate capsidiol, was constitutive. tion Ser. No. 12/182,000, ?led Jul. 29, 2008, now US. Pat. Hoshino et al. (Phytochemistry 38:609-613, 1995) added to No. 8,263,362 which is a continuation of US. patent appli the observations of Whitehead et al. (Phytochemistry 28:775 cation Ser. No. 10/097,559, ?led Mar. 8, 2002 (now issued 779, 1989) by directly measuring 3-hydroxylase-activity in US. Pat. No. 7,405,057), which claims the bene?t of US. microsomal preparations of arachidonic acid-elicited Capsi Provisional Application Nos. 60/274,421 and 60/275,597, cum annuum fruits and seedlings. These assays consisted of ?led on Mar. 9, 2001 and Mar. 13, 2001, respectively, all of incubating 5-epi-aristolochene with microsome preparations which are hereby incorporated by reference. and subsequently determining the amount of 1-deoxycapsid iol generated by a combination of thin-layer chromatography FIELD OF THE INVENTION (TLC) separations and gas chromatography (GC). Their evi dence demonstrated that the conversion of 5-epi-aris This invention relates to cytochrome P450s and uses tolochene to 1-deoxycapsidiol was dependent on both thereof. NADPH and 02, and that 1-deoxycapsidiol accumulation in vitro was arrested by the P450 antagonists carbon monox BACKGROUND OF THE INVENTION ide (Omura and Sato, J. Biol. Chem. 239:2370-2378, 1964), 20 ancymidol (Coolbaugh et al., Plant Physiol. 62:571-576, Cytochrome P450s encompass a superfamily of oxidases 1978), and ketoconazole (Rademacher, Arum. Rev. Plant responsible for the oxidation of numerous endobiotics and Physiol. Plant Mol. Biol. 51:501-531, 2000). thousands of xenobiotics. In addition, in plants, cytochrome Recent results suggest that the hydroxylation of 5-epi P450s play important roles in wound healing, pest resistance, aristolochene is an important regulated step in capsidiol bio signaling, and anti-microbial and anti-fungal activity. 25 synthesis. In studies to evaluate the effectiveness of methyl Capsidiol is a bicyclic, dihydroxylated sesquiterpene pro jasmonate as an inducer ofcapsidiol biosynthesis in tobacco duced by many Solanaceous species in response to a variety cell cultures, Mandujano-Chavez et al. (Arch. Biochem. Bio of environmental stimuli, including exposure to UV (Back et phys. 381 :285-294, 2000), reported that the modest accumu al., Plant Cell. Physiol. 389:899-904, 1998) and infection by lation of this phytoalexin was accompanied by a strong induc microorganisms (Molot et al., Physiol. Plant Pathol. 379-389, 30 tion of EAS. This result implied that steps before or after the 1981; Stolle et al., Phytopathology 78:1193-1197,1988; sesquiterpene cyclase reaction were limiting. Using an in vivo Keller et al., Planta. 205:467-476, 1998). It is the primary assay measuring the conversion rate of radiolabeled 5-epi antibiotic or phytoalexin produced in tobacco in response to aristolochene to capsidiol, a very limited induction of the fungal elicitation, and it is derived from the isoprenoid path hydroxylase activity was observed in cells treated with way via its hydrocarbon precursor, 5-epi-aristolochene (FIG. 35 methyl jasmonate relative to that in fungal elicitor-treated 1). Several of the biosynthetic enzymes leading up to 5-epi cells. This result pointed to the hydroxylase reactions as a aristolochene formation have been studied (Chappell, Annu. potentially limiting step in capsidiol biosynthesis. Rev. Plant Physiol. Plant Mol. Biol. 46:521-547, 1995), espe cially 5-epi-aristolochene synthase (BAS) (Vogeli and Chap SUMMARY OF THE INVENTION pell, Plant Physiol. 88: 1291 -1296, 1988; Back and Chappell, 40 Proc. Natl. Acad. Sci. USA. 93:6841-6845, 1996; Mathis et In one aspect, the invention features several isolated cyto al., Biochemistry 36:8340-8348, 1997; Starks et al., Science chrome P450 polypeptides (such as CYP71D20, CYP71D21, 277: 1815-1820, 1997). BAS commits carbon to sesquiter CYP73A27, CYP73A28, and CYP92A5, and P450s having pene metabolism by catalyzing the cyclization of famesyl substantial identity to these polypeptides), as well as isolated diphosphate (FPP) to 5-epi-aristolochene. However, until the 45 nucleic acid molecules that encode these P450s. present invention, the enzyme(s) responsible for the conver In related aspects, the invention features a vector (such as sion of 5-epi-aristolochene to capsidiol has yet to be fully an expression vector) including an isolated nucleic acid mol identi?ed and characterized. ecule of the invention and a cell (for example, a prokaryotic Biochemical evidence from previous studies in tobacco cell, such as Agrobacterium or E. coli, or a eukaryotic cell, (Whitehead et al., Phytochemistry 28:775-779, 1989) and 50 such as a mammalian, insect, yeast, or plant cell) including green pepper (Hoshino et al., Phytochemistry 38:609-613, the isolated nucleic acid molecule or vector. 1995) have suggested that the oxidation of 5-epi-aris In yet another aspect, the invention features a transgenic tolochene to capsidiol occurs in a two step process with one of plant or transgenic plant component including a nucleic acid the hydroxylation steps being constitutive and the other being molecule of the invention, wherein the nucleic acid molecule mediated by an elicitor-inducible cytochrome P450 55 is expressed in the transgenic plant or the transgenic plant (FIG. 1). Because 1-deoxycapsidiol had been isolated from component. Preferably, the transgenic plant or transgenic natural sources (Watson et al., Biochem. Soc. Trans. 11:589, plant component is an angiosperm (for example, a monocot or 1983), Whitehead et al. (Phytochemistry 28:775-779, 1989), dicot). In preferred embodiments, the transgenic plant or surmised that perhaps the biosynthesis of this intermediate transgenic plant component is a solanaceous, maize, rice, or was due to pathogen induction of a corresponding hydroxy 60 cruciferous plant or a component thereof. The invention fur lase. They therefore prepared synthetic 1-deoxycapsidiol and ther includes a seed produced by the transgenic plant or reported a modest conversion of this compound to capsidiol transgenic plant component, or progeny thereof. when fed to control or unelicited tobacco cell cultures. This In another aspect, the invention features a method of pro was further supported by their observation that radiolabeled viding an increased level of resistance against a disease 5-epi-aristolochene was only converted to capsidiol when fed 65 caused by a plant pathogen in a transgenic plant. The method to elicitor-induced cell cultures but not control cultures. involves: (a) producing a transgenic plant cell including the Whitehead et al. (Phytochemistry 28:775-779, 1989) there nucleic acid molecule of the invention integrated into the US 8,445,231 B2 3 4 genome of the transgenic plant cell and positioned for expres tively). Forpolypeptides, the length of comparison sequences sion in the plant cell; and (b) growing a transgenic plant from will generally be at least 16 amino acids, preferably at least 20 the plant cell wherein the nucleic acid molecule is expressed amino acids, more preferably at least 25 amino acids, and in the transgenic plant and the transgenic plant is thereby most preferably 35 amino acids. For nucleic acids, the length provided with an increased level of resistance against a dis of comparison sequences will generally be at least 50 nucle ease caused by a plant pathogen. otides, preferably at least 60 nucleotides, more preferably at In another aspect, the invention features a method for pro least 75 nucleotides, and most preferably 110 nucleotides. ducing an altered compound, the method including the steps Sequence identity is typically measured using sequence of contacting the compound with one or more of the isolated analysis software (for example, Sequence Analysis Software polypeptides disclosed herein under conditions allowing for Package of the Genetics Computer Group, University of Wis the hydroxylation, oxidation, demethylation, or methylation consin Biotechnology Center, 1710 University Avenue, of the compound and recovering the altered compound. Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX In still another aspect, the invention features a hydroxylat programs). Such software matches identical or similar ing agent including any of the isolated polypeptides disclosed sequences by assigning degrees of homology to various sub herein. stitutions, deletions, and/or other modi?cations. Conserva In yet another embodiment, the invention features an iso tive substitutions typically include substitutions within the lated nucleic acid molecule that speci?cally hybridizes under following groups: glycine, alanine; valine, isoleucine, leu highly stringent conditions to the complement of any one of cine; aspartic acid, glutamic acid, asparagine, glutamine; the sequences described in SEQ ID N012 (CYP71D20), SEQ serine, threonine; lysine, arginine; and phenylalanine, ID N014 (CYP71D21), SEQ ID N016 (CYP73A27), SEQ ID 20 tyrosine. N018 (CYP73A28), or SEQ ID N0112 (CYP92A5), wherein By an “isolated polypeptide” is meant a P450 polypeptide such a nucleic acid molecule encodes a cytochrome P450 (for example, a CYP71D20 (SEQ ID N011), CYP71D21 polypeptide. (SEQ ID N013), CYP73A27 (SEQ ID N015), CYP73A28 In another aspect, the invention features a host cell express (SEQ ID N017), or CYP92A5 (SEQ ID N011 1) polypeptide) ing a recombinant isoprenoid synthase and a recombinant 25 that has been separated from components that naturally cytochrome P450. In preferred embodiments, the host cell accompany it. Typically, the polypeptide is isolated when it is further expresses, independently or in combination, a recom at least 60%, by weight, free from the proteins and naturally binant acetyltransferase, methyltransferase, or fatty acyl occurring organic molecules with which it is naturally asso transferase. In other preferred embodiments, the host ciated. Preferably, the preparation is at least 75%, more pref expresses an endogenous or recombinant cytochrome reduc 30 erably at least 90%, and most preferably at least 99%, by tase. Preferably, the host cell is a yeast cell, a bacterial cell, an weight, a P450 polypeptide. An isolated P450 polypeptide insect cell, or a plant cell. may be obtained, for example, by extraction from a natural In a related aspect, the invention features a method for source (for example, a plant cell); by expression of a recom producing an isoprenoid compound, the method including the binant nucleic acid encoding a P450 polypeptide; or by steps of: (a) culturing a cell that expresses a recombinant 35 chemically synthesizing the protein. Purity can be measured isoprenoid synthase and a recombinant cytochrome P450 by any appropriate method, for example, column chromatog under conditions wherein the isoprenoid synthase and the raphy, polyacrylamide gel electrophoresis, or by HPLC cytochrome P450 are expressed and catalyze the formation of analysis. an isoprenoid compound not normally produced by the cell; By “derived from” or “obtained from” is meant isolated and (b) recovering the isoprenoid compound. In preferred 40 from or having the sequence of a naturally-occurring embodiments, the host cell further expresses a recombinant sequence (e.g., cDNA, genomic DNA, synthetic, or combi acetyltransferase, a recombinant methyltransferase, or a nation thereof). recombinant fatty acyltransferase. In other preferred embodi By “isolated nucleic acid molecule” is meant a nucleic acid ments, the host cell expresses an endogenous or recombinant molecule, e.g., a DNA molecule, that is free of the nucleic cytochrome reductase. Preferably, the host cell is a yeast cell, 45 acid sequence(s) which, in the naturally-occurring genome of a bacterial cell, an insect cell, or a plant cell. the organism from which the nucleic acid molecule of the In yet another aspect, the invention features an isoprenoid invention is derived, ?ank the nucleic acid molecule. The compound produced according to the above-mentioned term therefore includes, for example, a recombinant DNA methods. that is incorporated into a vector; into an autonomously rep By “P450 polypeptide,” “cytochrome P450,” or “P450” is 50 licating plasmid or virus; or into the genomic DNA of a meant a polypeptide that contains a heme-binding domain prokaryote or eukaryote; or that exists as a separate molecule and shows a C0 absorption spectra peak at 450 nm according (for example, a cDNA or a genomic or cDNA fragment pro to standard methods, for example, those described herein. duced by PCR or restriction endonuclease digestion) inde Such P450s may also include, without limitation, hydroxy pendent of other sequences. The term “isolated nucleic acid lase activity, dual hydroxylase activity, demethylase activity, 55 molecule” also includes a recombinant DNA which is part of or oxidase activity. Such enzymatic activities are determined a hybrid gene encoding additional polypeptide sequences. using methods well known in the art. By “speci?cally hybridizes” is meant that a nucleic acid By “polypeptide” is meant any chain of amino acids, sequence is capable of hybridizing to a DNA sequence at least regardless of length or post-translational modi?cation (for under low stringency conditions, and preferably under high example, glycosylation or phosphorylation). 60 stringency conditions. For example, high stringency condi By “substantially identical” is meant a polypeptide or tions may include hybridization at approximately 42° C. in nucleic acid exhibiting at least 80 or 85%, preferably 90%, about 50% forrnamide, 0.1 mg/ml sheared salmon sperm more preferably 95%, and most preferably 97%, or even 98% DNA, 1% SDS, 2>