Huntingtin DNA, protein and uses thereof

United States Patent [19] MacDonald et al.

USO0568628

[11] Patent Number: 5,686,288 [45] Date of Patent: Nov. 11, 1997

[54] HUNTINGTIN DNA, PROTEIN AND USES THEREOF

[75] Inventors: Marcy E. MacDonald. Lexington; Christine M. Ambrose. Charlestown; Mabel P. Duyao. Cambridge; James F. Gusella. Framingham. all of Mass.

[73] Assignee: The General Hospital Corporation. Boston. Mass.

[21] Appl. No.: 246,982

[22] Filed: May 20, 1994

Related US. Application Data

[63] Continuation-impart of Ser. No. 85,000, Jul. 1, 1993, aban doned, which is a continuation‘in-part of Ser. No. 27,498, Mar. 5, 1993, abandoned.

[51] Int. Cl.6 ........................... .. C12N 5/00; C12N 15/12; C07H 17/00; C07K 14/00

[52] US. Cl. ................................... .. 435/2401; 435/320.1; 435/691; 536/235; 530/350

[58] Field of Search ......................... .. 435/6. 69.1. 240.2. 435/3201. 252.3. 254.11; 530/350; 536/221.

23.1. 23.4. 23.5

[56] References Cited

PUBLICATIONS

Allitto. B.A. et al.. “Increased recombination adjacent to the Huntington disease-linked D48 10 mar .” Genomics 92104-112 (1991). Altherr. M.R. et al.. “Radiation hybrid map spanning the Huntington disease gene region of chromosome 4." Genom ics 13:1040-1046 (1992). Altschul. S.F. et al.. “Basic local alignment search tool." J. Mol. Biol. 215:403-410 (1990). Ambrose. C. et al.. “A novel G protein-coupled receptor kinase gene cloned from 4p16.3.” Hum. Mol. Genet. 1(9) :697-703 (1992). Anderson MA. and Gusella. J.F.. “Use of cyclosporin A in establishing Epstein-Barr virus-transformed human lym phoblastoid cell lines.” In Wtro 20(11):856—858 (Nov. 1984). Andrew S.E. et al.. ‘The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington’s disease.” Nature Genet. 4:398-403 (Aug. 1993). Ashizawa. T. and Epstein. “Ethnic distribution of myotonic dystrophy ” Lancet 338:642-643 (Sep. 7. 1991). Aslanidis. C. et al.. “Cloning of the essential myotonic dystrophy region and mapping of the putative defect." Nature 355:548-551 (Feb. 6. 1992). Bates. G.P. et al.. “Characterization of a yeast arti?cial chromosome contig spanning the Huntington's disease gene candidate region." Nature Genet. 1:180—187 (Jun. 1992). Bates. G.P. et al.. “De?ned physical limits of the Huntington disease gene candidate region." Am. J. Hum. Genet. 4917-16 (1991). Bates. G.P. et al.. “A yeast arti?cial chromosome telomere clone spanning a possible location of the Huntington disease gene.” Am. J. Hum. Genet. 46:762-775 (1990).

Baxendale. S. et al. "l‘he direct screening of cosmid libraries with YAC clones," Nucleic Acids Res. 19(23):6651 (Aug. 20. 1991). Biancalana. V. et al.. “Moderate instability of the trinucle otide repeat in spino bulbar muscular atrophy.” Hum. Mol. Genet. (4):255—258 (1992). Brook. J.D. et al.. “Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CI‘G) repeat at the 3' end of a transcript encoding a protein kinase family member.” Cell 681799-808 (Feb. 21. 1992). Brunner. H.G. et al.. “Brief report: revmse mutation in myotonic dystrophy.” New Engl. J. Med. 328(7):476—480 (Feb. 18. 1993). Bucan. M. et al.. “Physical maps of 4p16.3. the area expected to contain the Huntington disease mutation.” Genomics 6: 1-15 (1990). Buckler. AJ. et al. . “Exon ampli?cation: a strategy to isolate mammalian genes based on RNA splicing.” Proc. Natl. Acad. Sci. USA 88:4005-4009 (May 1991). Buxton. I. et al.. “Detection of an unstable fragment of DNA speci?c to individuals with myotonic dystrophy." Nature 355:547-548 (Feb. 6. 1992). Cheng. S.V. et al.. “Synteny on Mouse Chromosome 5 of Homologs for human DNA Loci Linked to the Huntington Disease Gene.” Genomics 4:419-426 (1989). Conneally. RM. et al.. “Huntington disease: no evidence for locus heterogeneity.” Genomic-s 5:304-308 (1989). Daly. C.B.. “Genetic cause is identi?ed for Huntington’s disease.” The Washington Post, Mar. 24. 1993. DeBoulle. K. et al.. “A point mutation in the F1V?{—l gene associated with fragile X mental retardation.” Nature Genet. 3:31-35 (Jan. 1993). Doucette-Stamm. L.A. et al.. “Generation and characteriza tion of irradiation hybrids of human chromosome 4.” Somat. Cell M01. Genet. 17(5):471-480 (1991). Duyao. M. et al.. "Irinucleotide repeat length instability and age of onset in Huntington’s disease.” Nature Genet. 4:387-392 (Aug. 1993). Fu Y.H. et al.. “An unstable triplet repeat in a gene related to myotonic muscular dystrophy." Science 255:1256—1258 (Mar. 6. 1992). Fu. Y.H. et al.. “Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.” Cell 67:1047-1058 (Dec. 20. 1991). Goldberg. Y.P. et al.. “Identi?cation of an Alu retrotranspo sition event in close proximity to a strong candidate gene for Huntington’s disease.” Nature 362:370-373 (Mar. 25. 1993).

(List continued on next page.)

Primary Examiner-Vasu S. Jagannathan Assistant Examiner-K. Cochrane Carlson Attorney Agent, or Firm—Sterne. Kessler. Goldstein. Fox P.L.L.C.

[57] ABSTRACT

A novel gene. huntingtin. is described. encoding huntingtin protein. recombinant vectors and hosts capable of express ing huntingtin. Methods for the diagnosis and treatment of Huntington’s disease are also provided.

7 Claims, 50 Drawing Sheets

5,686,288 Page 2

PUBLICATIONS Goodfellow, P.N.. “Planting alfalfa and cloning the Hun tington's disease gene.” Cell 722817-818 (Mar. 26. 1993). Gusella. J.F. “Chapter 3—Huntington’s disease." Adv. Hum. Genet. 202125-151 (1991). Gusella. J.F.. “Location cloning strategy for characterizing genetic defects in Huntington’s disease and Alzheimer's disease." FASEB J. 3:2036-2041 (Jul. 1989). Gusella. J.F. et a1. “DNA markers for nervous system diseases.” Science 225:1320-1326 (Sep. 21. 1984). Gusella. J .F. et al.. “A polymorphic DNA marker genetically linked to Huntington’s disease.” Nature 306:234-238 (Nov. 17. 1983). Gusella. J.F. et al.. “Precise localization of human [S-globin gene complex on chromosome 11.” Proc. Natl. Acad. Sci. USA 76(l0):5239-5243 (Oct. 1979). Gusella. JR and Macdonald. M.E.. “Hunting for Hunting ton’s Disease." In: Molecular Genetic Medicine, vol. 111. Ed. Friedmann. Academic Press (San Diego). pp. 139-158 (1993). Harley. H.G. et al.. “Unstable DNA sequence in myotonic dystrophy.” Lancet 339:1125-1128 (May 9. 1992). Harley. H.G. et al.. “Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy." Nature 355:545-546 (Feb. 6. 1992). Harley. H.G. et al.. “Detection of linkage disequilibrium between the myotonic dystrophy locus and a new polymor phic DNA marker.” Am. J. Hum. Genet. 49:68-75 (1991). Harper. P.S.. ‘The epidemiology of Huntington’s disease." Hum. Genet. 89:365-376 (1992). Hoogeveen. A.T. et al.. “Characterization and localization of the Huntington disease gene product.” Hum. Mol. Genet. 2(12):2069-2073 (1993). Jerome. R.. “Huntington's cornered.” The Sciences, p. 7 (May/Jun. 1993). Kremer. EJ. et al.. “Mapping of DNA instability at the fragile X to a ninucleotide repeat sequence p(CCG)n." Science 252:1711-1714 (Jun. 21. 1991). LaSpada. A.R. et al.. “Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy.” Nature 352:77-79 (Jul. 4. 1991). Lin. B. et al.. “Sequence of the murine Huntington disease gene: evidence for conservation. and polymorphism in a triplet (CCG) repeat alternate splicing.” Hum. Mol. Genet. 3(1):85-92 ( 1994). Lin. B. et al.. “Di?erential 3' polyadenylation of the Hun tington disease gene results in two mRNA species with variable tissue expression." Hum. Mol. Genet. 2(10):1541-1545 (1993). Lin. C.S. et al.. “New DNA markers in the Huntington’s disease gene candidate region.” Somat. Cell Mol. Genet. 17(5):481-488 (1991). Little, P.. "The end of the beginning.” Nature 362:408-409 (Apr. 1. 1993). MacDonald. M.E. et al. “A novel gene containing a trinucle otide repeat that is expanded and unstable on Huntington’s disease chromosomes.” Cell 72:971-983 (Mar. 26. 1993). MacDonald. M.E. et al.. “Gametic but not somatic instabil ity of CAG repeat length in Huntington's disease.” J. Med. Genet. 30:982-986 (1993). MacDonald. M.E. et al.. ‘The Huntington’s disease candi date region exhibits many different haplotypes." Nature Genet. 1299-103 (May 1992).

MacDonald M.E. et al.. “Complex patterns of linkage dis equilibrium in the Huntington disease region.” Am. J. Hum. Genet. 49:723-734 (1991). MacDonald, M.E. et al.. “Clustering of multiallele DNA markers near the Huntington's disease gene.“ J. Clin. Invest. 84:1013-1016 (Sep. 1989). MacDonald. M.E. et al.. ‘Recombination events suggest potential sites for the Huntington’s disease gene.” Neuron 3:183-190 (Aug. 1989). Mahadevan. M. et al.. “Myotonic dystrophy mutation: an unstable CI‘G repeat in the 3' untranslated region of the gene.” Science 25511253-1255 (Mar. 6, 1992). Martin. J.B. and Gusella. J.F.. "Huntington’s disease: patho genesis and management.” New Engl. J. Med. 315(20):1267-1276 (Nov. 13. 1986). McClatchey. A.I. et al.. "The genomic structure of the human skeletal muscle sodium channel gene.” Hum. Mol. Genet. 1(7):521-527 (1992). Merritt. A.D. et al.. “Juvenile Huntington’s chorea.” Excerpta Medica, Amsterdam. pp. 645-650 (1969). Morell. V.. “Huntington’s gene ?nally found.” Science 260:28-30 (Apr. 2. 1993). Myers. R.H. et al.. “Homozygote for Huntington disease.” Am. J. Hum. Genet. 45:615-618 (1989). Oudet C. et al.. “Linkage disequilibrium between the fragile X mutation and two closely linked CA repeats suggests that fragile X chromosomes are derived from a small number of founder chromosomes.” Am. J. Hum. Genet. 52:297-304 (1993). Pieretti. M. et al.. “Absence of expression of the FMR-l gene in fragile X syndrome." Cell 662817-822 (Aug. 23. 1991). Pohl. T.M. et al.. “Construction of a NotI linking library and isolation of new markers close to the Huntington’s disease gene.” Nucleic Acids Res. 16(19):9185-9198 (1988). Pritchard. C. et al.. “Recombination of 4p16 DNA markers in an unusual family with Huntington disease." Am. J. Hum. Genet. 50:1218-1230 (1992). Richards. R.I. et al.. “Evidence of founder chromosomes in fragile X syndrome." Nature Genet. 1:257-260 (Jul. 1992). Rubinsztein. D.C. et al.. “Site of (CCG) polymorphism in the HD gene.” Nature Genet. 5:214-215 (Nov. 1993). Sanger. F. et al.. “DNA sequencing with chain-terminating inhibitors.” Proc. Natl. Acad. Scl. USA 74(12):5463-5467 (Dec. 1977). Snell. R.G. et al.. “Relationship between trinucleotide. repeat expansion and phenotypic variation in Huntington‘s disease." Nature Genet. 4:393-397 (Aug. 1993). Snell. R.G. et al.. “A recombination event that rede?nes the Huntington disease region.” Am. J. Hum Genet. 5 1:357-362 (1992). Snell. R.G. et a1. “Linkage disequilibrium in Huntington’s disease: an improved localisation for the gene.” J. Med. Genet. 26:673-675 (1989). Suthers. G.K. et al.. “Instability versus predictability: the molecular diagnosis of myotonic dystrophy.” J. Med. Genet. 29:761-765 (1992). Taylor. S.A.M. et al.. “Cloning of the ot-adducin gene from the Huntington’s disease candidate region of chromosome 4 by exon ampli?cation.” Nature Genet. 2:223-227 (Nov. 1992). Theilmann. J. et al.. “Non-random association between alleles detected at D4895 and D4898 and the Huntington’s disease gene.” J. Med. Genet. 26:676-681 (1989).

5,686,288 Page 3

Thompson. L.M. et a1. . “A gene encoding a ?broblast growth factor receptor isolated from the Huntington disease gene region of human chromosome 4,” Genomics 11:1133-1142 (1991). ,

Tsil?dis. C. et aL. "Correlation between CI‘G trinucleotide repeat length and frequency of severe congenital myotonic dystrophy.” Nature Genet. 1:192-195 (Jun. 1992). Verkerk. A.J.M.H. et al., “Identi?cation of a gene (FMR-l) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syn drome," Cell 65:905-914 (May 31, 1991). Wexler, N.S. et a1 “Homozygotes for Huntington’s disease." Nature 326:194-197 (Mar. 12. 1987).

Whaley. W.L. et 3.1.. "Mapping of oosmid clones in Hun tington’s disease region of chromosome 4,” Somat. Cell MoL Genet. l7(1):83-91 (1991).

Wolff. G. et aL. “New mutation to Huntington‘s disease" J. Med. Genet. 26:18-27 (1989).

Youngman. S. et al., ‘The telomen'c 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p. 15p. 21p and 22p." Genomics 14:350 356 (1992).

Yu. S. et a1., “Fragile-X syndrome: unique genetics of the heritable unstable element." Am. J. Hum. Genet. 50:968-980 (1992).

US. Patent Nov. 11, 1997 Sheet 1 of 50 5,686,288

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US. Patent Nov. 11, 1997 Sheet 4 of so 5,686,288

nccrcrcrc AGGCAGMCC TGCGGGGGGA cococococr ccrrcccmc ccrcccmro so

ccmmrccc CAGGCTAGGG crcrcmca rccroccccc ccrocccccc ccrccccccc no

0000000000 ccrccccccc CGGACGTCIG GGACGCAAGG cccccrcccc ccrocccoor I80

CGGGTCCMG ATGGACGGCC GCTCAGGUC TGGTTTIACG TGCGGCCCAG AGCCCCATIC 240

ATTGCCCCGG TGCTGAGGGG CGCCGCGAGT GGGGCGGAGG CCTCCGGGGA crccccrccc 300

GGGCGGGAGA CCGCC AIG GCG ACC CIG GM MG GIG ATG MG GCG NC GAG 351 Mel Alo Thr Leu Glu Lys Leu Met Lys Alo Phe Clu

I 5 l0

ICC CTC MG TCC TTC CA6 CAO CAG GAG CA6 CAG CAO CAG GAG (JAG GAG J99 Ser Leu Lys Ser Phe Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

I5 20 25

CAG CAO CA6 CNS CA6 CAG CA6 CAG CAB CAB CM CAO CCG CCA CCG (ICC 447 Cln Cln Gln Gln Gln Gln Cln Gln Gin Cln Gin Gln Pro Pro Pro Pro

50 35 40

CCG CCG CCG CCG CCG CCT CCT CAG CIT CCT CA6 CCG (ICC CCC CM; CCA 495 Pro Pro Pro Pro Pro Pro Pro Gin Leu Pro Gln Pro Pro Pro Gln Ala 45 50 55 60

CAG CCG CTG CFC GCI CAO CCG CAG CCG C00 C06 CCG CCG CCC C00 C06 543 Sin Pro Leu Leu Pro Gln Pro Gln Pro Pro Pro Pro Pro Pro Pro Pro

65 70 75

CCA (ICC (36C CCG GCT GTG GCT GAG GAG CCG GIG CAC CGA CCA MG MA 591 Pro Pro Gly Pro Alo Vol Alo Glu Glu Pro Leu His Arq Pro lys Lys

80 85 90

GM CIT TCA GCT ACC MG MA GAG COT GTG AAI CAT IGT GIG ACA MA 659 Glu Leu Ser Alo Thr Ly‘s Lys Asp Arg Vol Asn His Cys Leu Thr lie

95 I00 I05

TGT GM MC ATA GTG GCA CM; TCT GTC AGA MI ICT CCA GM UT CM} 687 Cys Glu Asn He Vol No Cln Ser Vol Arg Asn Ser Pro Glu Phe Gln

H0 I15 120

US. Patent Nov. 11, 1997 Sheet 5 of so 5,686,288

AAA CII CIC CCC AIC CCI AIC CAA CII III CIC CIC ICC ACI CAI CAC 7J5 Lys Leu Leu 61y [1e Alo Mel Clu Leu Phe Leu Leu Cys Ser Asp Asp 125 130 135 140

CCA CAC ICA CAI CIC ACC AIC CIC CCI CAC CAA ICC CIC AAC AAA CII 783 A10 Clu Ser Asp Vol Arg Mel Vol Alo Asp C1 u Cys Leu Asn Lys Vol

145 150 155

AIC AAA CCI IIC AIC CAI ICI AAI CII CCA ACC IIA CAC CIC CAC CIC 831 1 1e Lys Alo Leu Mel Asp Ser Asn Leu Pro Arg Leu Cln Leu Clu Leu

160 165 170

IAI AAC CAA AII AAA AAC AAI CCI CCC CCI CCC ACI IIC CCI CCI CCC 879 Iyr Lys Clu 1 le Lys Lys Asn Cly Alo Pro Arq Ser Leu Arg A10 A10

175 180 185

CIC ICC ACC III CCI CAC CIC CCI CAC CIC CII CCC CCI CAC AAA ICC 927 Leu Irp Arg Phe Alo Clu Leu Alo His Leu Vol Arq Pro Cln Lys Cys

190 195 200

ACC CCI IAC CIC 616 MC CII CIC CCC ICC CIC ACI CCA ACA ACC AAC 975 Arg Pro Iyr Leu Vol Asn Leu Leu Pro Cys Leu Ihr Arg Ihr Ser Lys 205 210 215 220

ACA CCC CAA CAA ICA CIC CAC CAC ACC IIC CCI CCA CCI CII CCC AAA 1023 Arg Pro Clu Clu Ser Vol Cln C1 u Ihr Leu A10 A10 A10 Vol Pro Lys

225 230 235

AII AIC CCI ICI III CCC AAI III CCA AAI CAC MI CAA AII AAC CII 1071 1 1e Mel Alo Ser Phe Cly Asn Phe Alo Asn Asp Asn Clu I la Lys Vol

240 245 250

IIC IIA AAC CCC IIC AIA CCC AAC CIC AAC ICA ACC ICC CCC ACC AII 1 1 19 Lou Leu Lys Alo Phe 1 le Alo Asn Leu Iys Ser Ser Ser Pro Ihr 1 lo

255 260 265

C00 C06 ACA CCC CCI CCA ICA CCA 616 ACC AIC ICC CAC CAC ICA ACA 1 167 Arg Arg Ihr A10 A10 Cly Ser A10 Vol Ser 1 1e Cys Cln His Ser Arg

270 275 280


AGG ACA CAA TAT TTC TAT ACT TGG CTA CTA AAT CTG CTC TTA GGC TTA 1215 Arq Thr Cln Tyr Phe Tyr Ser Trp Leu Leu Asn Vol Leu Leu Gly Leu 285 290 295 300

CTC GTT CCT GTC GAG CAT 6AA CAC TCC ACT CTC CTG ATT CTT GGC GTG 1263 Leu Vol Pro Vol Cl u Asp Glu His Ser Thr Leu Leu I la Leu Gly Vol

305 310 315

CTC CTC ACC CTG AGG TAT TTG CTG CCC TTG CTG CAC CAO CAC CTC MG 131 1 Leu Leu Thr Leu Arg Tyr Leu Vol Pro Leu Leu Gln Gln Gin Vol Lys

320 325 330

GAC ACA ACC CTC AAA CCC ACC TTC GGA GTG ACA ACC AAA 6AA ATG GM 1359 Asp Thr Ser Leu Lys Cly Ser Phe Gly Vol Thr Arq Lys Glu Met Clu

355 340 545

GTC TCT CCT TCT GCA GAG CAO CTT CTC CAG GTT TAT 0AA CTC ACC TTA 1407 Vol Ser Pro Ser Alo Glu Gln Leu Vol CTn V0! Tyr Clu Leu Thr Leu

550 - 355 360

CAT CAT ACA CA6 CAC CAA GAC CAC AAT GTT GTG ACC GGA CCC CTC GAG 1455 His His Thr Gln His Gin Asp His Asn Vol Vol Thr Gly Alo Leu Giu 365 370 375 380

US TTG CAB CAG CTC TTC ACA ACC CCT CCA (ICC GAG CTT CTG CAA ACC 1503 Leu Leu Gln Cln Leu Phe Arg Thr Pro Pro Pro Glu Leu Leu Cln Thr

385 390 395

CTC ACC GCA CTC GCC GCC ATT CCG CAC CTC ACC GCT GCT AAG GAG GAG I551 Leu Thr Alo Vol Cly Gly l le Gly Gln Leu Thr Mo A10 Lys Glu Glu

400 405 410

TCT SGT 66C CGA ACC CGT ACT 0G0 ACT ATT CTG 6AA CTT ATA CCT CGA 1599 Ser Gly Ciy Arg Ser Arg Ser Cly Ser I Te Vol (3| u Leu I Ie Alu Cly

4T5 420 425

CBC GGT TCC TCA ICC AGC CCT GTC CTT TCA AGA MA CAA AM 600 MA 1647 Gly Gly Ser Ser Cys Ser Pro Vol Leu Ser Arg Lys Gln Lys Gly Lys

430 435 440


CTC CTC TTA CCA CAA CAA CAA CCC TTC CAC CAT CAC TCT CAA TCC ACA 1695 Vol Leu Leu Cly Clu Clu Clu Alo Leu Clu Asp Asp Ser Clu Ser Arg 445 450 455 460

TCC CAT CTC ABC ACC TCT CCC TTA ACA CCC TCA CTC AAC CAT CAC ATC T743 Ser Asp Vol Ser Ser Ser Alo Leu Thr ATo Ser Vol Lys Asp Cl u I I e

465 470 475

ACT CCA CAC CTC CCT CCT TCT TCA CCC CTT TCC ACT CCA CCC TCA CCA 179! Ser Cly Clu Leu Alu Alo Ser Ser Cly Vol Ser Thr Pro My Ser Ala

480 485 490

CCT CAT CAC ATC ATC ACA CAA CAC CCA (ICC TCA CAC CAC ACA CTC CAC T839 Cly His Asp I le T Ie Thr Clu Cln Pro Arg Ser Cln His Thr Leu Cln

495 500 505

CCC CAC TCA CTC CAT CTC CCC ACC TCT CAC TTC ACA ACC TCT CCC ACT T887 Alo Asp Ser Leu Asp Leu Alo Ser Cys Asp Leu Thr Ser Ser Alo Thr

510 515 520

CAT 666 CAT GAG CAC CAT ATC TTC ACC CAC ACC TCC ACC CAC CTC ACC T955 Asp Cl y Asp Clu Clu Asp I la Leu Ser His Ser Ser Ser Cln Vol Ser 525 550 535 540

600 GT0 CCA TCT CAC CCT CCC ATC CAC CTC AAT CAT 606 ACC CAC CCC T983 Alo Vol Pro Ser Asp Pro Ala Met Asp Leu Asn Asp Cly Thr Cln Alo

545 550 555

TCC TCC CCC ATC ACC CAC ACC TCC CAC ACC ACC ACC CAA CCC CCT CAT 2051 Ser Ser Pro | le Ser Asp Ser Ser Cln Thr Thr Thr Clu Cly Pro Asp

560 565 570

TCA CCT CTT ACC CCT TCA CAC ACT TCT CAA ATT CTC TTA CAC CCT ACC 2079 Ser Alu Vol Thr Pro Ser Asp Ser Ser Clu 1 le Vol Leu Asp Cly Thr

575 580 585

CAC AAC CAC TAT TTG GCC CTC CAC ATT CCA CAC CCC CAC CAT CAA CAT 2127 Asp Asn Cln Tyr Leu Cly Leu Gln I la Cly Cln Pro Cln Asp Clu Asp

590 595 600


6A6 CM 606 ACA CCT ATT CTT CCT CAT CM 666 T66 CA6 CCC TTC A66 2175 Clu 6|u Alo Thr Cly I To Leu Pro Asp Clu ATo Ser Clu Alo Phe Arg 605 610 ' 615 620

AAC TCT TCC ATC CCC CTT CAA CA6 GCA CAT TTA TTC AAA AAC ATG ACT 2223 Asn Ser Ser Mel Alo Leu Cln Cln Alo His Leu Leu Lys Asn Mel Ser

625 ' 636 635

CAC T66 A66 CA6 CCT TCT CAC ACC ACT CTT CAT AAA TTT CTC TTC ACA 227! His Cys Arg Cln Pro Ser Asp Ser Ser Vol Asp Lys Phe Vol Leu Arg

640 645 650

CAT 6AA CCT ACT 6AA C66 CCT CAT CM CM M6 M6 CCT TCC CCC ATC 23H? Asp Clu Alo Thr Clu Pro Cly Asp Cln 6|u Asn Lys Pro Cys Arq I lo

655 660 665

AAA CCT CAC ATT 66A CA6 TCC ACT CAT CAT CAC TCT 66A CCT CTT CTC 2367 Lys Cly Asp I To Cly Cln Ser Thr Asp Asp Asp Ser Alo Pro Leu Vol

670 675 680

CAT TCT CTC CCC CTT TTA TCT CCT TCC TTT TTC CTA ACA 66C CCA AAA 2415 His Ser Vol Arg Leu Leu Ser Alo Ser Phe Leu Leu Thr Cly Cly Lys 685 690 695 700

MT CTC CTC CTT CCC 6A6 A66 CAT 6T6 A66 CTC ACC CTC MC 666 CTC 2463 Asn Vol Leu Vol Pro Asp Arg Asp Vol Arg VoT Ser Vol Lys Alo Leu

705 710 715

CCC CTC ACC TCT 6T6 66A 66A 6CT 6T6 6C6 CTC CA6 CCC 6AA TCT TTC 251 1 Mo Leu Ser Cys Vol 61y Alo Alo Vol Alo Leu His Pro Clo Ser Phe

720 725 730

TTC ACC AAA CTC TAT AAA CTT CCT CTT CAC ACC AC6 6AA TAC CCT CA6 2559 Phe Ser Lys Leu Tyr Lys Vol Pro Leu Asp Thr Thr Clu Tyr Pro Clo

735 740 745


CAA CAG TAT CTC TCA CAC ATC TTG AAC TAC ATC CAT CAT GCA CAC CCA 2607 , Clu Cln Tyr Vol Ser Asp l le Leu Asn Tyr T Ie Asp His Cly Asp Pro

750 755 760

CAG CTT CGA CGA CCC ACT CCC ATT CTC TCT GGC ACC CTC ATC TCC TCC 2655 Cln Vol Arg Gly Ala Thr Alo I la Leu Cys Cly Thr Leu I la Cys Ser 765 770 775 780

ATC CTC AGC AGC TCC CCC TTC CAC CTC GCA CAT TCC ATC CCC ACC MT 2703 [ Ie Leu Ser Arq Ser Arg Phe His Vol GTy Asp Trp Mel CTy Thr I Is

785 790 795

ACA ACC CTC ACA CCA AAT ACA TTT TCT TTC CCC CAT TCC ATT CCT TTC 275T Arg Thr Leu Thr Cly Asn Thr Phe Ser Leu Mo Asp Cys I la Pro Leu

800 805 810

GT6 CGG AAA ACA CTC AAC CAT GAG TCT TCT CTT ACT TCC AAC TTA CCT 2799 Leu Arg Lys Thr Leu Lys Asp Glu Ser Ser Vol Thr Cys Lys Leu AI 0

815 820 825

TGT ACA CCT GTG AC6 AAC TCT CTC ATC ACT CTC TCC ACC ACC ACC TAC 2847 Cys Thr Alo Vol Arg Asn Cys Vol Met Ser Leu Cys Ser Ser Ser Tyr

830 835 840

ACT CAC TTA CCA CTC CAC CTC ATC ATC CAT GT6 CTG ACT CTG AGC AAC 2895 Ser Cl u Leu Gly Leu Cln Leu l le | Te Asp Vol Leu Thr Leu Arg Asn 845 850 855 860

ACT TCC TAT TCC CTC CTG AC6 ACA GAC CTT CTC CAA ACC CTT CCA CAC 2943 Ser Ser Tyr Trp Leu Vol Arg Thr Glu Leu Leu Clu Thr Leu Alo Clu

865 870 875

ATT GAC TTC ACC CTC CTG ACC TTT TTG CAC CCA AAA GCA CAA AAC TTA 2991 I le Asp Phe Arg Leu Vol Ser Phe Leu Clu ATo lys Ala Clu Asn Leu

880 885 890

CAC ACA COG GCT CAT CAT TAT ACA GGC CTT TTA AAA CTC CAA CAA CCA 3039 His Arg Ciy Alo His His Tyr Thr G ly Leu Leu Lys Leu Gln Clu Arg

895 900 905

FIG.4F


CTC CTC AAT AAT CTT CTC ATC CAT TTC CTT CGA CAT CAA CAC CCC ACC 3087 Vol Leu Asn Asn Vol Vol I le His Leu Leu Cly Asp Clu Asp Pro Arg

910 915 920

GT6 CCA CAT CTT CCC GCA CCA TCA CTA ATT ACC CTT CTC CCA AAC CTC 3135 Vol Arg His Vol Alo ATo Aln Ser Leu | le Arg Leu Vol Pro Lys Leu 925 930 935 940

TTT TAT AAA TCT CAC CAA GCA CAA CCT CAT CCA CTA CTC CCC CTC CCA 3183 Phe Tyr Lys Cys Asp Cln Cly Cln Ala Asp Pro Vol Vol Ala Vol Alo

945 950 955

ACA CAT CAA ACC ACT CTT TAC CTC AAA CTT CTC ATC CAT CAC ACC CAC 3231 Arg Asp Gln Ser Ser Vul Tyr Leu Lys Leu Leu Mel His Clu Thr Cln

960 965 970

CCT CCA TCT CAT TTC TCC CTC ACC ACA ATA ACC ACA ATA TAT ACA GCC 3279 Pro Pro Ser His Phe Ser Vol Ser Thr T le Thr Arg l Te Tyr Arg Cly

975 980 985

TAT AAC CTA CTA CCA ACC ATA ACA CAC CTC ACT ATC CAA AAT AAC CTT 3327 Tyr Asn Leu Leu Pro Ser | Te Thr Asp Vol Thr Mel Glu Asn Asn Leu

990 995 I000

TCA ACA CTT ATT CCA CCA CTT TCT CAT CAA CTA ATC ACA TCA ACC ACC 3375 Ser Arq Vol I la M0 M0 Vol Ser His Clu Leu I la Thr Ser Thr Thr 1005 T010 T015 1020

AGA CCA CTC ACA TTT GGA TCC TCT 6AA CCT TTC TCT CTT CTT TCC ACT 3423 Arg Alo Leu Thr Phe Cly Cys Cys Clu Alu Leu Cys Leu Leu Ser Thr

T025 1030 I035

CCC TTC CCA CTT TCC ATT TCC ACT TTA CGT TCC CAC TCT CCA GTC CCT 3471 Ala Phe Pro Vul Cys I is Trp Ser Leu Cly Trp His Cys Gly Vol Pro

T040 1045 1050

CCA CTC ACT BCC TCA CAT GAG TCT AGG MG ACC TCT ACC CTT CGG MC 3579 Pro Leu Ser Alo Ser Asp Clu Ser Arg Lys Ser Cys Thr Vol Cly Mel

I055 I060 I065

FIG.4G


CCC ACA AIC AII CIC ACC CIC CIC ICC ICA CCI ICC IIC CCA IIC CAI 3567 Ala Ihr Mel I Ie Leu Ihr Leu Leu Ser Ser Alo Irp Phe Pro Leu Asp

I070 I075 I080

CIC ICA CCC CAI CAA CAI CCI IIC AII TIC CCC CCA AAC IIC CII CCA 3615 [cu Ser AIo His Cln Asp Alo Leu I la Leu Alo Cly Asn Leu Leu Alo I085 I090 I095 I I00

CCC ACI CCI CCC AAA ICI CIC ACA ACI ICA ICC CCC ICI CAA CAA CAA 3663 Ala Ser AIo Pro Lys Ser Leu Arg Ser Ser Irp Alo Ser Clu Clu Clu

I I05 I I I0 I I I5

CCC AAC CCA CCA CCC ACC AAC CAA CAC CAC CIC ICC CCA CCC CIC CCC 37I I Ala Asn Pro Alo Alo Ihr Lys CIn Clu Clu Vol Irp Pro AIo Leu Cly

H20 1125 I I30

CAC CCC CCC CIC CIC CCC AIC CIC CAC CAC CIC IIC ICI CAC CIC CIC 3759 Asp Arg AIo Leu Vol Pro Mel Vol Clu CIn Leu Phe Ser His Leu Leu

H35 I I40 I I45

AAC CIC AIT AAC AII ICI CCC CAC CIC CIC CAI CAC CIC CCI CCI CCA 3807 Lys Vol I la Asn I Ie Cys Alo His Vol Leu Asp Asp Vol AIo Pro Cly

II50 I155 I I60

CCC CCA AIA AAC CCA CCC ITC CCI ICI CIA ACA AAC CCC CCI ICI CIA 3855 Pro Alo I la Lys M0 M0 Leu Pro Ser Leu Ihr Asn Pro Pro Ser Leu I165 H70 I175 I I80

ACI CCC AIC CCA CCA AAC CCC AAC CAC AAA CAA CCA CCA CM CM CCA 3903 Ser Pro IIe Arg Arg Lys CIy Lys Clu Lys Clu Pro Cly Clu Cln Alo

H85 I I90 I I95

ICI CIA CCC IIC ACI CCC AAC AAA CCC ACI CAC CCC ACT CCA CCI ICI 395I Ser Vol Pro Leu Ser Pro Lys Lys CIy Ser CI u AIo Ser AIo Alo Ser

I200 I205 I210

ACA CAA ICI CAI ACC ICA CCI CCI CTI ACA ACA ACI AAA ICC ICA ICA 3999 Arg CIn Ser Asp Ihr Ser CIy Pro Vol Ihr Ihr Ser Lys Ser Scr Scr

I215 I220 I225

FIG.4H

U.S. Patent Nov. 11, 1997 Sheet 12 of 50 5,686,288

CTC CCC ACT TTC TAT CAT CTT CCT TCA TAC CTC ACA CTC CAT CAT GTC 4047 Leu Cly Ser Phe Tyr His Leu Pro Ser Tyr Leu Arg Leu His Asp Vol

1230 I235 I240

CTC AAA CCT ACA CAC CCT AAC TAC AAC CTC ACC CTC CAT CTT CAC AAC 4095 Leu Lys Alo Thr His ATo Asn Tyr Lys Vol Thr Leu Asp Leu Cln Asn 1245 1250 1255 1260

ACC ACC CAA AAC TTT CCA CCC TTT CTC CCC TCA CCC TTC CAT CTT CTT 4143 Ser Thr Clu Lys Phe Cly Cly Phe Leu Arg Ser Alo Leu Asp Vol Leu

1265 1270 1275

TCT CAC ATA CTA CAC CTC CCC ACA CTC CAC CAC ATT CCC AAC TCT CT I' 4191 Ser Cln 1 Te Leu Cl u Leu Alo Thr Leu Clo Asp I la Cly Lys Cys Vol

1280 I285 1290

CM CAC ATC CTA CCA TAC CTC AAA TCC TCC TTT ACT CCA CAA CCA ATC 4239 Clu Clu l Te Leu Cl y Tyr Leu Lys Ser Cys Phe Ser Arg Clu Pro Mel

I295 T300 T305

ATC CCA ACT CTT TCT CTT CAA CAA TTC TTC AAC ACT CTC TTT CCC ACA 4287 Mel Alu Thr Vol Cys Vol Cln CTn Leu Leu Lys Thr Leu Phe Cly Thr

T310 1315 1320

MC TTC CCC TCC CAC TTT CAT CCC TTA TCT TCC AAC CCC ACC AAC TCA 4335 Asn Leu Alo Ser Cln Phe Asp Cly Leu Ser Ser Asn Pro Ser Lys Ser 1325 1.330 1335 I340

CAA CCC CCA CCA CAC CCC CTT CCC TCC TCC ACT CTC ACC CCA CCC TTC 4383 Cln Cly Arg Aio Cln Arg Leu Cly Ser Ser Ser Vol Arg Pro Cly Leu

T345 I350 I355

TAC CAC TAC TCC TTC ATC CCC CCC TAC ACC CAC TTC ACC CAC CCC CTC 4431 Tyr His Tyr Cys Phe Mel Mo Pro Tyr Thr His Phe Thr Cln Ala Leu

I360 T365 T370

CCT CAC CCC ACC CTC ACC AAC ATC CTC CAC CCC CAC CAC CAC AAC CAC 4479 Ala Asp A10 Ser Leu Arg Asn Mel Vol 6m Alo Clu Cln Clu Asn Asp

T375 1380 I385


ACC TCC CCA TCC TTT GAT OTC CTC CAG AAA GTG TCT ACC CAG TTG A110 4527 Thr Ser Gly Trp Phe Asp V01 1211 CM Lys Vul Ser Thr Cln Leu Lys

1390 1395 1400

ACA AAC CTC ACC ACT GTC ACA AAC AAC CGT CCA CAT AAC AAT GCT A11 4575 Thr Asn Leu Thr Ser V01 Thr Lys Asn Arg A10 Asp Lys Asn A10 1 le 1405 1410 1415 1420

CAT AAT CAC ATT CGT TTG TTT 6AA CCT CTT GTT ATA AAA CCT TTA AAA 4623 His Asn His I le Arg Leu Phe Clu Pro Leu V01 1 le Lys A10 Leu Lys

1425 1430 1435

CAG TAB ATXS ACT ABA ACA TGT CTG CAG TTA CAG AAG CA6 CTT TTA CAT 4671 Cln Tyr Thr Thr Thr Thr Cys Vol Cln Leu Cln Lys Cln Vol Leu Asp

1440 1445 1450

TTG CTC CCC CAC CTG CTT CA6 TTA C66 CTT AAT TAC TGT CTT CTC CAT 4719 Leu Leu Ala Cln Leu V01 Gln Leu Arg V01 Asn Tyr Cys Leu Leu Asp

1455 1460 1465

TCA GAT CA6 GT6 TTT ATT GGC TTT GTA TTC AAA CAG TTT 6AA TAC ATT 4767 Ser Asp Gln Vol Phe 1 le Gly Phe Vol Leu Lys Cln Phe Glu Tyr 1 1c '

1470 1475 1480

GAA CTG CGC CAO TTC AC6 CAA TCA GAB CCA ATC ATT CCA AAC ATC T11 4815 Clu Vol 61y Cln Phe Arg Clu Ser Clu A10 1 1e 1 le Pro Asn 1 1e Phe 1485 1490 1495 1500

TTC TTC TTC CTA TTA CTA TCT TAT CAA OCC TAT CAT TCA AAA CAG ATC 4865 Phe Phe Leu Vol Leu Leu Ser Tyr Clu Arg Tyr His Ser Lys Cln 1 1c

1505 1510 1515

A11 GCA ATT CCT AAA ATC ATT CAC CTC TGT CAT 660 A10 A10 GCC AC1 491 1 1 1e 01y 1 11: Pro Lys 1 le 1 1e G111 Leu Cys Asp Gly 1 11: Meet Me Ser

1520 1525 1530

SSA AGC AAC GCT GTO ACA CAT CCC ATA CCC CCT CTC CAC CCC ATA CTC 4959 01y Arg Lys A10 V01 Thr His A10 1 le Pro Alo Leu Cln Pro I 1e Vol

1535 1540 1545


CAC GAC CTC TTT CTA TTA ACA CGA ACA AAT AAA CCT CAT CCA CGA AAA 5007 His Asp Leu Phe Vol Leu Arq Cly Thr Asn Lys AIo Asp Ale Cly Lys

I550 I555 I560

CAC CTT GM ACC CM AAA GAG CTC CTC CTC TCA ATC TTA CTC ACA CTC 5055 Clu Leu Clu Thr Clrl Lys Clu Vol Vol Vol Ser Mel Leu Leu Arg Leu I565 I570 I575 I580

ATC CAC TAC CAT CAC CTC TTC CAC ATC TTC ATT CTT CTC CTC CAC CAC 5103 I la Cln Tyr His Cln Vol Leu Clu Met Phe I Ie Leu Vol Leu Cln Cln

I585 I590 I595

TCC CAC AAC CAC AAT CAA CAC AAC TCC AAC CCA CTC TCT CCA CAG ATA 5151 Cys His Lys Clu Asn Clu Asp lys Trp Lys Arg Leu Ser Arg CIn I is

1600 I605 I610

CCT CAC ATC ATC CTC CCA ATC TTA GCC AAA CAC CAC ATC CAC ATT CAC 5I99 Alu Asp I la I he Leu Pro Mel Leu Alo Lys Cln CIn Mel His I I9 Asp

I615 I620 I625

TCT CAT CAA CCC CTT CCA CTC TTA AAT ACA TTA TTT CAC ATT TIC CCC 5247 Ser His CI u AIo Leu CIy Vol Leu Asn Thr Leu Phe Clu I le Leu Ala

1630 I635 I640

CCT TCC TCC CTC CCT CCC CTA CAC ATC CTT TTA CCC ACT ATC TTC CTC 5295 Pro Ser Ser Leu Arg Pro Vul Asp Mel Leu Leu Arg Ser Mel Phe VoI I645 I650 I655 1650

ACT CCA AAC ACA ATC CCC TCC CTC ACC ACT CTT CAA CTC TCC ATA TCC 5343 Thr Pro Asn Thr Mel Alu Ser Vul Ser Thr Vol Cln Leu Trp I la Ser

I665 I670 I675

CCA ATT CIC CCC ATT TTC ACC CTT CTC ATT TCC CAC TCA ACT CAA CAT 539I Cly I Ie Leu Alo I le Leu Arg VuI Leu | Te Ser Cln Ser Thr C I u Asp

I680 I685 I690

ATT CTT CTT TCT CCT ATT CAC CAC CTC TCC TTC TCT CCC TAT TIA ATC 5439 I la Val Leu Ser Arq I le CIn Clu Leu Ser Phe Ser Pro Tyr Leu I la

I695 I700 I705


ICC 167 ACA CIA AII AAI AGG TIA ABA CAT 600 CAC ACT ACT TCA ACO 5487 Ser Cys Thr Vol I Is Asn Arg Lcu Arg Asp CIy Asp Ser Ihr Ser Ihr

I7I0 I715 I720

CIA CM CM CAC ACT CM 060 AAA CAA ATA AAC MI US CCA 0AA 0AA 5535 Leu GIu Clu His Ser Clu Gly Iys Gln I le Lys Asn Leu Pro Clu Clu I725 I730 I735 I740

ACA III TCA ACO III CIA ITA CM 076 GT7 CGI ATT CIT TIA CAA CAC 5583 Ihr Phe Ser Arg Phe Leu Leu Cln Leu VoI Cly I Ie Leu Leu CIu Asp

I745 I750 I755

ATI GI'I ACA AAA CAC CTG AAC GIC 0AA ATG ACI CAC CAC CAA CAI ACI 563I I Ie VoI Ihr Lys Gln Leu Lys Vol Glu Met Ser CIu Cln Cln His Ihr

I760 I765 I770

ITC IAT ICC CAO 0AA CIA 00C ACA CIC CIA AIC ICI CIC AIC CAC AIC 5679 Phe Iyr Cys Gin CI u Leu Cly Ihr Leu Leu Met Cys Leu I le His I le

I775 I780 I785

IIC AAC 7C7 GGA AIG TIC C00 ACA ATC ACA CCA CCI GCC ACT ACC CIC 5727 Phe Lys Ser Cly Mel Phe Arg Arg I Ie Ihr Ala Aio Alu Ihr Arg Leu

I790 I795 I800

TIC CGC ACT CAI CCC TC? 060 CGC AGI TIC IAC ACC CIC CAC AGC TIC 5775 Phe Arg Ser Asp Cly Cys Gly CIy Ser Phe Iyr Ihr Leu Asp Ser Leu I805 I8I0 I815 I820

AAC TIC CCC CCI CGT ICC A70 A70 ACC ACC CAC CCC (ICC CTC GIC CIC 5823 Asn Leu Arq Alu Arg Ser Met I la Thr Thr His Pro Al 0 Leu Vol Leu

I825 I830 I835

CTC I00 707 CAG ATA CIC CIC CII GTC AAC CAC ACC CAC IAC CGC ICC 5871 Leu Irp Cys Cln I Ie Leu Leu Leu Vol Asn His Ihr Asp Iyr Arg Irp

I840 I845 I850


TCC CCA CAA CTC CM; CAC ACC CCC AAA ACA CAC ACT CTC TCC ACC ACA 59I9 Trp Ala Clu Vol Cln Cln Thr Pro Lys Arg His Ser Leu Ser Ser Thr

I855 T860 1865

MC TTA CTT ACT CCC CA6 ATC TCT CCA CAA CAC GAG CAT TCT CAC TTC 5967 Lys Leu Leu Ser Pro Cln Mel Ser Cly Clu Clu Clu Asp Ser Asp Leu

1870 I875 I880

CCA CCC AAA CTT CCA ATC TCC AAT ACA CAA ATA CTA CCA ACA CCC CCT 60L‘) Alo Ala Lys Leu Cly Mel Cys Asn Arg Clu 1 le Vol Arg Arg Cly Alo T885 1890 1895 I900

CTC ATT CTC TTC TCT CAT TAT CTC TCT CAC AAC CTC CAT CAC TCC CAC 6063 Leu ] Ie Leu Phe Cys Asp Tyr V0! Cys Cln Asn Leu His Asp Ser Clu

I905 I910 I915

CAC TTA ACC TCC CTC ATT CTA AAT CAC ATT CAA CAT CTC ATC ACC CTT 6| I I His Leu Thr Trp Leu 1 le Val Asn His | Te Cln Asp Leu I la Ser Leu

1920 I925 I930

TCC CAC CAC CCT CCA CTA CAC CAC TTC ATC ACT CCC CTT CAT CCC AAC 5159 Ser His Clu Pro Pro Vol Cln Asp Phe I Ie Ser Ala Vol His Arg Asn

T935 T940 1945

TCT GCT CCC ACC CCC CTC TTC ATC CAG CCA ATT CAC TCT CCT TCT CAA 6207 Ser Alo Alu Ser Cly Leu Phe I la Cln Alo I la Cln Ser Arg Cys Clu

1950 T955 T960

AAC CTT TCA ACT CCA ACC ATC CTC AAC AAA ACT CTT CAC TCC TTC CAC 6255 Asn Leu Ser Thr Pro Thr Mel Leu Lys Lys Thr Leu Cln Cys Leu Clu I965 1970 1975 I980

CCC ATC CAT CTC ACC CAC TCC CCA CCT CTC CTC ACC CTC TAT CTC CAC 6503 Cly T le His Leu Ser Cln Ser Cly Alo Vol Leu Thr Leu Tyr Vol Asp

T985 T990 1995

ACC CTT CTC TCC ACC CCT ‘TTC CCT CTC CTC CCT CCC ATC CTC CAC ATC 535T Arq Leu Leu Cys Thr Pro Phe Arq Vol Leu Alo Arg Mel Vol Asp I Is

2000 2005 2010


011 001 101 000 C00 01A 0AA A10 011 C10 001 00A AA1 11A 0110 A00 6399 Lou Alo Cys Arg Arg Vol 0111 AM Leu Leu A10 A10 Asn Leu Gln Ser

2015 2020 2025

A00 A10 000 0A0 T10 CCA A10 CM CM C10 AAC AGA A10 CAO 0AA IAC 6447 Ser Met A10 010 Leu Pro Me101u 01o Leu Asn Arg He 0ln 0Iu1yr

2030 2035 2040

C11 CAO A00 A00 000 01C 001 CAO AGA CAO CM A00 010 1A1 10C 010 6495 Leu 0In Ser Ser 01y Leu A10 Gln Arg His 01n Arg Leu 1yr Ser Leu 2045 2050 2055 2060

C10 0A0 A00 111 C01 01C 100 A00 A10 CM 0710 10A 011 A01 C00 101 6543 Lou Asp Arg Phe Arg Leu Ser 1hr Mel 0In Asp Ser Leu Ser Pro Ser

2065 2070 2075

C01 CCA 01C 101 100 0A0 C00 010 GM) 000 0A1 000 CAC G10 10A C10 6591 Pro Pro V01 Ser Ser His Pro Leu Asp 01y Asp 01y His V01 Ser Leu

2080 2085 2090

GAA ACA 010 A01 000 0A0 AAA 0A0 100 1A0 011 CAT C11 010 MA 100 6639 01u_1hr Vol Ser Pro Asp Lys Asp 1rp Tyr Vol His Leu Vol Lys Ser

2095 2100 2105

0A0 101 100 A00 A00 10A 0A1 101 0011 C10 010 CM 001 00A 0A0 C10 6687 Cln Cys Trp 1hr Arq Ser Asp Ser A10 Leu Leu 0|u 01y Alo 01u Leu

2110 2115 ’ 2120

010 M1 000 A11 001 001 CM 0A1 A10 AA1 00C 110 A10 A10 MC 100 6735 Vol Asn Arg He Pro Alo 01u Asp Mel Asn Alo Phe Mel Mel Asn Ser 2125 2130 2135 2140

0A0 11C AAC 01A A00 C10 01A 001 00A 100 11A A00 01A 000 A10 A01 6783 01o Phe Asn Leu Ser Leu Leu Alo Pro Cys Leu Ser Leu 01y Mel Ser

2145 2150 2155

GAA A11 101 0G1 000 CAG M0 A01 000 C11 111 GM 00A 000 C01 0A0 6831 Glu 1|e Ser 01y 01y 01n Lys Ser A10 Leu Phe 0lu Alo Alo Arg 0lu

2160 2165 2170

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Huntingtin DNA, protein and uses thereof