AU2004235635B2 - Novel lipoxygenase proteins and polynucleotides encoding the same - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

APPLICANT:

Invention Title: Lexicon Genetics Incorporated NOVEL LIPOXYGENASE PROTEINS AND POLYNUCLEOTIDES ENCODING THE SAME The following statement is a full description of this invention, including the best method of performing it known to me:

O

SNOVEL LPOXYGENASE PROTEINS AND SPOLYNUCLEOTIDES ENCODING THE SAME 0 The present application claims priority to United States Provisional SApplication Serial Nos. 60/128,817 and 60/150,454, all of which are Incorporated herein by reference in their entirety for any purpose.

mi. INTROUCflN ec O The present invention relates to the discovery, Identification, and 'n characterization of novel human polynucleotides that encode proteins that r share sequence almilarity with lipoxygenases. The invention encompasses o 10 the described polynudeotides, host cell expression systems, the encoded Cl proteins, fusion proteins, polypeptides and peptides, antibodies to the encoded proteins and peptides, and genetically engineered animals that lack the disclosed genes or over-express the disclosed genes, or antagonists and agonists of the proteins, and other compounds that modulate the expression or activity of the proteins encoded by the disclosed polynucleolides that can be used for diagnosis, drug screening, clinical trial monitoring, or the treatment of physiological or behavioral disorders.

2. BACKGROUND OF THE INVENTION Upoxygenases are enzymes that mediate the oxidation of lipid substrates. As such, lpoxygenases are Involved in the synthesis of leukotrenes. Leukotrienes influence a variety of biological processes, and can serve as, Inter aia, potent chemotactic agents, and mediators of inflammation, smooth muscle contraction, etc. Accordingly, lipoxygenases represent a key target for the regulation of a variety of biological pathways and conditions.

3. SUMMARY OF THEINVENTION The present Invention relates to the discovery, identification, and characterization of nudeotides that encode novel human lipoxygenase proteins, and the corresponding amino acid sequences of these proteins.

The novel human proteins (NHPs) described for the first time herein share structural similarity with animal and plant lipoxygenase proteins. As such, the 1- -2 o ~~~novel genes represent a ntew class Of IlPOxygenase proteinswiharn.o homologues and orthologs that transcend a broad range of phyla and 0 speche In vention conmprises pal ypeptudes wifth SeQ ID NOS:2, 4,6.,8, o ~10. 12, 14, 1a. 18. 20, fl. 24, 26, and 28; homologues and aflio Variants of SE=Q ID NOSQ2, 4, 6. S, 10, 12, 14, 18, 18, 2C0,22, 24, 26, and 28; (c) 'fl fragments of SEQ I0 NOS&2, 4, 6, 8, 10, 12, 14,16,18,20,22,24,.28, and INDZ28of any size, for example, from 4 amino acids to less than the full-ength of en 8 rlruQItid of SEQ 10 N082, 4,86, 8, 10, 12Z 14, 16, 18, 20, 22, 24, 28, or 28 and any number betwn; (di) fragments of SEQ ID NOez 4. 68, 8 10. 12, 14, 16. 18, 2,2.24, 28, and 28 that correspond to a functicnal domain (for example, a catalytic domain, a signal sequence. a liound binding domain,.a regulatory domain, etc.); fusion proteins comprising a poiypepwie aequence ofarny one of through mutant poiypeptijes (including is engineered and naturally occurring mutants) compuising a Pciypeptjde sequence of any one of through Including, but not limited to. deletion Mutants, insertion mutants, substiution mutant, and mutant pO~ypeptie 5 in which all or a part of at least one of the domailns is deleted or altered mutant of the active site with altered substrt specicity).

The invenjtion further comprises Pclynucleotwes with SEQ

ID

NO$:i1. 3. 6. 7. 9, 11. 13, 15, 19. 21, 23, 26, 27, and 29: (hi) POlNucleouie encoding any one of the poiypeptide of the invention including, but not Umitec to, polypeptides specifcally described in through M(1) abo; V) PolYnucte capable of hybsicnzing to a 8eon 26 polynuclweo. thi is comnplesmntsw to a Polynucieoude described In (g) and/or (h above under cond itions of low. medium, or high stigency; 0 Oligonucjoide corresponding to a segment of a polynucbeotide described In through above and such oligonucloesi having any size from 2 nucfteuc through less then the full-lengthln, ceo ad any lenpt inbetweon.

in oertain embodimets, the novel human nucleic acid sequences o dG*cribd herein, encode proteins/open reading frames (ORFa) of 7,11, 481D, 56,- 334,8615,480, 291, 69, 139195. 110887,845, anid 771 amino acids in length, (see SEQ ID NOS-2z 4, 6, 8, 10, 12, 14. 16, 18. 20, 22, 24, 25, and 28 o respectively).

The invarntion further comprises antgbodies to any one of the P0typaptides. or Po1lugflde of "h inventiorn. The invention also comprises host cells that are engineered to contain and/or express any one of enthe Po~~I80ues~ and/or polypeptides of the invention.

t 10 The lnven*00 also compPIse agoni ats and antagonists of the 0 descded Nips, including small molecules, large molecules, mutant NHPs, or port!ions thereof that Compete With native NHP, and antibodies, The invention further comprises nucleotide sequenCes that can be used to Inhibit 16 the expmssjon of the decibed NHPs anUsense and ribozyre 16Oligonucleotijcl anfor polynuedes~, and gene or reguary sequence replacent cOnstructs) or to enhance thea express ion Of the descibed flI4r genes exprn~ constructs that place t desouibed gene under t control of.a strung Promoter system), and transgenic: animals that express a transgene, or w oc-outs" (which can be conditonal) that do not expreSs functionar

NHR.

Further, the Present invention also relates to Methods for identiyng compounds that Modulate, i act as agonuals or anagonit of, ?*Ip xprenlOn and/or NHP product activty that uh1be purified PreParatlons of the NHPs and/or NI-P product, or cells expressing the Same. Such Compoundsg can be used as therapeutic agents for the treatment or any of a Widevaretyof inpo~~associated with biological disorders or Imbalances.

4. DECITON OFTEIFENELSIG N

IUE

The Sequence Listing provides the sequencs of 14 lIpoxygenasg..f 1 e ORFS tht are encoded by the descibed NHP poynUcleofige (SE=Q ID 3,5, 7,,q.11a, 5,17 921,23.5 27, and 29) and the amino O- -4- 0 acid sequences (SEQ ID NOS:2, 4, 6,8, 10, 12, 14, 16, 18,20.22,24,26, and 28) encoded thereby.

DETAILED DESCRIPTION OFTHE INVENTIO SULpoxygenases oxidize, or oxygenate, lipids to produce leukotrenes.

6 Depending on the leukotriene synthesized, a wide variety of biological in functions can be affected. Typically, leukotrienes will bind cognate receptors n an trigger a biological effect (such as, for example, signal transductlon).

Interfering with poxygenase activity ultimately effects leukotlene production and downstream leukotriene-medlated processes. Alternatively, enhancing ftpoxygenase activity n vivo, can boost the effects/activity levels the 0 corresponding biological processes. Various lipoxygenase activities can be found in a variety of cells and tissues in both animals and plants. Three predominant types of lpoxygenases include the 12-, and lipoxygenases, and each type of lipoxygenase can have additional forms depending upon the tissues or cells In which they are expressed.

The 12-, and 15-ipoxygenases, and the leukotrienes they produce, have been implicated with a variety of diseases and disorders. Given that leukotrienes can modulate Inflammatory reactions, they have been associated with a spectrum of mammalian diseases including, but not limited to, asthma, eye diseases, anaphylaxis, lung disease, hematological disorders, infectious diseases, granulomatosis, abscess, pacreatitis, prostattljs, hepatitis, atherosclerosis, heart disease, graft rejection, thrombosis, restenosis, ulcers, kidney disease, hypertension, dermatoses, cramping, autommune disorders (lupus, scleroderma, Crohn's disease, rheumatoid arthritis, etc.), granulomatoss, hyperproliferative diseases, cancer, nausea, headache, metastases, inflammatory bowel disorder, allergy, cancer, arthritis, eczema, melanoma, erythema, acne, psoriasis, shingles, infectious disease, and diabetes. Accordingly, one embodiment of the present invention are processes for identifying compounds useful for the treatment of one or more of the above diseases and disorders that Include the use of one or more of the described ipoxygenase4ike genes, proteins, or a novel portion thereof.

0 N Given the biological importance of ipoxygenases, the genes encoding such proteins (and the proteins encoded thereby as well as inhibitors thereof) 0 have been subjected to intense scientific ommercial scrutiny (see, for Sexample, U.S. Patents Nos. 5,036,105, 5,162,365, 5.504,097, 5,086,679, 5,830,453, 4.761,403, 5,589,506, 5,026729, and 5,861,268) (all of which are Sherein inoorporated by reference In their entirety).

SThe presently described NHPs share significant similarity with n previously described human Ipoxygenases. Expression studies using RT- N PCR detect NHP transcripts In. intare Aa, neural tissue brain, spinal cord, etc.), skin, testis, prostate, adrenal gland, cervix, salivary gland, pancreas, C heart, lymphoid cells (lymph node, spleen, thymus), and mammary glands.

Northem analysis showed a predominant signal in testis, with less predominant but longer, transcripts detectable in testis, lymph node, and spinal cord. A full length cDNA of a NHP coding region (with 5' and 3' extensions) was Isolated from a human brain cDNA library (Edge BoSystems, Galthersburg, MD) and sequenoed (SEQ ID NO: 29). A possible murine ortholog of the described NHPs is predominantly expressed in skin (Kinzig et al., 1899, Genomics 58:158-164).

The invention encompasses the use of the described NHP nudeotides, NHPs and peptides, as well as antibodies, preferably monoclonal antibodies, or binding fragments, domains, or fusion proteins thereof, or anti-idiotypic variants derived therefrom, that bind NHPs, other antagonists that inhibit binding activity or expression, or agonists that activate NHP activity or increase NHP expression, In the diagnosis and/or treatment of disease.

In particular, the Invention described In the subsections below encompasses NHP polypeptides or peptides corresponding to functional domains of NHPa. mutated, truncated ordeleted NHPs NHPs missing one or more functional domains or portions thereof), NHP fusion proteins a NHP ore functional domain of a NHP fused to an unrelated protein or peptlde such as an Immunoglobulin constant region, IgFc), nuceotide -em 0 0 sequences encoding such products, and host cell expression systems that o can produce such NHP products.

SThe Invention also encompasses antibodies and antdi-dotypic Santibodies (including Fab fragments), antagonists end agonists of the NHP, as well as compounds or nucleotide constructs that inhibit expression of a NHP geno (tranecription factor inhibitors, antisense and ribozyme molecules, n or gene or regulatory sequence replacement constructs), or promote expression of NHP expression constructs in which NHP coding Ssequences are operatively associated with expression control elements such t 10 as promoters, promoter/enhancers, etc.). The invention also relates to host 0 cells and animals genetically engineered to express the NHPs (or mutant variants thereof) or to inhibit or "knock-out expression of an animal homolog of an endogenous NHP gene.

The NHPs or peptides. NHP fusion proteins, NHP nucleotide sequences, antibodies, antagonists and agonists can be useful for the detection of mutant NHPs or inappropriately expressed NHPs for the diagnosis of disease. The NHP proteins or peptides, NHP fusion proteins, NHP nucdeotide sequences, host cell expression systems, antibodies, antagonists, agonists and genetically engineered cells and animals can be used for screening for drugs (or high throughput screening of combinatorial libraries) effective in the treatment of the symptomatic or phenotypic manifestations of perturbing the normal function of NHP in the body.

The use of engineered host cells and/or animals offers an advantage In that such systems allow for both the identification of compounds that interact with an NHP. and also provide Information regarding the biological significance of the NHP.

Finally, NHP products (especially soluble derivatives such as peptides corresponding to the NHP), and NHP fusion protein products (such as NHP-lg fusion proteins, fusions of a NHP, or a domain of a NHP, to an IgFc), antibodies and anti-diotypic antibodies (including Fab fragments), antagonists or agonits (including compounds that modulate signal transduction which -7- 0N may act on downstream targets in a NHP-associated leukotriene pathway) o can be used to directly treat diseases or disorders.

0 Nudeotde constructs encoding such NHP products can be delivered e to host cells that subsequently express the products in viv: these genetically engineered cells function as "boreactos" in the body delivering a continuous supply of a NHP, a NHP peptide, or a NHP tuslon protein to the body.

en Nucleotide constructs encoding functional NHPs, mutant NHPs, as well as N antisense and itbozyme molecules can also be used in 'gene therapy Sapproaches for the modulation of NHP expression. Thus, the invention also encompasses pharmaceutical formulations and methods for treating o biological disorders.

Various aspects of the invention are described in greater detail in the subsections below.

5.1. NHP POLYNUCLETIDES The cDNA sequences (SEQ ID NOS: 1, 3. 5, 7. 9, 11, 13, 16, 17, 19, 21, 23, 25. 27, and 29) and deduced amino acid sequences (SEQ ID NOS: 2, 6.8, 10, 12. 14. 16, 18, 20, 22, 24, 26, and 28) corresponding to the described NHPs are presented in the Sequence Listing. The NHP ORFs were obtained from human testis and brain cDNA libraries using probes and/or primers generated from human gene trapped sequence tags.

The NHP sequences of the present invention include: the human DNA sequences presented in the Sequence Listing and addonally ontemplate any nucleotide sequence encoding a contiguous and functional NHP that hybridizes to a complement of the DNA sequences presented In the Sequence Usting under highly stringent conditions, hybridization to fiterbound DNA in 0.5 M NaHPO 4 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65C, and washing in 0.1xSSC/0.1% SDS at 68"C (Ausubel F.M. et a, eds., 1989, Current Prtocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley sons, Inc., New York, at p. 2.10.3) and encodes a functionally equivalent gene product. Additionally contemplated ae any nucleoide sequences that hybridize to the complement of the DNA r~f~ -8- 0 sequences that encode and express an amino acid sequence presented in the Sequence Listing under moderately staingent condimlons, washing In 0 0.2xSS/o.1% SDS at 42'C (Ausubel at al., 1989, sUpra). yet which still Sencode a functionally equivalent NHP product. Functional equivalents of o NHP Include naturally occuning NHPs present in other species, and mutant NHPa whether naturally occurring or engineered. The Invention also Includes degenerate variants of the disclosed sequences.

en IN The invention also ncludes nucleic acid molecules, preferably

DNA

en molecles, that hybridize to, and are therefor e the complements of, the 10 described NHP nucleotide sequences. such hybridization conditions maybe 0 highlystringent or less highly stringent as described above. In instances wherein the nucleic acid molecules are deoxyoligonucleotides r(DNA oligos), such molecules (and particularly about 16 to about 100 base long, about to about 80, or about 34 to about 45 base long, or any variation or 1 combination of sizes representd therein that Incorporate a contiguous region of sequence first disclosed in the present Sequence Listing, can be used In conlunction with the polymerase chain reaction (PCR) to screen libraries, isolate clones, and prepare cloning and sequencing templates, etc..

Altmrnatvely, the oligonucleotides can be used singly or in chip format as hybridization probes. For example, a series of the described

NHP

iiganucleade sequences, or the comrnplements thereof, can be used to represent all or a portion of the described NHP sequences. The Olgonucleotdes, typically between about 16 to about 40 (or any whole number within the stated range) nucleotides in length,. may partially ovedrlap each other and/or the NHP sequence may be represented using ollgnueoides that don o; ogonucleotis that do not overlap. Accordingly, the described

NHP

polynuceotide sequences shall typically comprise at least about two or three distinc a 0ndeotidet sequences of at least about 18, and Preferably about 23, fluceoes In length that are first disclosed In the described Sequence Lsting. Such OIg0nuctCtide sequences may begin at any nuceotide present wfthin a sequerce in the Sequence Listing and proceed In either a sense 0 to4') oentatij'js-vis4M the described sequence or In an antisense o ~~orientaton. For ollgonucleotide probes, highly stringent condition. may refer, 0e.g., to washing in BxS$C/0.05% sodium pyrophosphate at 37 0C (for 14-base oligos), 480 (for 17-base oligos), 55tC (for 20.baae oligos), and 604C (for o 23-base oligos). These nucle acid molecules may encode or act as I'JIP gone antimense molecule., useful, for example, In NHP gene regulation (for and/or as antisense primers in amplification reactions of NHP gene nucleic

IND

acid sequences). WNII respect to NKP gene regulation, such techniques can be used to regulate biological functions. For example, H has been reported ,'71 10 that Apoxygonase mRNA carn bm trunslationaliy ~sII ned" by a differntiation.

o control element In the 3' untranslarted region (LJTR) of the liinscuipt in erid cells (Ostareck of ga/, 1997. Cell, W.:597-606). Further, such sequences may be used as part of ribozyrne and/or triple helix sequences, also useful for NHP gene regulation.

lb Upoxygenase antisense oligonuoleotldes may comprise at least one modied base moity which Is selected from the group Including but not lhnhte to 5-fiucrouracil, 5-bromnourejil, 5-chlorouracil, hypoxantidne, xantine, 4-acetylcytosine, &-(oarboxyhydroxylniethyl) ucl, S' arboxymetlaminomethyk2niounidin.

S-carboxymethyamiomenhyumcul dihydrourean, beta-D-galactosyiqueosiu, Inosine, N6s-iopentenyladenine, l-mnethylguarune, 1-rnethylinoalne, ZZ2-dimetflguanjne, 2-methytedenine, 2-mthylguanine, 3-methycytslne, S-methylcytosina, N6-adenirie, 7-methylguanine, 5 -methylamlnomernyfuraci, &metoxyaninomethyl-24tlouai3l, beta-D-mannosylqueouine, fl'-methoxycarbox~metnYlumdnJ 5-methoxyuracir, 2-methythlo-S Isopentenyladenjne, uracil--xyaceuc acid wybutoxosine, pseudouracE, qusosine, 2-thiocytosins, 5-methyl-2-thloureci. 2-thiouraci, 4-thiouracl fl-Mnwfiyracii, uracil-5-Oxyaceflc acid methylester, uracll- 5-.oxacequo acid S-methy1-2-thlou ral. S4(3smln )-N-2-curboxypwpy) uracil, (acp3)w, and so Z.6-diaminopurine.

0 1 0 The antisense olgonucleotude may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 0 2-fluroarabinose, xylulose, and hexose.

0 la In yet another embodiment, the antisense aligonucleotide comprises at Sleast one modified phosphate backbone selected from the group consisting of a phosphorothloate, a phosphordithloate, a phosphoramldothbate, a en phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a fonnacetal or analog thereof in yet another embodiment, the anltisonse ollgonuleotide is an u-anomeri cligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual -unts. the strands run parallel to each other (Gautier et al., 1987, Nuc.

Acids Res. 158-6625-8841). The oiigonucleotide is a (Inou et al.. 1987, Nuc. Acids Res. 15:1 3 1-6148), or a chineric RNA-DNA analogue (Inoue et al., 1987. FEES LetL. 215:327-330).

Oliganucleotides of the present invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Blosystems. etc.). As examples, phosphorothioate ollgonucleotides may be synthesized by the method of Stein et al. (1988., Nuct. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sal. U.S.A.

85:7448-7451), etc.

Low stringency conditions are we known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example. Sambrook ofet al., 1989, Molecular Cloning,

A

Laboratory Manual (and periodic updates thereof), Cold Springs Harbor Press, and Ausubel t eat. 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interaclence,

N.Y.

Altrnlivjy sitably labuleds "HP nucleowd Probes may be uimed to (N screen a human genonved library using aPPropritely flin9en conditonsor bYPCR. The Ideni fication and chaten~tln of human genomic clones is 0 ~~~~helpful for identifyng Po y o p ju de e in n th g no f s r ct e a en 5given locusijase and desigruing diagnostic teats. For example, foquenoes derivd fcm region. adjacent to the lntmrvtx~ boundaries of the human gee cwn be Used to design primers for use In amplScaflon as~ odtc ICm u tatlo n s w ih n te eX O S, intm ns, sp i e s t p i e et W de h or donor Site's) 6 e-tc., that can be used in diagnostic and phanneg~, a M eemybeioae rmnuceicso Further, a H oehomojogth 0Organism Of interest by Performing may? using te tw o m gnujolgonadd ofthe N p i m e p o ls esrg ed n t e b sisO f a m in o a cid se q u e n ces w thin th e N H P Product disclosed herein. The template for the reacnion may be total RNA, MrIJ*A# and/or cDVA obtained by revrse transcripfion Of rrRNA Prepared IS fibm for e a pe hu a or om ur C ll lines or t ssu such as cho vr jcj peskOwn Or suspected to express is NHP gene allele.

The PCI? Product may be auboboned anrd sequenced to ensure that the ampified Sequene represent the sequence of the. desired NHP gene. The PCI? fragment may then be used to Isolate a full length CONA clone by a variey of methods. For example, the anipliflgcj fagment may be labeled ands used to screen a cDNA librry, such as a bactohage CONA library.

Aftemativey, the labeled fraggrwn may be usedi to Isolate genomlo dlones via the screening Of 8 e~ri library.

MCIPOIR technology may also be utilized to Isolate full length cONA aquencs, For example, RNA may be isolated, followIng standard P~cedun, frm an pwpaz cellular or (issue 6ouroe qie., one knowt, or £taSPonjmj to exPress: NHP gene, such as, for example, skin, teso. ri 3 0 U i n g j a OSf rnS 9 P Ur n R e acti o n f M a y b e P r f o n e d o n t h e R N A Usi g a of gon ci ua, pri er pec c or the m ost 51 end of the am plified for the Priming Of first strand synthesis. The resulting

RNAIDNA

hybrid may then be ftapleds using a Stan dard terminal transferalse reaction. the -12- Shybrid may be digested with RNase H. and second strand synthesis may then be primed with a complementary primer. Thus, cDNA sequences upstream of Sthe amplified fragment may easily be isolated. For a review of cloning C, strategies which may be used, see Sambrook ef aL, 1989, supra.

6 A cDNA of a mutant NHP gene may be isolated, for example, by using PCR. In this cae, the first cDNA strand may be synthesized by hybridizing an oligo-dT ollgonuctotide to mRNA Isolated from tissue known or suspected ito be expressed in an individual putatively carrying a mutant NHP allele, and n by extending the new strand with reverse transcrptase. The second strand of 10 the cDNA is then synthesized using an ollgonucleotide that hybridizes Sspedfcically to the 5' end of the normal gene. Using these two primers, the product is then amplified via PCR, optionally cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill In the art. By comparing the DNA sequence of the mutant NHP allele to that of the normal NHP allele, the mutation(s) responsible for the loss or alteration of function of the mutant NHP gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtained from an individual suspected of or known to cany the mutant NHP allele. or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express the mutant NHP allele. A normal NHP gene, or any suitable fragment thereof, can then be labeled and used as a probe to identify the corresponding mutant NHP allele in such fibraries. Clones containing the mutant NHP gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill In the art.

Additionally, an expression library can be constructed utlizing cDNA synthesized from for example, ANA isolated from a tissue known, or suspected, to express a mutant NHP alele In an Individual suspected of or known to carry such a mutant allele. In this manner, gene products made by the putatively mutant tissue may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against 13- 0 0 the normal NHP product, as described, below, In Section 5.3. (For screening 0 techniques, see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A 0 Laboratory Manual", Cold Spring Harbor Press. Cold Spring Harbor.) n Additionally, screening can be accomplished by screening with labeled NHP fusion proteins, such as, for example, AP-NHP or NHP-AP fusion proteins. In cases where a NHP mutation results in an expressed gene m product with altered function as a result of a missense or a frameshflt in mutation), a polyaonal set of antibodies to NHP are likely to cross-react with e the mutant NHP gene product Library clones detected via their reaction with 10 such labeled antibodies can be purified and subjected to sequence analysis Saccording to methods well known to those of skill in the art.

An additional method of "screening" for NHP-related sequences (both nucleotlde an amino acid) involves electronic methods of storing, retrieving, and analyzing the described sequences and derivatives thereof. Accordingly, an additional embodiment of the present invention includes computer readable electronic data storage medium, or any system incorporating the same, that comprises a representation of any contiguous stretch of sequence first disclosed in the Sequence Listing.

The invention also encompasses nucleotde sequences that encode mutant NHPs, peptide fragments of the NHPs, truncated NHPs, and NHP fusion proteins. These include, but are not limited to nucleotide sequences encoding mutant NHPs described in section 5.2 infra polypeptides or paptides corresponding to one or more domains of the NHP or portions of these domains; truncated NHPs In which one or more of the domains Is deleted, or a truncated nonfunctional NHP. Nucleotides encoding fusion proteins may Include, but are not limited to, full length NHP sequences, truncated NHPs, or nucleotides encoding peptide fragments of a NHP fused to an unrelated protein or peptide, such as for example, a NHP domain fused to an Ig Fc domain which Increases the stability and half life of the resulting fusion protein NHP-Ig) In the bloodstream; or an enzyme such as a fluorescent protein or a luminescent protein which can be used as a marker.

-14- 0 The invention also encompasses: DNA vectors that contain any of 0 the foregoing NHP coding sequences and/or their complements 0 antisense); DNA expression vectors that contain any of the foregoing NHP (r coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; genetically engineered host cells that contain any of the foregoing NHP coding sequences m operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell; and genetically engineered host eC cells that express an endogenous NHP gene under the control of an exogenously Introdued regulatory element gene actvation). As used o herein, regulatory elements include but are not limited to inducible and noninducible promoters, enhancers, operators and other elements known to those sklted in the art that drive and regulate expression. Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, regulatable, viral (particularly retrovira LTR promoters) the early or late promoters of SV40 adenovirus, the lac system, the itp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase (PGK). the promoters of acid phosphatase, and the promoters of the yeast a-mating factors.

5.2. NHP POLYPEPTIDES NHPs, polypeptides, peptide fragments, mutated, truncated, or deleted forms of the NHPs, and/or NHP fusion proteins can be prepared for a variety of uses, including but not limited to the generation of antibodies, as reagents in diagnostic assays, the Identification of other cellular gene products related to a NHP, as reagents In assays for screening for compounds that can be as pharmaceutical reagents useful in the therapeutic treatment of mental, biological, or medical disorders and disease.

The Sequence Usting discloses the amino acid sequences encoded by the described NHP genes. The NHPs have initiator methlonines In DNA sequence contexts consistent with translation initiation sites. The sequence I_ c data presented herein indicate that alternative forms variants arising o from alternative splicing, promoters, etc.) of the NHPs may exist (which may 0 or may not be tissue specific).

e The NHP amino acid sequences of the invention include the amino acid sequences presented in the Sequence Usting as well as analogues and derivatives thereof. Further, orresponding NHP homologues from other n species are encompassed by the invention. In fact, any NHP protein encoded in by the NHP nucleotide sequences described in Section 6.1, above, are within en the scope of the invention, as are any novel polynucleotide sequences encoding all or any novel portion of an amino acid sequence presented in the o Sequence Listing. The degenerate nature of the genetic code is well known, and, accordingly, each amino acid presented In the Sequence Usting, Is generically representative of the well known nucleic acid "triplet" codon, or In many cases codons, that can encode the amino acid. As such, as contemplated herein, the amino acid sequences presented in the Sequence Listing, when taken together with the genetic code (see, for example, Table 4- 1 at page 109 of "Molecular Cell Biology", 1986, J. Damell et al eds..

Scientific American Books, New York, NY, herein Incorporated by reference) are generically representative of all the various permutations and combinations of nucleic acid sequences that can encode such amino acid sequences.

The invention also encompasses proteins that are functionally equivalent to the NHPs encoded by the nuceotide sequences described in Section 5.1. as judged by any of a number of criteria, including, bul not limited to, the ability to mediate lipoxygenase activity, the ability to effect an identical or complementary leukotriene pathway, a change in cellular metabolism Ion flux. tyrosine phosphoryfation. etc.), or change in phenotype when the NHP equivalent Is imilarly expressed or mutated In an appropriate cell type (such as the amelioration, prevention or delay of a biochemical.

biophysical, or overt phenotype. Such functionally equivalent NHP proteins Include, but are not limited to, additions or substitutions of amino acid -16o residues within the amino acid sequence encoded by the NHP nucleotide o sequences described above, in Section 5.1, but which result in a silent change, thus producing a functionally equivalent gene product Amino add C substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobfty, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include C, alanine, leucine, isoleucine, valine, prollne, phenylalanine, typtophan, and Va methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosne, asparagine, and glutamlne; positively charged (basic) t 10 amino acids include arginine, lyslne, and histidine; and negatively charged o (acidic) amino acids include aspartic add and glutamic acid.

While random mutations can be made to NHP DNA (using random mutagenesis techniques well known to those skilled in the art) and the resulting mutant NHPs tested for activity, site-directed mutations of the NHP coding sequence can be engineered (using site-drected mutagenesis techniques well known to those skilled in the art) to generate mutant NHPs with Increased function, higher lipoxygenase activity, decreased function, and/or Increased physiological hal-life. One starting point for such analysis is to align the disclosed human sequences with corresponding gene/protein sequences from, for example, other mammals In order to Identify amino acid sequence motifs that are conserved between different species. Nonconservative changes can be engineered at variable positions to alter function, signal transduction capability, or both. Alternatively, where alteration of function is desired, deletion or non-conservative alterations of the conserved regions identical amino acids) can be engineered.

Other mutations to the NHP coding sequence can be made to generate NHPs that are better suited for expression, scale up, etc. in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid in order to eliminate disuffide bridges; N-linked glycosyation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that Is more easily recovered and -17-

O

o purified from yeast hosts which are known to hyperglycosylate4inked stes.

o To this end, a variety of amno acid substitutions at one or both of the first or 0 third amino acid positions of any one or more of the glycosylation recognition Ssequences (N-X-S or and/or an amino acid deletion at the second position of any one or more such recognition sequences will prevent glycosyleton of the NHP at the modified trpeptide sequence. (See, e.g..

SMiyaJima et aL, 1986, EMBO J. 5(6):1193-1197).

ti Peptides corresponding to one or more domains of a NHP, truncated or deleted NHPs, as well as fusion proteins In which a full length NHP, a NHP t 0 peptide, or truncated NHP is fused to an unrelated protein, are also within the o scope of the invention and can be designed on the basis of the presently disclosed NHP nucleotide and NHP amino acid sequences. Such fusion proteins Include, but are not limited to, tgFc fusions which stabilize the NHP protein or peptide and prolong half-life h viv:o or fusions to any amino acid S sequence that allows the fusion protein to be anchored to the cell membrane; or fusions to an enzyme, fluorescent protein, or luminescent protein which provide a marker function.

White the NHPs and peptides can be chemically synthesized see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H.

Freeman Co., large polypeptides derived from a NHP and full length NHPs can be advantageously produced by recombinant DNA technology using techniques well known In the art for expressing nucleic acid containing NHP gene sequences and/or coding sequences. Such methods can be used to construct expression vectors containing a NHP nucleotide sequences described in Section 5.1 and appropriate transcriptional and translational control signals. These methods include, for example, in viro recombinant DNA techniques, synthetic techniques, and In viM genetic recombination.

See, for example, the techniques described in Sambrook at al. 1989, supra.

and Ausubel et 1989, supm. For example, recombinant llpoxygenase has been successfully produced in insect cells (using baculo virus) and purified using nickel affinity chromatography. Altematively, RNA corresponding to all -18- Q or a portion of a transcript encoded by a NHP nucleotlds sequence may be o chemically synthesized using, for example, synthesizers. See, for example, Sthe techniques described in "Otigonucleotide Synthesis", 1984, Gait, M.J, ed., r IRL Press, Oxford, which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems may be utilized to express the NHP nudeotide sequences of the Invention. Where the NHP peptide or Spolypeptlde is a soluble derivative, the peptide or polypeptide can be IV recovered from the culture, or from the host cell in cases where the NHP C peptide or potypeptide Is not secreted, end from the culture media in cases t 10 where the NHP peptide or polypeptide has been engineered to be secreted o by the cells. However, such expression systems also encompass engineered host cells that express a NHP, or functional equivalent, h/ situ, anchored in the cell membrane. One study has Indicated that the majority of one type of 16S-lipoxygenase protein can typically be found in soluble cytoplasmic cell fractions, but the majority of lipoxygenase activity can be found in the membrane fraction.

Purification or enrichment of NHP from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such engineered host cells themselves may be used in situations where it is important to not only to retain the structural and functional characteristics of the NHP, but to assess biological activity, In drug screening assays.

The expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria E. coi, 8. aubiis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing NHP nucleotide sequences; yeast Sacoheromyces. Pichla) transformed with recombinant yeast expression vectors containing NHP nucleotide sequences; Insect cell systems infected with recombinant virus expression vectors baculo virus) containing NHP sequences; plant cell systems infected with recombinant virus expression vectors cauliflower mosaic virus, CaMV; -18- 0 tobacco mosaic virus, TMV) or transformed with recombinant plasmid expres- Ssion vectors Ti plasmid) containing NHP nudeotide sequences; or 0 mammalian ceU systems COS, CHO, BHK, 293, 3T3) harboring en recombinant expression constructs containing promoters derived from the genome of mammalian cells metallothionein promoter) or from mammalian viruaes the adenovIrus late promoter; the vaccinia virus en 7.5K promoter).

In bacterial systems, a number of expression vectors may be e advantageously selected depending upon the use Intended for the NHP 10 product being expressed. For example, when a large quantity of such a Sprotein is to be produced for the identification of molecules that inhibit or enhance NHP activity, for the generation of pharmaceutical compositions comprising NHP, or for raising antibodies to a NHP, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coff expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequence may be ligated Individually into the vector in-frame with the IcZ coding region so that a fusion protein is produced; pIN vectors (Inouye Inouye, 1985, Nucleic Acds Res. 13:3101-3109; Van Heeke Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glulathione 8-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathloneagarose beads followed by elution in the presence of free glutathlone. The PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa caollmica nudear polyhldrasis virus (AcNPV) Is used as a vector to express foreign genes. The virus grows in Spodoptera fnglperda cells. A NHP gene coding sequence may be cloned tndividually into non-essential regions (for example the polyhedrln gene) of 1 o the virus and placed under control of an AcNPV promoter (for example the o polyhedrin promoter). Successful insertion of NHP gene coding sequence will result in inactivation of the polyhedrin gene and production of nonooccluded recombinant virus virus lacking the proteinaoeous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptora frugiperda cdlls In which tie inserted gene is expressed en see Smith et 1983, J. Virol. 46: 584; Smith. U.S. Patent No. 4,215,051).

I In mammalian host cells, a number of viral-based expression systems en may be utilized. In cases where an adenovirus is used as an expression 10 vector, the NHP nudeotide sequence of interest may be ligated to an o adenovlrus Iranscription/translation control complex, the late promoter and tripartite leader sequence. This chimeric gene may then be inserted In the adenovlrus genome by In vitro or in vivo recombination. Insertion In a non-essential region of the viral genome region El or E3) will result In a recombinant virus that Is viable and capable of expressing a NHP product In infected hosts See Logan Shenk. 1984, Proc. Natl. Acad. Scl. USA 81:3655-3659). Specffic Initiation signals may also be required for efficient translation of Inserted NHP nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire NHP gene or cDNA, Including its own initiation codon and adjacent sequences, Is inserted Into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of a NHP coding sequence is Inserted, exogenous translational control signals.

including, perhaps, the ATG Initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcripton enhancer elements, transcription terminators, etc. (See Blttner et al, 1987, Methods in Enzymol.

163:516-544).

S" -21- 0 O In addition, a host cell strain may be chosen that modulates the Sexpression of the inserted sequences, or modifies and processes the gene Sproduct in the specific fashion desired. Such modificatons Sglycosylation) and processing cleavage) of protein products may be o 5 important for the function of the protein. Different host cells have charactartetio and epecific mechanismn for the poetanslattonal processing and modification of proteins and gene products. Appropriate cell lines or host INO systems can be chosen to ensure the correct modification and processing of en the foreign protein expressed. To this end, eukaryotic host cells which 10 possess the cellular machinery for proper processing of the primary transcript, Sglycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and in particular, human cell lines.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the NHP sequences described above may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.

The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably Integrate the plasmid into their chromosomes and grow to fomn foci which In turn can be cloned and expanded Into cell lines. this method may advantageously be used to engineer cell lines that express a NHP product. Such engineered cell fines may be particularly useful in screening and evaluation of compounds thal affect the endogenous actMity of the NHP product.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wvgler, t al.. 1977, Cell o -22- 0 N 11:223), hypoxanthlne-guanine phosphodrbosyttransferase (Szybalska 0 Szybalsd, 1962, Proc. Natl. Acad. Sci. USA 48.2026) and adenine Sphosphorlbosyltransferase (Lowy, et 1980, Cell 22:817) genes can be o employed in tkc, hgprt or aprr cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr.

Iwhich confere resistance to methottexate (Wigler, et al., 1980, Natl. Acad, I Sc. USA 773567; O'Hare, et al., 1981, Proc. Natl Acad. Sl. USA 781527); Sgpt, which confers resistance to mycophenolic acid (Mulligan Berg, 1981, c Proc. Natl. Acad. Sd. USA 78:2072); neo, which confers resistance to the o 10 aminoglycoside G-418 (Colberre-Garapln, et at., 1981, J. Mol. Biol. 150.1); N and hygro, which confers resistance to hygromycn (Santerre, e al., 1984, Gene 30:147).

Altematively, any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht el al allows for the ready purification of nondenatured fusion proteins expressed In human cell lines (Janknecht, et al., 1991, Proc. Nail. Acad. Sci. USA 88: 8972-8978). In this system, the gene of interest is subdoned into a vaccinia recombination plasmid such that the gene's open reading frame is transtationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni'ntriloacetlc acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazolecontaining buffers.

NHP products can also be expressed in transgenic animals. Animals of any species, including, but not limited to. worms, mice, rats, rabbits, guinea pigs, pigs. micro-pigs, birds, goats, and non-human primates, baboons, monkeys, and chimpanzees may be used to generate NHP transgenic animals.

Any technique known in the art may be used to introduce a NHP transgene into animals to produce the founder lines of transgenic animals.

Such techniques Include, but are not limited to pronuctear microinjection -23c (Hoppe, P.C. and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovlrus 0 mediated gene transfer Into germ lines (Van der Putten et 1985, Proc.

SNatl. Acad. Si., USA 826148-6152); gene targeting in embryonic stem cells Thompson et 1989, Cell 56:313-321); elecroporation of embryos (Lo, 1983, Mol Cell. Biol. 31803-1814); and sperm-mediated gene transfer In (Lavitrano ft at, 1980, Cell 57:717-723); etc. For a review of such Stechniques, see Gordon, 1989, Transgenic Animals, ntl. Rev. Cytol. 115:171tn 229, which is incorporated by reference herein In its entirety.

Ci The present Invention provides for transgenic animals that carry the NHP transgene in all their cells, as well as animals which carry the tranogene Sin some, but not all their cells, mosaic animals or somatic cell transgenic animals. The transgene may be integrated as a single transgene orln concatamers, head-to-head tandems or head-to-tall tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et 1992, Proc.

Natl. Acad. Scl. USA 89:6232-6236. The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill In the art.

When it is desired that the NHP gene transgene be Integrated into the chromosomal site of the endogenous NHP gene, gene targeting is preferred.

Briefly, when such a technique is to be utilized, vectors containing some nucteotide sequences homologous to the endogenous NHP gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nuceotide sequence of the endogenous NHP gene "knockout" animals).

The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous NHP gene in only that cell type, by following, for example, the teaching of Gu et al., 1994, Science, 265:103-106.

The regulatory sequences required for such a cell-type specific Inactivation will depend upon the particular cell type of Interest, and will be apparent to those of skill In the art -24-

O

0 Once transgenic animals have been generated, the expression of the o recomblnant NHP gene may be assayed utilizing standard techniques. Initial 0 screening may be accomplished by Southern blot analysis or PCR techniques CC. to analyze animal tissues to assay whether integration of the transgene has 6 taken place. The level of mRNA expression of the transgene in the tissues of the tranagenlo animals may also be assessed using techniques which include Cc but are not limited to Northern blot analysis of tissue samples obtained from IN the animal, In situ hybridization analysis, and RT-PCR. Samples of NHP gene-expressing tissue, may also be evaluated Immunocytochemically using antibodies specific for the NHP transgene product S5.3. ANTIBODIES TO NHPF Antibodies that specifically recognize one or more epitopes of a NHP, or epitopes of conserved variants of a NHP, or peptide fragments of a NHP are also encompassed by the invention. Such antibodies include but are not limited to polydonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

The antibodies of the invention may be used, for example, in the detection of NHP in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal amounts of NHP. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described.

below, In Section 5.5, for the evaluation of the effect of test compounds on expression and/or activity of a NHP gene product Additionally, such antibodies can be used in conjunction gene therapy to, for example, evaluate the normal and/or engineered NHP-expressing cells prior to their Introduction into the patient Such antibodies may additionally be used as a method for the Inhibition of abnormal NHP activity. Thus, such antibodies, domains thereof, or peptides therefrom, may be utilized as part of treatment methods.

0 c For the production of antibodies, various host animals may be SImmunized by Injection with the NHP, an NHP peptide one 0 corresponding to a functional domain of an NHP), tnuncated NHP polypeptides (NHP in which one or more domains have been deleted), functional equivalents of the NHP or mutants of the NHP. Such host animals may include but are not limitod to rbbita, mice, goats, and rats, to name but a en few. Various adjuvants may be used to increase the immunological in response, depending on the host species, including but not limited to ec C Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysoedithin, pluronic polyols, 0 polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dintrophenol, and potentially useful human adjuvants such as BCG (badlle Calmette-Guerin) and Corynebacteriumparvum. Polyconal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495497; and U.S. Patent No.

4,378,110). the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72: Cole t al., 1983, Proc. Nail. Acad. Sci. USA 80;2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, igA. IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs In vivo makes this the presently preferred method of production.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et at., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger e al., 1984, Nature, 312:604-608; Takeda t al.. 1985, Nature, S -26- 0 N 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody 0 molecule of appropriate biological activity can be used. A chimeric antibody r is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulln constant region.

en Alteratively, techniques described for the production of single chein t' antibodies Patent 4,946,778; Bird, 1988, Science 242:423-426; Huston CN 1988, Proc. Natl. Acad. Scl. USA 85:5879-5883; and Ward et ae, 1989, o 10 Nature 334:544-546) can be adapted to produce single chain antibodies cl against NHP gene products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments Include, but are not limited to: the F(ab') fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the fragments.

Alternatively, Fab expression libraries may be constructed (Huse et 1989.

Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Antibodies to a NHP can, In turn, be utilized to generate anti-Idiotype antibodies that 'mimic" a given NHP, using techniques well known to those skilled in the art. (See, Greenspan Bona, 1993, FASEB J 7(5):437- 444; and Nisslnoff, 1991, J. Immunol. 147(8):2429-2438). For example antibodies which bind to a NHP domain and competitively inhibit the binding of NHP to its cognate receptor can be used to generate antidlotypes that mmimic" the NHP and, therefore, bind and activate or neutralize a receptor.

Such anti-idiotypIc antibodies or Feb fragments of such anti-diotypes can be used in therapeutic regimens involving the NHP signaling pathway.

-27- 0 5.4. DIAGNOSIS OF ABNORMALITIES RELATED TO A NHP SA vadety of methods can be employed for the diagnostic and 0 prognostic evaluation of disorders related to NHP function, and for the n Identification of subjects having a predisposition to such disorders.

Such methods may, for example, utilize reagents such as the NHP nuceotide sequences dosoribed in Section 5.1, and NHP antibodies, as e described, in Section 5.3. Specifically, such reagents may be used, for 'n example, for the detection of the presence of NHP gene mutations, or the detection of either over- or under-expression of NHP mRNA relative to a given 10 phenotype; the detection of either an over- or an under-abundance of NHP 0 gene product relative to a given phenotype; and the detection of perturbations or abnormalities in any potential signal transductlon, metabolic, or catabolic pathway mediated by a NHP.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific NHP nucleotide sequence or NHP antibody reagent described herein, which may be conveniently used, in clinical settings, to diagnose patients exhibiting inflammatory disorders.

For the detection of NHP mutations, any nucleated cell can be used as a starting source for genomic nucleic acid. For the detection of NHP gene expression or NHP gene products, any cell type or tissue in which the NHP gene is expressed, such as, for example, sidn, testis, or brain cells, may be utilized.

Nucleic acid-based detection techniques are described, below, in Section 5.4.1. Peptide detection techniques are described, below, in Section 5.4.2.

6.4.1. DETECTION OF NHP POLYNUCLEOTIDES Mutations within a NHP gene can be detected by utilizing a number of techniques. Nucleic add from any nucleated cell can be used as the starting point for such assay techniques, and may be isolated according to standard S-28- 0 c- nucleic acid preparation procedures which are well known to those of skill In 0 the art 0 DNA may be used in hybridization or amplification assays of biological g samples to detect abnormalities involving NHP gene structure, including point 6 mutations, insertions, deletions and chromosomal rearrangements. Such assays may include, but are not limited o, Soutmemr analyses, single en stranded conformatlonal polymorphism analyses (SSCP). and PCFt analyses.

~S 8uch diagnostic methods for the detection of NHP gene-specific c- mutations can involve for example, contacting and incubating nucleic acids Including recombinant DNA molecules, cloned genes or degenerate variants 0 thereof, obtained from a sample, derived from a patient sample or other appropriate cellular source, with one or more labeled nucleic acid reagents Including recombinant DNA molecules, cloned genes or degenerate variants thereof, as described in Section 5.1, under conditions favorable for the specific annealing of these reagents to their complementary sequences within a given NHP gene. Preferably, the lengths of these nucleic acid reagents are at least 15 to 30 nucleotfdes. After Incubation, all non-annealed nucleic acids are removed from the nuceic acld:NHP molecule hybrid. The presence of nucleic acids which have hybridized, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from the cell type or tissue of interest can be Immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a mlcrotiter plate or polystyrene beads. In this case, after Incubation, non-annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed.

Detection of the remaining, annealed, labeled NHP nucleic acid reagents is accomplished using standard techniques well-known to those in the art. The NHP gene sequences to which the nucleic add reagents have annealed can be compared to the annealing pattern expected from a normal NHP gene sequence in order to determine whether a NHP gene mutation Is present 0 Alternative diagnostic methods for the detection of NHP gene specific nucleic acid molecules, In patient samples or other appropriate cell sources, S-29- 0 Smay involve their amplification, by PCR (the experimental embodiment set forth In Mullis, 1987, U.S. Patent No. 4,683,202), followed by the Sdetection of the amplified molecules using techniques well known to those of g skill In the art. The resulting amplified sequences can be compared to those which would be expected if the nucleic acid being amplified contained only nonnal copies of a NHP gene In order to determine whether a NHP gene em mutation exists.

NO

IV Additionally, well-known genotyping techniques can be performed to identify Individuals carrying NHP gene mutations. Such techniques include, for example, the use of restriction fragment length polymorphisms (RFLPs), Swhich involve sequence variations In one of the recognition sites for the specific restriction enzyme used.

Additionally, Improved methods for analydzng DNA polymorphisms which can be utilized for the Identification of NHP gene mutations have been described which capitalize on the presence of variable numbers of short, tandemly repeated DNA sequences between the restriction enzyme sites. For example, Weber Pat No. 5.075.217. which is incorporated herein by reference in its entirety) describes a DNA marker based on length polymorphisms In blocks of (dC-dA)n-(dG.dT)n short tandem repeats. The average separation of (dC-dA)n-(dG-dT)n blocks is estimated to be 30,000- 60,000 bp. Markers which are so closely spaced exhibit a high frequency co- Inheritance, and are extremely useful In the identification of genetic mutations, such as, for example, mutations within a given NHP gene, and the diagnosis of diseases and disorders related to NHP mutations.

Also, Caskey at at Pat No. 5,364,759, which is incorporated herein by reference in its entirety) describe a DNA profling assay for detecting short tri and tetra nucleotide repeat sequences. The process includes extracting the DNA of interest, such as the NHP gene, amplifying the extracted DNA, and labeling the repeat sequences to form a genotypic map of the indivdual's

DNA.

0 C An additional embodiment of the present invention Involves identifying the association between NHPs, or NHP variants, and disease. Using such Sassociations, Individuals can be Identified that harbor NHP variants or display SNHP expression profiles that correlate with a given disease dermatoses, asthma, IBD, etc.). Once such a genetic diagnosis has been established Susing single nuleotide polymorphisms (SNPs), coding SNPs (cSNPs), etc., e an appropriate treatment regimen can be tailored to the patient.

i' The level of NHP gene expression can also be assayed by detecting CI and measuring NHP transcription. For example, RNA from a cell type or tissue known, or suspected to express the NHP gene, such as skin, testis, or 0 brain, may be Isolated and tested utilizing hybridization or PCR techniques such as are described, above. The isolated cells can be derived from cell culture or from a patient The analysis of cells taken from culture may be a necessary step In the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the NHP gene. Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the NHP gene, Including activation or inactivation of NHP gene expression. A preferred method of conducting such screening assays uses the described sequences as part of a larger array of sequences microchip arrays, etc.).

In one embodiment of such a detection scheme, cDNAs are synthesized from the RNAs of interest by reverse transcripton of the RNA molecule into cDNA). A sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The ncdeic acid reagents used as synthesis initiation reagents primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the NHP nucleic acid reagents described In Section 5.1, The preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides. For detection of the amplified product, the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified 0 -31.

C product may be made such that the product may be visualized by standard o. ethidlum bromide staining, by utilizing any other suitable nucleic acid staining Smethod, or by sequencing.

SAdditionally, It is possible to perform such NHP gene expression assays "In stu", directly upon tissue sections (fixed and/or frozen) of patient Issue obtained from biopsies or resections, such that no nulelc acid e purification is necessary. Nucleic acid reagents such as those described in in Section 5.1 may be used as probes and/or primer for such In situ procedures C (See, for example, Nuovo, 1992, "PCR In Situ Hybridization: Protocols And Applications', Raven Press, NY).

SAlternatively, if a sufficient quantity of the appropriate cells can be obtained, standard Northern analysis can be performed to determine the level of mRNA expression of the NHP gene.

5.4.2. DETECTION OF NHP POLYPEPTIDES Antibodies directed against wild type or mutant NHP products or conserved variants or peptide fragments thereof, which are discussed, above, in Section 5.3, may also be used as diagnostics and prognostics, as described herein. Such diagnostic methods, may be used to detect abnormalities in the level of NHP gene expression, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular location of the NHP, and may be performed in vivo or in vitro, such as, for example, on biopsy tissue.

For example, antibodies directed to epitopes of an NHP can be used in vivo to detect the pattern and level of expression of the NHP in the body.

Such antibodies can be labeled, with a radio-opaque or other appropriate compound and Injected Into a subject in order to visualize binding to the NHP expressed in the body using methods such as X-rays, CAT-scans, or MRI. Labeled antibody fragments, the Fab or single chain antibody comprising the smallest portion of the antigen binding region, are preferred for this purpose to promote crossing the blood-brain barrier and permit labeling of NHPs expressed In the brain.

o -32- SAdditionally, any NHP fusion protein or NHP conjugated protein whose presence can be detected, can be administered. For example, NHP fusion or 0 conjugated proteins labeled with a radio-opaque or other appropriate 3 compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further such NHP fusion proteins (such as AP-NHP or NHP-AP) can be utilized fur in vttm diagnostic procedures.

SAlternatively, immunoassays or fusion protein detection assays, as in described above, can be utilized on biopsy and autopsy samples in vit to ci permit assessment of the expression pattern of the NHP. Such assays are not confined to the use of antibodies that define a NHP domain, but can Sinclude the use of antibodies directed to epitopes of any domain of a NHP.

The use of each or all of these labeled antibodies will yield useful Information regarding translation and intracellular transport of the NHP to the cell surface and can identify defects in processing.

The tissue or cell type to be analyzed will generally Include those which are known, or suspected, to express the NHP gene, such as, for example, epithelial cells, brain cells, etc. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press. Cold Spring Harbor. New York), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, altemratvely, to test the effect of compounds on the expression of a NHP gene.

For example, antibodies, or fragments of antibodies, such as those described, above, in Section 5.3, useful in the present invention may be used to quantitatively or qualftatively detect the presence of NHP products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by Immunofluorescence techniques employing a fluorescenty labeled antibody (see below, this Section) coupled with light microscopic, flow O -33- N ytometric, or fluorimetric detection. Such techniques are especially prefered o If such NHP products are at least transiently present on the cell surface.

0 The antibodies (or fragments thereof) or NHP fusion or conjugated g3 proteins useful In the present Invention may, additionally, be employed i histologically, as In Immunofluorescence immunoelectron microscopy or nonimmuno assays, for In situ detection of NHP gene products or conserved e variants or peptide fragments thereof, or to assay NHP binding (in the case of in labeled NHP-fuslon protein).

C In slu detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or fusion N protein of the present invention. The antibody (or fragment) or fusion protein Is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the NHP product, or conserved variants or 16 peptide fragments, or NHP binding, but also its distribution in the examined tissue. Using the present Invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified In order to achieve such in situ detection.

Immunoassays and non-immunoassays for NHP products, or conserved vaiants or peptide fragments thereof, will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested ells, or lysates of cells which have been incubated In cell culture, in the presence of a detectably labeled antibody capable of identifying

NHP

products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art Alternatively, the labeled antibody can be directed against an antigenio tag that has been directly or indirectly attached to a NHP.

The biological sample may be brought in contact with and immobiized onto a solid phase support or carrier such as nitrocellulose, or other solid 3 support which is capable of Immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by .34- 0 treatment with the detectably labeled NHP antibody or NHP receptor fusion Sprotein. The solid phase support may then be washed with the buffer a Ssecond time to remove unbound antibody or fusion protein. The amount of Sbound label on solid support may then be detected by conventional means.

By *solid phase support or carrer is intended any support capable of binding an antigen or an antibody. well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, IN natural and modified celuloses, polyacrylamldes. gabbros, and magnetite.

SThe nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have 0 virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the Inside surface of a test tube, or the external surface of a rod. Altematively, the surface may be flat such as a sheet, test strip, etc. Prefered supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of NHP antibody or NHP ligand fusion protein may be determined according to well known methods. Those skilled In the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation With respect to antibodies, one of the ways In which the NHP antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, "The Enzyme Linked Immunosoment Assay (ELISA), 1978, Diagnostic Horizons 21-7. Microbiological Associates Quarterly Publication, Walkersvlle, MD); Voller, A. et al. 1978, J. Cln, Pathol, 31:507-520; Butler, 1981, Meth. Enrymol. 73:482-523; Magglo, E. 1980, Enzyme Immunoassay, CRC Press, Boca Raton. FL,; Ishikawa, E. et at, 1981. Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme that is bound to the antibody will react with an appropriate substrate, 0 preferably a chrmnogenre substrate, in such a manner as to produce a Schemical moiety which can be detected, for example, by spectrophotometic, fluodmetric or by visual means. Enzymes which can be used to detectably M label the antibody include, but are not limited to, malate dehydrogenase, 6 staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dohydrogenase, alpha-glycerophospate. dehydrogenase, trose phosphate Sisomrerase, horseradish peroxidase. alkaline phosphatase, asparaginase, IV glucose oxidase, betagalaclosidase, ribonuclease, urease, catalase.

Ml lucose6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric S methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate In comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of other 6 Immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it Is possible to detect NHP through the use of a radiolmmunoassy (RIA) (see, for example. Weintraub, Principles of Radioimmunoassays. Seventh Training Course on Radloligand Assay Techniques, The Endocrine Society, March, 1986, which Is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.

When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are flooresceln sothiocyanate, rhodamlne, phyoerythrin. phycocyanin, allophycocyanin, o- Phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as or others of the anthanlde series. These metals can be attached to the antibody using such metal chelating groups as -36-

O

o diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid o (EDTA).

The antibody also can be detectably labeled by coupling It to a (c chemiluminescent compound. The presence of the chemluminescent-tagged 6 antibody Is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemlluminescent labeling compounds are lumlnol, isolumlnol, N theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

n Likewise, a bioluminescent compound may be used to label the t 10 antibody of the present invention. Bfoluminescence is a type of o chemiluminescence found in biological systems In, which a catalytic protein increases the efficiency of the chemlluminescent reaction. The presence of a blolumlnescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

SCREENING ASSAYS FOR COMPOUNDS THAT MODULATE NHP EXPRESSION OR ACTIVITY The following assays are designed to Identify compounds that Interact with bind to) NHPs, compounds that Interfere NHP activity, compounds that modulate the activity of NHP gene expression modulate the level of NHP gene expression) or compounds that modulate the levels of NHP in the body. Assays may additionally be utilized that Identify compounds that bind to NHP gene regulatory sequences promoter sequences) and, consequently, can modulate NHP gene expression. See Platt, KA., 1994. J. Bio. Chem. 269:28558-28562, which Is incorporated herein by reference in its entirety.

The compounds that can be screened in accordance with the Invention include but are not limited to peptides, antibodies and fragments thereof, and other organic compounds peptldomimetics) that bind to a NHP and inhibit, hinder, or enhance lipoxygenase activity.

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o Such compounds may include, but are not lImited to, peptides such as, o for example, soluble peptides, including but not limited to members of random peptide libraries; (see, Lam, K.S. etal., 1991, Nature 354:82.84; SHoughten, R. et 1991, Nature 354:84-86). and combinatorial chemistryo 5 derived molecular libraries made of 0- and/or L- configuration amino adds, phosphapeptldes (including, but not limited to momber of random or partially degenerate, directed phosphopeptide libraries; see, Songyang, Z. etal., N 1993, Cell 72:767-778), antibodies (including, but not limited to, polyclonal, monodonal, humanized, anti-diotyple, chimeric or single chain antibodies, I0 and FAb, F(ab'% and FAb expression library fragments. and epitope-binding o fragments thereof), and small organic or inorganic molecules.

Other compounds which can be screened in accordance with the invention include but are not limited to small organic molecules that am able to cross the blood-brain barrier, gain entry into an appropriate cell in the chomld plexus, pituitary, the hypothalamus, etc.) and affect the expression of a NHP gene or some other gene involved In a NHP mediated pathway by interacting with the regulatory region or transcription factors Involved in gene expression or translation); or such compounds that affect NHPmediated leukotriene synthesis, or catabolic, inflammatory, or metabolic pathways.

Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate NHP expression or activity. Having identified such a compound or compostion, the active sites or regions are identified. Such active sites might typically be Ilgand or substrate binding sites. The active site can be identified using methods known in the art including, for example, from the amino add sequences of peptides, from the nudeotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand Is found.

1 -38- N Next, the three dimensional geometric structure of the active site is o determined. This can be done by known methods, including X-ray 0 crystallography, which can determine a complete molecular structure. On the m other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can bo unod to obtain partial or complete geometric structures, m The geometric structures may be measured with a complexed Ilgand, natural i' or artificial, which may Increase the accuracy of the active site structure Cdetermined.

If an incomplete or Insufficiently accurate structure is determined, the Smethods of computer based numerical modeling can be used to complete the structure or improve Its accuracy. Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statihtical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.

Finally, having determined the structure of the active site (or binding site), either experimentally, by modeling, or by a combination, candidate modulating compounds can be identified by searching databases containing compounds along with Information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that Interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential NHP modulating compounds.

Altematively, these methods can be used to Identify improved modulating compounds from an already known modulating compound or I I -So-

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o! ligand. The composition of the known compound can be modified and the o structural effects of modification can be determined using the experimental Sand computer modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or Interaction results. In this manner systematic variations in composition, such as by

V

cvarying side groups, can be quickly evaluated to obtain modified modulating tir compounds or Ilgands of improved specificity or activity.

Further experimental and computer modeling methods useful to t 10 identify modulating compounds based upon identification of the active sites O (or binding sites) of a NHP, and related transduction and transcription factors will be apparent to those of skill in the art.

Examples of molecular modeling systems are the CHARMm and QUANTA programs (Polygen Corporation. Waltham, MA). CHARMm performs the energy minimization and molecular dynamics functions.

QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows Interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs Interactive with specific proteins, such as Rotivinen, at at, 1988, Acta Pharmaceutical Fennica 97:159-166; Ripka, New Scientist 54-57 (June 16, 1988); McKinaly and Rossmann, 1989, Annu. Rev. Pharmacol. Toxiclol. 29111-122; Perry and Davies, OSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R.

Sac. Lond. 236:125-140 and 141-182; and, with respect to a model receptor for nuceic acid components, Askew, et 1989, J. Am. Chem. Soc.

111:1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BloDesign, Inc. (Pasadena, Allellx, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc- (Cambridge, Ontario). Although these are primarily designed for application

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0 to drugs specific to particular proteins, they can be adapted to design of drugs 0 apecific to regions of DNA or RNA, once that region is Identified.

0 Although described above with reference to design and generation of Scompounds which could alter binding, one could also screen libraries of known compounds, Including natural products or synthetic chemicals, and biologically active matorials, including proteins, for compounds which are nm inhibitors or activators.

Cael-based systems can also be used to identify compounds that bind C (or mimic) NHPs as well as assess the altered activity associated with such binding in living cells. One tool of particular interest for such assays is green fluorescent protein which Is described, infer aia, In U.S. Patent No.

6,625,048, herein Incorporated by reference. Cells that may be used in such cellular assays Include, but are not limited to, leukocytes, or cell lines derived from leukocytes, lymphocytes, stem cells, including embryonic stem cells, and the like. In addition, expression host cells 895 cells, COS cells, CHO cells, OMK cells, flbroblasts, Sf9 cells) genetically engineered to express a functional NHP of interest and to respond to activation by the test, or natural, ligand, as measured by a chemical or phenotypic change, or Induction of another host cell gene, can be used as an end point in the assay.

Compounds Identified via assays such as those described herein may be useful, for example, In elucidating the biological function of a NHP product.

Such compounds can be administered to a patient at therapeutically effective doses to treat any of a variety of physiological or mental disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in any amelioration, Impediment, prevention, or alteration of any biological symptom.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures In cell cultures or experimental animals, for determining the LD, (the dose lethal to 50% of the population) and the ED, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects Is the Stherapeutic index and it can be expressed as the ratio LDaEDR.

o Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of 0 5 affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

SThe data obtained from the cell culture assays and animal studies can N be used in formulating a range of dosage for use In humans. The dosage of n such compounds lies preferably within a range of circulating concentrations that include the ED. with little or no toxicity. The dosage may vary within this O range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC the concentration of the lest compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses In humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Pharmaceutical compositions for use in accordance with the present Invention may be formulated in conventional manner using one or more physiologically acceptable carriers or exciplents. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, intracranial, intrathecal, topical (skin creams, ointments, etc.), or rectal administration.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable exclpients such as binding agents pregelatinised maize starch, polyvinylpyrolidone or hydroxypropy methytcellulose): fillers lactose, microcrystalline cellulose or calcium S-42- 0 N hydrogen phosphate); lubricants magnesium stearate, talc or slcal); o disintegrants potato starch or sodium starch glycolate); or wetting Sagents sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the fomn of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional In means with pharmaceutically acceptable additives such as suspending C agents sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents lecithin or acacia); non-aqueous vehicles 0almond oil, oily esters, ethyl alcohol or fractionated vegetable oUl); and preservatives methyl or propyl-p-hydroxyberzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by Inhalation, the compounds for use according to the present invention are convenlently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane. carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by Injection, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, in ampoules or In multidose containers, with an added preservative. The compositions may take -43such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stablizing 0 andlor dispersing agents. Altematively, the active Ingredient may be in o powder form for constitution with a suitable vehicle, stedle pyrogen-free water, before use.

In The oompounds may also be formulated In rectal compositions such as suppositories or retention enemas, containing oonventional suppository n bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds O 10 may also be formulated as a depot preparation. Such long acting C formulations may be administered by Implantation (for example subcutaneously or Intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

5.5.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS

THAT

BIND TO NHPs In vtm systems may be designed to identify compounds capable of Interacting with binding to) NHPs. The compounds identified can be useful, for example, in modulating the activity of wild type andfor mutant NHP products; can be useful in elaborating the biological function of the NHP; can be utilized In screens for Identifying compounds that disrupt normal NHP interactions; or may themselves disrupt or activate such interactions.

The principle of the assays used to identify compounds that bind to NHPs, involves preparing a reaction mixture of an NHP and the test -44- C compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be Sremoved and/or detected In the reaction mixture. The NHP species used can o n vary depending upon the goal of the screening assay. For example, where compounds that directly interact with the NHP are sought full-length NHP.

paptidee corresponding to the NHP. or fusion proteins containing NHPs can Sbe used.

n' The screening assays can be conducted in a varety of ways. For Ci example, one method to conduct such an assay would involve anchoring the 0 10 NHP, polypeptide, peptide, or fusion protein therefrom, or the test substance C onto a solid phase and detecting NHP/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the NHP reactant may be anchored onto a solid surface, and the test compound, which Is not anchored, may be labeled, either directly or Indirectly.

In practice, microtlter plates may conveniently be utilized as the solid phase. The anchored component may be Immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and dryIng.

Alteratively, an immobiltzed antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction Is complete, unreacted components are removed by washing) under conditions such that any complexes formed will remain Immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component Is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonlmmoblilzed component Is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; using a labeled antibody specific for the previously nonimmoblized component (the antibody, in turn, may be Sdirectly labeled or indirectly labeled with a labeled anti-ig antibody).

SAternatlvely, a reaction can be conducted in a liquid phase, the c reaction products separated from unreacted components, and complexes detected; using an immobilized antibody specific for a NHP protein, potypeptde, peptide or fuslon protein or the test compound to anchor any Scomplexes formed in solution, and a labeled antibody specific for the other In component of the possible complex to detect anchored complexes.

C Atematively, cell-based assays can be used to identify compounds that interact with NHP. To this end, cell lines that express a NHP or cell lines COS cells, CHO cells, fibroblasts, etc.) that have been genetically engineered to express NHP by transfecion or transduction of suitably engineered NHP DNA) can be used. Interaction of the test compound with, for example, a NHP expressed by the host cell can be determined by 16 comparison to cells that do not express the NHP.

5.5.2. ASSAYS FOR INTRACELLULAR PROTEINS THAT ARE ASSOCIATED WITH NHP Any method suitable for detecting protein-protein Interactions may be employed for identifying membrane proteins or intracellular proteins that directly or indirectly interact with a NHP. For direct interactions, the traditional methods that can be employed include, but are not limited to, co-mmunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns of cell lysates or proteins obtained from cell lysates and a NHP to identify proteins in the lysate that interact with the NHP. For these assays, the NHP component can be a full length NHP, a soluble derivative of a NHP, a NHP peptide, or a NHP fusion protein. Once isolated, such an intracellutar protein can be identified and can, In turn, be used, in conjunction with standard techniques, to identify proteins with which It interacts. For example, at least a portion of the amino acid sequence of an Intracellular protein which interacts with a NHP can be ascertained using techniques known In the art, such as Edman degradation. (See, e.g..

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SCrelghton, 1983, "Proteins; Structures and Molecular Principles", W.H.

O Freeman Co., pp.34-49). The amino acid sequence obtained may be Sused as a guide for the generation of oligonudeotide mixtures that can be n used to screen for gene sequences encoding such Intracellular proteins.

Screening may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide nixtures e and the screening ar well-known. (See, Ausubel, supra, and PCR n Protocols: A Guide to Methods and Applications, 1990. Innis, M. et eds.

e Academic Press, Inc., New York).

Additionally, methods can be employed that result In the simultaneous Sidentification of genes that encode transmembrane or Intracellular proteins that interact with the NHP. These methods include, for example, probing expression libraries, in a manner similar to the well known technique of antibody probing of Agtl I libraries, using labeled NHP protein, or an NHP polypeptde, peptide or fusion protein, an NHP polypeptide or NHP domain fused to a marker an enzyme, fluor, luminescent protein, or dye), or an Ig-Fc domain.

One method that detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation.

One version of this system has been described (Chien etal., 1991, Proc. Natl.

Acad. Sci. USA, 8&9578-9582) and is commercially available from Clontech (Palo Alto, CA).

Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one plasmid consists of nudeotides encoding the DNAbinding domain of a transcription activator protein fused to a nucleotlde sequence encoding a NIP, or NHP polypeptide, peptide, or fusion protein therefrom, and the other plasmid consists of nucleotides encoding the transcription activator protein's activation domain fused to a cDNA encoding an unknown protein which has been recomblned Into this plasmid as pat of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saochemmyces cerevislae that 1 -47- N contains a reporter gene HBS or lacZ) whose regulatory region contains C the transcription activators binding site. Either hybrid protein alone cannot Sactivate transcription of the reporter gene: the DNA-blnding domain hybrid Scannot because it does not provide activation function and the activation domain hybrid cannot because it cannot localize to the activator's binding n seites. Intoraotlon of the two hybrid proteins rconstitutes the functional v. activator protein and results In expression of the reporter gene, which Is e detected by an assay for the reporter gene product s The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bar gene product. By way of example, and not by way of limitation, a NHP can be used as the bait product. Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait NHP gene product fused to the DNA-binding domain are 16 cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, and not by way of limitation, a bait NHP gene sequence, such as the open reading frame of a NHP (or a domain of a NHP) can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA.bindlng domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing Is then used to identify the proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact with bait NHP gene product are to be detected can be made using methods routinely practiced In the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4. This library can be co-transformed along with the bait NHP gene-GAL4 fusion plasmid Into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GALA transcriptional activation domain, that Interacts with bait NHP gene -48-

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0 product wll reconstitute an active GAL4 protein and thereby drive expression Sof the HIS3 gene. Colonies which express HIS3 can be detected by their Sgrowth on petri dishes containing semi-solid agar based media lacking Shistidlne. The cDNA can then be purified from these strains, and used to produce and isolate the bait NHP gene-interacting protein using techniques routinely pmctioed in the art.

C 5.6.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH NO NHP/INTRACELLULAR MACROMOLECULE OR NHPITRANSMEMBRANE MACROMOLECULE

INTERACTION

CN "1 Macromolecules that interact with NHPs are referred to, for purposes o of this discussion, as "binding partners". These binding partners are likely to be involved in NHP mediated biological pathways. Therefore, it is desirable to identify compounds that interfere with or disrupt the Interaction of such binding partners which may be useful In regulating or augmenting NHP activity In the body and/or controlling disorders associated with NHP activity (or a deficiency thereof).

The basic principle of the assay systems used to identify compounds that interfere with the interaction between a NHP or NHP receptor (collectively, the NHP moiety), and its binding partner or partners involves preparing a reaction mixture containing NHP, or NHP polypeptides, peptides or fusion proteins as described in Sections 5.5.1 and 5.5.2 above, and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In orderto test a compound for Inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound may be Initially included in the reaction mixture, or may be added at a time subsequent to the addition of the NHP moiety and its binding partner. Control reaction mixtures are Incubated without the test compound or with a placebo. The formation of any complexes between the NHP moiety and the binding partner Is then detected.

The formation of a complex In the control reaction, but not in the reaction mixture containing the test compound, Indicates that the compound interferes -49- 0 Swith the interaction of the NHP moiety and the Interactive binding partner.

o Additionally, complex formation within reaction mixtures containing the test Scompound and normal NHP protein may also be compared to complex Sformation within reaction mixtures containing the test compound and a mutant O 5 NHP. This comparison may be important in those cases wherein it is desirable to idontify compounds that specifically disrupt Interactions of n mutant or mutated, NHPs but not normal NHPs, orr er lipoxygenases 0 The assay for compounds that Interfere with the interaction of the NHP Sand binding partners can be conducted in a heterogeneous or homogeneous t" 10 format. Heterogeneous assays involve anchoring either the NHP moiety or o the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction Is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different Information about the compounds being tested. For example, test compounds that Interfere with the Interaction by competition can be identified by conducting the reaction in the presence of the test substance; by adding the test substance to the reaction mixture prior to, or simultaneously with, a NHP moiety and interactive binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly beJow.

In a heterogeneous assay system, either a NHP moiety or an Interactive binding partner, is anchored onto a solid surface, while the nonanchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Noncovalent attachment may be accomplished simply by coating the solid surface with a solution of the NHP moiety or binding partner and drying. Alternatively, an immobilized antibody

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o specific for the species to be anchored may be used to anchor the speces to o the solid surface. The surfaces may be prepared in advance and stored.

SIn order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed by washing) and any complexes formod will remain immobilized on the solid surface. The e detection of complexes anchored on the solid surface can be accomplilshed in a number of ways. Where the non-immobilized species Is pre-labeled, the c n detection of label immobilized on the surface Indicates that complexes were '4 10 formed. Where the non-immobilized species is not pre-labeled, an Indirect 0 label can be used to detect complexes anchored on the surface; using a labeled antibody specific for the Initially non-immobilized spedes (the antibody, In turn, may be directly labeled or Indirectly labeled with a labeled anti-lg antibody). Depending upon the order of addition of reaction 16 components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted In a liquid phase In the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; using an immobilized antibody specific for one of the binding components to anchor any complexes formed In solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an altemate embodiment of the Invention, a homogeneous assay can be used. In this aplproach, a preformed complex of a NHP moiety and an Interactive binding partner Is prepared In which either the NHP moiety or its binding partners Is labeled, but the signal generated by the label is quenched due to formation of the complex (see, U.S. Patent No. 4.109,496 by Rubensteln which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from -51-

O

Sthe preformed complex will result In the generation of a signal above Sbackground. In this way, test substances which disrupt NHP/intracellular C binding partner Interaction can be Identlfied.

In a particular embodiment, a NHP fusion can be prepared for immobilization. For example, a NHP or a peptide fragment can be fused to a glutathlon-S-trnsforaoo (GST) gene using a fusion vector, such as pGEX- M 5X-1, in such a manner that its binding activity Is maintained in the resulting fusion protein. The interactive binding partner can be purified and used to n raise a monoclonal antibody, using methods routinely practiced In the art and S 10 described above, In Section 5.3. This antibody can be labeled with the Sradioactive isotope 2i, for example, by methods routinely practiced in the art.

In a heterogeneous assay, the GST-NHP fusion protein can be anchored to glutathione-agarose beads. The interactive binding partner can then be added In the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between a NHP moiety and the interactive binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-NHP moiety fusion protein and the interactive binding partner can be mixed together in liquid In the absence of the solid glutathlone-agarose beads. The test compound can be added either during or after the species are allowed to interact This mixture can then be added to the glutathione-agarose beads and unbound material is washed away.

Again the extent of inhibition of the NHP moiety/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.

S -52- N In another embodiment of the invention, these same techniques can be Semployed using peptide fragments that correspond to the binding domains of Sa NHP moiety and/or the interactive or binding partner (in cases where the o binding partner is a protein), in place of one or both of the full length proteins.

Any number of methods routinely practiced in the art can be used to identify Sand isolate the binding sites. Those methods include, but are not limited to, M mutagenesis of the gene encoding one of the proteins and screening for en disruption of binding in a co-immunopredpitation assay. Compensatory Smutations in the gene encoding the second species in the complex can then 0 10 be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding. Altematively, one protein can be anchored to a solid surface using methods described above, and allowed to Interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a relatively short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing.

Also, once the gene coding for the Intracellular binding partner Is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.

For example, and not by way of limitation, a NHP moiety can be anchored to a solid material as described, above, by making a GST-NHP moiety fusion protein and allowing it to bind to glutathione agarose beads.

The interactive binding partner can be labeled with a radioactive isotope, such as and cleaved wittha proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-NHP moiety fusion protein and allowed to bind. After washing away unbound peptldes, labeled bound material, representing the intracellular binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known S-53- Smethods. Peptides so Identified can be produced synthetically or fused to 0 appropriate faciltative proteins using recombinant DNA technology.

Refemnce to MicmoamantelDeos n The following plasmid has been deposited at the American Type 0 Culture Colection (ATCC), Manassas, VA. USA, under the terms of the Budapest Treaty on tho International Recognition of the Deposit of n Microorganisms for the Purposes of Patent Procedure and Regulations vI thereunder (Budapest Treaty) and is thus maintained and made available e acording to the terms of the Budapest Treaty. Availability of such plasmid Is not to be construed as a license to practice the invention in contravention of o the rights granted under the authority of any government in accordance with Its patent laws.

The deposited plasmid has been assigned the Indicated ATCC deposit number.

Pasmid ATCC No.

LEXENZI7D PTA-503 The present invention Is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of Individual aspects of the invention, and functionally equivalent methods and components are within the scope of the Invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended daims. All publications, patents, and patent applications referenced herein are incorporated by reference In their entirety.

Claims (13)

1. 3. An isolated antibody that specifically recognizes at least one epitope of a polypeptide encoded by a first polynucleotide capable of hybridizing to a second polynucleotide under highly stringent conditions, wherein the first polynucleotide encodes a polypeptide having lipoxygenase activity, and wherein the second polynucleotide is complementary to the sequence of SEQ ID NO:1.
2. 4. The isolated antibody of claim 3, wherein the antibody is a polyclonal antibody. The isolated antibody of claim 3, wherein the antibody is a monoclonal antibody.
3. 6. The isolated antibody of claim 3, wherein the antibody is a chimeric antibody.
4. 7. The isolated antibody of claim 3, wherein the antibody is a single chain antibody.
5. 8. The isolated antibody of claim 3, wherein the antibody is a fragment of an antibody.
6. 9. The isolated antibody of claim 3, wherein the antibody is humanized. IO An isolated anti-idiotype antibody that specifically recognizes at least one epitope oj of the antibody of claim 3.
7. 11. A cell engineered to contain a polynucleotide of claim 1. In ¢n 12. A transgenic mouse engineered to contain a polynucleotide of claim 1.
8. 13. A method of screening for compounds that bind to the polypeptide of claim 2, the Smethod comprising: combining the polypeptide of claim 2 with at least one test compound in a reaction mixture; and detecting whether a complex comprising the polypeptide of claim 2 and the at least one test compound is formed.
9. 14. The method of claim 13, wherein the polypeptide of claim 2 or the at least one test compound is anchored onto a solid phase. A method of screening for a compound that alters the lipoxygenase activity of the polypeptide of claim 2, the method comprising: exposing a cell engineered to express the polypeptide of claim 2 to at least one test compound; and detecting a change in leukotriene production. EDITORIAL NOTE APPLICATION NUMBER 43400/00 The following Sequence Listing pages 1 to 26 are part of the description. The claims pages follow on page "54". SEQUENCE LISTIN Turner. C. Alexander, Jr. zambrowics. Brian Roble, #4chae1 rriedrich. Glenn Sands, Arthur T. -c120'. Novel Lipozygnsfc Proteins and Polynuontides encoding the lam 423M. 770S.0009-00304 c160, 29 4170* FastSBQ for Windows Version <2.10:k 1 (211:' 2701 4C212)- DNA ic2133h Boec Sapiens 1 atggcagtgt ecCCttg aacatctctg tcaoacogt atgggoaggg aettogocc Ctgg~gttc ttgctgct ttttggtact gtagcoct tgotatcagt ggattgaagg *tttgtoagg actatattoc Oaagaatget acegotsgna tg~tttcsggq agatggagta gacesgggtg acagcagtgg Ccatccctga tgtacatgga cattoaatg ceatoccego ggctootgga saagctgga sa"oatatg taacagagca ggtpcoaatc cog toatgct *atgaoatvgg tggccccott gggaaoatet toctagagga ggOegocagc agftacgtggc ctggtgccct tggccatcca Mcoaccgaat ccgaatqgga Otggtgoaog aaaacaacac aLtoggceogo tgcgCCagCt aetwgataca cgctgcaggt ctogtggece aggtcacgta atgcaact toacetacac Otggotatc Ocasotacca agottt-gtct cgAAatogt toghagoec aggectgguc talgtttcc ceageoggot ttcttcaatt gotctgccca tgatgecoa atgctccatc tgtgaooa ct ggqcaogtgt tggtcggta geggtacac CtgttcCcc CtactgcaCC ccta-ctcctg gAtCtatgcc a40*caagat* g*&tcggta gcccaatgtt gtacttggga tgaLoatgoag Ctggtgtg3aa CCtgGatc gotgggacag Ott ctggata ogoccactg gctcagccag atggctgctg gcactttctg gccgctctgc gaacaccatc catagggtgg @attectge atacegagac gct act at tggcgagatt gtgoaoooo-a gcaogatgct atocatgagg ggtccctacc ggtgaaagcc cagaagtaoa aaggagcgct gacqggtg gtggagctg& gateacagga cctggcttco ttgcettgt CtgcCegct cgatactcag atgaagette aacatcttct gatcaettct tctagCttgC gacaoatgce "9909gg4g9 tgcctgctgt QeC ccCgggc gccagacgt tscacgcAtt caccocatot gcgagggfcca caaggcotca attooggaCa gacggcctga tatoccagtg tttganog ggjagagatgg gtoaaoagtg cagcoccac tgAgggceg cotagoagog aggtgogttg acgotttCtt gtatttat C ggccaggaac cacgggact cctgcatggt cfl"gacgac tcCCOttgt ocacomagac gagggetgtt ggtgetata ttgggtacoa coagoansgt tgcagacaga coccoacoca figotoagoco ctgacagccc gggtgcgoaa tgctgtgcga acasgotoat cgcogctcaa teotacatoat gcctgcggC sgatctggg Acjoatatge cgttcctgg tq&attoct ggcagcatga occagaccas CaCtCtgfC gatagatoga cacagogag ccaaggac cot tococ Ag"Aagaact ooggaaega agacgtcaae tact tgtgta aattgacatc gatcagLg gatottCaC9 gtmcctgamat gCCtgtCcc gctagagag ctgcctm~aac ccagggggagg catcttcctg cCttgttC qgcottCgcc ActcaCCa&C ccocgaggoc gagcacgggo cegoggcgtc ggoCattgag gcagcaggat coggamgc eaictgcaato atttggggce ;gggaccacc 120 160 240 300 360 42D 490 540 600 660 720 710 840 900 960 L020 lose 1140 1200 310 1320 USC0 1440 1500 1360 1620 168D 1740 1000 1860 acactgaaga otcttetggt gagonottos aagatotcaa gaccotOec tccetgttct tggatatgac ggttaaaago ttcactta tt ggtttctg tttotacgtt gcctggaaac tagcaccttt t ottacotaga cacocteect tggttagCA agaacocaag caa99gac coogaggog; gggacat-cc& ggagcggaac tcattgagaa oagcgtctco tacaugcatg agaggacca cagttaOCt. gaacottoto atcataaaf ctgggcoctg cgotgaccaa. agtcoaatgc gcaccaattg gggoagactc cttttctccc toatoctec ctotttctto C8*4cctSga tggaoaga aggatgtgt aaggggaggg gaagaattgg gaaqtgaaa gacoagagge gOcatogoag caggvgcctgg atesaccac gttccccgg ttctgcacat agctgtgaga BCfltagCCC ttgttecago caatoceceas gcaacaago gectggct&&k agggcagrt tcagotgtsa caacctcctc ccctgggcac otaceagat cottocagag cages tggco cactgcccta acOLctbCCtg ccccamatac oaccaaa tectcoagae eattcCatoC ggagactttt gcagccaaga 900&905aCa goagogtcca ctcogaaagg aaggaaccgc cctttggaat gcccagettg aatcottctc ctacoatg@o oaqottcctt ctetuga& catgtggcc ectttgtcac goctggacCCcataasowotg 1320 19B00 2040 2100 2190 2220 2280 2340 2400 2460 2520 3580 2640 2700
10. 2701. -c2tC> 2 '211>, 711 'C212P. PRT
11. 4213.>% Rom sap~eni 440@:p 2. Mitt Ala Val Tyr Arg Lou Cys Val Thr Thr Sly Pro Tyr Lou Arg Ala Gly 7b: Lou Asp Bar Pr" Lys OIL Ann Ile Ber Val Thr Lou Val sly Thx eye Gly Sin Ala Pro Sly Arg LOU ASP Gly Arg AUp Pbe Eu: Val so Lou Lou SE Cur Try 03= Ly YS Tyey AMg Cyl Thr Ala Gin Lsa Sly Giu Lou Lou Arg Val His 70 Lys Gtu Arg Tyr Ala Ph. Pha Mxg Ty Cys icr m9 Ile eye Val Olu Pro Asp Sly Lye Asp so Bar Val Bar tie Pb. Len Mrg nvo 12S Len LOU ASP 130 Ber Phe aln 'Uhr Thr Cy. Sly Phe Pro 295 Msa Val Mrg 210 12e Pro Ala 225 Gly $or Trp Gym Tyr Cla Tfly Thr Ala Mrg Thr 120 Arg Thr Ar; GMu 135 Tyr Ala Pro lily Ile 105 Lie 7-eu Phe Olin sly Tyr cy. Cys Ola Asp Bar 125 Arg Ala Arg Gln 140 pro C9'S met Val RlIG Ile liar Val Ulu 110 LOU Pro Lou Olu Lye Tyr Asp Val AO1n Ito Thr Lye Tt: 175 150 glu Not Ola 165 Vol Asp ala 155 Lys The 3cr Asp Lys Sly Asp Bar 185 Asp Ile Pro Ala LOU Oly Lun.Ar; Lye Ile Bor LOU Met Tyr Lou Pro 190 Not Glu Pro Phe Ann Ala Tyr Ser Ala Tr Ile ser Scr Lou Lye Lys Gly Met Lys Lou Azg 230 Lou Asp Usp Hot Gin 250 Lys Tyr Val Thr Olin Lea Asp Arg Nun 11. Ph* Trp Cys His 355 Riou Trp Lye ala Asgp His Lys Tbr Phe Thr Tyr Gn Tlyr 265 Lau Asa ly 270 Val met Lou His rho Phe Gly Val Ann Pro 275 250 Cys Ilo gar Her Leu Pro Sor Lye 390 Ala Pro Lau Lou Sly Gin 295 Amp 305 sly Ann Ile UtSe Hip Ca LOU ABU 340 Lou Tsp Lou Se 355 Sar Gin Thr Pro 170 Mu Trp Asp Trp Lou Val Ki Gilu lu Ala Phe Ala 420 Sic Tyr Lys Lau 435 Thr Sic Mla flg 450 Val Thr Set Ile 465 Lou Ala His Phe Ala Arg sly Tel 500 Lau Lye Tie Trp 525 Tyr Tyr Tyr Pro 530 Ala Trp Thr sly 545 Be sly Phe Pro Lau Tkw Ala Ile 5610 Ear Sly Gin ia 595 Mot Arg az Pro 410 "vs Lou Asp Thr 625 Lau roba Trp Leu 1 Thr Tyr Pro Asp 660 Ala Ala Ph* Gin I 675 $10 LOu Jl6a asp 325 Gly Mg din Pro Gin Sly ely Pro Asp 375 LOu Lau Ala 390 hmn Asn Th 405 Het Ala Thr Lai Lou Pro Ala Thr Lou 45 Sly rg Gln 470 mxr Tyr Thr Lou Ala Ile Ala A-la ioe Ser Asp Ala 535 GIU lie Phe 550 Bar lg [jcu 565 lie Pho As= Asp Phe sly Pro Pro Gin 615 Le Pro lu 630 Val Bar Gin C 145 flu Kis P. h Thr Tyr Gin Ala 360 Bar Lye la LOu His 440 Lau Sly An Pro, Gu 520 hr la cym 600 rhr Pal 1lu P 7hr a 285 Lou Pro Yak Thr- An Asp Met Val 300 Cys Lau Gin 71w 014 Lou lu Arg 315 320 Trp lie Leu Ala flu Ala Pro Thr 320 335 Tyr Val Ala Ala Pro Lou Cys Leu 345 350 Lau Va1 Pro Lou Ala si Gin Lau 263 Pro Ile Phe Lou Pro Thr Asp Bnz Thr Trp Val Arg Ann Sar Glu Mho 395 400 Phe LOu Qyn Thr Hi Lou Lou Cys 410 415 Arg 0n LOu Pro Lou Cys His Pr 425 430 Thr Mg Tyr Thz Lou ala Va3 An 445 Ass Pro Olu Sly Lou Val Asp Gln 460 Lou Ri Tyr Lou Nat Bar Tbr Sly 475 480 Phe Dye Lou Pro Asp Bar LOU Ag 490 495 Ann Tyr 1e4 Tyr Arg Asp Amp sly 505 510 Her Pbe Val Sor 43u Rio Val sly 52 Val Gln G3n Asp Sr GIU Leu Gln 540 Sin Ala Phe Lou Gly Arg Glu Bar 555 560 T'r Pro Sly (lu Mot Val Lye Ph. 510 575 9cr Ala Gla Ris A& Ala Yal Asn 185 590 rrp Het Pro Asa Ala Pro Bar Bar 605 ye sly Thr Thr Thr Lou Lys Thr 620 Lzn 11o bar Cys Asia Amn LOu Lou 635 640 'ro Lys AMp Gin Arg Pro Lou sly 650 655 lu Mlu Ala Pro Arg Arg Ser lie rem 670 In lie nor Ax g ^p i1e ala Gig aet Ar; Lu Ala a 680 Ag An S in Sly Lou Ala Leu Pro Tyr Tar [yr 690 695 685 Lou Asp Pr Pro Lou 700 Ilo oim Ama sow Val Bar Ile 705 710 dc210, 3 4211;. 1470 -c212:a DNA 42112, Ra sapienas C400w 3 atggcagtot aaoatotc tg atgggcaggg @tgggtga tattggtact tgotatoagt atttgtcagg caaatgct agctteagg aoateoatga Oc~ttoatg ggotootgsa *CAmagtatg ggtgtcnatc aatgaoatgg gycogacago ctggtgccct aacaotgact otwtraog *tqggaag agaqgcttg acagoatgtg tcacuctggt aottgecme tcttgotgct gtaoogcat ggattgaagg actatottoc accgetggaA 49"9990gt aagcagtgg tgtaicatgga coatoectgc agaagctgga toacagagca eegtoacget eggec-ct t toactagagga agt-acgtg tggacatca ee-gaatgyga asaacaacac tgcgcaat ogatgcaggt aagatotggg tgtgaccact gggcacgtgt tggatcggta gegtgtacao atgtgtcacc cta~ctgcacc ccteeteetg gatatatc agaceagasa6 gaatcggtatc gaeaattt gtccttggg& tgaootgcag ctggtgtgaa ccactgcat getggaea ctaetggata cgfcccaotg ggt@oata@O tgagggaogg ggtgaaac acagcageg oagaamtaca aqgtqegtq aaggaggt aagctttortt Suacoggat; gtagtgtate gtggagcoa ga tcacaga. a taugttoc tttgoottga vtgocoggct egattatoag atgaagatto sacatottat gatotettet tctagcttgo g3acacatgc ctggag tgectgctgt ggccaggaac eaegggapot: aotgoatggt eaaagacgac tccccatgaa cescceagac gagggtgt c ggtgeoataa ttgggtacca ocagaaget tgcagaeaga eccccaCcOs4 ggatcaqoc Ctgacagac gggtgcgcaa tgatgtgoga acsagatect cgatyctnaa acesactaca cacaatggac gctagatcga eaacagag cagoaaggac ccsottocca agaagnact cogggoecga tgflCgtcaac aacttgtgta aattgac-atc gatataget; gtcguaag gcttcaag gtacatgjaat gct-asagg acaggggc~g aatcttoctg etotgagttc ggCtogcc actoocecac ccescaggc actacogaga 240 300 360 420 400 540 G0o 66D 720 700 900 960 1020 10500 1140 1200 1260 1320 1350 1440 1470 got cagocag acccccgggc ctggctgctg geeaagaogt gcactttctg tgeaegcatt gccgctetge caccecatat glacaccatc gcgagggcca cacgoggegt cctggctatc cgcattga -K2ioP 4 4211.,, 489 t422 M -c213:b Uomnaapiens C400:0 met A2a Val I @17 Tbx Lou Bar Pro Lye Bar Val 0i4 4 Tyr Mrg Lau Cy. Val Thr 5 Asp Asn Ile ser V/al Th: 25 Gin Ar; Lau Asp Ar; Met Thr Gly 10 Lou V/al Oly Ar; Pro Tyr Lou Ary Ala. is OWy Tbr Ops Gly Gin Axp ph. Ala Pro OWy Lys Tyr Lys A" Cya Thr Ala (flu Gly Glii Lou so Lou Leu mcr nwp Bar NiB Lau An Val Tyr Cyw Bar PMe Pro eye Gin Ar; Tyr Phe Phie Art Lye Ila cyw Val Pro Asp Gly ear vai Val. Cii Tyr Gin Trjp Sly Tyr Cop Pbs Pb* sly 275 Cy Ile Ber 290 Ala Pro Le 305 Gly ABU Ile fun Cys Lou Len Trp Ian 355 Bar 0Th Thr 370 Glu ?rp Asp 385 Len Val His cla Ala Phe Ie Tyr Lye 435 flr Il. Ala 450 Pro Mle Sly Arg Arg Pro Gly Thr Ala Arg hz 120 aima Arg Thr Arg Olm 135 Ile Tyr Ala Pro fly 150 Glu Mat Glu Ser Asp 165 Val Asp la Gly Asp 130 Not Lye Ils Asp 11 200 tyr Bar Aim Thr Lys 215 Bar Lau Gly Met Lys 230 Lye Lye Lgu Asp Asp 245 Thr Thr Lye Tyr Val 260 Tyr Gin Tyr Lou Awn 280 Bar Lou Pro Ser Lye 295 Le Uly eta Asp Tfl 310 Phe Lem Ala Asp Tyr 325 Aan Sly Arg Gn Gin 340 Bar Pro Gin Oly Ala 360 Pro Sly Pro Asp Scr 275 Trp Lou Lou Ala Lye 390 Gin Ann Amn Tkr Bin 405 Ala Met Ala Thr Lou 420 Leu Leu ItI Pro Rio 440 Avg Ala 'Thr Lou ieu 455 Pro Arg Ag Pro Sly 470 Gtu Asp Lou sly Gly 485 Ile Cys 01 Asp LOu Arg Ma A1s 140 Pfl Pro CyR met 115 Lye Lys phe Ala 170 Sor Bar Gly Aga 105 Pro Gar Lou Met 7Tr Ile Sir Lou 220 Lou Arg Cly Lou 235 Met aln Awn Ile 250 Thr Olu His Trp 265 Gly Val Asn Pro Lau Pro Val Thr 300 Cys LOu Gin Thr 315 Tsp IIe Lou Ala 330 Tyr Vat Ala Ala 345 Lou val Pro Lcu Pro Ile Phe LOu 280 TkW Trp Val Arg 395 Ph. LOU Cya Ttr 410 Arg Ca Let Pro 425 Thr Arg Tyr Thr Bar 125 01* Vai Asp Lou Thr Arg Tyr 190 Tyr met 205 Lou Ph Lau Asp Phe fTp Cy: Olu 270 Val Met 285 Ann Amp Gin LOu Clu Ala Pro Lau Val An 160 Lys Thr 175 Lou Pro Slu Pro san Ala Arg Lye 240 Oys Hi. 255 Asp Hi. Leu His Not Val Slu Arf 320 Pro Tbr 335 Cys Lau Leu Pro Lou Glu cym Tyr Ala 365 Pro Asp AeS Lou Lou 445 Len Pro 3so 1le 0in Lea Tb3r AIp Sir Per Olu Phe 400 Lou Lou Cym 415 Cym Rio Pro 430 Oln Val Anni Val Asp ain Lou Pro Arg 480 Asm Pro Tyr Pro Him ely 460 Leu c210z c211 2236 .C*122, am MEmo sapiens 4400: w acggtagaou acgaoaatt atgamaattg agegatot ctottgguto eatmagacet taeeagtact aagctgoctg acagagetag acaeaotgcc agooaoagg agcc~atot tgcmiactctg tgogaggcct otoetactoc otoaacceg ctoatgagca cgggcccgcg tgggoggccu tetgtgeagc ctgggccggg ttcotcaRctg oatfactttg aaaggga tngtaaeaaec ggcacctscc cagagacgc cectacct aatacoucc cagaccatto cttttgcagc goaeagoag9c aaaaawae ggaacgccca ttatcgtaac tacttctctg ggtcccttt facccctmac tcaacagctt gtgtagacca acaccata cgcctet gcaaggc ccaegacaa tgaatggtgt toaccaatga taaaggccg gggagotggt toctgccac agttaotggt tcgccatggc ccacactaog agggcctcgt agggaotggt gcgtcctggc ttgugagctt aggattogga aaagctoagg ClAtcatott gggcotggat ccacc acoct tcctcctctt cagatgage&. tggocccagat acctggacoc aag~agatg catctct caagatggot gtcoaggrta aesgttocac gcttgggctt atgcctttct. gaaaagcotg grcccragca gectt tcaggagatg gagccagacs. gggtgacagc agtgggaatc cctgatatc atggagccca caatgcaatc cctgogtocc cegqnageag ctggatgaca. gtatgtcaca gagcaatggrt cntoocgta atgeteaC&t catggeggcc ccettgaeg c-atattocta goggactact coagCagtGC gtggccgoc gcccttgoc atccagctca tgaotccgaa :gggactggc gcacgaaaac acceact cacgctgcgo 0890 40 @90 atacacgctg @&ggtg~a gg&ccaggta acgtccatog comottocc taoaccaatt tatcccsac tacac&tace egtcccaaa at ogtgggat gctgcaggcc tggaotggcg cttccaago cggctgtgca caattgctc ot eccagegc gcccaatgct ocatoaceca gaagattan etagacacco ctggttggtt agccaagaac ottoacagag gaggoocoga ctcaagggac atocaggage tOOcatcatt gageacagog aaaggtccaa gcatgaggag gttctc-agtt cacctgaacc otgacatcat acaaactggg anagoccng aecaaagtcc ttottgeacc acttgggga ctgtgctttt otoaocato aogttctott tcttccaagc goaactgggc acagoaggac cctttaagg gaggggaaga agrkatttgc gqtacotgcc itgttagata tgggaatgaa tgcsgaftcat gtgamgatca goat etetag gacagcac ggatoctffe gcugtmecc tgctggccas ttotgtgcaLc totgocn~cc cacogagag ggaggamagg9 totifoottoc gaa~ogg actattatoc agatttttgc coocaggaga otgctgtcaa tgaggeagco tcoctgaagt caaggucca ggoggageat. ggaaccaggg totccateta gacagttoC ttctcttctg cootgagctg aatgaaata goctcttgtt otccesato atagagecac cttgacaaag eggottocco ctcagcoao gottcgaggg cttctggtga cecettggg cttgccago atgcctgaag ggaggoccce gctgtggctc cgggcctgac gagtgggtg gcatttgotg catctaaag ggoacgotg ecteatotac ggacagcctg cttgaagatc cagtgacgca tcaggcgttc gatggtgaag aagtgggcag ccaccccag gaacateaga gaggcccctg ogeagctte tatggcsctg accaccoca teaggtectc earAtggaga tgsgmgacca agcccctccg ccagcctt cccaaafltac agoocagat 220 180 240 300 360 420 400 540 400 660 720 1180 1140 g00 060 1020 1080 1140 L200 1260 1220 130 1440 1600 1560 1740 ISG0 1860 1920 1980 3040 2100 250 3220 2236 tgtgtgqcctg rtaatut &ttggagggc agcttgectg 6 c221, 556 ,c22: PRT c213: Homo sapiens 4400 6 Val Asp Val tAn Bar Ph. Gin Gin. met (flu Bar Amp Lys Lays Phe is AIDa Lou Thr Lys Thr Thr Thr CYc Val 25 Phe Pro Met Ann Arg Tyr Lou Pro 3S 36t 1Wr Me Ginu Pro Gay Loeu Lou 40 Amu Val Arg Tyr Ile Pro Ala Oar Asp 01n lily Amp Bar Oar lily Lyu Ile Asp Ile Pro Sir Lou 46 Sar Ala Tb: Ly. flr Ile Bar Lau Gly Met Lys Len Arg lily PhD Amu M.a 75 so 0 C) C), en c Iou Lou Asp Arg Lyr, Gly So: Trp Lys Lye Lou Asp Asp Hot Gji Asa lie Phe Tip Cy 100 Trp Cy. Glu Asp Lis Pro Val net Lau 130 Thr "a Asp met 145 Thr Glu Lou alu Ala Gus Ala Pro 180 Ala Pro Lou Cys 195 34U Al lie Gin 210 Len Pro Thr Asp 325 Ag Len Ber Giu Thr Xis Lau LOu 260 'to Lau CyR HIS 275 Thr Lou Gin Val 290 ly Lou Va Asp 305 LOu Not tar lhr Pro Amp Bar Lou 340 tyr Arg Amp Asp' 355 BOW alu l Val 370 Asp &or cim LOU Lem fly Ag lu 4 Glu fat Val Lye 420 Bim Ala Ala Val 435 His Lys Th His Ph. Ph His Gym 11 13 Val Ala Psi 150 Arg Gly Asl 195 Thr Hiso Cy Le Le Tq Leu $or 0i1 21! Bar Glu Trj 230 Phe Lou VaI 245 eye Giu Aa Pro 3le Ty Ann thr Ile 2R1 CIA Val Thr 310 Sly Lau Ala 325 M~g Ala irg Gly Lou Lye Sly Tyr Tyr 175 Dii Ala Tip 390 so: Sar ly Phe Lau Tb: ksn Bar Gly x Pile Thr Thr Lye Tyr VI Thr 31 105 110 e Oly Tyr Gla Tyr Le Au sly Va 120 325 Se: BOr Leu Pro Bar Lye L.eu Puc 5 140 D Iau Lou fly Gin Amp Thr Cys Lev 135 Ile Phe LOu Ala Asp Tyr Tip Ile 170 17! Lou An Oly Arg Gin Gin Tyr Val 185 IS0 LOu Sar Pro Gin Gly Ala Iu Val 200 205 a Thr Pro 0ly Pro Asp Bar Pro Ile 9 220 3,Asp Try Lou Len Ala Lye Thy Txp 235 Lis Gin Ass Asn 7hz His lbs Lou 250 355 Pie Ala Met Ala Tbt Leu nrg Gin 265 270 Lye Lau Lou Lou Pro Rim Thr Mg 280 385 Ala Arg Ala Thr Lou Leu Ann Pro 300 Sax Ile Sly Arg Gin Gly Iou Ile 315 *HiS Pbs Thr Tyr Til Asn Pie Cy 330 335 ly Val Lau Ali% Ile Pro Asa Tyr 345 350 I1e Tp Ala Ala Ile Glu Br Phe 360 365 Tyr Pro 6eo Asp Ala Bar Val Gin 380 Tht Gly 6lu Ile Pe Ala Gin Ala 395 Phe Pro Ser Arg Lou Cys Thr Pro 410 415 Ala Ile 11. Phe ADD Cya Bar Ala 425 430 Gin His Asp Phe Gly Ala Trp met 440 445 Oln Pro Pro Pro Gin Thr Lye Sly 450 Asp Thr Lou Pro Glu Val Ann Ilt 475 Trp Len Val Bar Gin Slu Pro Lye 490 Pro Asp Glu His Phe Thr Glu Glu A His Val a ln 160 Ala Pro Phe Val 240 Eye Len Tyr lu Tyr 320 Lau His Val Gla The 400 Oly Gla Pro lir Bar 410 Asp Ala Aug Ala Pro sar Bar Met 450 Thr Til Lou Lye 7hr Tyr 465 470 Cym Ann ASA Lu Le Leu Gin Mg Pro Leu auy Thr P00 1'ro ~s 1 Azg et lie Ala Arg 455 LOu Phe Tyr 505 510 Ala Phe Gin Boar Ag Lou Ala Gin Ile er 515 S20 525 US hasp Z1* Gin Glu Arg Ann Gin Oly Lou, Ala Lon Pro Tyr Thr Tyr 530 535 540 Loeu Ap Pro Pro Lou Ile Cii Ansa t ar a or f'lu 545 550 4210om 7 c21t2, L005 4c212I MA c-213p Nowa sapiens cCO00p 7 fqgtagacg acgaamactt atgamtg magas gatct ot~ttogac catnagaect taooagtaoo asgotgaetg moagagotag ugacaagg agooccatet gcqa8Atctg tgsggct atoctactoc atcasocceg Otaceacoae taaacagatt gtgcagacea acatccate ogetqctctt gjcamqggcta tagaaa tgasbtggtgt taaccaatga agaggMgau tmflcggaeg gngogtggrt toargeecac agttcctggt tcgaeatgga coacacteg agggcategt egagagueg4 taaggagatg gggtgacac cctgatgtac caatgooatc ctggaagaag gtacgtaaca caatccagt~c catggtggec catattocts CcagCagtac gocatggee tpaotoogno gcacgaakaac OaOgcrtgogc acaoacgctg ggacoagcct gcotgaagat gagtaaca agagatttga agtgggaacc ggtaccctgc aLtggagcca4 atgttcgata coegcget tgggamtgaa otggatgaca tgcagaaaat gageactggt gtgaagatoa atgctc coot goatonctag accttgctgg gacaggacac gaggactaet ggatootggc gtglgccgcce cactg3tgoct atacagoto. gaaagaaoe tgggactggc tgctgaoaa acagact ttcttgOSC cagctgaegc titgoaacca caggtgacS ccetgegag ggcaocgc ggcgtcctgg ctgggcggoc attga attgacaaaa oggttcca oteagocaco gattcgaggg cttctggftga ottctttggg attgccctgc acctgaag ggaggeccac gctgtggct@ cgggeetgae gmogtgggtg goftttgctg catotacSa ggccaScgctg atateaccaa 120 100 240 300 360 420 480 540 600 660 720 780 H4D 500 960 100S c2102- .C211;- c212> e213:p S 334 F=y ROta Sapiens 4400b. Val Asp Va Asn flux Pb. Gin 0Th Mat Glii Ser Asp Lys LY0 Ph. is LAU Thr Lys Thr Thx Tbr Cys Ass, Arg Met Tyr so Lou Lau Tyr Lau Pro Met Glii Pro PhD ABn Ala Val Asp 2S M4et Lys Sin Sly le Any Gly Pba Pro Asa Vaol Axg Tyr Car Ala Asp Set Ber Lily Ile Pro $,er Lou Lye Thr Ile Ser Lys Lieu Mrg Gly s0 ASP Met Gin Ann Ile Pro jAa Sex Lieu Lou lIeu Usp Arg Lys sly Ser as 11e Pla T zp Cys ii Lys Thr Trp Lys Lys Lou Asp Ciii Hi. WF &Y Pro Val 130 200 C0li Sap 215 Met Lieu Rig PhD rh. His eye Ile lag Ph. Th~r 106 Oly Tyr 120 Bar Sir Thr Lye Tyr Val flax Gin Tyr Lou Lou Pro Ser 140 110 Gly Val AGM Lieu Pro Val AmL UmP Met Val Glu Leu. Gin. Ala pro Lou ISO Oiy Asa Ile Kin cys Leu Lou lily Girt Asp 155- Rho Loa Ala ASP 170 Asa Lly "g Gin Ber Pro Gin sly h cys 3.n aim 160 Tyr Tip Ile Lou 1.75 Ma QIU Ala Mia Pro Lou 195 LOU Ale Ile 03-a Tyr Vel A-1a ISO Ala Lu Vai Pro LOU LOU TtV 21.0 Lau Pro Th: Gin Stau Bar Gin 215 Asp Her Gin. Trp 230 Gin Pus Lan Val Pro lily Pro Pro Ile Pb. Ann Bar AUp Trp LeU His Glu Ago 250 Lou Ala Lyen Thr Tip Val 235 240 Asa Thr Wes Ph& Lou Cyn Thr His Lou Lou 260 Pro Lau Cy. Him2 375 Thr Loa n Va An Gin Gin Lou Cin Ala Pho Ala Nat 256 Ile Tyr Lys Lou Lou zo0 Thr le Ala Arg Aia 295 Pro Ala (Ely Pro Avg Ala Thr Lea Arg 290 Sir LOU Lou Pro Thr Lou Ar; Pro 315 Leu Gly 270 Rio T1w An TYr USs Lau Asn Pro 01u ValI Asp 310 Lau Pro Lau Pro Az-q Arg Afl Pro Gin 325 Ap 330 Gly Tyr Pro 13ly His e2io.* 9 4211.% 1640 -c2i2:P mA 'C2133, Hobapiens 9 atfgoagtgt aot to. *tgggeaggg act cegggtagc tot tcttggtaot gta tgotatcagc Sga &tttgtaag e0t caagaatgct ac agctttcmg aga saccagggtg ace eeataaatga tgt @tattoamag ca ggcteetgga age *caagtatg to. 39tgtcatc cog *Btqacatgg tfl gnastat toe ggoogcoagc agt Otggtgcct tgg cccactgect =og atggtgcaeg a-u &tggcacgc tga actogataca ago Ctogtggacc agg gctgltg cactggt ~tcgcccc :tgotgat igcgscat ittgaagg !getggas 'tggagt a igcegtqg tccctgc agotgga icagagca tAatgot tgtgaccact gggcaogtgt tggatoggta gegtgtacac ctgtgtcaca otacogeaoc ccc t ctg gatatatgc agaceagaa gaatcggtsc gcccaatgtt gtccttggga tgacatgosg atggtgtgaa cc&Cctgcata ggtaoctacc ggtgaaagcc cegagtaca aaggagogot gaacC~ggatg gtggagotga gatoaflgga ccggcttco tttgocttga otgcooggot cgatsctcag &tgflgatta eacatottet gatcacttot totacttgc gacaaatgca a tgggagg cgectgctgt aocccgggC goaacge tgcacgcatt cacccatot gcgagggca caagg~tca' tgagggcogg aacaotgsa cosagoageg gotagatoga aggtgcgttg cmaaggag aogcttcctt cegeaaggac gtagtgtatc cot toco ggooaggaac agoaasgast oaggagct. cogggcoga cctgoatggt eaaegeegac toaaaatgea ccaoosagao gagggctgtt ggtgooataa ttggtaco Coaggeagot tgoagacaga caccoaoooa ggotcagccc Otgacagcc agaagteaaec aaottgtgtm mattgacatc gatctogctg ggatcgcaag gacottosog gtecotgaat. gctgqtcaoc gctaga~gagg o tgctaa Cc&ggggg9cg aatctg 120 230 240 300 360 420 4800 540 600 600 720 760 sac 900 960 1030 1080 1140 1200 1260 1320 1180 1440 ccoont gctgggaoag tagegga otaatggatc aotggo atggga acamo gcooqot tgcaggt tcacgtc cgccoactg gatcagocag ctggctqctg gctotttatg gaegcactga gaacacoatc cat ogggagg gggtgcea otetgagrttc tgotgtgoga ggaoga aoaagcteet ectccosac Ogatbatcea ccoagagggo tctactoat gagekAgggc otgacosoct reabctao oaatttotgo ofloaca gootgogga oawggnte otggotatcc coaaCtBCC& otacegagac geoggocuga agaottgoc ggccattgag agotttgtct cagaaatogt ggamtat tatcaagtg aogostctgt. gcagcaggmt teggagngoc aggctggao tggcgagatt tttgoeagg egttoctggg oogggaaago tcaggtttoc oagagget gtgcacceca ggagagatgg tgaagttcct cUetgCamtc atattoatt gcctccca gcsegcgct gcacagt ggaaggmogg flgaggrggt atoagggstg gtaagaggg aggrtpatact caactatgg oaaotga cflO2 c211j 615 c212:i PRT c22 iSlowas sapiens 1500 1560 1620 16a0 1740 1800 148 4400b Met Ala Val Tyr I Sly Thr IOU Asp 3cr Pro Lys Gin Gar Vol Gin Lys so LeuLO LOU Arg 61 Box Tx-p Tyr Cym Bar Nis Whe Pro Ica Lou Arg Pro Sly 115S Lou Lou Asp His 130 ArgTp Lye 1ia 145 Bar Pits Gin Ulu Tar Thr cys Vral too Gly Pbs Pro Met its Am Val AMg Tyr 210 Ile Pro Ama Bet 235 Cly Bar Try Lye Arg Lau Cys Vali Tin Thr Oly Pro Tyr Lou Arg Mla 5 10 is Aun Il. gar Val Wax Lau Vai Gly Tbr Cym Gly Clii 25 Arg Lau asp Mrg Met Gly Arg Asp Pbs0 Ala Pro Cly 40 as Tyr Lye Val Arg "y War Ala Glu Lau Oly OIju Lail 55 Vat His Lye Ulu Mg Tyr Ala Pho Ph AM Lys Asp 70 75 so SexAr Ile Sin V7 al Tar Oiu Pro Anp Gly Ber Val as go Cys Tyr Gin Trp Its aW l0y Tyr 07' Thr Val Glu 105 110 Thr Ala Ar Thr Ile Cysp Gin Asp Sew Lou Pro Lau 120 125 agV Thr Arg (flu Leg Mrg Alm Arg Gin Ului Cys Tyr 135 140 Tyr Ala Pro Gly Phe pro Cym Met val. Asp Val han 150 155 110 met Gin 8cr Asp Lys Lye Whe Ala Lou Thr Lys Thr 165 170 1.75 Ap Gin Sly Asp Sex Set Gly Ann Arg Tyr Lau Pro 185 190 Lys Ie Ap Iie Pro Ser loeu Mot Tyr Not Uilu Pro 200 205 gaw Ala Thr Lye Thr Ile Nor Lou Lou Phe Ann Mla 21.5 220 Lou fly Met Lys LCU Arg Cly Lau Lou Asp Mrg Lye 230 23S 240 Lys Lev Asp Asp Hot Gin hAn Ila Who Trp Cyn Nis 24S 250 355 War Lys Tyr Val Thr Glu His Trp Cyg hits Amp Etc 265 270 Gin Tyr Lou Ann Gly Val Ann Pro Val Net Lou Xis 290 285 LOU Pro $ow Lys Leu Pro Val Thr An, Asp Net Vol 295 3100 fly Gin Asp Thr Cys Leu Gin Thr Gin Lou Ula Arg 310 315 320 Lys War ph* lb bs. f~ly 275 Cys Ilo ser 230 Mla Pro Leu Thr 260 Tyr Lau 308 Gly Ann Ile Ph. Leu Alm Asp Tyr TrP 1ie Lea Ala Ulu Mla Pro Tbr 325 His Cys Laeu Asfl Oly Arg 340 Lf Try Lou $or Pro Gin 355 Ser Gin Thr Pro Gly Pro 270 Gin Tzy Asp Tzrp Lou LOU 385 390 Lou Val His Gin Ann Asn 405 (auw Ala Ph. Ala Net Ala 420 fle Tyr Lys Lou Lau Leu 425 iTt~ Ile la Avg Ala Thr 450 Val Thr Bar Il. ely Arg 470 330 Gin 0Th Tyr Val Oly AMa 360 Amp Bar 375 Mla Lys Thr Hin Leu Val Pro Ile Thr lrp Ph* Lau 236 Ala Ala Pro Lou Cym Lenu 250 Pro Lou Ala Ile 0Th Lou 355 Ph. Lou Pro Thr Asp ser 360 Val nrg Asn Urw Gin Ph. 309 400 Cys Thr His LOU Lou Cye 415 410 Thr Lou An Gin 429 Pro His Thzr Mg 440 Lou P" Len Tyr Thr Lou Cys ai. Pro 430 Gin Val Amn Val Asp Gin Lou Lau Asn Pro Gin 455 Gin aly Lou Ile Tyr 445 OlyV Len 460 Lau Met 8cr Tbr His Ph. Ala Arg Gly Va2 Soo Lou Lyn Iie Trp Thr Lau Ala Tyr Ttir hae Phe Cys 490 Ala XIe Pro hAn Tyr SOS Ala le Giu ter toe 520 Asp Ala Bar Val Gin 535 11e Phe Ala Gin Mla Pm6 ASP Bar LOU Arg His Tryr Ari Tyr Tyr 530 Ala Tip Pro Ser Val Bar Gin Asp 540 Ph. La Oly GinU Asp Asip Gly 510 Ile Val cly Gin Lau Gin Thr Gly Gin Gly Arg Glu Pro 03lY Who Pro Lou Cys Thx not Val Lyn flo 575 Lou Thr Ala Bar Gly Gin 595 App Thr Pro 610 Ile Soo hap Phe ABU LYS Gin His Ala Ala Vl Asn Sao Asp MY ly Gin GSly Gly 605 Cly Am, Sly Mg9 Lou Lou Mla t2l03 21
12. 42111. 113 '412> va 4213: Romo sapients cGOO, 11 £tggtagaog toanoagat t tcaggaqat.9 togacaatt gtgtagacca ggtgagc atgmaagttg Aagagatat otgttggatc cOSSagacot taccaf taco aagatgaotg *cSBOgtag @cccatgc c AgC@Cceas *catoacatc ogotgorctt gcaagggatc tocogacasa tgatcgm tecaatga agaggg taceoggoog gggctgg; cot gacgtac caatgecatc ctggaagaag gtatgtcaca caatcccgatc catggtga eat at tets acawotao gcccttggcc gagtcaga agtgggaata atggagccca cctgcgtaa t ctggatgaoa gagcactggt atgo tocact ccc ttqctgg geggactact gtggogccc atccagctea agaaattto ggtaCCtgac atgttogat* tgg&tga tgcagaacat gtgB*9&tca gcatctctag cttgacaaag cggcttccco ctcagecacc gattogag ottctntgC cttatttggg cttoccagc ganaggaeca toetgcag ggatcatggo gaggeceec caatgtgcot gctgtggcta gocagaCocC CgggCCtgaC ageccatct tactgocoso og*0msatg agttoctggt tgcg&ggeot togocatggo ctcctactco occa&actcg ctflflacco agggcatcgt etoaugagea cgggcotggO cggcegg gogtctga tgggcgcca ttgsgagott totgtgoaga agggattcggS atgggog uagecagg ttootcactg castcatott gaaggoagag gtggaatcag tga tg&ctOCgfl goacuaAs oacgotgec ataoacgovg ggaccagtc ccacttcaoc tategoc tqtct~oagaa gctgoaggcc tttccaagc caattgotct, ggatqtgaa zgggaCtggc aacaogcact cagctOcgO oaggt~gsca acgtecatcg taoaccamtt taccaCtOCc atogngct tegnotwagv o~ggctgtgca gcoagc"~ gagggaggtg tgoctggccaa gacgtgggtg ttctgtgcae gc~tttgotg tetgcoacco catctaca ocatogogag ggcoactg ggaggcaag totgootteC mtattatOC agatutttc cocoaggaga ctgotgto~b atactoccet ggacageetg eotgaagato cagrgacgoa tcmggottc gatgggtag oagtgggCag tctggooaac 720 640 900 560 1020 1030 1140 1200 1260 1320 1310 1313 c2203- 12 421.% 460 c2125- PIT
13. 42133. HMO sapiensa c400;- 12 Not Val Asp Val 1 Ala Lou Thr LYS Amn Ar; Tyr Lou 33 met TZyr Not Glu so Lea LOU) rh Ann ZeOU LOU Asp Are Anna 88: Pbs Gin Gju Met Stu So: Asp LyS LYS Who is Tb? Tiat Tb: Cym Pro Gly the Pro 40 Asp Gin Gly Lys It* Asp it* Pro Bar Lau Lys Thr Ile gar iro Afl Vra1 Ala Ile Pro Ar; T'yr Bar Ala Ala tSr Lenu Sly Lys LoU Arg Uly Bar Trp Lys Zia Ph. trp Tzv Oyu Gin 2S Pro Val met 220 Itr ABU Asp 145 thx Olta Lou Ala QitU Ala Ala Pro Lou 195 Lau Ala Ilu His Lys Thr Phe Lys Lou !§o Thr Lys Gi1n TYr Usp ASP MOC Gin Amn Tyr V/al Thr (flu. His Lau Asa Gly Val Amn Hits Ph& Who 12S Lou Pro Set Lys Lou His Gym Ile 135 mat 1/al MA Pro 150 Gin Arg Sly An 165 Pro flit His Cys gor Lou Lou aly Ile the Lau Asp Thr ASP Tyr lou Pro V/al 160 TrR Ile Lou :175 Lou A9ma Ar; Gin Gin Tzp Leu Bar Lau Lou Pro Gin Gly 190 Lou V/al Pro Pro 11. Ph& Gin La 210 Lou pro 225 Arg Aen Th: use Pro LOU Thr Asfp Ber gar Gin Pbo 245 bell Lou cyz gar Gin Thr Pro dly Pro 21! Gin Trp AUP Trp Lou Lou 230 235 Lou 1Val Rio Glu Asa Asa Lys Thr lTp Thr His Pbs Olu Ala the Ila Tyr Lys 250 Ala Ho0t Lau Gym 255 Alia Thi Lau cy's Pro 270 Thr Arg Tyr C" ProLu Leu Pro His Thkr LoU 290 aly Lou 275 QICn Val Vail AUP 20a Ann Thr Ile Ala Arg 295 Gin Val mhr 5cr Ile Lou Not Sor Tbr Pro Asp Sor Loa 340 Tyr Jag Asp Asp 355 Ber Giu Ilo Val Ala His Ph& 28S Ala Thr Lou 1413 300 ely Arg G1u Gly a's Tltr Tyr Thr Asa 330 Lou Jaa 1206 Pro Ala Aia Ile Ila Sex Alp Aim Ser 300 AND Pro Gin LOU Ile Tyr 120 PMe cys LOu 335 ABC Tyr His 360 set Ph. Val Val Gin Gin AM9 Ala Arg Gay Losu Lye dly Val 345 Ile Trp 360 Tlyr Pro air Tyr 370 Asp Bar Gin Lou Gin Ala 390 Ug Gin Bar Ser Tbr Gly Giu 11. Phe Pro Bar Ara 410 Put AIRalGi Ala Gly GlUy LOU Cys Thr Pro air 41.5 Gin Noe Val Rio Ala Ala 435 Sly Uiu Giu 450 405 She Lou A0n% Sot Tr Ala Ile 425 my Gin Asp 440 flat Pro Lou Ile Phe Asa Gly Mrg Sly LOU Ait Aim 460 eye Ber Ala Gin 430 Sly le AMg Asp 44S ely Gly ASP .c=2, c211 c2U.2 .233- 23 1441. IWA Hlow sapiens 4400:p atggcacg! acgcataea ctcgtgga@C otggeceout ctggotatcc agotttgtct tcggagetgc toaggtttoc atottcaan tggatgacca aaootgaag~a atottatggt gagoacttca cagatctca aaoteacc gB**gaagg tcctgttot tggotetgac ggutaaaagc ttoaottott ggtttotgag tttctacgtt 900tggaaao tagoaottt t 13 tgogooagot ogetgoAggt aggtoacgta toactacac ocac"tacca oagaaatcgt &ggcctggao cangoogget gctctgc cor atgctccatc CtrACotaga. tggttagca eaagc ggcatcca tcattgaga. tacaagcatg cagttcaact atoatacaa cgatgccOa gcacccttg @ttttctocc otctttctt a tgggcacaga aaggggaggg gcogctctgc cacao Oat Ct gamoacoata gogagggcca oatcgggagg ernaggoCtcft aaatttctgc ottoggaa ottO~gagaC gacgtctg& ggtactat titcccagtg tggcgagatt ttrgecagg gcgcacacca ggagagatgg gca~cgctgat gtcaacagtg atcrtgagg osgaccccac eacactcot gaagtgaaca agaacocaag gaccagaggc accgaggcgg agoatogoeg acaigetoct actecccc&O ogctgccusa cccgagggc tetaoctoat ;ftgeagfl gcctgcgggc eogcggcgtc &gatctgggo ggccattgag acgcatatgt gagoaggat cgttatgg cogggaaaga tgaagtcact caatgcmatc ggaagcatg* ctttggggcc cacaguccaa ggggaacacc tcagotgtaa afaccto ccctgggeaC ataaocaqat oat tacagag ccgctggcc cactgocctS cacctaactg ccmeastac cacccaga tcatecagaeccattocatoc ggagactttt gnagccaaga gacctgcaC& goagcgtcca ctccaagg aaggaaccgc ctcttggat gcocagcttg actcottoto otacatgoc cagcttcatt ctctggaaaa catgtggtcc cctttgtoce gcctggOccc caflggctg ta0 180 240 300 360 420 480 540 600 660 720 780 040 900 960 1020 1000 1140 1200 1260 2320 1310 1440 1441 ggaggac aagcgtat cc aggaggacca guacattata atgggcoctg agtccaatgc ggpaactO tatatca aagactaga mggactgt gaagaattgg cagggtctgg atatnoCaC& gttcctcagg tctgcacat &gctgtgflga acaatagca ttgtccgc attaccaa gacaCccac gaatggctaa agggoagat t c2l20m c2llb 212. <213 3 it 291 PAT Homo sapilen 4400a 14 Met Ali Thr Leu Axj ala Lou 1 5 Lau Leu Pro ais Thr Ag Tyr Ala Thr LOu Leu Asp Pro Glo Sly Arg Gla Sly Lau lie Tyr so 55 ThV Tyr hr Ann Phe Cye Leu 70 LOU Ala Ile Pro Ann Tyr Hta Ala Ala lie Clu. nor Phe Vl 100 Nor asp Ala Ber Vl Gin Gin 115 Gl%1&la P1he A Gin Ala Ph. 130 135 Bar ng9 LeU eye Thr Pro Gly 145 10 Ile Ph* Asm Cy; Sor Ala Gin 165 Asp Ph*oly Ala Trp Net Pro Pro Pro Gin Thr Lye Gly Thr 195' Lau Pro lu Val hen lie oar 210 2%5 Val Bar Gin Oli Pro Lye ASp 225 230 GIu Hiu Phta Thr alu alu Ala 245 Bar AMg Lou Mla Gin 11 5er 260 LfU A4 Lau Pro Tyr Thryr 275 Val nr Ile 290 Pro Lau eye Nib Pro Xie Tyr Lye La 10 Thz Lou Sin Val hen ?hr .la Ala Mrg 25 Sly Leu Vil Asp GIn Val Thr Bar fle 40 Lau Net Bcr Thr Sly Iau Ala Mis Ph. d0 Pro Asp Ber Le Arg Ala Arg Gly Val 75 Tyr Arg Asp Asp Gly Lau Lye Ilo Trp 90 ser lu 11 val Gly Tyr Tyr Tyr Pro 105 110 Asp Her Oti Lou Gin Ala Trp Thr ly 120 125 Lau Gly Arg Gla Ser Bar OiV Rho Pro 140 Olu Met Val Lys; Phe Lou Tbr AL Ile M5 160 Bis Ala Ala Val Ast Ber (ly Gla Kin 170 175 Ann Ala Pro er er Met Arg Sin Pro Ids Thr Tfhi Leu Lys Thr Tyr Lou Asp Thr 200 205 Cys Ann Ann Lou Lou Lou Ph. hw Le 220 Gin Mrg Pro Lem Gly Thr Tyr Pro Asp 23S 240 Pro Mrg Arg Scr le Ala A.l1a Phe Gin 250 255 Mg ASP le Gin Gin Ag hen 0in Gly 265 270 Lou Asp Pro Pro Lou Zi* Ole hen Nor 280 285 4210) I c21i3 210 1212;3 DEL -2 133 DcHo sapiens atggccacgc tgagooagot gccgctctgc caccacatat acaagetcct actcaoeac aetgataa cgetgcaggt gaccacocac qc~agggcca. goetgaccaa acccgagggc Ctcgtggacc egoctgcggg cocgcggcgt cctggctatc cecaotaco actacogaga cgmcggoeag aagatctggg cggccattga <210.- Is <211:0 %23-23, DR? 4213.- ROMG Bfl~tSh 4400x 16 Net Maa Thr Laut Arg Gi Leu Pro Leu Cys His Pro Ile Tyr Lys Lau 1 5 10 is Leu Leu Pro His liar Mrg Tyr Thr leu Gin Val Ann Tbr Use Ala Ag 25 Ala Thr Lou Lou Asn Pro Cflu dly Leu Val Asp Gin Pro Ala Oly Pro 40 Mrg Mrg Pro Gly Tyr Pro Gin Loau Pro Lou Pmo arg Ag Arg Pro 0it so S5 Asp iau dly 121y His c210 1 17 4211;. 420 t212w DEM 4213:b Rosm sapiens 4400;- 17 atggtgaagt toctaactgc atatotto aattgctctg occagaog tgotgtcanc agtggogc etgaatttgg ggcotggatg ccoaatgotc catoatoost gaggoegocc COCCoCaga ecaaggggaa CaoaCCctg aagact taco tagacaccat cagaagug saCatoagot gtaacaacct cctcctcttc tggttggttat gcaagaac oaggaag aggcoctgg goecetacca agatgagec tccacagagg aggoccogag gagagoate 9ggC ttw agagCCegoct ggcccagato tcauggacat tocaggagog gacagggt ctgomatgc cotacacata cctggaccct cccctcatttg agaacaqagt atacatotna 4210:b 16 4h11S 139 4l22. PAT 4213;. Romi sapiens 4400;. 19 Nan Vol LYe Pht Lcu Thr Ala IC Ile Pbs Mgn Cys Bar A Gin Rio 1. 5 10 Is Ala kla Val Aun Ber Sly Gin Him Asp vk* my Mla Trp mat Pro Aen 25 Ala Pro $Or Bor Met Arg Gin pro pro Pro OLD Thr Lye Sly Tbr Thr 40 49 Thr [mu Lye Thr Tyr Lau Asp Thr Leu Pro Glu Val Ash Ile Bar Cys so 55 Ann ABn Lou Leu Lou Pht Trp Lieu Val Sex Gin Cit pro Lys Asp Gin 1! 70 75 so Arg Pra ou atly Thr Tyr Pro Asp Cit Ili$ phe ThX 0hz alt Aim pro as 90 Arg Arg 8cr 11o Ala Ala Pbt Gin icr Arg Lou Mla Gin Ile $or Axg 100 205 110 Asp, Ile GiU Giu n A n Gin Sly Lou Ala Lou Pro Tyr Thr Tyr Lou ii 1.20 125 UaP Pro Pro LU Ile Sit AnA Bar Val Ser Ile 130 135 4210:p 13 czti,0 583 -c2R23 DNA 4213h, HMO S&POnD CftO0P 19 atggcoagc atagataca ctgae otwc cat otggctatao agcttgtot tcggagctgc tcaggtttcc Atcttoaatt ataagggutg tgcgoaagct gocctctgc caccocatot cgotge&gjgt gaftoaccate gcgagggccm aggtoacgt@ toacatacac ccaactaOOO cagasatcgt aggcctgpac caaagccgc8t gctctScOC gtgaagaggg aatagggagg caacttctga otaccgagac gggctactat tggagagatt gtgcacccc& geaegctgct Awgtbatact caaggcctca ottoeggaca gaaggeag tatcooagtg tutgotcagg ggagagatgg gtcaaaagtg ;cccttctgg acaagctCet cgctqatCUa tctaootoat gcctgcgggc agatatgggo aagoatctgt cgttcctggg tgmagtt ggcaggaog ccaaotga actccacac ccgagggo gmgoACgggc CoCgg909c ggcOattgag cactgcaatc ca9pggtgg <210, C2ii: 135 c2i2: PRI t433 HMOc sapiens c4003 20 Xat ala Thr Lau Ar9 Gin 1 5 Leu Leu, Pro His Thr Arg Lou Pro Laeu Cya Hi. 10 Tyr 2Tr Lou Gin Val Pro Ile Tyr Lye Leu Is Anh Thr le Ala krg Oi: Val Ttr Snr le Ala Thr Len 33 lieu Afn Pro Gin ben VatI Asp Gly Uzg Gin sly Ln le so Thz Tyr Thr Msn Ph* Cys 70 Tyr Leu Net tSr Thr sly LOU Ala His Ph. S5 so Lou Aia Ile Pro Ann Tyr Ser Pho Leu Pro Asp ear Laeu 75 His Tyr Arg Asp Asp 90 Val nor Gin Ile Val 105 Afl Mla Arg Sly Val Sly Lou Lys Ie 'Tpy MlY Tyr Tyr Tyr P"o 110 Gln Mla Trp Thr Sly Ala Ala Ear Asp Gln Ile 130 der Mrg 145 Ile Pbe Sly Axg Ile Ala val Gin Gin Sar Gin Lieu Sly Arg 03ti Ph. Ala Oin Ala Ph. 135 Leu Cys Thr Pro Sly 150 Amu Cys Ser Ala Gin 165 Sly Sly Ila Arg Asp 125 $or Sex 140 Gly Phe pro Gin. met Val Lys Phe Lau Thr 155 Hi. Mea Ala Val A Bar Gly 170 sly (flu Gin Gly Gly Asp Thr lBS 29D Ala Ile 160 Gin Asp 173 Pro -Lou IeU Mei Asn 295 c210- 21 4cR11> 190 cR2>3 EmA c213>1 Xauc sapiens -c400;p 21 atgcooaatg atgaagactt ttotggttgg cactteca1 atctcaaggg atcccwa agamnggtcc- cgctttcag atatgacato taaaagaaga aettattgco ttcgagott ctacgttcta tgaaetgg atoatoato aootagaoaa ttmgooaage agsaggoaco sL~caoa ttgagaacmg acatgagg ttaaootgaa atacamacco tgacoaaagt cocttggg ttatcacta tttcttcaa groaoagaagg catgaggaag aotaoctg4A accaaggac qaggoggaga geggeaccag cgtotocatc aggaccaqtt oottctattc ugocugago ccaatglcaoa cagatottg tctoaooaa occoaccoc agmo oaaggg gtgaacatoa getgtaaoaa cagaggooc tgggoaccta atogoogoet teesagoog gCcggoao tgcotaoac taaaCCCO C&AaCaCaC ootomggtoo toongfaact tgcaoatgga gaottt rgoa gacoacoaco cotcotctt oooagatgag cotggoooag ataoctae Comageaga. tccatootao gooaagatgg gogtocagpt gamoogotto oagotegggt coaatgottt tggaaaagec ttgtoootag aoggotgt 120 ISO 240 300 360 420 490 540 600 660 720 790 840 ae tgtgagagac aragocooco ttaoagooto tcaccoaact oagoacagcoa C-gM~ggaag ttggaatgc Cttotocta ottcottete gtggto moot tggaeceta gootagagce aecagocoag aotgcgtgce tggetBaaot gaattggagg gcagcttgco Oaotttaag gggaggggaa 4210;, 22 t211N 12.0 c42Si PR! .c2i3p. Rom sapiens c00, 22 Pro Asa Ala Pro Bar Bar not Arg Gin Pro P= ro O Gn Thr Lys J. @13' Thr Tb: Thr Lou Lyn Thr Tyr Zle $or Cys Asa Ann Lou Lctu beu 40 LYN Amy Gin Arg Pro Lou Gly Tbr so 5 10 Lou Asp Thr Lou pro Gin Val Asa Gin Gin Pro Pb. fTp Lou Val Bar Tyr Pro Asp Olu His Ph$ Tbr Glu Arg Lou Ala Gin GIU Ala Pro ArM Arg Ser 110 Ala Ala Pbs Gin 70 75 Ile Ber arg Amp 2l71 Gin ub Arg Asa Gin Gly as 90 Sex. LOU Ala LOU so Pro Tyr Tb: Tyr LOU Asp Pro Pro Lou Ile Glu 100 105 Ann Ser Val Bar t2103. 21 <211P. 2604 c23-2:P DMA -c.2ib Haow sapiens 23 atggggagga cagt@cgc tttcatct @tgggtgsgc Oegfctgtg UOCcaggaac otagoactoo Ctgctgatgq OgCCtgtgtg acaot-ggtgg ttegoccatg acagatettg, cog-tfaccag cagoatctga cagccagagg 9goggagagla ataccagaga wcccagaaac ggooagagtt t§&ccactgg goaagtgtgg gatcggtaca ggggacattg goggagggca tocatceetc gaocggccgt ggaagcccga gaguteggoc Ccagccctgt gccggaggat coaatacctg tgaaagcco gaag'tabaag gggagtgggc gtgtcocaat Ogomgtggaa gtcagaggoa gccgccagoa catcctCaga coggocgc CaCttCCtcC 4gggcoggoa aageagcggc gcgcgttgca ggacaagrcac tccagggoat tatacaggog ttacctoott eccaggcco tgratatccat agcaaacaag tattagagtg gggagcttcg gagagagaa octaoamigo coagoagoc gotattotoo tooaggccg tteecatcat ggcagtgtac caCtggacaa catetctgt v tagatogoat gggcagggac cagoggagot gqgtgagotc GO 120 100 240 300 360 420 400 540 600 6so ttgctgc gtgtaoaoaa ggagcgcac agcgoatat gtgtoacoa aooggatggt attgaaggct *ctgno~cgt tortcttcoO toctectgga cgctggaaga tatgaocco atqgagt cag aceagasat t agcatggga atogytacot taoatggagececaacgtueg *tCCtgagt. Ccttgggaat aagotgga4tg acatgcagaa. akcag~aot ggtgtgaaga gtoatgccce Setgcatetc gcaocttga tgggaoafla Obagaggact aotggatoct taagcg oaacactgtg gcatacage tcagaeagac gaatggact ggatgctgge "aaaaaoofae actttotgtg ccagctgo agctctgcoaL tgcaggtga acaooatogo 9teaagtocn tagggaggoa acatacaccat atttctgcct aaotaccaut a1cogagaga gaaatcgtgg gctactatta gcatggactg gcgagatttt agaaggott goacoaagg totgccago tcgotgct' gotacatoat ooatgaggoa t&Ocagaca cctoctga Sttagoaag aacacaagga gaggaggoco cgraggaggag gDCatcagg agoggasocaj &ttgflgaaea gegtetcaat ggagctgragg tcacaggaca tggcttLene tgcctcgaoa gaceggette getttCttao godaggoct agtgtatca attcocg caggatag~ zsagauctat cygggoc gggcccgaea tgeatggtag acgtasacag aagagaaaa ottgtgtaga eceatgana cegacatao0 ttggtactgt atatefsgtgg ttgtaaggaC agaatgctao Cttteaggag cagggtao atcatgatg atactoagoc aoCaagg tcogctgcc guasocoga gggotgttgg atogaaaggg oatottctgg tgaoataga eetCaogac tcaorccc gggcaccagt aotgaatgg ;agcttgooc sgcaagatgo otgtoeocaa aaeatgeC~t cagacagago tagagagggg gggaso coaccact goctaaaagg oatgctgtgg ctagccccC agggggegct occogggoot gacagacca tcttoctgcc oaag~acgtgg gtgCgcaact Ctgagtcot aacgaatttyg ctgtgcgagg aettogocat CoCoatotac aagctcocao tOaccccR gagggcc ctgctaaoo ccggggcct aggoacato tacctcatga goaogggoot teeggacase ctgcgggaeo geggt cggcctgaqg atgggogfg coattgagag tccaagtgac gcatotgcgc agoaggatto tgotcaggeg ttcotgggco gggaaagoco agagatggtg tagttcecca etgeaancat caacagtggg cugoatgact ttggggcctg 9ccOccaOCcc cagaccaagg ggaccacaac agtgaacato agctgftaoa aecteCtoct omgaggoc otgggeaeae. accagatga 3atacgoc ttecagagcc goctggcac wgtctggaa ctgocctfca cotacctgga :taa atotogaag aaagtatct tgacacggtg gaatcatctt C Cagfocaag cactgactc ggtgagn t agataccog cgtggaccag ggaoaac~tto ggatatco cc ctttgrt tc a ggagctgoag mggtttecca cttcaattgo ;atgancaat cctgaagact ot tctgttg ;caotcaca ;atctCaag :cotcccctc 720 780 M" 900 960 1020 1000 1W.4 1200 1260 1320 1380 1440 1300 1620 1690 1740 1900 1360 1920 2980 2040 2100 2160 2230 2290 2340 2400 2460 2520 2580 2604 C210M 24 42i11z 567 c212 prr C233 sao sapiens c4002, 24 Met MlY AM An Arg Ber rrp 1 9 Sly Thr Lou Sly 3cr Ith Pro Gly Rix Gin Lau Tyr LrO Bar &CU Arg Bar so Ala AMg Sly ITh Gin hop "Cy' ly Az g Olu Ly1e Ica Gin Pro, Tyr pro Gin SUr Sly pro Arg Tyr Lau Lau Arg Aig Ser le er 13ly Ag ITh Bar AMg Ala Va1. r Ala Bar Amp Pro Lou Sly Sin Pro mar Ila Arg Val Sly Thr Glu Mla Pro ASp X2e Sly Cy. V/al Mrg Gly Lys 70 Zig9 Mg (flu Cit Ala Arg as 0 Ala Gin Slu Rlls Pro AMg Ala Oar "g Glu Len 105 Pro LOU Ala Pro Gin a Ala Sin Bar Trp LCu M~g Pro Ala Hst Pro 110 Gin Thr Sin 11 Pro Cys Pro Ala Cya 130 Pro Ala Lau Pro Gly 145 Arg Lou Cym Vol Thr 1ES Agn Ile Str Val Thr 180 Arg Lou Asp Arg Hot 135 Ty Lym Val Arg Cye 210 Vol Sit Lye Glu Azg 225 fcr Ar" Ile Cy. Va1 245 eye Tr Gn Tip Ile 260 Tbr Ale Arg Thra Ila 275 Arq Tb Arg Gu Lou 390 Tyr Ala Pro Gly Phe 305 nt lu Bar Asp Lye 325 ADp Gn Gly Asp Nor 340 Lys fIb Asp Ile Pro 355 Sex Ala Thr Lye hr 370 Le Oly Net We Lou 305 Lym Lou Asp Asp Net 401 Thr Lye Tvr Val Thr 420 Gin Tyr Lou Aga sly 433 LOU Pro Bar Lye Lou 1 450 ly Gin Asp Thr eye I 465 4 Lou Ala Asp Tyr Tzp 1 485 Sly Arg 3in Gln Tyr V 120 Arg 3cr Ser Pro Pro 125 Sly Arg Lau 135 His Pro 2SO Thr Oly Len Val Gly Arg Thr Ala 215 Tyr Ala 230 Thr Olu Glu Gly Cys Gln Axg AMa 295 Pro cys Ph Pri al~ Asj 201 all Phe Prc Tyr Amp 230 Arg mEt 140 a Le Lou Pro Xle Mot 15 3 Tyr Lou Ar; Ala Oly 170 r Thr Cys ly Glu Bar 185 PhO Ala Pro Gly Ser 205 A Lou Sly Gin Le Lou 220 a Ph ng Lys Asp Set 235 Asp Gly So Val Bar 250 Cy. Tht Val Olu Lou 26S Bet Lau Pro Lau Lou 285 mn Olu Cys Tyr Mg 300 val Asp val A"s ser 315 Le Thr Lye Thr Thr 330 Arg Tyr Lou Pro ely 345 Tyr net lu Pro Aens 365 Leu Ph. Asa Ala Ile 1 380 Lau Amp Ar; Lys ly j 3,5 Phe Trp cys His Ly: I 410 Cys Olu Amp Big Ph 1 425 4 Val met Leu His Cys I 445 Ass Amp Nat val Ala 060 Gin Lau Oh Arg Oly A 476 Glu Ala Pro Thr Rig C Lou Lou Mg Ala Val Tyr 160 Tht Lou .Asp 175 Pro Lye aln 190 Vat Gin lays Lau Lou Arg TrP Tyr Cys 240 His Ph. Pro 235 Arg Pro ly 270 Lau Asp His ?rp Lye Ilo Pht Gin Glu 320 Thr Cys Val 335 Ph. Pro Not Ifa Al g Tyr rxo Ala cr Tip LYe 400 hr Pho Thr 415 'he Oly Tyr Is Ser Bar Io Lau Lau sn Il Phe 450 ye Lou Ann 495 33.0 Lqa Ph. Ala Per Gly Ann kcr Lou net 360 Ile Ser Lou 37$ rGOly Lou 190 31n An Ile flu His Trp aul Ann Pro 440 'ro Vol Thr 455 41k Gla Thr 70 is Leu Ala al Ala Ala 490 Soo Pro al Sly Ala OSy Pro lsp Sr 530 Lau Lou Ala Lys Pro Lou 505 Lou Val Pro Ile Thr Trp 550 Pro Lau Ala 520 tos Lou Pro 535 Val Alrg Ass Cys Lou Lou Trp Leu 510 Cl Lou ar Gin 'hr 525 Asp 2ar flu frp Asp 540 Glu Phe Lu Val Hls 55S Ser Pro Trp Glu 560 Ago Ann Tbr io Ph. Luau Cye Thr Hisi Lou Lou Cys ala. Al1a Pho Ala met Ala STir Lau 550 La LOU Pro His SRS Ala ITk Lou Lou si0 ciW Axg Gin Sly 425 Thr Tyr Thr Mgn Lau la. lio pro 660 Mla Ala lie Qiu 673 Bar amp Ala Bar GlU 110 Pb. Mla 703 Bar arg Lou aye Il. Ph. Ann Cys 740 Asp Ph. Sly Ala 755 Pro Pro Gin Thr 770 Lou Pro Gia Val. 715 Val Ear Gin Giu (au Him Pho STar sex Arg Lou Ala 815 Lou Ala Lou Pro 850 Val Bar Il. !570 Arg Gin Lou Pro Lou CYS His Thr Arg Tyr T'hr Lou Gin Val 600 Asn Pro Glu air Lou Val Asp 'Is Lou Ile Tyr Lou Met mar Thr 630 635 Phe Cym Lou Pro Asp Bar Lou 645 650 Asa. Tyr Kim Tyr Arg Asp Asp 665 $or Ph* Val Car Glu Iie Val 680 Val. Gin Gin Asp Sex Gin Lou 69S Sin Ala rho Lou Gly Mgq Olt 710 71S Thr Pro Sly Gin Mat Val. Lys 725 730 Bar Ala Gin His Mla Mla Val 745 rrp Met Pro Amn Ala Pro Bar 760 Lys Gly Thr ITh Thr Lau Lye 775 Asa 11o Sar Cys Aan Ann Lou 790 795 Pro Lys Asp Gin Arg Pro Leu 905 810O Glu Glma Ala Pro Arg Arg Bar 825 Gin Xlo Sex Arg Asp Ie gin 840 Tyr Thr Tyr Lou Asp Pro Pro 955 575 Pro leTyr Lye Lou 590 Aen Thr Ile A1a Arg 605 Gin Val Thr Ser Ile 620 ely Lou Mla His Ph. 640 arg Aa& Arg Oly Val 155 sly Lau Lye I~o Try 670 Gly Tyr Tyr Tyr Pro gas Gin Ala Trp Thr Giy 700 Bar Bar Sly Pho Pro 720 Ph. Lau Thr Ala Ile 735 Ann er Sly Gin His 750 tar met Mrg Gin Pro 765 Thr Tyr La Asp mxr Lou Leu Who Try Lou 000 sly Thr T'yr Pro Asp li e ala Ph. Gln 500 Giu Arg Ann Gin sly Lou ZIl Gbz An Bar 860 866 <21lba- 2 .c2X1938 'dial. c2112. Romi sapiens 400) £tg ggja ucmgatottg ggggacattg gggagtggga ggaoaagcac taaagggeat Cagtcogggc cgctgaccag gcggagcga rsgtccoaat tanacmags ntacotcatt ttotoCatct Cagc-atotga toOCtoCCtC Cgcflgtggaa accaggoto tgmtauocat Otguetago cagaaagagg gee eggetgt gtoaqagea ageassenag tattagagtg caagaotgug ggoggagaga ggaagoooga gCCgCcagca gggagottcg gagagagasa iccoaggase atcoagaga gagctgggoe cateotag ostacacogec cccgcagcc ctagccctee gcccagaasc ccagrccctgt ccggogtgoc gotettctcc tccaggoogg atgargergc ggocagcgtt gccggggcat coottectec teeeatcat goagtgeac egcotgtgtg aoaotggtgg ttaaaotg ttgctgctgc agcogcatat attgsaggot tototuacc agatggna atggagtcag kagtgg taaatgug atcctgogt aagotggag Acagageact gtcatgotc gceocttgc otagcogot taogtggaog gOO4t@O"go gaatggact £*@asacgc cgccagotgc Ctgoaggtgm cctgggo gatatgggcg tgacoactgg geacgtgtgg gatcggtaa gtgtaaaaa qgtcaccga aotgoacot teotcatgga tctatgccoc aaaagaaatt ataggtcat Ccaatgttcg cttgggaat acatgcagaa ggtgtaaga act goat at tgggaaagga flctggatcct ceecaatgtg toagocagac ggotgotgga actttctgtg cgetccca acaCoat ago cgcggagt oc gccattga tccctacctg ugagooco gaagtaaaag ggagcgotac aeeggatggt ggagctgagg toacaggaca tggcttoaoo tgccttgaca ocoggotto atactoagac gadgottoga cat ettctg tcaottcttt tagattgcc agggecggca aagcngcggc gtgettgca gctttattcc agtgtatcc ocaggaoag egggagcec tgcatggtag aagacgacaa caoatgaaaa accasgacga gggctgrttgg tgeeataaga gggtaccagt agoaago tgc cactggaoaa caatcgut cagategmat gggcagggmac cagcoagot gggtgagata gcaaggactc ttggjtactgt acttcccotg CtflCagtg cagacQtat ttgtcaggac ggcccgaca agaatgctac acgtcaacag entaggag attgtgagaa ccaggfgtgac ctgacacco atccctgatg tot cgctgat attoaatgcc atcgoaaggg ctcctggaag inatt@cga aaagtatgto mcctgaatgg tgtcaatccc ogteaoeaa tgaoatggftg tagagagggg gaacatette ;cctaaacgg cgocagoag tggggoct ggtgoccttg :ccteetgcc eseegactac :tgagttoot ggtgcacgaa cttcocat goacgotg :CCCCCA&c tcgatacacg :agaggcot cgtggaccag :accgagacg acactgaa 540D 600 660 720 780 840 900 560 102a 1080 1140 2.200 1260 1120 1a 00 1440 1500 1560 1630 1610 1740 1800 1960 1920 1I3B oacatgootg cagacagago ggcggaggcc ccea ccact cctgccg otoagococ eccegggeet gacagececa oaagacgtgg gtgagoaact cacgoatttg Ctgtgcgagg coccatatac Bagotoctac gagggccaog otgctcaacc tggatntccc cactaccac C C .c21Oi, 26 4211P. 645 <212., PAT dG413A UtacO sapinS 44000w 26 Net sly AMg Ann Arg 1 5 Thr Pro sly min Gin gar Trp Oly Tftr Lou Oly Bar 10 Ser Sly Pro LCU Thr Mrg Avg Sly Avg Thr Bar Arg Ale Val Bar Ala gar Asp Pro Gin Lsu Tyr Mrg Arg Tyr Lou Lau 25 Pb. Ser Z1. gar Sar Lou so Ala Mgs 6S Arg Bar sly rbr Gla Ala Prio Ap 110 His so Olt Thr Lou Sly Cia Pro Sly Thr Sly Mrg Gly Lys Bar 11., axv G1n Amip "y sly Mrg Arg Glu Glu Ala Arg Ala Ala aer AMg (flu Laeu 9S Amy "g Gi Gi3t fto Tyr Ala Gin Gin His Mla Pro Gin Pro AMg U05 Lou Ala Pro Pro Glu So: Trp Ala Big Pro 110 Lieu Arg Pro Gin Ttr Gin Pro eye Pro Al1a Cys Arg Pro Ala Lou Pro air mine 145 250 AM [LU CYm Val Thr Thr 125 Sly Mgy Lou loeu Lou Phe Lieu Lev Met Ai Val Gly pro Tyr Lou 170 ThT Cys AMg Ala Sly Thr Sly Gig Sex Pro Lau Asp 17S Lys Gin A le Bar Val LuVlSy Lau Val Gly Ari Tyr Val 225 Bar cyl Thr Tyr 305 met Asp per lag Lys 196 Lys DIii Zig Cys. Sin nrp 260 "sgTbr. 275 Mrg 0hz Pro cay Bar Asp sly Asp Asp Ila 355 Not Gly CyU Th: Arq Tyr 230 Val Thxr 24S Ile 0l2u Zig Cys Lou Arg Pho Pro Lys Lye Bar nor Pro Ser 18! Arg Asp Pb. 200 Mla Glii Lau 21IS Ala Pb. Ph. Glit Pro Asp Oly Tyr Cys 265 Gin Asp Ser 230 Ala Avg Cia 295 Cys Net Val PhG Ala Lou Sly Ann A9g 346 Lou Met Wi 380 Bar Lou Lou 175 aly Lou Lau Ann Ile Who Hsis Tly Cys 425 Arn Pro Val Ala Thr Lys Thr Ie 370 Sly Net Lye Lou. Ag 35 Laul Asp asp nee Gin I ISO Ala Pro Gay Bar Val. Gin Lys 205 Gip Glu. LeLou Lou et Lou Arg 220 Ar; Lye A8p Bor frp Tyr Cys; 235 240 Oly Sir Val Sew Him Pb. Pro 250 255 IThr Val Gin Lou Arg fto Sly 270 Led Pro Lou Lou leu Asp Ri. 285 Gld Cys Tyr Arg Trp Lys Ile 300 AND Val ABU Her Ph* Gin Glu 315 320 Thr Lye Thy Tbr Th: Cys va3. 310 335 Tyr Lau Pro Sly Phe Pro Mot 350 MWe Gin Pro Ann Val Mxg Tyr 365 lbs Aga Aim Ile Pro Aa der 380 Asp Arg Lyn Sly Bor Tip Lye 395 400 TrP Cye His Lys Thr Phe Thx 410 416 Oli Asp His Who Ph& 0ly~ Tyr 430 Met Lou His Cys 11e gar gar 445 Asp Net Val Ala Pro Lou Lou 460 Lou DIU "r Sly Aso Ile Ph. 475 480 Ala Pro Thr His Cyg Lou Aga 490 495 Lou Gym Leta Lou Trp Lou Per 510 116 Gin LeU Nor Gin Thz- pro 525 Thr Asp $er Gin nop Asp Tnp 540 8cr Mui Phe Leta Val Hi. Gig 565 560 Lou Lou Cym Gin u W ho ALa. 570 575 Gym Rio Pro le Tyr Lye, Lou 590 Gin Vai Ann Tb: Ile AJa Ar; 605 Val Asp CIA Pro Aa Sly Fro 620 Thr Lye Tyr V&3i 420 Gin TYr Lou Amu 435 Lea Pro $or Lrye ego GlY SIM Asp Tbr 465 Lou Ala Amp Tyr 017 AMg Gin Gein g00 Pro Ci3c Sly Ala 515. Ohy Pro Amp Ber Lou Lou Ala Lye 545 Aga Ann Thx His Met Ala flr Lou 580 Len LoVU Pro Rio 599 Ala Thr Lou Lou 610 405 Thr Glii Sly Val Lou Pro eye Lou 470 Nrp Ie 485 Tyr Vai Lou Val Pro le Th! Tip 550 Ph. Lou Ar; Gin Thr Arg Asa Pro 440 Vai '?br Las 455 Gin Tar Ciii Lou Ala GlU Ala Ala Pro SOS Pro Lou Ala 520 Ph. Lou Pro SIB Val "r Ass Cys Tuy Rig Lou Pro Lou 55 rYr ith Lou 600 SIta Sly Lou 491 Arg Pro Gly Tyr Pro Gin Lou Pro Lou PrO Arg Arg Arg PrM Oh 630 635 640 Lea Gly Oly His 645 4210O 27 c23,2316 q%212. 3, c2lJ; Herni sapiens 000p 27 atgcgagga aeagaeottg ggggaattg caqtaagMga Cgetgaocag gegqaqggaa ctotceatat amgaattg tccotcactc Obtgggc C~gCaqAgag gaCggotgt @GAgctgtg ggogmgaga ggaagcaga goooa99Gaetacocagaga gagctgggc atagecec geecagaaac ccagceetgt otetcta ggoo~agtt gaaggggot obaatgtgtg tgscomctgg tocctacctg aaaotggtgg gaotggg tgaaagccoc ttefoncctg gacggtaca gatagtaoaag ttgctgctge gtgtacacaa 99agcgctac agoagoatot gtgtaaccga aocggatggt attucaagot £Ctgcaccgt ggagatgagg totottocac tatcatgga toacaggaca agctqgaaga totatgoccoc tggcttcaoc atgagcag *Ceaagaaatt tbcottgaca acagtgg utcggtaact gccaggcttc- tacatggagc caatgttag atactoagec atccctg~gt cettggga~n giagattega agcggag acatgcagaa catcttctgg acaggact ggtgtgaaga tcacttcett StOatgctac actgcatctc tagattgoaa gccc-ttgc tgggacagga cacatgcctg a9tagogact actggatat ggaggecc tacgtggacg caccactgtg Cctgatgtgg c 9acatacuga toagecagac cccggct g asateggact gctgotgqce caagacgtgg 2 aacaaCacge £ctttatgtg cacgaatttg c Ogaaagctgo Cgotctoca ecatcaac a Otucaggtga acac S~ agggcaag c Btctcgtaaa togggaggom aggoctcatc t actacacue *tttlctgaat tooggacago a aactacgnt acoagagfaga aggocgaag a gaaatcgtgg3 0t&atta tccoagtgao g Rcc t ggaetg gagaattct tgetcag~q t agcoggotgt BSccmcagg agagat~ggtga tatgScccagc "cotgotgt aeacagtggg c 99aega~gStgatacec cettotggc al 4211a 712 C2; PM '232 Namo sapiens; gggagtgggo gtgtceeaat ccagtggaa gtoagaggca. gccgccagca catcctcagc ceggggoc oat teoto &gggooggca aAacc gtgagjttgoa gettettec agtgtatec= ggaocagoiao toogqggac tatacaggog ttaoctaatt agoaaaaag ggagettog ctacocage 9etottotoc ttaccataat Cactggacaa tagatogaat caaggacca ccaggaaeag casgaatac cgggagctao gggooogaca rnagacgaoaa CCOatgraaaa acaaagaaga jggctgttqg tgcoataaga. ;ggtaocagrt Igoaagctga :agacagagc cceaeeact ;tcagcacco raaagacca rtgagcanet 'tgtgoag agcectac tifotonace acctcatga tgegggeec aagcacag ottgtgtaga ttgaoatcc teteget43et Stagaaaggg ccttcaogaa adotga~atgg ctgtcaccna tagagagggg gootaaacg *gggggect tcttactgcc ccttcgccat toccoacacI Cc9&gggaot 5 ;ccgggcat 9 tgatatocat tattaqagtg gagagagaa ocegoeagec tccaggoagg Wflgtgtac Catctcgtc ggogggac gggtgagatc ttggtaatg~t ctatoagtgg ttgtcaggao agaatgctsc etteaggag aagtgac Btcactgmtg at toaatgec otoatgg-aag taagtatgtc Lgtcaatacc gacatqqtg ;aaoatattc :cgacagaag rgtgccaeg :acrtgaatoc rstgaacgaa cgltflcaag gtggaccag geccact to gctatcaoa tttgqtctca jagut gcag ;gttteca ttcmattqc aggAtggt ISO 340 300 360 420 480 840 S00 660 720 760 040 M0 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1630 1620 1740 1600 log0 1920 2980 3040 2100 3360 2220 2280 2316 tctggggg coattgagag oatctgtge ascaggatta tctggcc gggaaagetc tgttactca Ctgcaatcet Iotga 400'* 28 Hot Gly Arg Agn iTh Pro MEY His 01: Lou Tyr Ar; 8ar Leu Arg ear so Al4 Arg Gly flax 61 GIB asp cys 015' Ari Amg Gin Lys 106 G3s Pro Tyr Pro 115 Pro eye Pro Ala 130 Pro Ala Lou Pro 145 Ang LOU Lys Val ma Ile Bar Val 190 Mrg Lou Asp Arg 19s Tyr Lye Val At9 210 Val Hi* Lys Gin 225 Ber Rig Ile Cye Cys Tyr ali Tip 260 ITh Ala Arg Thr 275 Arg hr "g Glu. 290 Tyr Ala Pro Sly 305 Not Gin. ser Asp Asp GIBn Gly Asp 340 LyO Ile Asp 114 365 Bar Ala Thyx Lys 370 Lau± (Ely Mt Lyn 205 &Y@n LOU Asp Asp Thr 4's~ T Val 420 ARg Set Trp (Ely Thr Lou Sly Bar Sly "i Thr Ber 10 Gin Ser 015' Pro Lou Thr Aig ARg rg A& Val 8cr 2S ARg Tyr Leu Leu Phe Sey Ile Sex M.a Bar Asp Pro 40 sly Thr Gin Ala Pro Asp Ile His Leu Sly (flu Pro 55 SO (Ely eye Vai Avg 013 LyE Olm Tbr Sew Ile Arg Val 70 75 so Rig Arg Gin Gin Ala Arg Ala Ala $er Asp Sin Leu s0 Ala ala. Gin Hi. Pro Rig Gin Sax Tip Mla His Pro 205 110 Ala Pro Gin. Pro Lou Ala Lau Ang Pro Glu Thr Ola 3.20 125 Cyr, Arg Ser Bar Pro Pro Sly Arg Lou Leu Lou Rig 235 140 ely His Pro rho Lau Lou Pro Ile Met Ala Vol Tyr 150 15 ISO iTt Thr sly Pro Tyr Lou Arg Mla (Ely Thr 14u Asp 170 175 7tt Leu. Val sly Thr Cys Gly Gin Set Pro Lys Gin 115 190 Met 01y Arg Asp Pb. Ala Pro Sly Bar Val Gin Lye 200 205 Cys Thi Ala Gin Lou (Ily ain Leu Lou Lou Len. Rg 22.5 220 Arg Tyr Ala Phe Pbs M9g Lys Asp Bar Tarp Tyr Ova 230 235 240 Val iTr Ciii Pro Asp (Ely Bar Vai 8cr His Pb. Pro 245 250 255 Ile Gin (Ely Tyr eye Thy Val Gln Lou Mrg Pro Oly 265 270 Ile Cyn Sin Asp Sex Lau Pro Leu Loeu Lau Asp &to 290 205 Lou Arg Ala Arg Gin Gin. Cys Twi Ary Tip Lye Ile 295 300 rite Pro Cys Met Val Asp Val Asm $or Ph* Gin Gin 310 315 320 Lys Lys Phe Ala Lou Thi Lys Ty iTh Thr eye Val 325 330 33S ear Her Sly Am rg T'yr Lou Pro (Ely Phe Pro "et 345 350 Pro Bear Lou Het Tyr Met Gin Pro Mua Val Arg Ty'r 360 365 Thr Ile $or Lau Lou Ph. Asa Ala Ile Pro Ala Ber 375 Leu Mrg Sly Lou Len Asp Arg Lya Cly Aar fTp Lye 380 395 400 Met Slit Ann Ile Flat Tip Cys His Lys Thx Ph. Thi 405 410 415 Thr Gin Rie Trp Ca Gin Asp Hsm Phe Phe sly Tyr 425 430 Gin Tyr Lou. 435 LoU Pro Ser 450 Sly 31a Asp 465 14U Maa M9p sly Arg Gin Pro 02M Sly Oly Pro Asp Asn OWy Val Lye Lou Pro Tilt Cys Lou 470 Tyr lip lie 49 Gin Tyr val. S00 Ala Lon Val Bar Pro 1i. Pro Val. Met Lou 440 Tilt Ann Amp Not Thr Gin Lou Gin 475 Alia Gin Ma.s Pro 490 Ala Pro Lou Cys SOS Lou Ala Il. Gins 520 Lou Pro Thr Amp jig Gyml lie 445 via Mla Pro 460 Arg Gly Ago Thr His Cys Lem LOU trp Lou $cr Sic 525 Bar Giu Trp LIE 491 TI" AN' 520 535 LOU Lem Ala Lye Tilt Trp V&.1 545 550 Awn A-ma Tilt Rio Pb., Lou Cym Net. Ala iTh Lou Ar Gin Lsu Soo Lou Lou Pro Kim Tilt Ag Tyr SOS Mla Tar Lou Lou A-sn pro Sin 610 G15 Sly Mrg Gin Gly, Lau Ile Tyr 623 630 Thr Tyr Thr An Pile eyn Len 645 Lou Ala Xi4 Pro Aen Tyr Rim 660 Am Len Thr His Pro Lou 355 Tbr Lem Sly Lou Lau wet Pro Asp Tyr Arg Sor Lou 570 C,, Gin Val hier Bar 650 Amp Uiu Pile Lou Lenu "y Gin Hiei Pro lie Val Ann Tilt 605 Asp Gin Val £420 72tr 91y Lanu 635 Lou Arg Ala Amp Sly Lou Val mia Ala Pie 575 Tyr Lye Soo Ilo Ala Thr Ber &rg Gly 655 Lye no4 r Sew Ph*e 480 Pro TXp 540 Ala Liou Arg 12e Ph* 640 Val fTp Pro Sly Pro 720 no0 Asp Lau Alia Ala Ies Gin or Pho Val scr Gin 675 $or Amp Mla 690 Giu, lie Ph. 705 her Leu Zig ph. Amn *iy Arg Sly 755 LiOU Al1a Ann 770 SAO Bar Val Gi.n Gin Asp 695 ALa Gin Mla Pile Lou 710 Cyob Tilt Pro Gly Gin 725 Cym her Ala Gin min 740 Gly Ile Arg Amp Ciy 760 Ile Vol fly Tyr Tyr Tyr Gin Lou Gin Ale Trp Tilt 700 Arg Gin her Bar Sly Pile 715 Val Lye Pile Lou Tilt Ala 730 735 A-ia Val Ann Bar sly Gin 750 Gin Sly sly Asp Tilt Pro 765 .42i0. 29 C2ll. 3384 .c2l2.w Dfl -C233- 3kM0 aeptema c4Db 29 aaggtgcooct steotoatet ctgoctggga atggggagga *cagatcttg Bgggattg gqgatg*gg aoaecac tcagggcat cagtaogggecvgctgaacag ffg9*gggc qtgtceoa~n tetacaggog ttacatcot; ttctccatct cascatctyc tocotoocte CgoaLggna cac*aggt tsatatcgatt ctgjgtgago Cagceagagg gaecggetgt 180 240 ctoctoagea *ogg0aggc agaag gtfiottoca agtgtagoco Comwaoag ogggagtoo tgfttgg O@@ltgaaaa £ooaagaoga1 Iguottctgg taceatasga Yggtacoagt agoasgoege bagaoagago cacocactj Ctcagacoc a Iaoagcoccoa t gtgcoaact~ a otgtgogagg 0 aagctctac t tg~ttamco c tacotoatga g Ctgdgggcco gi atctgggogg ac go-actgtgc st ttcctgggoc g ttaotca C OAgatgaot t. gg AlUetgtaaca 8 *tsggaoot s tt-aoagagoc go ctWO~cotacM coI acaaatacea cc Ct*aagAocq ttl gactnrge *g COtgIoacagc ag~ C~gaSagqaa 99m Cttggnatgo ca tacteotace akc 9Cttcattat atg Catgtggtce ac UcctqGgco 0 ata. Stctgtattt etc tttttttott tot1 4Snmaa aa Rgcaaaoaag tattagagtg caagactgtg ssw9ea9cg0 ga9gaan scacaggflc ectacecago Cocgaagcaa. Otagucceocc botottataC tcoaggocgg ctgotgotgc ttcocateat ggcg Mac ago ctgrgtg oactggaaaa catotctgtcaeaotggqtg cagatcgaa: 9gogaggac ttcgccatg 0agoggagt ufgtgagctc ttgatgctgo gosaggacte ttggtaccgt ageogeact acttecag otatoagtgg at tgaaggt1 caagacat. ttgtcaggac tctottacca gggcaca agaatgctac cmatggaaga acgtoaaoag ctttoaggag atggagtcag orttgtgtaga CCalggtgac &*agggai ttgacaeecc atoaotgatg taceeggago c botcgctget Ottoaatg a*toaatgagt c Btocaaggg Ctcccggaag aagacggatg a :attagac aaagtatgtc *caaggocact Icctgaatgg tgtaaeccc gtcatgotoc a ztoacoas tgracatggtg gcccccttga t agagagggg gasosecte cageggact- a rtotaaacgg Ccgccagcag taogtggocg c ogeggegt ggtgoc-tg gcatcac t4 *tt scoca Cctgaotgga ggaoctg tgagttct Ogtgcaa ca s a m ettogocar BOMCegeg Ccagatgac Cccaoac tCgatac~qg ctgoaggtga ac ogagggc Ogtggaaq gtcacgtccm tc 'acggact 9gcocac-tta acoe~Sacca at iggogtoot ogotatecac ametacoact at ;ttgegag ctctqcctoa qaaatcgStgg go poaggattc 99agctgcag 9cctggaotg! go Igaaagct a ggtttgoca ascoggtrt *o gomatet Ottcmattgc tctgocaagc aq ggggoctg OAtgeccaat getceatcat cc~ acC2'ccaCtlasgact tacotagaca ec otootact Cttc~tg Pttagcaafag sac acagatga 9catteana gaggaggec ag Otggecca gtcetcag gEacattocagg *gc tskcotga coctcoc~t Uttgagaaoa Mg c4fl9Aaga aagmggqto Oucaetgag gag ccatcctC Oetgttctcm gttcacgg act ~caagtg 9CLtracat Catacfaeac g :9tooagg ttaaaagccg Ctgaooaaag too Lccgctt CcttottgC ccactgg 9caj Llttggg tttctsgrt ttcecto atoc Otgaott tot scgtct Ctttettaoa agog4 gam~ago ctgg'aaactgJ 9cacagaag gmat ttgtgcoc tagoaoottt 8aggggaggg g&ag ac-gatg tt Otoaggag caggttecca ggcc oasgat ttcaccaac tcaatttea teec gtocttg agccagcgg Stoaataaa saco rn44a Maa gga~ggagaga tooagagp b3Occagaac ggdeafget tgaccaotgg gatoggta 9tgtacaeaa gtgcaooga tctgoaoopt tea tcctgga totatgao kflgaaatt1 9gmagacog ccagccetg tccctacotj toamagoaci gaagtaoaa 1 99agagotac £aggetgg tOcaggaca tggottocce ItCggrt6A !Caatgtl lattggg LCatgoat gtgtgaa Otgaatc gggaoag ceggaca occaatg Oagccag ;otgac 9 ;tttctgi ltqtgog aceacog :;9aggc ttctgoc Ogaqoeg tactatt gagattt acooag Ctgctgr Etgaggo :tocctg mOcaggA l99aaccI tocat Smocast ttctet t Ocoegag aatgcac ,ctctt :tocoa :tAgagc :gtgtgc Iaattgg Itggggt Aotce aaaata tcottqaca C0t 3 0 0 0flettc tog fltaotoagoe aSt tgnCttagn gatcatt.-tt to tagcngooc, gIn CaCatgcctg Ct 9g9flagge tg Cotgotgtg go eoaggct ioceaagaopggq bg Ccccatttg ~a Occcototac KC slgubeceog 24 Uggacctc :t teoggacage ra cggcctgag" A tcccagtgac pagagatg a agtgaacatc acoagaggecc I Cflagccgoaj ggctggoa Ctaooccoc ttcteaggtc Ctgrcacatgg Ctgrtgagaga attqastect gttcoagect ttocccengc Cacoagoca OtBOwtotma Ahggagottc 9tt~ttgmg tttcate0 tttggcaaaa a 300 t 420' L 480 540 3 600 660 720 7eO 040 960 1020 logo 1640 1200 1260 1320 2380 1440 1300 1860 1620 1600 1740 1000 1860 1930 in 0 2040 2100 2160 3220 2280 2340 3400 2460 3820 2510 2640 2700 2760 2920 2800 2940 3000 3060 3120 lIeD 3240 3300 3360 3394