(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2003/0104596 A1
`Mukerji et al.
`(43) Pub. Date:
`Jun. 5, 2003
`
`US 2003.01.04596A1
`
`(54)
`
`(76)
`
`(21)
`(22)
`
`(60)
`
`HUMAN DESATURASE GENE AND USES
`THEREOF
`
`Inventors: Pradip Mukerji, Gahanna, OH (US);
`Amanda Eun-Yeong Leonard,
`Gahanna, OH (US); Yung-Sheng
`Huang, Columbus, OH (US); Tapas
`Das, Worthington, OH (US)
`Correspondence Address:
`STEVEN F. WEINSTOCK; ABBOTT
`LABORATORIES
`100 ABBOTT PARK ROAD
`DEPT. 377/AP6A
`ABBOTT PARK, IL 60064-6008 (US)
`Appl. No.:
`10/191,513
`
`Filed:
`
`Jul. 9, 2002
`Related U.S. Application Data
`Division of application No. 09/227,613, filed on Jan.
`8, 1999, now Pat. No. 6,432,684, which is a continu
`ation-in-part of application No. PCT/US98/07422,
`
`filed on Apr. 10, 1998, which is a continuation-in-part
`of application No. 08/833,610, filed on Apr. 11, 1997,
`now Pat. No. 5,972,664.
`
`Publication Classification
`
`(51) Int. Cl. ............................ C12P 21/02; C12N 5/06;
`C07H 21/04; C12P 17/04;
`C12N 9/04
`(52) U.S. Cl. ..................... 435/190; 435/69.1; 435/320.1;
`435/325; 536/23.2; 435/134
`
`(57)
`
`ABSTRACT
`
`The Subject invention relates to the identification of a gene
`involved in the desaturation of polyunsaturated fatty acids at
`carbon 5 (i.e., "human A5-desaturase') and to uses thereof.
`In particular, human A5-desaturase may be utilized, for
`example, in the conversion of dihomo-y-linolenic acid
`(DGLA) to arachidonic acid (AA) and in the conversion of
`20:4n-3 to eicosapentaenoic acid (EPA). AA or polyunsatu
`rated fatty acids produced therefrom may be added to
`pharmaceutical compositions, nutritional compositions, ani
`mal feeds, as well as other products Such as cosmetics.
`
`ATGGCCCCCGACCCGGTGGCCGCCGAGACCGCGGCTCAGGGACCTACCCCGCGCTACTTCACCTGG
`GACCACGTCCCCCAGCGCTCAGGGTGCGAGGAGCCGTCCCTAGTGATCGACCGTAAGGTGTACAAC
`ATCAGCCAGTTCACCCGCCGGCATCCAGGGGGCTCCCGGGTCATCAGCCACTACGCCGGGCAGGAT
`GCCACCGATCCCTTTGTGGCCTTCCACATCAACAAGGGCCTTGTGAAGAAGTATATGAACTCTCTC
`CTGATTGGAGAACTGTCTCCAGAGCAGCCCAGCTTTGAGCCCACCAAGAATAAAGAGCTGACAGAT
`GACTTCCCCGAGCTGCGGGCCACAGTGGAGCCGATGGGGCTCATGAACGCCAACCATGTCTTCTTC
`CTCCTCTACCTCCTCCACATCTTCCTCCTGGATGGTCCAGCCTGGCTCACCCTTTGGGTCTTTGGG
`ACCTCCTTTTTCCCCTTCCTCCTCTGTGCGGTGCTGCTCAGTGCAGTTCACGCCCAGGCTGGCTCG
`CTCCACCATGACTTTGGGCACCTGTCGGTCTTCAGCACCTCAAAGTGGAACCATCTCCTACATCAT
`TTTCTGATTCGCCACCTGAAGGGGGCCCCCGCCAGTTGGTGGAACCACATCCACTCCAGCACCAT
`GCCAAGCCCAACTCCTTCCCCAAAGACCCAGACATCAACATGCATCCCTTCTTCTTTCCCTTGGGG
`AAGATCCTCTCTGTGGAGCTTGGGAAACAGAAGAAAAAATATATCCCGTACAACCACCACCACAAA
`TACTTCTTCCTAATTGGGCCCCCAGCCTTGCTGCCTCTCTACTTCCAGTGGTATATTTTCTATTTT
`GTTATCCAGCGAAAGAAGTGGGTGGACTTGGCCTGGATGATTACCTTCTACGTCCCCTTCTTCCTC
`ACTTATCTCCCACTATTGGGGCTGAAAGCCTTCCTGGGCCTTTTCTTCATAGTCAGGTTCCTCCAA
`AGCAACTGGTTTGTGTGGGTGACACAGATGAACCATATTCCCATGCACATTGATCATGACCGGAAC
`ATGGACTGGGTTTCCACCCAGCTCCTGGCCACATGCAATGTCCACAAGTCTCCCTTCAATGACTGG
`TTCACTCCACACCTCAACTTCCAGATTGACCACCATCTTTTTCCCACGATGCCTCGACACAATTAC
`CACAAAGTCCCTCCCCTGGTGCAGTCCTTGTGTCCCAAGCGTGGCATAGAGTACCAGTCCAAGCCC
`CTCCTGTCAGCCTTCGCCGACATCATCCACTCACTAAAGGAGTCAGGGCAGCTCTCGCTAGATGCC
`TATCTTCACCAATAA
`
`Human Power of N Company
`EX1047
`Page 1 of 95
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`
`JWWLIDOWVOIDOVIVOWIDLLOWIDIOLVOLVW991991DWWOIOLOLIIOLOOOWWW1LVOIOOLLVOLSOLLWW
`
`
`
`
`
`WWOWWOODOWLOVIOIVOQIDVOIOLWWOQIOOLOLLOLLESLSIOWIOLOOLVOWWOLOLVIOOOLVIOVVOVIOLLOLY
`
`
`
`LLIVLOLVOQOOLVOLIILIIIIOIIIIOLLVOLIOLLOLovLWWOOWIOVIOWOLWOWLID091IOVIVYVOLIOWWS
`
`
`
`
`
`L9Q0DDLVOWLOWLIOVILIOIIIILDODLODVIOLODLWWOWWLVIOLOOLVOLVOOWOLVOLVOLVOVIOWLOVOO
`
`
`
`
`
`
`JOVILDV!LOSLOWOLIIOWLOQDOVOOWVIOVILLWOVOOLWOLIDIVOVILVVOLVOVOWOVSILISOLOLOL119
`
`
`
`
`
`
`JIWWIILIDILILIOWVIIOLILOVWOWWOLDDILIWOLVOLVOVIDLIOLIIIIINVILOWLODIDIVVOVIOVIOV
`
`
`
`
`
`JQQIIIOWLVIQWILVOLOIDIIILOIIOIWIOLVIIOIIIIIOVILLOVIDIVILVOWWOWLLSOWLIVI
`
`
`
`
`
`JLVILVLODOLIIODVIOVIIILIIOVILIDIOVIQOIOLIDVIOVIDILIOVILLOW199099990LIDVIOD
`
`
`
`
`
`IILDOVIOLIIOVVOIIOLOQDVOVIDIIIOIIOVOWIOOVVOLVWVOVILL9DV19LOLLLODOLOLLOLVISLLOLVODIOVOIOVILIDDVIOLYLOLIOLIOWOVOIILILIGIWILILLOLLLL9991VOLWVIOVOVIOIOLVOL
`
`
`
`
`
`
`
`
`
`
`
`DIDWOLYIIDWOLIDIOVILODILIDIDLODVIIILVILIOILODWVIDWLVIDIDILIAVIVILLILOLLLL99L9199
`
`
`
`
`
`Page 7 of 95
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`Page 7 of 95
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`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 7 of 39
`
`US 2003/0104596 Al
`
`50
`40
`.
`30
`20
`10
`Ma29 pep MGTDQGKT- --FTWEELAAHNTKDDLLLAIRGRVYDVTKFLSRHPGGVDTLLLGAGRDVT
`Hl Uebbes
`or
`Eh
`lbssh TL TEE
`QGPTPRYFTWDEVAQRSGCEERWLVIDRKVYNISEFTRRHPGGSRV I SHYAGQDAT
`10
`20
`30
`40
`50
`
`253538a
`
`Ma29.pep
`
`253538a
`
`110
`100
`90
`80
`70
`60
`PVFEMYHAF-GAADATMKKYYVGTLVSNELPIFPEPTVFHKTIKTRVEGYFTORNIDPKN
`sds TET EL TE
`|:
`DPFVAFHINKGLVKKYMNSLLIGEL -SPEQPSF - EPTKNKELTDEFRELRATVERNGLMK
`60
`70
`80
`90
`100
`110
`
`120
`
`130
`
`140
`
`150
`
`160
`
`170
`
`Ma29.pep
`2030384
`
`RPEIWGRYALTFGSLTASYYAQLFVPFVVERTWLOVVE-ATIMGFACAQVGLNPLHDASH
`ANHVF--FLLYLLHTLLLDGAAWLTLWVFGTSFLPFLLCAVLLSAVQAQAGULO- HDYGH
`120
`130
`140
`150
`160
`170
`
`180
`
`190
`
`200
`
`210
`
`220
`
`Ma29 .pep
`253538a_—_
`
`FSVTHNPTVWKILGATHDF- -- -FNGASYLVWMYQHMLGHHPYTNIAGADPDVSTSE--
`Alot
`de ttt |
`AE
`dosed dP
`TE OT
`LSVYRKPK-WNHL--VHKFVIGHLKGASANHNNHRH-FQHHAKPNI FHKDPOVNMLHVFV
`180
`190
`200
`210
`220
`
`280
`270
`260
`250
`240
`230
`Naz9.pep ----PDVRRIKPNOKUIF- VNHINQHMFV- - PFLYGLLAFKVRIQDINILYFVKTNDAIRV
`Pos
`des
`Up sch |
`|
`Is fd:
`253538a_LGEWOPIEYGKKKLKYLPYNHQHEYFFLIGPPLL IPMYFQYQI- --- IMTMIVHKNWVDL
`230
`240
`250
`260
`270
`280
`
`Ma29.pep
`
`253538a
`
`340
`330
`320
`310
`300
`290
`NPISTWHTVMFWGGKAFFVWYRLIVPLQYLPLGKVLLLFTVADMVSSYHLALTFQANHV
`|
`Heb
`che
`|
`Rb
`eddess
`ts
`depes
`tf Ted
`~~ --AWAVSYYI---RFFITY---IPF-YGILG-ALLFLNFIRFLESHWFVWVTOMNHIV
`290
`300
`310
`320
`330
`
`390
`380
`370
`360
`350
`EEVQWPLPDENGI 1QKDWAAMQVETT- - - -QDYAHDSHLWTSITGSLNYQAVHHLFPNVS
`I:
`ess
`the deh
`dee
`bh
`ED ETE TELE:
`253538a_NEI-----DQEAY--ROWFSSQLTATCNVEQSFFND- - -WFS - -GHLNFOTEHHLFPTMP
`340
`350
`360
`370
`
`Ma29. pep
`
`440
`430
`420
`410
`400
`Ma29.pep QHHYPDILAIIKNTCSEYKVPYLVKDTFWQAFASHLEHLRVLGLRPKEEX
`tfJe
`J
`oottfe
`fers
`ts
`|
`|
`RHNLHKIAPLVKSLCAKHGIEYQEKPLLRALLDI IRSLKKSGKLWLDAYLHKX
`380
`390
`400
`410
`420
`430
`
`253538a
`
`FIG.9
`
`Page 8 of 95
`
`Page 8 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 8 of 39
`
`US 2003/0104596 Al
`
`Ma524.pep
`
`253038a
`
`Ma524. pep
`
`253538a
`
`10
`20
`30
`40
`50
`=.
`«59
`MAAAPSVRTFTRAEVLNAEALNEGKKDAEAPFLMI IDNKVYDVREFVPOHPGGSV ILTH-
`|
`||
`its
`SAP Ube:
`The WP ed
`QGPTPRYFTWDEV--------AQRSGCEERWLVIDRKVYNISEFTRRHPGGSRVISHY
`10
`20
`30
`40
`50
`
`60
`70
`80
`90
`100
`110
`VGKOGTOVFDTFHPEAAW- -ETLANFYVGDIDE---SDRDIKNDDFAAEVRKLRTLFQSL
`Rel Tih:
`scsfie
`for ft | Tp:
`AGODATOPFVAFHINKGLVKKYMNSLL IGELSPEQPSFEPTKNKELTDEFRELRATVERM
`60
`70
`80
`90
`100
`110
`
`
`
`120 130=:140 150 160 170
`
`
`GYYDSSKAYYAFKVSFNLCTWGLSTV IVAKWGQTSTLANVLSAALLGLFHQQCGHLAHDF
`Ma524.pep
`Js oritint:
`:
`AHL cl ere ET:
`GLVKANHVFFLLYLLHILLLOGAAWLTLWVEG-TSFLPFLLCAVLLSAVQAQAGHLQHDY
`120
`130
`140
`150
`160
`
`253538a
`
`Ma524 . pep
`
`203038a
`
`230
`220
`210
`200
`190
`180
`LHHOVFQORFWGDL FGAFLGGVCOGFSSSWWKDKHNTHHAAPNVHGEDPD IDTHPLLTWS
`Peles
`de Pde dh deed ch TP Te Tbe
`2]
`GHLSVYRKPKWNHLVHKFV IGHLKGASANWWNHRHFQHHAKPN I FHKDPDVN- - -ML- - -
`170
`180
`190
`200
`210
`220
`
`Ma524. pep
`
`253038a
`
`Ma524.. pep
`
`2535384
`
`Ma524 . pep
`
`293538a
`
`Ma524 . pep
`
`2935384
`
`290
`=
`280
`270
`260
`250
`240
`EHALEMFSDVPDEELTRMWSRFMVLNOTWFYFPILS- - -FARLSWCLOSILFVLPNGQAH
`Jr: oritt
`|
`[:
`dd:
`7
`|
`|:
`‘|
`-HVF -VLGEWOPTEYGKKKLKYLPYNHQHEYFFLIGPPLLIPMYFQYQLIMTNI- ---VH
`230
`240
`250
`260
`270
`
`300
`310
`320
`-
`330
`340
`349
`KPSGARVPISLVEQLSLAMHWTWYLATMFLFTK- -DPVNMLVYFLVSQAVCGNLLALVES
`|
`‘AL
`ref:
`:|
`[::
`|:
`K----+------NWVDLAWAVSYVTRFFITYIPFYGILGALLFLNFIRFLESHMFVAVTO
`
`280
`
`290
`
`300
`
`310
`
`320
`
`409
`400
`390
`380
`370
`360
`350
`LNHNGMPVSKEEAVDMDFFTKQI ITGROVHPGL FANWFTGGLNYOTEHHL F PSMPRHINF
`Abd dh bebe heh Te PETE ETT
`MNHIVMEI - -DQEAYR-DkIFSSOL-TATCNVEQSFFNOMFSGHLNEQEHHLFPTMPRHNL
`330
`340
`350
`360
`370
`380
`
`A410
`420
`430
`440
`450
`SKIQPAVETLCKKYNVRYHTTGMIEGTAEVFSRLNEVSKAASKMGKAQK
`I]t feed)
`feescf:
`Jr: |
`HKTAPLVKSLCAKHGIEYOEKPLLRALLDI IRSLKKSGKLWLDAYLHKX
`390
`400
`410
`420
`430
`
`FIG.10
`
`Page 9 of 95
`
`Page 9 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 9 of 39
`
`US 2003/0104596 A1
`
`||"|{JIONIVXSHYSOI00
`g-ueoe]=sfg-urgi]
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`Page 10 of 95
`
`Page 10 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 10 of 39 US 2003/0104596 A1
`
`ATGGCCCCCCACCCCGTCCCCCCCGAGACCGCCGCTCAGCCACCTACCCCCCCCTACTTCACCTCC
`GACCACGTGGCCCAGCGCTCAGGGTGCGAGGAGCCGTCCCTAGTGATCGACCGTAAGGTGTACAAC
`ATCAGCCAGTTCACCCCCCGGCATCCAGGGGGCTCCCGGGTCATCAGCCACTACGCCGGGCAGGAT
`GCCACCGATCCCTTTGTCGCCTTCCACATCAACAAGGGCCTTGTGAAGAACTATATGAACTCTCTC
`CTGATTGGAGAACTGTCTCCAGACCACCCCAGCTTTGAGCCCACCAAGAATAAACACCTGACAGAT
`GAGTCCCCGACCTCCGGGCCACAGTCGAGCCGATCCGCCTCATGAACGCCAACCATGTCTTCTTC
`CTCCTCTACCTCCTCCACATCTTCCTCCTCCATCGTCCACCCTCCCTCACCCTTTCCCTCTTTCCC
`ACGTCCTTTTTCCCCTTCCTCCTCTGGCCGTGCTGCTCAGTGCAGTTCACGCCCAGGCTGGCTCC
`CTCCACCATGACTTTGGGCACCTCTCCGTCTTCACCACCCAAAGTGGAACCATCTCCTACATCAT
`TTTGTGATTGGCCACCTGAACGGGGCCCCCGCCAGTTGGTGGAACCACATCCACTCCAGCACCAT
`CCCAAGCCCAACTCCTTCCCCAAAGACCCAGACATCAACATGCATCCCTTCTTCTTTCCCTTGGGG
`AAGATCCTCTCTGTCGAGCTTGGGAAACAGAAGAAAAAAIATAGCCGTACAACCACCACCACAAA
`TACTTCTTCCTAATTCGGCCCCCAGCCTTCCTGCCTCTCTACTTCCAGTCGTATATTTTCTATTTT
`GTTATCCAGCGAAAGAAGTGGGTGGACTTGGCCTGGATGATTACCTTCACGTCCCCTTCTTCCTC
`ACTTATCTCCCACIATTCCCCCTGAAAGCCTTCCTGGGCCTTTTCTTCATAGTCACGTTCCTCCAA
`ACCAACTGGTTTCTCTCCCTGACACAGATGAACCATATTCCCATGCACATTGATCATGACCGGAAC
`ATCGACTGGGTTTCCACCCAGCTCCTCGCCACATGCAATGTCCACAAGTCTCCCTTCAATGACTGG
`TTCACTCCACACCTCAACTTCCAGATTGACCACCATCTTTTTCCCACCATCCCTCCACACAATTAC
`CACAAAGTGGCTCCCCTGGTGCACTCCTTGTGTCCCAAGCGTGGCATAGAGACCAGTCCAAGCCC
`CTGCTCTCAGCCTTCGCCGACATCATCCACTCACTAAAGGAGTCAGGGCAGCTCTCGCTAGATGCC
`TATCTTCACCAATAA
`
`FIG. 12
`
`1 MAPDPWAAET AAOGPTPRYF TWDEVAORSG CEERWLVIDR KWYNSEFTR
`
`51 RHPGGSRWIS HYAGODATDPFWAFHINKGL WKKYMNSLL CELSPEOPSF
`
`101 EPTKNKELTDEFRELRATVE RMGLMKANHV FFLLYLLHL LLDGAAWLL
`
`151 WFGTSFLPF LLCAVLSAW OAQAGWLOHD FGHLSWFSTS KWNHLLHHFW
`
`201 ICHLKGAPAS WNHMHFOHHAKPNCFRKDP DNMHPFFFA LGKILSVELG
`
`25 KOKKKYMPYN HOHKYFFLIG PPALLPLYFQ WYIFYFWIQR KKWDLAWM
`
`301 TFYVRFFTY WPLLCLKAFL CLFFVRFLE SNWFWTQM NHPMHDHD
`
`351 RNMDWSTO LATCNVHKSA FNDWFSGHLN FQIEHHLFPT MPRHNYHKVA
`
`401 PLWOSLCAKR GEYOSKPLL SAFADHSL KESGOLWLDA YLHQ
`F.G. 13
`
`Page 11 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003. Sheet 11 of 39
`
`>—————
`
`
`
`890| 01 LQG '0/-3Wºd
`
`>————————————
`
`>—————
`
`
`
`****----¬)________
`
`~---------------——————?___
`
`Page 12 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003. Sheet 12 of 39 US 2003/0104596 A1
`
`10
`
`20
`
`50
`
`40
`s:
`CTC CTG GAG CCC GTC AGT ATC GGC GGA ATT CCG GCA GTT CAG GCC CAG
`Leu Leu Gu Pro Vol Ser I e Giy Gly le Pro Alo Vol Gin Alo Ginx
`0 0 0
`OTRANSLATION OF PRAE-7 MV A. O.
`O. O.
`O.
`>
`60
`70
`80
`90
`
`50
`
`s
`CCT CGC TGG CTG CAG CAT GAC TTT GGG CAC CTG TCG GTC TTC AGC AC
`Alo Gly Trp Leu Gin His Asp Phe Giy His Leu Ser Vol Phe Ser Thr)
`0 0-0 0-TRANSLATION OF PRAE-7 MV Al-O-O-O-O >
`100
`110
`120
`30
`40
`t
`:
`k
`TCA AAG TCC AAC CAT CTG CTA CAT CAT TTT GTG ATT GGC CAC CTG AAG
`Ser Lys Trp Asn His leu Lleu. His His Phe Vol Ile Giy His leu LysX
`0 0-0 0-TRANSLATION OF PRAE-7 MV A. O.
`O.
`O. O >
`150
`160
`170
`180
`190
`
`k
`
`GGG GCC CCC GCC AGT TGG TGG AAC CAC ATG CAC TTC CAG CAC CAT GCC
`G|y Aio Pro Alo Ser Trp Trp Asn His Met His Phe Gin His His Alo)
`0-0 0 OTRANSLATION OF PRAE-7 MV A) 0-0-0-0-->
`200
`2O
`220
`250
`240
`
`k
`AAC CCC AAC TGC TTC CGC AAA GAC CCA GAC ATC AAC ATG CAT CCC TTC
`Lys Pro Asn Cys Phe Arg Lys Asp Pro Asp lie Asn Met His Pro Phex
`0 0 0 O_TRANSLATION OF PRAE-7 MV A)
`250
`260
`270
`280
`t
`k
`TTC TTT CCC TTC CCG AAG ATC CTC TCT GTG GAG CTT CCG AAA CAG AAG
`Phe Phe Ao Leu Giy Lys lie Leu Ser Vol Gu Leu Giy Lys Gin Lys>
`0 0 0-0 TRANSLATION OF PRAE-7 MV A 0-0 0-0-->
`500
`310
`320
`330
`
`290
`
`AAA AAA TAT ATG CCG TAC AAC CAC CAG CAC AAA TAC TTC TTC CTA ATT
`Lys Lys Tyr Met Pro Tyr Asn His Gin His Lys Tyr Phe Phe Leu Ilex
`O_0 0 OTRANSLATION OF PRAE-7 MV A 0- 0. 0-0->
`FIG.15A
`
`Page 13 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 13 of 39 US 2003/0104596A1
`
`380
`
`540
`
`350
`
`560
`370
`k
`k
`k
`CCG CCC CCACCC TTG CTG CCT CTC TAC TTC CAG TCG TAT ATT TTC TAT
`Gly Pro Pro AIO Leu Lleu Pro Leu Tyr Phe Gin Trp Tyr le Phe Tyrx
`0-0 0-0-TRANSLATION OF PRAE-7 MV Al-O-O-O-O->
`390
`400
`410
`420
`430
`k
`k
`TTT GTT ATC CAG CGA AAG AAG TCG GTG GAC TTG GCC TGG ATG ATT ACC
`Phe Vol Ile Gin Arg Lys Lys Trp Vol Asp Leu Alo Trp Met Ile Thrx
`O-O-O OTRANSLATION OF PRAE-7 MV Al-O O.
`O.
`O.-->
`440
`450
`460
`470
`480
`k
`k
`TTC TAC GTC CCC TTC TTC CTC ACT TAT GTG CCA CTA TTG CCG CTG AAA
`Phe Tyr Val Arg Phe Phe Leu Thr Tyr Vol Pro Leu Lleu Giy Leu LysX
`0
`0 10
`OTRANSLATION OF PRAE-7 MV A_0 0 10
`O >
`490
`500
`510
`520
`
`530
`
`s:
`t
`:
`k
`t
`t
`GCC TTC CTG GGC CTT TTC TTC ATA GTC AGG TTC CTG GAA AGC AAC TCG
`Alo Phe Leu Giy Leu Phe Phe lie Vol Arg Phe Leu Glu Ser Asn Trp>
`0-0 0-0 TRANSLATION OF PRAE-7 MV A. O.
`O.
`O. O >
`540
`550
`560
`570
`k
`k
`k
`
`k
`
`k
`
`t
`
`k
`
`TTT GTG TCG GTG ACA CAG ATG AAC CAT ATT CCC ATG CAC ATT CAT CAT
`Phe Vol Trp Vol Thr Gin Met Asn His lie Pro Met His Ile Asp His>
`0-0-0 0-TRANSLATION OF PRAE-7 MV A. O.
`O.
`O.
`O. Y
`580
`590
`600
`610
`620
`k
`k
`
`GAC CGG AAC ATG GAC TGG GTTTCC ACC CAG CTC CAG GCC ACA TGC AAT
`Asp Arg Asn Met Asp Trp Vol Ser Thr Gin Leu Gin Alo Thr Cys Asn)
`a
`O
`o O TRANSLATION OF PRAE-7 MV A. O.
`O.
`O.
`O >
`630
`640
`650
`660
`670
`k
`k
`k
`GTC CAC AAG TC GCC TTC AAT GAC TCG TTC AGT CGA CAC CTC AAC TTC
`Vol His Lys Ser Alo Phe Asn Asp Trp Phe Ser Cly His Leu Asn Phex
`0 0 0 OTRANSLATION OF PRAE-7 MV A) 0 0 0 0
`}
`
`k
`
`FIG.15B
`
`Page 14 of 95
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`

`

`Patent Application Publication
`
`Jun. 5, 2003. Sheet 14 of 39 US 2003/0104596 A1
`
`690
`
`700
`
`70
`
`720
`
`770
`
`760
`
`810
`
`680
`k
`CAG ATT GAG CAC CAT CTTTTT CCC ACC ATG CCT CCA CAC AAT TAC CAC
`Gln lie Gu His His Leu Phe Pro Thr Met Pro Arg His Asn Tyr His>
`O
`o
`O OTRANSLATION OF PRAE-7 MV A
`730
`740
`750
`k
`k
`AAA GTG GCT CCC CTG GTG CAG TCC TTG TGT GCC AAG CAT GCC ATA GAG
`Lys Vol Alo Pro Leu Voi Gln Ser Leu Cys Alo Lys His Gly lie Glux
`0 0 lo OTRANSLATION OF PRAE-7 MV A
`780
`790
`800
`k
`k
`k
`TAC CAG TCC AAG CCC CTG CTG TCA GCC TTC GCC GAC ATC ATC CAC TCA
`Tyr Gln Ser Lys Pro Leu Lleu Ser Ao Phe Alo Asp lie lie His Ser)
`0 0 0 OTRANSLATION OF PRAE-7 MV A. O.
`O.
`O.
`O. X
`820
`850
`840
`850
`860
`
`k
`
`CTA AAG GAG TCA GGG CAG CTC TGG CTA GAT GCC TAT CTT CAC CAA TAA
`Leu Lys Glu Ser Gly Gin Leu Trp leu Asp Alo Tyr Leu His Gin k >
`0 o O O-TRANSLATION OF PRAE-7 MV Al-O-O. O.
`O
`>
`
`FIG.15C
`
`Page 15 of 95
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`

`

`Patent Application Publication
`
`Jun. 5, 2003. Sheet 15 of 39 US 2003/0104596 A1
`
`pRAE-7. pep
`
`Ma29, pep
`
`40
`3O
`2O
`10
`LLEPVSIGGIPAVOAQAGWLO-HDFGHLSV-FSTSKWNHL--LH
`:
`: :
`.
`.
`.
`ASYYAOLFVPFVVERTWLOVVFAIIMGFACAQVGLNPLHDASHFSVTHNPTVWKILGATH
`140
`150
`160
`170
`18O
`190
`
`90
`8O
`70
`60
`50
`pRAE-7. pep HFVIGHLKGAPASWWNHMH-FQHAKPNCFRKDPDINM-HPFFFALGKILSVELGKQKKK
`. .
`.
`. . .
`.
`.
`.
`. .
`.
`; :
`.
`. .
`DF----FNGASYLVWMYOHMLGHHPYTNIAGADPDVSTSFP- - - - - - - - - DVRRIKPNOK
`2OO
`210
`220
`230
`
`Ma29, pep
`
`pRAE-7. pep
`
`Ma29, pep
`
`149
`140
`130
`120
`110
`100
`YMPYNH--QHKYF-FLIGPPALLPLYFQWYIFYFWIQ----RKKWVDLAWMITFY--VRF
`::
`||
`|| |
`| |
`|
`| .
`.
`.
`.
`.
`. .
`.
`.
`WF-VNHINGHMFVPFLYGLLAFKVRIODINILYFWKTNDAIRVNPISTWHIVMFWGGKAF
`240
`250
`260
`270
`28O
`290
`
`pRAE-7. pep
`
`Ma29 pep
`
`190
`18O
`170
`160
`150
`FLTY---VPL--LGLKAFLGLFFIVRFLESNWFVWVTOMNHIPMHID---HDRN----MD
`: |
`|
`|
`|
`|
`|
`. .
`. .
`.
`. .
`.
`.
`.
`. . .
`.
`.
`FVWYRLIVPLOYLPLGKVLLLFTVADMVSSYWLALTFOANHVVEEVOWPLPDENGIIQKD
`300
`31 O
`32O
`330
`340
`350
`
`250
`2AO
`230
`220
`210
`2OO
`pRAE-7. pep WVSTOLOATCN-VHKSAFNDWFSGHLNFQIEHLFPTMPRHNYHKVAPLVOSLCAKHGIE
`. .
`. . .
`.
`.
`.
`.
`. .
`.
`. .
`.
`.
`.
`. . . .
`. . .
`.
`WAAMQVETTODYAHDSHLWTSITGSLNYOAVHHLFPNVSQHHYPDILAIIKNTCSEYKVP
`360
`370
`38O
`390
`400
`410
`
`Ma29.pep
`
`pRAE-7. pep
`
`Ma29, pep
`
`280
`27O
`260
`YOSKPLL-SAFADIIHSLKESGQLWLDAYLHOX
`.
`.
`.
`. .
`.
`YLVKDTFWOAFASHLEHLRVLGLRPKEEX
`42O
`- 430
`440
`FIG.16
`
`Page 16 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003. Sheet 16 of 39 US 2003/0104596 A1
`
`pRAE-7. pep
`
`Ma524. pep
`
`40
`30
`2O
`10
`LLEPVSIGGIPAVOAOAGWLOHDFGHLSVFSTSKWNHLLHHFVIG
`|
`|
`| |
`|
`.
`.
`.
`.
`.
`GLSTVIVAKWGOTSTLANVLSAALLGLFWOOCGWLAHDFLHHOVFODRFWGDLFGAFLGG
`140
`150
`160
`17O
`18O
`190
`
`pRAE-7. pep
`
`Ma524.pep
`
`90
`80
`70
`60
`50
`HLKGAPASWWNHMHFOHHAKPNCFRKDPDINMHPFF----FALGKILSV---ELGKOKKK
`; :: || ||
`:
`:
`::
`: .
`.
`. .
`. .
`VCOGFSSSWWKDKHNTHHAAPNVHGEDPDIDTHPLTWSEHALEMFSDVPDEELTRMWSR
`200
`210
`220
`230
`240
`250
`
`pRAE-7. pep
`
`Ma524.pep
`
`140
`130
`12O
`110
`1OO
`YMPYNHOHKYFFLIGPPALLPLYFOWYIFYFV------------- IQRKKWVDLAWMITF
`:
`::
`:
`:
`:
`I.
`: ::
`:
`FMVLN-OTWFYFPILSFARLSWCLOSILFVLPNGOAHKPSGARVPISLVEQLSLAMHWTW
`260
`27O
`280
`290
`300
`310
`
`pRAE-7, pep
`
`Ma524, pep
`
`2OO
`190
`18O
`170
`160
`150
`YVRFFLTYW--PLLGLKAFLGLFFIWRFLESNWFWWVTOMNHIPMHI---DHDRNMDWVS
`.
`. .
`. .
`.
`.
`. .
`.
`.
`.
`. .
`.
`. .
`.
`.
`. .
`.
`.
`.
`YLATMFLFIKDPV----NMVYFLVSOAVCGNLLAIVFSLNHNGMPVISKEEAVDMDFFT
`32O
`330
`340
`350
`360
`370
`
`pRAE-7. pep
`
`Ma524, pep
`
`260
`250
`240
`230
`22O
`210
`TQLOATCNVHKSAFNDWFSGHLNFQIEHHLFPTMPRHNYHKVAPLVOSLCAKHGIEYOSK
`|: .
`.
`.
`.
`.
`:
`: || || || || || :
`:
`::
`::::::
`KQIITGRDVHPGLFANWFTGGLNYOIEHHLFPSMPRHNFSKIOPAVETLCKKYNVRYHTT
`380
`390
`400
`410
`420
`430
`
`pRAE-7. pep
`
`Mas24. pep
`
`280
`270
`PLLSAFADIIHSLKESGQLWLDAYLHOX
`. .
`.
`. . .
`.
`GMIEGTAEWFSRLNEVSKAASKMGKAQX
`440
`450
`
`FIG. 17
`
`Page 17 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 17 of 39.
`
`~US 2003/0104596 Al
`
`pRAE-7. pep
`
`2535
`
`/
`
`pRAE-7.pep
`
`2535
`
`50,
`40
`30
`20
`10
`LLEPVSIGG I PAVQAQAGWLQHDFGHL SVFSTSKWNHLLHHFVIGHLKGAPA
`HTTP EEET TEEPE]
`VF YFGNGWIPTL I TAFVLATSQAQAGWLOHDYGHL SVYRKPKWNHLVHKFVIGHLKGASA
`10
`20
`30
`40
`50
`60
`
`110
`100
`90
`80
`70
`60
` SWHNHMHFQHHAKPNCFRKDPDINM-HPFFFALGK IL SVELGKGKKKYMPYNHOHKYFFL
`STTE TETTTTETE ETHEL Ee ETE TEE
`NWWNHRHFQHHAKPN IFHADPDVNM_H—VFVLGEWOP IEYGKKKLKYLPYNHOHEYFFL
`70
`80
`90
`100
`110
`
`170
`160
`150
`140
`130
`120
`pRAE-7.pep_IGPPALLPLYFQWYIFYFVIORKKWWVDLAWMI TFYVRFFLTYVPLLG-LKAFLGLFFIVR
`HEAL deb deeb PT se ee EEE EL
`IGPPLL IPMYFQYQ! IMTMI VEKNNVDLAWAVSYYIRFF ITYIPFYGILGALLFLNF I-R
`120
`130
`140
`150
`160
`170
`
`2535
`
`230
`220
`210
`200
`190
`180
`pRAE~7.pep—FLESNWFWWVTQMNHI PMH DHORNMOWVSTOLQATCNVHKSAFNDWFSGHLNFQIEHHL
`PUTS Petes TE TET TPEL Ee EEE TTP
`F LESHWF VWVTQMNH | VME IDQEAYRDWFSSQL TATCNVEQSFFNOWFSCHL.NFQI EHHL
`180
`190
`200
`210
`220
`230
`
`2535
`
`280
`2/0
`260
`250
`240
`pPRAE-7.pep—FPTMPRHNYHKVAPLVQSLCAKHG I EYQSKPLLSAFADI IHSLKESGQLWLDAYLHQX
`LETTE TESTE PEEP TEP Es
`TPES EET ETE |
`F PTMPRHNLHK | APLVKSLCAKHG I EYQEKPLLRALLDI IRSLKKSGKLWLDAYLHKXSH
`240
`250
`260
`270
`280
`290
`
`2935
`
`2935
`
`SPRDTVGKGCRWGDGORNDGLLFXGVSERLVYALL TDPMLDL SPFLL SFF SSHLPHSTLP
`300
`310
`320
`330
`340
`350
`
`F1G.18
`
`Page 18 of 95
`
`Page 18 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 18 of 39°
`
`~US 2003/0104596 Al
`
`pRAE-7. pep
`
`38
`
`39
`30
`20
`10
`LLEPVS1GG IPAVQAQAGHLQHDFGHL SVF STSKWNHLL
`> LTTE TET TT
`-~—_LHILLLOGAAWL TLWVFGTSFLPFLLCAVLLSAVQAQAGWLQHDFCHL SVF STSKWNHLL
`130
`140
`150
`160
`170
`180
`
`99
`90
`80
`70
`60
`50
`40
`pRAE-7. pep—HHFVIGHLKGAPASWWNHMHFCHHAKPNCFRKDPDI NMHPFFFALGK ILSVELGKQKKKY
`HVPET TETTTEEE EET EEE EEE TEEPE
`HHFV [GHLKGAPASAWNHMHF QHHAKPNCF RKDPD INM-PFF F ALGK ILSVELGKQKKKY
`190
`200
`210
`220
`230
`240
`
`150189
`140
`130
`120
`110
`100
`pRAE~7.pep—MPYNHOKKYFFLIGPPALLPLYFQWY IFYFV I QRKKWVDLAWMI TFYVRFFLTYVPLLGL
`LELTETT TEETER ET
`MPYNHQHXYFFL IGPPALLPLYFQAY IFYFV1QRKKWVDLAWI SKQEYDEAGLPLSTANA
`250 260
`270
`280
`290
`300
`
`38
`
`219
`210
`200
`190
`180
`170
`160
`“pRAE-7.pep—KAFLGLFF I VRFLESNWFVWVTOMNH I PMHI DHDRNMDWVSTOLQATCNVHKSAFNOWFS
`
`38
`
`SK
`
`FIG.19
`
`A-1.pdt
`
`CYB5_HUMAN
`
`980
`9/0
`260
`390
`240
`930
`XLDLPTNMMEXRKAAAELXAAE TAAQGPTPRYF TWDEVAQRSGCEERWLV1 DRKVYNISE
`shef [eter [fee pecs:
`AEQSDEAVKYY TLEE [QKHNHSKSTWL ILHHKVYDLTK
`10
`20
`30
`
`640
`630
`620
`610
`600
`590
`F TRRHPGGSRVI SHYAGQDATDPFVAFHINKGLVKKYMN~SLL IGELSPEQPSF EPTKNK
`|
`::
`ee UP Te |i ot
`de sd ed DU
`CYBS_HUMAN=FLEEHPGGEEVLREQAGGDATENFE—DVGHSTDAREMSKTF I ]GELHPDD-—RPKLNK
`40
`30
`60
`70
`80
`90
`
`A-1.pdt
`
`A-1.pdt
`
`700
`690
`680
`670
`660
`650
`EL TDEFRELRATVEQRFPVXFL TCTGAHGFFSLEVPGLPDSNKXFSWTSRPI XWNKGKRP
`
`CYBS_HUMAN=PPETLI TT IDSSSSWWTNWV]PAISAVAVALMYRLYMAED
`100
`110
`120
`130
`
`FIG.20
`
`Page 19 of 95
`
`Page 19 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003. Sheet 19 of 39 US 2003/0104596 A1
`
`A
`
`330
`359
`349
`359
`569
`379
`589
`CCCGACCAAATGATGGAATAAAGGAAAGCCGCCGCTGAATTATAGGCCGCCGAGACCGC
`
`OC004228
`
`CCCGGCGCGCGGCGTCGCCAGGCCAGCTATGGCCCCCGACCCGGTGGCCGCCGAGACCGC
`60090
`60 100
`601 10
`60 120
`60130
`60140
`
`A
`
`Oc004228
`
`A-1
`
`0C004228
`
`A
`
`Oc004228
`
`A-1
`
`OC004228
`
`270
`279
`289
`299
`309
`319
`329
`GGCTCAGGGACCACCCCGCGTTACTTCACATGGGACGAGGTGGCCCAGCGCTCAGGGTG
`||
`|| || || || || ||
`||
`|| || || ||
`GCCTCACGGACCACCCCGCGCTACTTCACCTGGGACGAGGTGGCCCAGCCCTCAGGGTG
`60 150
`60 160
`6O170
`6080
`6090
`60200
`
`210
`219
`229
`259
`249
`259
`269
`CGAGGAGCCGTGGCTTGTGATCGACCGTAAGGTGTACAACATCAGCGAGTTCACCCGCCG
`|| || ||
`|| || || || ||
`|| ||
`||
`CGAGGAGCCGTCGCTAGTGATCGACCGTAACGTGTACAACATCAGCGAGTTCACCCGCCG
`60210
`60220
`60250
`60240
`6O250
`60260
`
`150
`159
`169
`179
`189
`199
`209
`GCATCCAGGGGGCTCCCGGGTCATCAGCCACTACGCCGGGCAGGATGCCACGGATCCCTT
`||
`||
`||
`CCATCCAGGGGGCTCCCGGGTCATCAGCCACTACCCCCCGCAGGATGCCACCGTGAGCGC
`60270
`60320
`60280
`60290
`60300
`60310
`
`90
`99
`109
`19
`29
`139
`49
`CGTCCCCTTCCACATCAACAAGGGCCTTGTGAAGAAGTATATGAACTCTCTCCTGATTGG
`AGCCAGGCCGGGGCACAGGAGAGGGCGGGACCGGAGGCTCACTCCAGGGGAGACACAGTT
`60550
`60540
`60350
`60360
`60370
`60380
`
`FIG.21
`
`Page 20 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 20 of 39 US 2003/0104596A1
`
`3-5
`
`Oc004228
`
`3-5
`
`OCOO4228
`
`3-5
`
`OcC04228
`
`3-5
`
`Oc004228
`
`3-5
`
`OcOO4228
`
`3-5
`
`oc004228
`
`70
`60
`50
`40
`30
`20
`AATACGACT CACTATAGGCCTCGAGCGGCCCCCCGGGCACGTCCCGACCTGCCAACGTGA
`|
`|
`|
`| | || || || ||
`CCCCCCCCCACACCCCGCACACTTACAGGGCCCGGGGCTG-CCGGACCTGCCAACGTGA
`6174O
`61750
`61760
`61710
`61720
`6 1730
`
`130
`120
`10
`100
`90
`80
`ATCTTATCCCCATGGACCTTACCTTGCACAACCCAAAGTACCTCCCTTGGGGCACCGGGT
`|| || || ||
`||
`|| || || || || || ||
`|| || ||
`ATCTTATCCCCATCGACCTTACCTTGCACAACCCAAAGTAGCTCCCTTGGGGCAGGGGGT
`61770
`61780
`61790
`61800
`61810
`61820
`
`190
`18O
`170
`160
`150
`140
`GGCCAGAGTCCTTAGGGAAATGTGGAGCCCTACCCAGAACAACGGTGGAGGGAAAGGGAA
`|
`|
`|
`|
`|| || || || || ||
`|| || ||
`GGCCAGAGTGCTTAGGGAAATGTGGAGCCCTACCCAGAACAACGGTGGAGGGAAAGGGAA
`61830
`61840
`61850
`6 1860
`6 1870
`61880
`
`250
`240
`230
`220
`210
`200
`GAAACGCAGAAGTGCCCCAGTTCGGACGTAGGGAAGTCTTCCTCTTCGTGGTTTTTGGAG
`|| || || || || ||
`|| || || || || || ||
`|| || ||
`GAAACGCAGAAGTGCCCCAGTTCGGACGTAGGGAAGTCTTCCTCTTCGTGGTTTTTGGAG
`61890
`61900
`61910
`6 1920
`6 1930
`6 1940
`
`310
`300
`290
`280
`270
`260
`AACCCTAGCTAAGAGAGGAAAGGGACTTATTGAAAGACCCGCAAGAAGGGACGGAAGTCT
`|| ||
`|| || ||
`||
`||
`|| ||
`AACCCTAGCTAAGAGAGGAAAGGGACTTATTGAAAGACCCGCAAGAACGGACGGAAGTCT
`61950 -
`6 1960
`6 1970
`61980
`6 1990
`62000
`
`370
`360
`350
`340
`330
`320
`CATAGCCCTGAGAGGATCCCTTTGTGGCCTTCCACATCAACAAGGGCCTTGTGAAGAAGT
`||
`|| ||
`CATAGCCCTGAGAGGTGAAGCCAGCTGGAGTTGATGGGTCGAATCGGGACCTAGAGAACT
`62010
`62O20
`62030
`62040
`62050
`62060
`
`FIG.22
`
`Page 21 of 95
`
`

`

`Patent Application Publication
`
`Jun. 5, 2003 Sheet 21 of 39 US 2003/0104596 A1
`
`A-10
`
`Oc004228
`
`A-10
`
`oc004228
`
`A-10
`
`Oc004228
`
`A-10
`
`Oc004228
`
`89
`80
`70
`60
`50
`40
`30
`TATAGGGCTCGAGCGGCCGCCCGGGCACGTCCCCGGAGGCGCCTCATCATACCTGTTGCC
`
`CGAGCCAAACACCGACTAATTCGGAGGAAAGCCCC