HEPATITIS C ASSAY UTILIZING RECOMBINANT ANTIGENS TO NS1

DEPISTAGE DE L'HEPATITE C A L'AIDE D'ANTIGENES RECOMBINANTS DE LA REGION NS1

English Abstract

2115926 9304088 PCTABS00020
The present invention provides unique recombinant antigens
representing distinct antigenic regions of the NS1 region of the HCV
genome which can be used as reagents for the detection of
antibodies and antigen in body fluids from individuals exposed to
hepatitis C virus (HCV). The present invention also provides an assay for
detecting the presence of an antibody to an HCV antigen in a
sample by contacting the sample with the recombinant antigens.
Preferred assay formats include a screening assay, a confirmatory
assay, a competition or neutralization assay and an immunodot assay.

Claims

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122
CLAIMS
1. A recombinant fusion protein SEQ. ID. NO. 1.
2. A recombinant fusion protein SEQ. ID. NO. 2.
3. A recombinant fusion protein SEQ. ID. NO. 3.
4. A recombinant fusion protein SEQ. ID. NO. 4.
5. A recombinant fusion protein SEQ. ID. NO. 5.
6. A polypeptide SEQ. ID. NO. 1.
7. A polypeptide SEQ. ID. NO. 2.
8. A polypeptide SEQ. ID. NO. 3.
9. A polypeptide SEQ. ID. NO. 4.
10. A polypeptide SEQ. ID. NO. 5.

11. An assay for identifying the presence of an antibody immunologically
reactive with an HCV antigen in a fluid sample comprising:
contacting the sample with at least one polypeptide selected from the group
consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID.
NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2,
SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5 under conditions suitable for
complexing the antibody with the polypeptide; and detecting the antibody-
polypeptide complex.

12. In a confirmatory assay for identifying the presence of an antibody in
a fluid sample immunologically reactive with an HCV antigen wherein the sample is
used to prepare first and second immunologically equivalent aliquots and the first
aliquot is contacted with at least one polypeptide selected from the group consisting
of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ.
ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID.
NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5 under conditions suitable for complexing the
antibody with the polypeptide and wherein the first antibody-antigen complex is
detected, and:
contacting the second aliquot with a polypeptide selected from the group
consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID.
NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2,
SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5 under conditions suitable to form a
second antibody-antigen complex; and detecting the second antibody-antigen
complex; wherein the polypeptide selected in the first aliquot is not the same as the
polypeptide selected in the second aliquot.


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123
13. In an immunodot assay for identifying the presence of an antibody
immunologically reactive with an HCV antigen in a fluid sample wherein the sample
is concurrently contacted with at least two polypeptides separately bound to distinct
regions of the solid support, each containing distinct epitopes of an HCV antigen
under conditions suitable for complexing the antibody with the polypeptide; and
detecting the antibody-polypeptide complex, and
wherein said polypeptides are selected from the group consisting of
recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID.
NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3,
SEQ. ID. NO. 4, SEQ. ID. NO. 5.
14. In a competition assay for identifying the presence of an antibody
immunologically reactive with an HCV antigen in a fluid sample wherein the sample
is used to prepare first and second immunologically equivalent aliquots wherein the
first aliquot is contacted with a polypeptide bound to a solid support under
conditions suitable for complexing the antibody with the polypeptide to form a
detectable antibody-polypeptide complex, and wherein the second aliquot is firstcontacted with unbound polypeptide and then contacted with said bound polypeptide
wherein the polypeptide is selected from the group consisting of recombinant fusion
proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO.
5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4,
SEQ. ID. NO. 5.
15. In a competition assay for identifying the presence of an antibody
immunologically reactive with an HCV antigen in a fluid sample wherein the sample
is used to prepare first and second immunologically equivalent aliquots wherein the
first aliquot is contacted with a polypeptide bound to a solid support under
conditions suitable for complexing the antibody with the polypeptide to form a
detectable antibody-polypeptide complex and wherein the second aliquot is first
contacted with unbound polypeptide and then contacted with said bound polypeptide
wherein the polypeptide is selected from the group consisting of recombinant fusion
proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO.
5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4,
SEQ. ID. NO. 5; wherein the second aliquot is contacted with unbound and bound
polypeptide simultaneously.
16. In a neutralization assay for identifying the presence of an antibody
immunologically reactive with an HCV antigen in a fluid sample wherein the sample
is used to prepare first and second immunologically equivalent aliquots wherein the

WO 93/04088 PCT/US92/07188

124
first aliquot is contacted with a polypeptide bound to a solid support under
conditions suitable for complexing the antibody with the polypeptide to form a
detectable antibody-polypeptide complex wherein the bound polypeptide is selected
from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ.ID.NO.2,SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ.
SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 6;
and wherein the second aliquot is first contacted with unbound polypeptide
and then contacted with said bound polypeptide wherein the unbound polypeptide is
selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1,
SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ.
ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3,SEQ. ID. NO.4, SEQ. ID. NO. 5 and wherein
the bound polypeptide selected is not the same as the same as the unbound
polypeptide selected.
17. In a neutralization assay for identifying the presence of an antibody
immunologically reactive with an HCV antigen in a fluid sample wherein the sample
is used to prepare first and second immunologically equivalent aliquots wherein the
first aliquot is contacted with a polypeptide bound to a solid support under
conditions suitable for complexing the antibody with the polypeptide to form a
detectable antibody-polypeptide complex wherein the bound polypeptide is selected
from the group consisting of recombinant fusion proteins SEQ. ID. NO.1, SEQ. ID.NO. 2 ,SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO.5, and polypeptides SEQ. ID. NO.1,
SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO.5;
and wherein the second aliquot is first contacted with unbound polypeptide
and then contacted with said bound polypeptide wherein the unbound polypeptide is
selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1,
SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ.
ID. NO. 1, SEQ. ID. NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID. NO.5;
and wherein the bound polypeptide selected is not the same as the unbound
polypeptide selected;
and wherein the second aliquot is contacted with unbound and bound
polypeptide simultaneously.
18. An immunoassay kit comprising:
a polypeptide containing at least one HCV antigen selected from the group
consisting of recombinant fusion proteins SEQ. ID NO. 1, SEQ. ID. NO. 2, SEQ. ID.
NO. 3, SEO. ID. NO. 4, SEO. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEO. ID. NO. 2,
SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5;
one or more sample preparation reagents;

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125
and one or more detection and signal producing reagents.
19. A kit of claim 18 wherein the polypeptides are bound to a solid
support.

Description

i WO 93/04088 . . n ~ PCl`/US92/07188
.~ L 1 c~ cJ ;~ ~)




HEPATI~IS C ASSAY UTILZING RECOMBINANT ANTIGENS TO NS1

This is a cont~nualion-~n-part application of U.S. Ser~al No. 07/572,822,
filed Augusl 24, 1990 and U.S. Ser~al No. 07,614,069, filed November 7, 1990,
5 which enpy common ownership and are incorporated herein by reference. This
awlicalion also is related to co-filed pa1ent applicationS entitled ~HEPATITIS CAæAY UTIUZING RECOMBINANT ANTIGENS FROM NS5 REGION~(U. S. Serial No.
748,565) and ~HEPATITIS C ASSAY UTILIZING RECOMBINANT ANTIGENS TO C-100
REGION~(U. S. Serial No. 748,566) which enpy common ownership and are
10 incorporated herein by reference.
This inventbn relates generaUy to an assay for identilying the presence in a
sample of an antibody which is immunologically reactive with a hepatitis C virusantigen and specifically to an assay for ~electing a complex of an an~ibody and
recombinant anti~ens representing distinct regions of the HCV genome. Recombinant
15 antigens derived from the molecular cloning and expression in a heterologous
expressbn system of the synthstic DNA sequences representing dist~nct antigenic
regiions of the HCV g~nome can be used as reagents for the detection of an1ibodies and
antben in body fluWs from individuals exposed to hepa1itls C virus (HCV).

2 0 ~4CKGK)UNDOFlHE INVENT~J
Acu1e viral hepatitis is clinically diagnosed by a well-defined set of patient
symptoms, including jaundice, hepatic 1endemess, and an incr~ase in the serum
levels of alanine aminotransferase (ALT) and aspartate aminotransferase.
Additional serologic immunoassays are generally performed to diagnose the specific
25 type of viral causative agent. Historically, patients presenting clinical hepatitis
symptoms and not otherwise infected by hepatitis A, hepati~is B, Epstein-Barr orcytomegalovirus were clinically diagnosed as having non-A non-B hepatitis
(NANBH) by default. The disease may result in chronic liver damage.
Each of the well-known, immunologica!ly characterized hepatitis-inducing
30 viruses, hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis D virus
(HDV) belongs to a separate family of viruses and has a distinctive viral
organization, protein structure, and mode of replication.
Attempts to identify the NANBH virus by virtue of genomic similarityl to one
of the known hepatitis viruses have failed, suggesting that NANBH has a distinctorganization and structure. ~Fowler, ~L~L.. J- Med. Virol., 12:205-213 (1983)
and Weiner, ~L~L.~I Med. Virol., 21:239-247 (1987)].
Progress in developing assays to detect anlibodies specific for NANBH has

. ~:

WO 93~041N8 PCI~/US92/07188


be~n panicularly hamp~r~d by difficultles in corr~clly Identtfying antlg~ns
associated wi1h NANBH. See, for example, Wands, J., ~L~.. U.S. Patent 4,870,076,Wands, et al.. Proc. Nat~l. Acad. Sci., 83:6608-6612 (1986), Ohori, ~L, J. Med.
Virol., 12:161-178 (1983), Bradley, ~, Proc. Nat'l. Acad. Sci., 84:6277-
6281, (1987), Akalsuka, T., ~LaL.J- Med.~ , 20:43-56 (1986), Seto, B-, Qt
~, U.S. Patent Application Number 07/234,641 (available from U.S. Department
of Commerce National Technical Information Service, Springfield, Virginia, No.
89138168), Takahashi, K., et al., European Patent Application No. 0 293 274,
published November 30, 1988, and Seelig, R., ~LaL. in PCT Application
1 0 PCT/EP88/00123.
Recently, another hepatitis-inducing virus has been unequivocally identified
as hepatilis C virus (HCV) by Houghton, M., ~Lal.. European Patent Application
publication number 0 318 216, May ~, 1989. Related papers describing this
virus include Kuo, G., ~.LaL, ~i~n~, 244:359-361 (1989) and Choo, Q., ~
1 5 ~icience, 244:362-364 (1989). Houghton, M., ~ reported isolating cDNA
sequences from HCV which encode an1igens which react immunologically with
antibodies present in patients infected with NANBH, thus establishing that HCV is
one ot the viral a~ents causing NANBH. The cDNA sequenc~s associatcd wi~h HCV
were isolated from a cDNA library prepared from the RNA obtained from pooled
2 0 se-um from a chimpanzee wilh chronic HCV infection. The cDNA library contained
cDNA sequences of approximate mean size of about 200 base pairs. The cDNA
library was screened for encoded epitopes expressed in clones that could bind toantibodies in sera from patients who had previously experienced NANBH.
In the European Patent Application, Houghton, M., i~LaL also described the
2 5 preparation of several superoxide dismutase fusion polypeptides ~SOD) and lhe use
of these SOD fusion polypeptides to develop an ~CV screening assay. The most
complex SOû fusion polypeptide described in the European Patent Application,
designated c100-3, was described as containing 154 amino acids of human SOD at
lhe aminoterminus, 5 amino acid residues derived from the expression of a
3 0 synthetic DNA adapter containing a restriction site, EcoRI, 363 amino acids derived
from 1he expression of a cloned HCV cDNA fragment, and 5 carboxyl terminal aminoacids derived from an MS2 cloning vector nucleotide sequence. The DNA sequence
encoding this polypeptide was transformed into yeast cells using a plasmid. The
transformed cells were cultured and expressed a 54,000 molecular weight
3~ polypeptide which was purified to about 80% purity by differential extraction.
Olher SOD fusion polypeptides designated SOD-NANBs 1 1 and SOD-
NANB81 we(e expressed in recombinant bacteria. The .~!i fusion polypeptides

' WO93/04088 .~ ) PCr/US92/07188


were puritied by differential extraction and by chromatography using anion and
catbn exchange columns. The purification proc~ures were able to produce SOD-
NANBs 1 1 as about 80% pure and SO~-NAN38, as about 50% pure.
The recombinant SOD fusion polypeptides described by Houghton, M., ~,
5 were coated on microtiter wells or polystyrene beads and used to assay serum
samples. Briefly, coated microtiter wells were incubated with a sample in a
diluent. Atter incubation, the microtiter wells were washed and then developed
using either a radioactively labelled sheep anti-human anti~ody or a mouse
antihuman IgG-HRP (horseradish peroxidase) conjugate. These assays were used to
1 0 detect both post acute phase and chronic phase HCV infection.
Due to the preparative methods, assay specificity required adding yeast or
i extracts to the samples in order to prevent undesired immunological
reactions with any yeast or ~,~ antibQdies present in samples.
Ortho Diagnostic Systems Inc. have developed a immunoenzyme assay to
1~ detect antibodies to HCV antigens. The Ortho assay procedure is a three-stage test
for serum/plasma carried out in a microwell coated with the recombinant
yeasVhepalitis C virus SOD fusion polypeptide c100-3.
In the first stage, a test specimen is diluted directly in the test well and
inaJbated for a specffled length of time. If antibodies to HCV antigens are present in
2 0 the speamen, antigen-antibody complexes will be f~rmed on the microwell surface.
If no antibodies are present, complexes will not be formed and the unbound serumor plasma proteins will be removed in a washing s1ep.
In the second stage, anti-human IgG murine monoclonal antibody horseradish
peroxidase conjugate is added to the microwell. The conjugale binds specifically to
25 the antibody portion of the antigen-antibody complexes. If antigen-antibody
oomplexes are not present, the unbound conjugate will also be removed by a washing
step.
In the third stage, an enzyme detection system composed of o-
pheny!enediamine 2HCI (OPD) and hydrogen peroxide is added to the test well. If
3 0 bound conjugate is present, the OPD will be oxidized, resultin~ in a colored end
product. After formation of the colored end product, dilute sulfuric acid is added to
the microwell to stop the color-forming detection reactîon.
The intensity of lhe colored end product is measured with a microwell
reader. The assay may be used to screen patient serum and plasma.
3 ~ It is established that HCV may be transmitted by contaminated blood andblood products. în transfused patients, as many as 10% will suffer from post-
transfusion hepatitis. Of these, approximately 90% are the result of infections

WO 93/04088 ,~ ~ ~ J j ~ ~ PCl~/US92/07188 ~




diagnosed as HCV. The prevention of 1ransmission of HCV by bbod and bbod
products requires reliable, sensitive and specific d~agnosis and prognostic tools to
identify HCV carriers as well as oontaminated blood and bbod products. Thus, there
exists a need for an HCV assay which uses reliable and efficient reagents and
methods to accura1ely detecl the presence of HCV antibod~es ~n samples.

SU ULUMY OF THE lNv~moN
The present invention provides an improved assay for detecting the presen¢e
of an anIibody to an HCV antben in a sample by contacting the sample with at least
1 0 one recombinant protein representing a distinct antigenic region of the HCV genome.
Recombinant an1igens which are derived from the molecular cloning and
expression of synthetic DNA sequences in helerologous hosts are provided. Briefly,
synthetic DNA sequences which encode the desired proteins representing distinct
antigenic regions of the HCV genome are optimked for expression in ~QIi by
1 5 specific codon selection. Specifically, recombinant proteins representing tive
distinct antigenic regions of NS1 of the HCV genome are described. The proteins are
express~d as chimeric tusbns with .QQli CMP-K~O synthetase (CKS) gene. The
flrst protein, expressed by plasm~d pHCV-77 (ident~f~ed as SEQ. ID. NO. 1)
represents am~no acids 365-579 of the HCV sequence of NS1 and, based on analogy
2 0 to the genomic or~anization of other fbviv~ruses, has been named HCV CKS-NS1S1.
Note that the tenn pHCV-77 will also refer to the fusion protein itself and thatpHCV-77' will be the designation for a polypeptWe represent~ng the NS1 region
-~ from about am~no ac~ds 365-579 of the HCV sequence prepared using other
recombinant or synthetic methodologies. Other recombinant methodologies would
indude the preparation of pHCV-77~, u~ilking different expression systems. The
methodology for the preparation of synthetic peptides of HCV is described in U.S.
Serial No. 456,162, filed December 22, 1989, and U.S. Serial No. 610,180, tiled
November 7, 1990, which enjoy common ownership and are incorporated herein by
reference. The next protein is expressed by plasmid pHCV-65, identified as SEQ.
3 0 ID. NO. 2, and represents ar,1ino acids 565-731 of the NS1 region of the HCV
genome, pHCV-65 has been named HCV CKS-NS1S2 and is expressed by the plasmi
pHCV-65. The fusion protein itself will also be referred to as pHCV-6S and pHCV-65~ shall be the designation for a polypeptide from the NS-1 region representingfrom about amino acids 565-731 of the HCV sequence prepared using other
3 5 recombinant or synthetic methodologies. The next recombinant antigen represents
amino acids 717-847 ot the NS1 region of the HCV sequence, and is expressed by
the plasmid pHCV-78 (identified by SEQ. ID. NO. 3). The fusion protein will be

WO 93/040~ PCI`/US92/07188




referred lo as pHCV-78 and pHCV-78' shall be the designation for a polypeptide
from the NS1 regbn representing from about amino acids 717-847 of the HCV
sequence prepared using other reoombinant or synthetb methodobgies. It has been
designated cbne HCV CKS-NS1S3 based on the strategy used in its construction.
Figure 44 illustrates the position of pHCV-77, pHCV-65 and pHCV-78 in the NS1
re~ion of the HCV genome. The recombinant antigen produced by pHCV-80is
identified as SEQ. ID.NO. 4 and ~s desbnated HCV CKS-NS1S1 -NS1 S2. The fusion
protein is also designated by pHCV-80 and pHCV-80' refers to the polypeptide
bcaled in 1he NS1 region of HCV, represen1ing amino acids 365-731 of 1he HCV
genome prepared using different recombinant methodologies. Figure 45 illustra1esthe posilion of pHCV-80 within the HCV genome. HCV CKS-Full Length NS1is 1he
designation for the recombinant pro1ein pHCV-92 (SEQ.ID. NO. 5). It represents
amino acids 365-847 of the HCV genome. The fusion proteins will be referred to as
pHCV-92 and pHCV 92' shall be the designation tor the polypeptide from the NS1
1 5 region representing amino acWs 365-847 of the HCV sequence prepared using
o1her recombinan1 or syn1hetic me1hodohgies. Figure 46 illustrates the position of
pHCV-92 in the HCV genome. These antigens are used in the inventive
immunoassays 10 de1ect 1he presencc ot HCV an1ibodies in samples.
One assay format acoording 10 the invention provides a screening assay for
2 0 iden1ifying 1he presence of an an1ibody 1ha1 is immunologically reactive wi1h an HCV
antigen. Briefly, a fluid sample is incubated with a solid support containing the
oommonly bound recombinant proteins. Finally, the antibody-antigen complex is
detected. In a modification of 1he screening assay 1he solid support additionally
oon1ains recombinant polypeptide c100-3.
Ano1her assay format provides a confirmatory assay for unequivocally
idenlifying lhe presence of an antibody that is immunologically reactive with an HCV
antigen. The confirmatory assay includes synthetic peptides or recombinant
antigens representing the epitopes contained within the NS1 region of the HCV
genome, which are the same regions represented by the recombinant proteins
3 0 described in the screening assay. These are pHCV-77, pHCV-65, pHCV-78, pHCV-
80 and pHCV-92. Recombinant proteins used in the confirmatory assay should have
a heterologous source of antigen to that used in the primary screening assay (i.e.
should not be an E~Qli-derived recombinant antigen nor a recombinant antigen
composed in part, of CKS sequences). Briefly, specimens repeatedly reactive in lhe
3 5 primary screening assay are relested in lhe confirmalory assay. Aliquols
containing identical amounts of specimen are contacted with a synthetic peptide or
recombinant antigen individually coated onto a solid support. Finally, the antibody-

W0 93/04088 W i L J ~ ~ PCl~/US92/07188

anligen complex is detected. The polypeptides Ot recombinant proteins can be
utilized as indicated or combined with other po~peptides and recombinant proteins
a described herein and also described ~n U.S. Serial No. 456,162 entitled "Hepatitis
C Assay", filed December 22, 1989, which enpys common ownership and is
5 incorporated herein by reference.
Another assay format provides a competition assay or neutralization assay
directed to the confirmation ~hat positive results are not false by identifying the
presenc~ of an antibody that is immunobgically reactive with an HCV anligen in aHuid sample where the sample is used to prepare first and second immunolo~ically10 equivalent aliquots. The first aliquot is contacted with solid support containing a
bound polypeptide which contains at least one epitope of an HCV antigen under
conditions suitable for complexing with the antibody to form a detectable antibody-
polypeptide complex and the second aliguot is first contacted with the same solid
support containing bound polypeptide. The preferred recombinant polypeptides
15 include pHCV-77, pHCV-65, pHCV-78, pHCV-80 and pHCV-92.
Another assay format provWes an immunodot assay for Wentifying the
presence of an aniibody that is immunologically reactive with an HCV antigen by
concurrently contacting a sample with recombinant polypeptides each containing
distinct epitopes of an HCV antigen under conditions suitable for complexing the2 0 antibody with at least one of the polypeptWes and detecting the antibodypo~peptide
complex by reacting the complex with cobrproducing reagents. The preferred
recombinant polypeptides employed include those recombinant polypeptides derivedfrom pHCV-77,pHCV-66, pHCV-78, pHCV-80, as well as pHCV-92.
In all of the assays, the sample is preferably diluted before contacting the
2 5 polypeptide absorbed on a solid support. Samples may be ob~ained from different
biological samples such as whole blood, serum, plasma, cerebral spinal fluid, and
Iymphocyte or cell culture supernatants. .Solid support materials may include
cellulose materials, such as paper and nnrocellulose, natural and synthetic
polymeric materials, such as polyacrylamide, poiystyrene, and cotton, porous gels
3 0 such as silica gel, agarose, dextran and gelatin, and inorganic materials such as
deactivated alumina, magnesium sulfate and glass. Suitable solid support materials
may be used in assays in a variety of well known physical configurations, including
microtiter wells, test tubes, beads, strips, membranes, and microparticles. A
preferred solid support for a non-immunodot assay is a polystyrene bead. A
3 5 preferred solid support for an immunodot assay is nitrocellubse.
Suitable methods and reagents for detecting an antibody-antigen complex in
- an assay of the present invention are commercially available or known in the
,,

WOg3/04088 ~ i') PCl'/US92/07188


relevant art. Represenlative methods may empby delection reagents such as
enzymatic, radbisotopic, fluorescent, lumineæent, or chemiluminescent rea~ents.
These re~ents may be used to prepare hapten-labelled antihapten detectbn systems acoordin~ to known procedures, for example, a biotin-labelled antibiotin system
may be used to detect an antibody-antben com~ex.
The present ~nvention also encompasses assay kits including polypept~s
whbh oontain at bast one epitope of an HCV antigen bound to a solid support as well
as needed sample preparatbn rea~ents, wash reagents, detection reagents and signal
producing reagents.
1 0 Other aspects and advantages of the invention will be apparent to those
skilled in the art upon consideration of the folbwing detailed description whichprovides illustrations o~ the invention in its presently preferred embodiments.
i strains containing plasmids useful for construcls of the invention have
been deposited at the American Type Culture Collection, Rockville, Maryland on
Au~ust 10, 1990, under the a~ssion Nbs. ATCC 68380(pHCV-23),ATCC68381
(pHCV-29), ATCC 68382(pHCV-31),ATCC 68383(pHCV-34) and on November
` 6,1990for~,~ strains conlaining ~lasmWs useful for constructs under the
- a~sshn Nos. ATCC 684~ (pHCV-~),ATCC68459(pHCV-57), ATCC 6~60
(pHCV-103), ATCC 68461 (pHCV-102),ATCC 68462(pHCV-51),ATCC 68463
(pHCV-105),ATCC 68464(pHCV-107),ATCC 68465(pHCV-104),ATCC 6~66
(pHCV-45),ATCC 6~67(pHCV~8),ATCC 68468(pHCV-49), ATCC 6~69
(pHCV-58) and ATCC 68470(pHCV-101). E. coli strains ~ntaining plasmids
uselul for constructs of lhe invention have been deposited at the A.T.C.C. on
September 26,1991 under deposit num~rs ATCC 68690(pHCV-77),ATCC
68696(pHCV-65), ATCC 68689(pHCV-78),ATCG 68688~pHCV-80) and ATCC
68695 (pHCV-923.

BREFDESCRlPTlONOFTHEDRAWlNq~
FIGURE 1 illustrates lhe HCV genome.
3 0 FIGURE 2 illustrates the use of recombinant polypeptides to identify the
presence of antibodies in a chimpanzee inoculated with HCV.
FIGURE 3 illustrates the sensitivity and specificity increase in using the
screening assay using pHCV-34 and pHCV-31 antigens.
FIGURE 4 illustrates the construction of plasmid pHCV-34.
FIGURE 5 illustrates fusion protein pHCV-34.
FIGURE 6 illustrates the expression of pHCV-34 proteins in
- FIGURE 7 illustrates the construction of plasmid pHCV-23.


"

WO93/04088 ~ PCr/US92/07188 ;~. 3

FIGURE 8 iilustrates the construction of plasmid pHCV-29.
FIGURE 9 il~ustrates ~he construction of plasmW pHCV-31.
FIGURE 10 illustrat~s the fusion protein pHCV-31.
FIGURE 11 illustrates the expression of pHCV-29 in ~j.
FIGURE 12 illustrates the expression of pHCV-23 in ,~.
FIGURE 13 illustrates the expression of pl~CV-31 in .~-
FIGURE 14 illustrates the increased sensitivity usin~ the sueening assay
utilizing the pHCV-34.
FIGURE 15 illustrates the increased specificity with the screening assay
10 utilizing pHCV-34 and pHCV-31.
FIGURE 16 illustrates the results in hemodialysis patien~s usin~ the
screening and confirmatory assays.
FIGURE 17 illustrates earlier detection of HCV in a hemodialysis patient
using the screening assay.
FIGURE 18 illustrates the results of the screening assay utilizing pH(::V-34
and ptlCV-31 on samples from individuals with acute NANBH.
FIGURE 19 illustrates the results of the confirmatory assay of the same
population group as in Figure 18.
FIGURE 20 illustrates the results of the screening and confirmatory assays
2 0 on individuals infected with chronic NANBH.
FIGURE 21 illustrat~s preferred buffers, pH conditions, and spolting
concentrations for the HCV immunodot assay.
FiGURE 22 illuslrates the results of the H{:V immunodot assay.
FIGURE 23 illustrates 1he fusion protein pHCV-4~.
FIGURE 24 illustrates the expression of pHCV-46 in ~.
FIGURE 25 illustrates the fusion protein pHCV-48.
FIGURE 26 illustrates the expression of pHCV-48 in ~Qli
FIGURE 27 iliustrates the fusion protein pHCV-51.
FIGURE 28 illustrates lhe expression of pHCV-51 in E.co!i.
3 0 FiGURE 29 illustrates the fusion protein pHCV-50.
FIGURE 30 illustrales the expression of pHCV-50 in ~QIi-
FIGURE 31 illustrates the fusion protein pHCV-49.
FIGURE 32 illustrates the expression of pHCV-49 in ~li
FIGURE 33 illustrates an immunoblot of pHCV-23, pHCV-45, pHCV-48,
pHCV-51, pHCV-50 and pHCV-49.
FIGURE 34 illustrates the fusion proteins pHCV-24, pHCV-57, pHCV-58.
FIGURE 35 illustrates the expression of pHCV-24, pHCV-57, and pHCV-58

W0 93/04088 ~ PCI /US92/07188


in ~
FIGURE 36 illustrates the fusion protein pHCV-105.
FIGURE 37 illustrates the expression of pHCV-105 in E,~
FIGURE 38 illustrates the fusion protein pHCV-103.
FIGURE 39 ~llustrates the fusion protein pHCV-101.
FIGURE 40 illustrates the fusion protein pHCV-102.
FIGURE 41 illustrates the expression of pHCV-102 in ~.
FIGURE 42 illus~rates the fusion protein pHCV-107.
FIGURE 43 illustrates the fusion protein pHCV-104.
1 0 FIGURE 44 illustrates the NS1 region of the HCV genome, and in particular,
the locations of pHCV-77, pHCV-65 and pHCV-78.
FIGURE 45 illustrates the NS1 region of the HCV genome, and in particular,
the location of pHCV-80.
FIGURE 46 illustrates the NS1 region of the HCV genome, and in particlar,
1 5 the location of pHCV-92.
FIGURE 47A ilustrates the expression of pHCV-77 in ., ~oli; and FIGURE
47B illustrates an immunblot of pHCV-77 in E. coli.
Fl¢URE 48A illus1rates the expression of pHCV-65 in E. ~oli and FIGURE
48B illustrates an immunoblot of pHCV-65 in ~
2.0 FIGURE 49A illustrates the expression of pHCV-80 in E. coli and FIGURE 49B illustrates an immunoblot of pHCV-80 in ~.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to an assay to detect an antibody to an HCV
2 5 antigen in a sample. Human serum or plasma is preferably diluted in a sample
diluent and incubated with a polystyrene bead coated with a recombinant polypeptide
1hat represents a distinct antigenic region of the HCV g~nome. If antibodies arepresent in the sampie they will form a complex with tl e antigenic polypeptide and
beoorne affixed to the polystyrene bead. After the complex has formed, unbound
3 0 materials and reagents are removed by washing the bead and the bead-antigeh-
antibody complex is reacted with a solution containing horseradish peroxidase
labeled goat antibodies directed against human antibodies. This pe~oxidase enzyme
then binds to the antigen-antibody complex already fixed to the bead. In a finalreaction the horseradish peroxidase is contacted with o-phenylenediamine and
hydrogen peroxide which results in a yellow-orange color. The intensity of the
color is proportional to the amount of antibody which initially binds to the antigen
fixed to the bead.

WO 93/04088 ~ 1 ~ J ~ ~ ~ PCr/US92/07188

1 0

The prefarred recombinant polypeptides having HCV antigenic epitopes were
selected from portions of the HCV genome which encoded polypeptides which
possessed amino acid sequences similar to other known immunologically reactive
agents and which were identified as having some immunohgbal reactivity. (The
immunological reactivity of a polypeptide was initially identified by reacting the
cellular extract of E.ÇQIi clones which had been transformed with cDNA fragments of
the HCV genome with HCV infected serum. Polypeptides expressed by clone
containing the incorporated cDNA were immunologically reactive with serum known
to contain antibody to HCV antigens.) An analysis of a given amino acid sequence,
however, only provides rough guides to predicting immunological reactivity. There
is no invariably predictable way to ensure immunological activity short of
preparing a given amino acid sequence and testing the suspected sequence in an
assay.
The use of recombinant polypeptides representing distinct antigenic regions
1 5 of the HCV genome to detect the presence of an anbbody to an HCV antigen isillustrated in Figure 2. The course of HCV Infection in the chimpanzee, Pan, wasfollowed with one assay using recombinant c100-3 polypeptide and with another
improved assay, using the hNo recombinant antigens CKS-Core (pHCV-34)
(SEQ.ID.N0 6 and 7) and pHCV-33c-BCD (pHCV-31) (SEa.lD.NO 8 and 9)
- 2 0 expressed by the plasmids pHCV-34 and pHCV-31, respectively. The assay
utilizing the recombinant pHCV-34 and pHCV-31 proteins detected plasma antibody
three weeks prior to detection of anbbody by the assay using c100-3.
A summaly of the results of a study which followed the course of HCV
infection in Pan and six other chimpanzees using the two assays described above is
2 5 summarized in Figure 3. Both assays gave negative results before inoculation and
both assays detected the presence of antibodies after the animal had been infected
with HCV. However, in the comparison of the two assays, the improved screening
assay using pHCV-34 and pHCV-31 detected seroconversion to HCV antigens at an
earlier or equivalent bleed date in six of the seven chimpanzees. Data from these
3 0 chimpanzee studies clearly demonstr~ate that overall detection of HCV antibodies is
greatly increased with the assay utilizing the pHCV-34 and pHCV-31 proteins.
This test is sufficiently sensitive to detect seroconversion during the acute phase of
this disease, as defined as an elevation in ALT levels, in most animals. Equallyimportant is the high degree of specificity of the test as no pre-inoculation
- ~ 35 specimens were reactive.
The polypeptides useful in the practice of this invention are produced using
recombinant technologies. The DNA sequences which encode the desired polypeptides

~ .
. , .

' ~ WO g3/04088 .~, 1 L 3 J .~ ~ PCI/US92/07188
1 1
are preferably assembled from fragments of 1he total desired sequenee. ~ynthQtiCDNA fragments of Ihe HCV ~enome can be synthesked based on their eorresponding
amino acid sequences. Once the amino aeid sequence is chosen, this is then reverse
translated to determi!le the complementary DNA sequence using eodons optimized to
5 faeilitate expression in the ehosen system. The fragmenls are generally prepared
using well known automated processes and apparatus. After the complete sequence
has been prepared the desired sequenee is incorporated into an expression vectorwhieh is transformed into a host eell. The DNA sequenee is then expressed by thehost cell to give the desired polypeptide which is harvested from the host cell or
10 from the medium in which the host cell is cultured. When smaller peptides are to
be made using recombinant technologies it may be advantageous to prepare a single
DNA sequence which encodes several copies of the desired polypeptide in a connected
chain. The long chain is then isolated and lhe chain is cleaved into the shorter,
desired sequenoes.
The methodology of polymerase ehain reaction (PCR) may also be employed
to devebp PCR amplified genes from any portion of the HCV genome, which in turn
may then be ehned and expressed in a manner similar to the synthetie genes.
Vector systems whieh ean be used inelude plant, baeterial, yeast, inseet, and
mammalian expression systems. It is preferred that the codons are optimized for
2 0 expression in the system used.
A preferred expression system utilizes a carrier gene for a fusion system
where the reeombinant HCV proteins are expressed as a fusion protein of an .~1ienzyme, CKS (CTP:CMP-3-deoxy-m~nQ-oetulosonate cytidylyl transferase or
CMP-KDO synthetase). The CKS method of protein synthesis is disclosed in U.S.
Patent Applieations Serial Nos. 167,067 and 276,263 filed March 11, 1988 and
November 23, 1988, respectively, by Bolling (EPO 891029282) which enjoy
common ownership and are incorporated herein by reference.
Other expression systems may be utilized including the lambda PL vector
system whose features include a strong lambda pL promoter, a strong three-frame
3 0 translation terminator rrnBtl, and translation starting at an ATG codon.
In the present invention, the amino acid sequences encoding for the
recombinant HCV antigens of interest were reverse translated using codons
optimized to facilitate high level expression in ~ l- Individual oligonucleolides
were synthesized by the method of oligonucleotide directed double-stranded breakrepair diselosed in U.S. Patent Application Serial No. 883,242, filed July 8, 1986
by Mandecki (EPO 87109357.1) which enjoys common ownership and is
incorporated herein by reference. Alternatively, the individual oligonucleotides
'

J
WO g3/040~8 PCI~/US92/07188 '~
1 2
may be synlhesked on the Applied Biosystem 380A DNA synthesizer using methods
and rea~ents reoommended by the manufacturer. The DNA sequences of lhe
individual oli~onucleotid~s were confirmed usin~ the Sanqer dideoxy chain
termination method (Sanger et al., J. Mole. Biol., 162:729 (1982)). These
indivWual gene fragments were Ihen annealed and ligated logether and cloned as
EooRI-BamHI subfragmenls in the CKS fusion vector pJO200. After subsequent
DNA sequence confirmation by the Sanger dideoxy chain termination method, the
subfragments were di~ested with appropriate restriction enzymes, gel purified,
ligated and c~oned again as an EcoRI-BamHI fragment in the CKS fusion vector
pJ0200. The resulting clones were mapped to identify a hybrid gene consisting ofthe EcoRI-BamHI HCV fragment inserted at the 3' end of the CKS (CMP-KD0
synthetase) gene. The resultant fusion proteins, under control of the ~ promoter,
consisl of 239 amino acids of the CKS p~olein fused lo the various re~ions of HCV.
The synthesis, cbning, and characterization of the recombinant polypeptides
as well as the preferred formats for assays using these polypeptides are provided in
the following examples. Examples 1 and 2 describe the synthesis and cloning of
CKS-Core and CKS-33-BCD, respectively. Example 3 describes a screening assay.
Example 4 de~cribe~ a oonfirma~ory assay. Example 5 descrlbes a comp~lltion
assay. Example 6 describes an immunodot assay. Example 7 describes the
2 0 synth~sis ar,~d cbnin~ of HCV CKS-NS5E, CKS-NSSF, CKS-NS5G, CKS-NS5H and
CKSNS51. Example 8 describes the preparation of HCV CKS-C100 vectors.
Example 9 describes the preparation of HCV PCR derived expression vectors.
Example 10 desc.ribes the synthesis and characterization of pHCV-77 of NS1.
Example 1~ describes the synthesis and characterization of pHCV-65 of NS1.
Example 12 describes the synthesis and characterization of pHCV-78 of NS1.
Example 13 describes lhe synlhesis and characterization of pHCV-80 of NS1.
Example 14 describes the synthesis and characterization of pHCV-92 of NS1.

REAGENTS AND ENZrMES
Media such as Luria-Bertani (LB) and Superbroth ll (Dri Form) were
obtained from Gibco Laboratories Life Technologies, Inc., Madison Wisconsin.
Restriction enzymes, Klenow fragment of DNA polymerase 1, T4 DNA ligase, T4
polynucleotide kinase, nucleic acid molecular weight standards, M13 sequencing
system, X-gal (5-bromo-4-chloro-3-indonyl-~-D-galactoside), IPTG
3 5 (isopropyl-~-D-thiogalactoside), glycerol, Dithiothreitol, 4-chloro-1-naphthol
were purchased from Boehringer Mannheim Biochemicals, Indianapolis, Indiana; or
New England Biolabs, Inc., Beverly, Massachusetts; or Bethesda Research

~;W093/04088 ~ 1 ~ J Y i~ ~ PCr/US92/07188
1 3
Laboratorbs Life Technobgies, Inc., Gaithetsburg, Maryland. Prestained protein
molecular weight slandards, acrylamWe (crystallized, electrophoretic grade
~9996); N-N'-Methylene-bis-acrylamide (BIS); N,N,N',N',-
~etramethylethylenediamine (TEMED) and sodium dodecylsulfate (SDS) were
5 purchased from BioRad Laboratories, Richmond, California. Lysozyme andampicillin were obtained from Sigma Chemical Co., St. Louis, Missouri.
Horseradish peroxidase (HRPO) labeled secondary antibodies were obtained from
Kirke~aard 8 Perry Laboratories, Inc., Gaithersburg, Maryland. Seaplaque~
agarose (low melting agarose) was purchased from FMC Bioproducts, Rockland,
1 0 Maine.
TSOE10 contained 50mM Tris, pH 8.0, lOmM EDTA; 1X TG contained 100mM
Tris, pH 7.5 and 10% glycerol; 2X SDSIPAGE loading buffer consisted of 15%
glycerol, 5% SDS, lOOmM Tris base, 1 M B-mercaptoethanol and 0.8%
Bromophenol blue dye; TBS container 50 mM Tris, pH 8.0, and 150 mM sodium
15 chlorWe; Blocking solution consisted of 5% Carnation nonfat dry milk in TBS.

HOSTCQ I CULTUR,S. '~NAS~URCESANDVECTORS
.~QU JM103 cells, pUC8, pUC18, pUC19 and M13 clonin~ v~ctors were
purchased from Pharmacia LKB Biotechnology, Inc., Piscataway, New Jersey;
20 Competent EpicureanrM coli stains XL1-Blue and JM109 were purchased from
Stratagene Cbning Systems, LaJolla, California. RR1 cells were obtained from Coli
Genetic Stock Center, Yale University, New Haven, Connecticut; and ~ i CAG456
cells from Dr. Carol Gross, University of Wisconsin, Madison, Wisconsin. Vector
pRK248.clts was obtained from Dr. Donald R. Helinski, University of California,
25 San Diego, Califomia.

GENEF~I~llETtlODS
All restriction enzyme digestion were performed according to suppliers'
instructions. At least 5 units of enzyme were used per microgram of DNA, and
3 0 sufficient ihcubation was allowed to complete digestion of DNA. Standard procedures
were used for minicell Iysate DNA preparation, phenol-chloroform extraction,
ethanol precipitation of DNA, restriction analysis of DNA on agarose, and low
melting agarose gel purification of DNA fragments ~Maniatis et al., Molecular
Clonina. A Laboratory Manual ~New York: Cold Spring Harbor, 1982]). Plasmid
3 5 isolations from ~ j strains used the alkali Iysis procedure and cesium chloride-
ethidium bromide density gradient method (Maniatis et al., supra). Standard
buffers were used for T4 DNA ligase and T4 polynucleotide kinase (Maniatis et al.,

WO 93/04088 ~ i Pcl/US92/07188'i


supra) .

EXAMPLE 1. CKS CORE
A. Construction of the Plasmid pJ0200
The cloning vector pJO200 allows the fusion of recombinant proteins to the
CKS protein. The plasmid consists of the plasmid pBR322 with a modified lac
promoter fused to a KdsB gene fragment (encoding the first Z39 of the entire 248amino acids of the E~Ç~ CMP-KDO synthetase of CKS protein), and a synthetic
linker fused to the end of the KdsB gene fragment. The cloning vector pJO200 is a
1 0 modification of vector pTB210. The synt~etic linker includes: mu~tiple restriction
sites for insertion of genes; translational stop signals, and the trpA rho-
independent transcriptional terminator. The CKS method of protein synthesis as
well as CKS vectors including pTB210 are disclosed in U.S. Patent Application
Serial Nos. 167,067 and 276,263, filed March 11, 1988 and November 23,
1 5 1988, respectively, by Bolling (EPO 891029282) which enjoy common
ownership, and are herein incorporated by reference.

B. Preparation of HCV CKS-Core ~xpression Vector
Six individual nucleotides representing amino aclds 1-150 of the HCV
O genome were ligated together and cloned as a 4B6 base pair EcoRI-BamHI fragment
into the CKS fusion vector pJO200 as presented in Figure 4. The complete l)NA
sequence of this plasmid, designated pHCV-34, and the entire amino acW sequence of
the pHCV-34 recombinant antigen produced is presented in SEQ.ID.NO 6 and 7. The
resultant fusion protein HCV CKS-Core, consists of 239 arnino acids of CKS, seven
25 amino acids contributed by linker DNA sequences, and the first 15û amino acids of
HCV as illustrated in Figure 5.
The pHCV-34 plasrnid and the CKS plasmid pTB210 were transfonned into
~.coli K-12 strain xL-I (recAI, endAI, gyrA96, thi-1, hsdRI7, supE44, relAI,
lac/F', proAB, laclqZDM15, TN10) cells made competent by the calcium chl~ride
3 0 method. In these constructions the expression of the CKS fusion proteins was under
the control of the iac promoter and was induced by the addition of IPTG. These
plasmids replicated as independent elements, were nonmobilizable and were
maintained at approximately 10-30 copies per cell.

35 C. Characterization of Recombinant HCV-Core
In order to establish that clone pHCV-34 expressed the unique HCV-CKS
Core protein, the pHCV-34/XL-1 culture was grown overnight at 37C in growth

`~WO 93/04088 ~ 1 1 J ~ ~ ~ PCI`/US92/07188
1 5
media consisting of yeast extract, trytone, phosphate salts, glucose, and ampicillin.
When the culture reached an OD600 of 1.0, IPTG was added to a final concentratbnof 1mM to induce oxpressbn. Samples (1.5 ml) were removed at 1 hour intervals,
and cells were pelleted and resuspended to an OD600 of 1.0 in 2X SDS/PAGE loading
5 buffer. Aliquots (15ul) of the prepared samples were separa~ed on duplicate
12.5% SDS/PAGE gels.
One gel was fixed in a solution of 50% methanol and 10% acetic acid for 20
minutes at room temperature, and then stained with 0.25% Coomassie blue dye in asolulion of 50h methanol and 10h acetic acid for 30 minutes. Destaining was
1 0 carried out using a solution of 10% melhanol and 7% acetic acid for 3-4 hours, or
until a clear baclcground was obtained.
~ Figure 6 presents the expression of pHCV-34 proteins in ~QIL Molecular
weight standards were run in Lane M. Lane 1 contains the plasmid pJ0200-the CKS
vector without the HCV sequence. The arrows on the left indicate the mobilities o~
1 5 lhe molecular weighl markers from lop to bottom: 110,000; 84,000; 47,000;
33,000; 24,000; and 16,000 daltons. The arrows on lhe righl indicate lhe
mobilities of lhe recombinanl HCV proteins. ane 2 contains lhe ,~Qli Iysate
oontaining pHCV-34 expressing CKS-Core (amino acids 1 to 150) prior lo
induction; and Lane 3 after 3 hours of induction. The results show 1hat the
2 0 reoombinant prolein pHCV-34 has an apparent mobility correspondin~ lo a
molecular size of 48,000 daltons. This compares acceptably with the predicted
molecular mass of 43,750 daltons.
Proteins from lhe second 12.5% SDS/PAGE gel were eleclrophoretically
lransferred to nitrocellulose for immunoblotting. The nitrocellulose sheet
~ .
25 conlaining lhe lransferred proteins was incubated with Blocking Solution for one
hour and incubated overnight at 4C with HCV patients' sera diluted in TBS
containing E.coli K-12 strain XL-I Iysate. The nitrocellulose sheet was washed
three limes in TBS, then incubated with HRPO-labeled goat anti-human IgG, diluted
in TBS containing 10% fetal calf sera. The nitrocellulose was washed three times3 0 with TBS and the color was developed in TBS containing 2 mglml 4-chloro-1-
napthol, 0.02% hydrogen peroxide and 17% methanol. Clone HCV-34 demonstrated
a strong immunoreactive band at 48,000 daltons with the HCV patients' sera. Thus,
the major protein in the Coomassie stained protein gel was immunoreactive.
Normal human serum did not react with any component of pHCV-34.
3 5
EXAMPLE 2. HCV CKS-33C-BCD
AA Preearation of HCV CKS-33c-BCD Ex~ression Vector



,,

WO 93/0408& f~ 1 ~ PCI /US92/07188


The construction of this recombinant clone expressing the HCV CKS-33-BCD
antigen was carried out in three steps described below. First, a clone expressing
the HCV CKS-BCD antigen was constructed, designated pHCV-23. Second, a clone
expressing the HCV CKS-33 antigen was constructed, designated pHCV-29. Lastly,
the HCV BCD region was excised from pHCV-~3 and inserted into pHCV-29 to
construct a clone expressing the HCV CKS-33-BCD antigen, designated pHCV-31
(SEQ.ID.NO. 8 and 9).
To construct the plasmid pHCV-23, thirteen individual oligonucleotides
representing amino acids 1676-1931 of the HCV genome were ligated together and
cloned as three separate EcoRI-BamHI subfragments into the CKS fusion vector
pJ0200. After subsequent DNA sequence confirmation, the three subfragments,
designated B, C, and D respectively, were digested with the appropriate restriction
enzymes, gel purified, ligated together, a~d cloned as a 781 base pair EcoRI-BamHI
fragment in the CKS fusion vector pJO200, as illustrated in Figure 7. The
resulting plasmid, designated pHCV-23, expresses the HCV CKS-BCD antigen under
control of the lac promoter. The HCV CKS-BCD antigen consists of 239 amino acidsof CKS, seven amino acids contributed by linker DNA sequences, 2~6 amino acids
from the HCV NS4 region (amino acids 1676-1931, and 10 additional amino acids
contributed by linker DNA sequences.
To construct the plasmid pHCV-29 twelve individual oligonucleotides
representing amino acids 1192-1457 of the HCV genome were ligated together and
cbned as two separate EcoRI-BamHI subfragments in the CKS fusion vector
pJ0200. After subsequent DNA sequence cs)nfirrnation, the two subfragments were
digested with the appropriate restriction enzymes, gel purified, ligated together and
cloned again as an 816 base pair EcoRI-BamHI fra~ment in the CKS fusion vector -
pJO200, as illustrated in Figure 8. The resulting plasmid, designated pHCV-29,
expresses the CKS-33 antigen under control of the iac promoter. The HCV CKS-33
antigen consists of 239 arnino acids of CKS, eight amino acids contributed by linker
DNA sequences, and 266 amino acids from the HCV NS3 region (amino acids 1192-
1457~.
To construct the plasmid pHCV-31, the 781 base pair EcoRI-BamHI
fragment from pHCV-23 representing the HCV-BCD region was linker-adapted to
produce a Cla1-BamH1 fragment which was then gel purified and ligated into pHCV-29 at the Cla1-BamH1 sites as illustrated in Figure 9. The resulting plasmid,
3~ designated pHCV-31, expresses the pHCV-31 antigen under control of the lac
promoter. The complete DNA sequence of pHCV-31 and the entire amino acid
sequence of the HCV CKS-33-BCD recombinant antigen produced is presented in

WO g3/04088 ~ 2 ~ PCI`/US92/07188


SEQ.ID.NO. 8 and 9. The HCV CKS-33-BCD antigen consists of 239 amino acids of
CKS, ei~ht amino acids contributed by linker DNA sequences, 266 amino acids of the
HCV NS3 region (amino acids 1192-1457), 2 amino acids contributed by linker
DNA sequences, 256 amino acids of the HCV NS4 region ~amino acids 1676-1931),
and 10 additional amino acids contributed by linker DNA sequences. Figure 12
presents a schematic representation of the pHCV-31 antigen.
The pHCV-31 plasmid was transformed into E~coli K-12 strain XL-I in a
manner similar to the pHCV-34 and CKS-pTB210 plasmids of Example 1.

1 0 B. Characterization of Recombinant HCV CKS-33-BCD
Characterization of pHCV CKS-33-BCD was carried out in a manner similar
to pHCV CKS-Core of Example 1. pHCV-23, pHCV SDS/PAGE gels were run for
E.coli Iysates containing the plasmidspHCV-29 (Figure 11), pHCV-23 (Figure
12), and pHCV-31 (Figure 13) expressing the recombinant fusion proteins CKS-
1 5 33c, CKS-BCD, and CKS-33-BCD, respectively. For all three figures, molecular
weight standards were run in Lane M, with the arrows on the left indicating
mobilities of the molecular weight markers the from top to bottom: 110,000;
84,000; 47,000; 33,000; 24,000; and 16,000 daltons. In Figure 11, Lane 1
contained the ~Q~ Iysate containing pHCV-29 expressing HCV CKS-33c (arnino
acids 1192 to 1457) prior to induction and lane 2 after 4 hours induction. Theseresults show that the recombinant pHCV-29 fusion protein has an apparent
mobility corresponding to a molecular size of 60,000 daltons. This compares
acceptably to the predicted molecular mass of 54,911.
In Figure 12, Lane 1 contained the E.coli Iysate containing pJO200-- the
2 5 CKS vector without the HCV sequence. Lane ~, contained pHCV-20 expressing the
HCV C:KS-B (amino acids 1676 to 1790). Lane 3, contained the fusion protein
pHCV-23 (amino acids 1676-1931). These results show that the recombinant
pl~CV-23 fusion protein has an apparent mobility corresponding to a molecular size
of ~5,000 daltons. This compares acceptably to the predicted molecular mass of
3 0 ~5,070 daltons.
In Figure 13, Lane 1 contained the E.coli Iysate containing pJO200 the CKS
vector without the HCV sequences. Lane 2 contained pHCV-31 expressing the CKS-
33c-BCD fusion protein (amino acids 1192 to 1447 and 1676 to 1931) prior to
induction and lane 3 after 2 hours induction. These results show that the
3~ recombinant pHCV-31 (CKS-33c-BCD) fusion protein has an apparent mobility
corresponding to a molecular size of 90,000 daltons. This compares acceptably tothe predicted molecular mass of 82,995 daltons.

WO 93/04088 ~ PCI`/US92/07188 `
1 8
An immunoblot was also run on one of the SDS/PAGE gels derived from the
pHCV-31~ 1 culture. Human serum from an HCV exposed irldividual reacted
slrongly with lhe major pHCY-31 band at 90,000 daltons. Normal human serum
did not react wi1h any component of the pHCV-31 (CKS-33-BCD) preparations.




E)(AMPLE 3. SCREENING ASSAY
The use of recombinant polypeptides which contain epitopes within cl00-3
as well as epitopes from other antigenic regions from the HCV genome, provide
immunological assays which have increased sensitivity and may be more specific
10 than HCV immunological assays using epilopes within c100-3 alone.
In the presently preferred screenin~ assay, the procedure uses two ~
expressed recombinant proteins, CKS-Core (pHCV-34) and GKS-33-BCD (pHCV-
31), representing three distinct regions of the HCV genome. These recombinant
polypeptides were prepared following procedures described above. In the screening
1 5 assay, both recombinant antigens are coated onto the same polystyrene bead. In a
modification of the screening assay the polystyrene bead may also be coated with the
SOD-fusion polypeptide c100-3.
The polystyrene beads are first washed with distilled waler and propanol and
then incubated with a solution containing recombinant pHCV-31 dillJted to 0.5 to2 0 2.0 ug/ml and pHCV-34 diluted to 0.1 to 0.~ ug/ml in 0.1 M NaH2P04-H20 with
0.4M NaC1 and 0.0022% Triton X-100, pH 6.~. The beads are incubated in the
antigen solution for 2 hours (plus or minus 10 minutes) at 38-42C, washed in
PBS and soaked in 0.1% (w/v) Triton X-100 in PBS ~r 60 minutes at 38-42C.
The beads are then washed two times in phosphat~ buffered saline (PBS), overcoated
25 with a solution of 5.0% (w/v) bovine serum albumin (BSA) in PBS for 60 rr~nules
at 38-42C and washed one time in PBS. Finally, the beads are overcoated with 5%(w/v) sucrose in PBS, and dried under nitrogen or air.
The polystyrene beads coated with pHCV-31 and pHCV-34 are used in an
antibody capture format. Ten microiiters of sample are added to the wells of the3 0 reaction tray along with 400 ul of a sample diluent and lhe recombinant coated bead.
The sample diluent consists of 10% (vtv) bovine serum and 20% (v/v) goat serum
in 20 mM Tris phosphate buffer containing 0.1~% (v/v) Triton X^100, 1%(w/v)
BSA, 1% .s~Qli Iysate and 500 ug/ml or less CKS Iysate. When lhe recombinant
yeast c~00-3 polypeptide is used, antibodies to yeast antigens which may be
3 5 present in a sample are reacted with yeast extracts which are added to the sample
diiuent (typically about 200 ug/ml). The addition of yeast extracts to the sample
diluent is used to prevent false positive results. The final material is sterile

'.~WO g3/04088 PCI/US92/07188
1 9
filtered and filled in plas~ic bonles, and preserved with 0.1% sodium azide.
After one hour of incubation at 40C, the beads are washed and 200 ul of
conjugate is added to the wells of the reaction tray.
The preferred oonjugate is ~oat anti-human IgG horseradish peroxidase
conjugate. Concentrated conjugate is titered to determine a working concentration.
A twenty-fold concentrate of the working conjugate solution is then prepared by
diluting the concentrate in diluent. The 20X concentrate is sterile filtered andstored in plastic bottles.
The conjugate diluent includes 10% (v/v) bovine serum. 10% (v/v) goat
serum and 0.15% Triton-X100 in 20 mM Tris buffer. pH 7.5 with 0.01%
gentamicin sulfate, 0.01% thimerosal and red dye. The conjugate is sterile filtered
and filled in plastic bottles.
Anti-HCV positive control is prepared from plasma units positive for
antibodies to HCV. The pool of units used includes plasma with antibodies reactive to
1 5 pHCV-31 and pHCV-34. The units are recalcified and heat inactivated at 59-61C
hr 12 hours with constant stirring. The pool is aliquoted and stored at -20C or at
2-8C. For each lot of positive control, the stock solution is diluSed with negative
control containing 0.1% sodium azide as a preservative. The final material is
s!erile filtered and filled in plastic bottles.
2 0 Anti-HCV negative control is prepared from recalcified human plasma,
negative for antibodies to pHCV-31 and pHCV-34 proteins of HCV. The plasma is
also negative for antibodies to human immunodeficiency virus (HIV) and negative
for hepatitis B surface antigen (HBsAg). The units are pooled, and 0.1% sodium
æide is added as a presen/ative. The final material is sterile filtered and ~illed in
2~ plastic bottles.
After one hour of incubation with the conjugate at 40C, the beads are
washed, exposed to the OPD substrate for thirty minutes at room temperature and
the reaction terminated by the addition of 1 N H2SO4. The absorbance is read at
492 nm.
3 0 In order to maintain acceptable specificity, the cutoff for the assay should be
at least ~-7 standard deviations above the absorbance value of the normal
population mean. In addition, it has generally been observed that acceptable
specificity is obtained when the population mean runs at a sample to cutoff (S/CO)
value of 0.2~ or less. Consistent with these criteria, a "preclinical" cutoff for the
3 ~ screening assay was selected which clearly separated most of the presumed "true
negalive" from "true positiveN specimens. The cutoff value was calculated as thesum of 1he positive control mean absorbance value multiplied by 0.25 and the

WO g3/04088 ~ PCI`/US~2/07188'~

negalive control mean absorbance value. The cutoff may be expressed algebraically
as:
Cutoff value~0~25 PCx + NCx.
Testing may be performed by two methods which differ primarily in the
5 degree of automalion and the mechanism for reading the resulting color development
in the assay. One method is referred to as the manual or OuantumlM method because
C~uantum or Quantumatic is used to read absorbance at 492 nm. It is also called the
manual method because sample pipening, washing and reagent additions are
generally done manually by the technician, using appropriately calibrated pipettes,
1 0 dispensers and wash instruments. The second method is referred to as the PPC method and utilizes the automated Abbott Commander~ system. This system
employs a pipetting device referred to as the Sample Management Center (SMC) anda wash/dispenselread device referred to~as the Parallel Processing Center (PPC)
disclosed in E.P.O. Publication No. g1114072.~. The optical reader used in the PPC
15 has dual wavelength capabilities that can measure differential absorbencies (peak
band and side band) from the sample wells. These readings are converted into
results by the processo~s Control Center.

Screeninn Assav Performance
2 0 1. Serum/Plasma From Inoculated Chimeanzees
As previously described, Table I summarizes the results of a study which
followQd the course of HCV infectbn in seven chimpanzees using a screening assaywhich utilized 1he c1û0-3 polypeptide, and the screening assay which utilized
pHCV-31 and pHCV-34. Both assays gave negative results b~fore inoculation and
2 5 both assays detected the presence of antibodies after the animal had been infected
with HCV. However, in the comparison of the two assays, the assay utiiizing pHCV-
31 and pHCV-34 detected seroconversion to HCV antigens at an earlier or equivalent
bleed date in six of the seven chimpanzees. Data from these chimpanzee studies
clearly demonstrate that overall detection of HCV antibodies is greatly increased
3 0 with the assay utilizing the pHCV-31 and pHCV-34 proleins. This test is
sufficienlly sensi1ive to detecl seroconversion during the acute phase of this
disease, as defined as an elevation in ALT levels, in most animals. Equally important
is the high degree of specificity of the test as no pre-inoculation specimens were
reactive.
2. Non-A. Non-B Panel 11 (H. Alter. NIH!
A panel of highly pedigreed human sera from Dr. H. Alter, NIH, Bethesda,

W0 93/04088 ~ PCr/US92/07188
21
MD., containing infectious HCV sera, negative sera and o1her disease controls were
tested. A total of 44 specimens were present in lhe panel.
Six of seven sera which were ~proven ~nfectious~ in chimpanzees were
positive in both the screening assay using c100-3 as well as in the screening assay
5 utilking the recombinant proteins pHCV-31 and pHCV-34. These six reactive
specim0ns were obtained from individuals with chronic hepatilis. All six of the
reactive specimens were confirmed positive using synthetic peptide sp67. One
specimen obtained during the acute phase of NANB post-transfusion hepatitis was
non-reactive in both screening assays.
In the group labeled "probable infectious" were three samples taken from
the same post transfusion hepatitis patient. The first two acute phase samples were
negathe in both assays, but the third sample was reactive in bolh assay. The
disease control samples and pedigree~_negative controls were uniformly negative.All sixteen specimens detected as positive by both screening assays were
confirmed by the spll7 confirmatory assay (Figure 14). In addition, specimens 10and 29 were newly detected in the screening assay utilking the recom~inant pHCV-31 and pHCV-34 antigens and were reactive by the sp7~ confirmatory assay.
Spocimen 39 was initially reactive in the screening test utilizing pHCV-34 and
pHCV-31, but upon retesting was negative and oould not be confirmed by the
2 0 oonfirmatory assays.
In summary, both screening tests identified 6 of 6 chronic NANBH carriers
and 1 of 4 acute NANBH samples. Paired specimens trom an implicated donor were
non-reactive in the screening ~est utilizing c100-3 but were reac1ive in lhe
screening test with pHCV-31 and pHCY-34. Thus, the screening test utilizing the
2 5 recombinant antigens pHCV-31 and pHCV-34 appears to be more sensitive than the
scrcening assay u1ilizing c100-3. None of the disease control specimens or
pedigreed negative control specimens were reactive in either screening assay.

3 CBER Referencs Panel
3 0 A reference panel for antibody to Hepatitis C was received from the Center
for Biologics Evaluation and Research (CBE~). This 10 member panel consists of
eight reactive samples diluted in normal human sera negative for antibody to HCVand two sera that contain no detectable antibody to HCV. This panel was run on the
Ortho first generation HCV EIA assay, the screening assay utilizing c100-3 and lhe
3 5 screening assay utilizing pHCV-31 and pHCV-34. The assay results are presented
in Figure 15.
The screening assay utilizing pHCV-31 and pHCV-34 detected all six of the

WO 93/04088 ~ 6 P~/US92/071~


HCV positive or borderline sample dilulions. The two non-reactive sample dilutions
(709 and 710) appear 1O be diluted well iDeyond endpoint of anti~ody detectabili1y
for both screening assays. A marked inaease was observed in the sample to cutoffvalues for three of the members on the screening assay utilking pHCV-31 and
pHCV-34 compared to the screenin~ assay utilking c100-3 or the Ortho first
generation test. All repeatably reactive specimens were confirmed.

EXAMPIF4.CON IRMATORYASSAY
The confirmatory assay provides a means for unequivocally iden1ifying the
presence of an antii~ody that is immunologically reactive with an HCV antigen. The
confirmatory assay includes synthetic peptides or recombinant antigens
r~presenting major epitopes contained within the three distinct regions of the HCV
genome, which are the same regions represented by the two recombinant antigens
described in the screening assay. Recombinant proteins used in the confirmatory
assay should have a heterologous source of antigen to that used in the primary
screening assay (i.e. should not be ~n ~ derived recombinant antigen nor a
recombinant antigen composed in pan, of CKS sequences). Specimens repeatedly
reactive in the primary screening assay are retested in the confirmatory assay.
Aliquots containing Wentical amounts of specimen are contacted with a synthetic
2 0 peptide or recombinanl antigen individually coated onto a polystyrene bead.
Seroreactivity for epitopes within the c100-3 region of the HCV genome are
confirmed by use of the synthetic peptides sp67 and sp65. The synthetic peptide
sp117 can also be used to confirm seroreactivity with the c100-3 region.
Seroreactivity for HCV epitopes within lhe putative core region of HCV are
2 5 confirmed by the use of the synthetic peptide sp75. In order to confirm
seroreactivity for HCV epitopes within the 33c region of HCV, a recombinant
antigen expressed as a chimeric protein with superoxide dismulase ~SOD) in yeastis used. Finally, the antibody-antigen complex is detected.
The assay protocols were similar to those described in Example 3 above. The
3 0 peptides are each individually coated on~o polystyrene beads and used in an antibody
capture format similar to that described for the screening assay. Ten rnicroliters of
specimen are added to the wells of a reaction tray along with 400 ui of a specimen
diluent and a peptide coated bead. After one hour of incubation at 40C, the beads
are washed and 200 ul of conjugate (identical to that described in Example 3) is3 5 added to the wells of the reaction tray. After one hour of incubation at 40C, the
beads are washed, exposed to the OPD substrate for 30 minutes at room temperature
and the reaction terminated by the addition of 1 N H2SO4. The absorbance is read at

W093/04U88 23 PCI/US92/0718~


492 nm. The cutoff value for lhe peptide assay is 4 times the mean of the negative
control absorbance value.

1. Panels containino Seecimens "At Risk" for HCV Infec1ion.
A group of 233 specimens representing 23 hemodialysis patients all with
clinically diagnosed NANBH were supplied by Gary Gitnick, M.D. at the University of
California, Los Angeles Center for the Health Sciences. These samples which weretested in by the screening assay utilizing c100-3 were subsequently tested in the
screening assay which uses pHCV-31 and pHCV-34. A total of 7/23 patients
1 0 (30.44%) were reactive in the c100-3 screening assay, with a total of 36 repeat
reactive specimens. Ten of 23 patients (43.48%) were reactive by the screening
assay utilizing pHCV-31 and pHCV-34, with a total of 70 repea~able reactives
among the available specimens (Figure~16). Two specimens were unavailable for
testing. All of the 36 repeatedly reactive specimens detected in the c100-3
1 5 screening assay were confirmed by synthetic peptide confirmatory assays. A total
of 34 of these 36 were repeatedly reactive on HCV EIA utilizing pHCV-34 and
pHCV-31; two specimens were not available for testing. Of the 36 specimens
additionally detected by the screening assay utilizing pHCV-34 and pHCV-31, 9
were confirmed by the core peptide confirmatory assay (sp75) and 27 were
confirmed by the SOD-33c confirmatory assay.
In summary these data indicate that detection of anti-HCV by the screening
assay utilizing pHCV-31 and pHCV-34 may occur al an equivalent bleed date or as
many as 9 months earlier, when compared to the c100-3 screening assay. Figure
17 depicts earlier detection by the screening assay utilizing pHCV-34 and pHCV-31
2 5 in a hemodialysis patien1. --

5. Acute/Chronic ~lon-A. Noo-B ~ is
A population of specimens was identified from individuals diagnosed as
having acute or chronic NANBH. Specimens from individuals with acute oases of
3 0 NANBH were received from Gary Gitnick, M.D. at the Universily of California, Los
Angeles Center for Health Sciences. The diagnosis of acute hepatitis was based on the
presence of a cytolytic syndrome (ALT levels greater than 2X the upper normal
limit) on at least 2 serum samples for a duration of less than 6 months with or
without other biological abnormalities and clinical symptoms. All specimens were35 also negative for IgM antibodies to Hepatitis A Virus (HAV) and were negative for
Hepatitis B surface Ag when tested with commercially available tests. Specimens
from cases of chronic NANBH were obtained from two clinical sites. Individuals

WO 93/04088 PCI'/US92/07188
24
were diagnosed as having chronic NANBH based on the following criteria:
persistently elevated ALT levels, liver biopsy results, and/or lhe absence of
detectable HBsAg. Specimens w~lh biopsy results were further categorked as either
chronic active NANBH, chronic persistent NANBH, or chronic NANBH with
5 cirrhosis.
These specimens were test~d by both the c100-3 screaning assay and the
screening assay utilizing pHCV-34 and pHCV-31. The laner testing was performed
in replicates of two by both the Quantum and PPC methods.
~ommunity Acguired NANBH (Acute~
The c100-3 screening assay detected 2 of 10 specimens (20.00%) as
repeatedly reactive, both of which were confirmed. The screening assay utilizingpHCV-34 and pHCV-31 detected both of these specimens plus and additional 2
specimens (Figure 18). These 2 specimens were confirmed by sp75 (see Figure
19).
15 Acute Post-Transfusion NANBH
The c100-3 assay detected 4 of 32 specimens (12.50%) as repeatedly
reactive, all of which was confirmed. The screening assay utilizing pHCV-34 and
pHCV-31 detected 3 out of these 4 specimens (75/O) as reactive. The one sample
that was missed had an SJCO of 0.95 by the latter screening test. This sample was
2 0 confirmed by the sp67 peptide (Figure 18). In addition, the screening assay
utilking pHCV-34 and pHCV-31 detected 11 specimens not reactive in the c100-3
screening assay. Of the 9 specimens available for confirrnation, 8 were oonfirmed
by sp75 and 1 could not be confirmed but had an S/CO of 0.90 in the sp65
confirmatory test. (see Figure 19).
2 ~ Chronic NANBH
A summary of the results on these populations is shown in Figure 20.
Overail, 155 of 164 (94.5%) chronic NANBH samples were detected by the
screening test utilizing pHCV-31 and pHCV-34 using either Quantum or PPC. The
155 reactive samples were all confirmed in alternate assays using synthetic
.
3 0 peptides based on sequences from either the clO0, 33c or core regions of the HCV
genome. In contrast, only 138 of 164 (84.1%) specimens were positive by the
c100-3 assay. All but one of the 138 c100-3 samples were detected as positive bythe screening assay utilizing pHCV-31 and pHCV-34. The one discordant specimen
was nol confirmed by either synthetic or neutralization assays. Conversely, there
3~ were 17 confirmed specimens which were positive only by the screening assay
utilizing pHCV-34 and pHCV-31.
The results indicate that the screening assay utilizing pHCV-34 and pHCV-

WO93/04088 ~ ~ 1J~ PCr/US92/07188


31 is more sensitive than the current test in detecting HCV positive individualswithin chronically infected NANBH populations.

EXAMPLE 5. CompQtition ASSAY
The recombinant polypeptides containing antigenic HCV epitopes are useful
for competition assays. To perfonn a neutralization assay, a recombinant
polypeptide representing epitopes within the c100-3 region such as CKS-BCD
(pHCV-23) is solubilized and mixed with a sample diluent to a final concentration
of 0.5-50 ug/ml. Ten microlilers of specimen or diluted specimen is added to a
1 0 reaction well followed by 400 ul of the sample diluent containing the recombinant
polypeptide and if desired, the mixture may be preincubated for about fifteen
minutes to two hours. A bead coated with c100-3 antigen is then added to the
reaction well and incubated for one hour at 40C. After washing, 200 ulofa
peroxidase labeled goat anti-human IgG in conjugate diluent is added and incubated
1 5 for one hour at 40C. After washing, OPD substrate is added and incubated at room
temperature for thirty mirlutes. The reaction is terminated by the addition of 1 N
sulfuric acid and the absol~ance read at 492 nm.
Samples containing antibodies to the c100-3 antigen generate a reduced
signal caused by the competitive binding of the peptides to these antibodies in
2 0 solution. The percentage of competitive binding may be calculated by comparing the
absorbance value of the sample in the presence of a recombinant polypeptide to the
absorbance value of the sample assayed in the absence of a recombinant polypeptide
at lhe same dilution.

2 5 E)(AMPLE 6, IMMUNODOT AS~Y
The immunodot assay system uses a panel of purified recombinant
polypeptides placed in an array on a nitrocellulose solid support. The prepared
solid support is contacted with a sample and captures specific antibodies to HCVantigens. The captured an~ibodies are detected by a conjugate-specific reaction.3 0 Preferably, the conjugate specific reaction is quantified using a reflectance optics
assemb3y within an instrument which has been described in U.S. Patent Applications
Serial No. 07/227,408 filed August 2, 1988. The related U.S. Patent ApplicationsSerial Nos. 07l227,272, 071227,586 and 07/227,590 further describe specific
methods and apparatus useful lo perform an imrnunodot assay. The assay has also
been described in U.S. Application Serial No. 07/532,489 filed June 6, 1990.
Briefly, a nitrocellulose-base test cartridge is treated with multiple antigenicpolypeptides. Each polypeptide is contained within a specific reaction zone on the

W0 93/04088 ~ ; PCI/US92/07188 .~ ' '',~
26
lest cartridge. After all the antigenic polypeptides have been placed on the
nitrocellulose, excess bindin~ siles on the nitrocellubse are blocked. The test
cartridge is then contacted with a sample such that each antigenic polypeptide in
each reaction zone will react if lhe sample contains the appropriate antibody. After
5 reaction, the test cartridge is washed and any antigen-antibody reactions are
identified using suitable well known reagents.
As described in the patent applications listed above, the entire process is
amenaUe to automation. The specifications of these applications related to the
method and apparatus for performing an immunodot assay are incorporated by
1 0 reference herein.
In a preferred immunodot assay, the recombinant polypeptides pHCV-23,
pHCV-29, pHCV-34, and cl00-3 were diluted in the preferred buffers, pH
conditions, and sponing concentrations as summarized in Figure 21 and applied to a
preassembled nitrocellulose ~est cartridge. After drying the cartridge overnight at
1 5 room temperature 37C, the non-specific binding capacity of the nitro-cellulose
phase was bhcked. The blocking solution contained 1% porcine gelatin, 1% casein
enzymatic hydrolysate, 5% Tween-20, 0.1% sodium azide, 0.5 M sodium chloride
and 20 mM Tris, pH 7.5.
Forly normal donors were assayed by following the method described above.
2 0 The mean reflectance density value then was determined for each of the recombinant
proteins. A cutoff value was calculated as the negative mean plus six standard
deviations. Test cartridges were incubated with samples A00642 and 423 (see
Figure 22). Sample A00642 was from a convalescent non-A, non-B hepatitis
patient, diluted in negative human plasma from 1:100 to 1:12800. The other
2 ~ sample, 423, was from a paid plasma donor which ~ested positive in an assay using
a recombinant c100-3 polypeptide, diluted in negative human plasma from 1:40 to
1:2560. After sample incubation, sequential incubations wi~h a biotin-conjugatedgoa~ anti-human immunoglobulin-specific antibody, an alkaline phosphatase-
conjugated rabbit anti-biotin specific antibody, and 5-bromo-4-chloro-3-indolyl
3 0 phosphate produced a colored product at the site of the reaction. Sample to cutoff
values (S/CO) were determined for all HCV recombinant proteins. Those S,'CO
values greater than or equal to 1.0 were considered reactive. The limiting dilution
was defined as the lowest dilution at which the SICO was greater than or equal to
1Ø As seen in Figure 22, each sample tested positive for all HCV recombinant
3 ~ proteins. The data demonstrate that reactivity for sample A00642 was greatest
with pHCV-29, and decreased for the remaining antigens pHCV-23, c100-3, and
pHCV-34. Sample 423 most strongly reacted with the recombinant proteins

WO 93/04088 ~ PCI'/US9Z/07188
27
expressing pHCV-29 and pHCV-34, and to a lesser extent with pHCV-23 and c100-
3.

E XAMPLE 7. HCV CK~NS5 E)(PRESSION VECTORS
5 A. Preparation of HCV CKS-NSSE
Eight individual oligonucleobdes representing amino acids 1932-2191 of
the HCV genome were ligated together and cloned as a 793 base pair EcoRI-BamHI
fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated
pHCV-45 (SEQ.ID.NO 8), expresses the HCV CKS-NS5E antigen under control of the
1 0 lac promoter. The HCV CKS-NS5E antigen consists of 239 amino acids of CKS, nine
amino acids contributed by linker DNA sequences, and 260 amino acids from the
HCV NS41NS5 region (amino acids 1932-2191). Figure 23 presents a schematic
representation of the recombinant antigen-expressed by pHCV~5. SEQ.ID.NO. 10
and 11 presents the DNA and amino acid sequence of the HCV CKS-NS5E recombinant
1 5 antigen produced by pHCV-45. Figure 24 presents the expression of pHCV-45
proteins in ~Q!l. Lane 1 contained the .~ Iysate containing pHCV45
expressing the HCV CKS-NS5E antigen (amino acids 1932-2191) prior to
induction and lanes 2 and 3 after 2 and 4 hours post induction, respectively. These
results show that the pHCV-45 fusion protein has an apparent mobility
2 0 conesponding to a molecular size of 55,000 daltons. This compares acceptably to
the predicted molecular mass of 57,597 daltons.

B. Preparation of HCV CKS-NS5F
Eleven individual oligonucleotides representing amino acids 2188-2481 of
2 6 the HCV genome were ligated t~gether and cloned as a 89~ base pair EcoRI-BamHI
fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated
pHCV-48, expresses the HCV CKS-NS5F antigen under control of the lac promoter.
The HCV CKS-NS5F antigen consists of 239 amino acids of CKS, eight amino acids
contributed by linker DNA sequences, and 294 amino acids from the HCV NS5 region(amino acids 2188-2481). Figure 25 presents a schematic representation of the
recombinant antigen expressed by pHCV-48. SEQ.ID.NO. 12 and 13 presents the
DNA and amino acid sequence of the HCV CKS-NS5F recombinant antigen produced by
pHCV-48. Figure 26 presents the expression of pHCV-48 proteins in ~Q!I- Lane
1 contained the E.coli Iysate containing pHCV-48 expressing the HCV CKS-NS5F
antigen (amino acids 2188-2481) prior to induction and lanes 2 and 3 after 2 an
4 hours post induction, respectively. These results show that the pHCV-48 fusionprotein has an apparent mobility corresponding to a molecular size of 65,000

WO 93/04088 w i ~ PCI`/US92/07188 ~,
28
daltons. This compares acceptably to the predicted molecular mass of 58,985
daltons.

C. Preparation of HCVCKS-NS5G
Seven individual oligonucleotides representing amino acids 2480-2729 of
the HCV genome were ligated togethe~ and cloned as a 769 base pair EcoRI-BamHI
fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated
pHCV-51(SEQ.lD.NO.10), expresses the HCV CKS-NS5G antigen under control of
the lac promoter. The HCVCKS-NS5G antigen consists of 239 amino acids of CKS,
eight amino acids contributed by linker DNA sequences, and 250 amino acids from
~e HCV NS5 region (amino acids 2480-2729). Figure 27 presents a schematic
representation of ~e recombinant antigen expressed by pHCV-51. SEQ.NO.ID NO.14
and 15 presents the DNA and amino acid sequence of the HCV CKS-NS5G recombinant
antigen produced by pHCV-51. Figure 28 presents the expression of pHCV-51
1 5 proteins in ~ i- Lane 1 contained the E.coli Iysate containing pHCV-51
expre~ing the HCV CKS-NS5G antigen (amino acids 2480-2729) prior to
induction and lanes 2 and 3 after 2 and 4 hoùrs post induction, respectively. These
results show that the pHCV-51 fushn protein has an apparent mobility
corresponding to a molecular ske of 55,000 daltons. This compares acceptably to
2 0 the predicted molecular mass of 54,720 daltons.

D. preparation of HCV CKS-NS6H
Si% individual oligonucleotides representing amino acids 2728-2867 of the
HCV genome were ligated together and cloned as a 439 base pair EcoRI-BamHI
fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated
pHCV-50 (SEQ.NO.11~.11) expresses the HCV CKS-NS5H antigen under control of
the lac promoter. The HCV CKS-NS5H antigen consists of 239 amino acids of CKS,
eight amino acids contributed by linker DNA sequences, and 140 amino acids from
the HCV NS5 region (amino acids 2728-2867). Figure 29 presents a schematic
3 0 representation of the recombinant antigen expressed by pHCV-50. SEQ.ID.NO. 16
and 17 presents the DNA and amino acid sequence of the HCV CKS-NS5H recombinant
antigen produced by pHCV-50. Figure 30 presents the expression of pHCV-50
proteins in E.coli. Lane 1 contained the E.coli Iysate containing pHCV-50
expressing the HCV CKS-NS5H antigen (amino acids 2728-2867) prior to
induction and lanes 2 and 3 after 2 and 4 hours post induction, respectively. These
results show that the pHCV-50 fusion protein has an apparent mobility
corresponding to a molecular size of 45,000 daltons. This compares acceptably to

W0 93/04088 ~ PC~r/US92/07188
29
the predicted molecular mass of 42,783 daltons.
E. Preparation of HCV CKS-NS51
Six individual oligonucleotides representing amino acids 2866-3011 of the
HCV genome were ligated together and cloned as a 460 base pair EcoRI-BamHI
5 fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated
pHCV~9 (SEQ.NO.ID.NO. 12), expresses the HCV CKS-NS51 antigen under control
of the lac promoter. The HCV CKS-NS51 antigen consists of 239 amino acids of CKS,
eight amino acids contributed by linker DNA sequences, and 146 amino acids from
the HCV NS5 region (amino acids 2866-3011). Figure 31 presents a schematic
10 representation of the recombinant antigen expressed by pHCV49. SEQ.ID.NO. 18
and 19 presents the DNA and amino acid sequence of the HCV CKS-NS61 recombinant
antigen produced by pHCV-49. Figure 32 presents the expression of pHCV49
proteins in E.coli. Lane 1 contained thq E.coli Iysate containing pHCV-49
expressing HCV CKS-NS51 antigen (amino acids 2866-3011) prior to induction
15 and lanes 2 and 3 after 2 and 4 hours post induction, respectively. These results
show that the pHCV-49 fusion protein has an apparent mobility corresponding to amolecular size of 42,000 daltons. This compares acceptably to the predicted
molecular mass of 43,497 daltons.

2 0 F. Immunoblot of HCV CKS-NS5 Antigens
Induced E.coli Iysates containing pHCV-23, pHCV~5, pHCV-48, pHCV-51,
pHCV-50, or pHCV-49 were individually run on preparative SDSIPAGE gels to
separate the various HCV CKS-NS5 or HCV CKS-BCD recombinant antigens assay
from the majority of other E.coli proteins. Gel slices containing the separated
2 5 individual HCV CKS-NS5 or HCV CKS-BCD recombinant antigens were then
electropheretically transferred to nitrocellulose, and the nitrocellulose sheet cut
into strips. Figure 40 presents the results of a Western Blot analysis of various
serum or plasma samples using these nitrocellulose strips. The arrows on the right
indicate the position of each HCY CKS-BCD or HGV CKS-NS5 recombinant antigen,
30 from top to bottom pHCV-23 (HCV CKS-BCD), pHCV45(HCV CKS-NS5E),pHCV-
48~HCV CKS-NS5F),pHCV-51 ~HCV CKS-NS5G),pHCV-50(HCV CKS-NS5H),
pHCV-49tHCV CKS-NS51), and pJO200 ~CKS). Panel A contained five normal
human plasma, panel B contained five normal human sera, panel C contained twentyhuman sera positive in the Abbott HCV EIA test, panel D contained two mouse sera3 5 directed against CKS, and panel E contained two normal mouse sera. Both the HCV
CKS-NS5E antigen expressed by pHCV45 and the HCV CKS-NS5F antigen
expressed by pHCV-48 were immunoreactive when screened with human serum

WO 93/04088 ~ i ~ 3 PCI`/US92/07188
samples containing HCV antibodies.

EXAMPLE 8. HCV CKS-C100
A. Preparation of HCV CKS-C100 Vectors
Eighteen individual oligonucleotides representing amino acids 1569-1931
of the HCV genome were ligated together and cloned as four separate EcoRI-BamHI
subfragments into the CKS fusion vector pJ0200. After subsequent DNA sequences
confirmation, the four subfragments were digested with the appropriate restriction
enzymes, gel purified, ligated toge!her, and cloned as an 1102 base pair EcoRI-
1 0 BamHI fragment in the CKS fusion vector pJ0200. The resulting plasmid,
designated pHCV-24, expresses the HCV CKS-C100 antigen under control of the lac
promoter. The HCV CKS-c100 antigen consists of 239 amino acids of CKS, eight
amino acids contributed by linker DNA seguences, 363 amino acids from the HCV
NS4 region (amino acids 1569-1931) and 10 additional amino acids contributed
1 5 by linker DNA sequences. The HCV CKS-c100 antigen was expressed at very low
levels by pHCV-24.
- Poor expression levels of this HCV CKS-c100 recombinant antigen were
overcome by constructing two additional clones containing deletions in the extreme
amino terrninal portion of the HCV c100 region. The first of these clones,
des`ignated pHCV-57 (SEQ.ID.NO. 20 and 21), contains a 23 amino acid deletion
(HCV amino acids 1575-1597) and was constructed by deleting a 69 base pair Ddel
restric~on fragment. The second of these clones, designated pHCV-58 (SEQ.ID.NO.
22 and 23), contains a 21 amino acid deletion (HCV amino acids 1600-1620) and
was constructed by deleting a 63 base pair NlalV-Haelll restriction fragment.
2 5 Figure 34 presents a schematic representation of the recombinant antigensexpressed by pHCV-24, pHCV-57, and pHCV-58. SEQ.ID. NO. 13 presents the DNA
and amino acid sequence of the HCV-C100D1 recombinant antigen produced by
pHCV-57. SEQ.ID.NO.14 presents the DNA and amino acid sequence of the HCV-
C100D2 recombinant antigen produced by pHCV-58. Figure 35 presents the
expression of pHCV-24, pHCV-57, and pHCV-58 proteins in E.coli. Lane 1
contained the E.coli Iysate containing pHCV-24 expressing the HCV CKS-c100
antigen (amino acids 1569-1931) prior to induction and lanes 2 and 3 after 2 and4 hours post induction, respectively. Lane 4 contained the E.coli Iysate containing
~ ~ pHCV-57 expressing the HCV-CKS-C100D1 antigen (amino acids 1669-1574 and
5~ 35 1598-1931) prior to induction and~lanes 5 and 6 after 2 and 4 hours induction,
respectively. Lane 7 contained the E.coli Iysate containing pHCV-58 expressing the
HCV CKS-C100D2 antigen (amino acids 1569-1599 and 1621-1931) prior to

:,

`3 WO 93/04088 PCI lUS92/07188


induction, and lanes 8 and 9 after 2 and 4 hours induction, respectively. These
results show that both the pHCV-57 and pHCV-58 fusion proteins express at
significan~y higher levels than the pHCV-24 fusion protein and that both the
pHCV-57 and pHCV-58 fusion proteins have an apparent mobility corresponding to
5 a molecular size of 65,000 daltons. This compares acceptably to the predicted
molecular mass of 64,450 daltons for pHCV-~7 and 64,458 daltons for pHCV-58.

EXAMPLE 9. HCV PCR DERIVED EXPRESSION VECTORS
A. Preparation of HCV DNA Fraçlments
RNA was extracted from the serum of various chimpanzees or humans
infected with HCV by first subjecting the samples to digestion with Proteinase K and
SDS for 1 hour at 37 centigrade followed by numerous phenol:chloroform
extractions. The RNA was then concentrated by several ethanol precipitations andresuspended in water. RNA samples were then reverse transcribed according to
1~ supplier's instructions using a specific primer. A second primer was then added
and PCR amplification was performed according to supplier's instructions. An
aliquot of this PCR feaction was then subjected to an aWitional round of PCR using
nested primers located internal to the first set of primers. In general, these
primers also contained restriction endonuclease recognition sequences to be used for
2 0 subsequent cloning. An aliquot of this second round nested PCR reaction was then
subjected to agarose gel electrophoresis and Southern blot analysis to confirm the
specificity of the PCR reaction. The remainder of the PCR reaction was then
digested with the appropriate restriction enzymes, the HCV DNA fragment of
interest gel purified, and ligated to an appropriate cloning vector. This ligation was
2 5 then transformed into E.coii and single coionies were isolated and plasmid DNA
prepared for DNA sequences analysis. The DNA sequences was then evaluated to
confirm that the specific HCV coding region of interest was intact. HCV DNA
fragments obtained in this manner were then cloned into appropriate vectors for
expression analysis.
3 ~ B. PreDaration ot HCV CKS-NS3
Using the methods detailed above, a 474 base pair DNA fragment from the
putative NS3 region of HCV was generated by PCR. This fragment represents HCV
amino acids #1473-1629 and was cloned into the CKS expression vector pJ0201
by blunt-end ligation. The resulting clone, designated pHCV-105, expresses the
3 ~ HCV CKS-NS3 antigen under control of the lac promoter. The HCV CKS-NS3 antigen
consists of 239 amino acids of CKS, 12 amino acids contributed by linker DNA
sequences, 1~7 amino acids from the HCV NS3 region ~amino acids 1473-1629),

W093/04088 ~ i i PCr/US92/07188


and 9 additional amino acids contributed by linker DNA sequences. Figure 36
prosents a schematic representation o~ the pHCV-105 anbgen. SEQ.ID.NO. 24 and
25 presents the DNA and amino acW sequence of the HCV CKS-NS3 recombinant
antigen produced by pHCV-105. Figure 37 presents the expression of pHCV-105
5 proteins in E.~:QIj. Lane 1 contained me ~Q~ Iysate containing pHCV-105
expressing the HCV CKS-NS3 antigen (amino acids 1472-1629) prior to induction
and lanes 2 and 3 after 2 and ~ hours induction, respectively. These results show
that the pHCV-105 fusion protein has an apparent mobility corresponding to a
molecular mass of 43,000 daltons. This compares acceptably to the predicted
1 0 molecular mass of 46,454 daltons.
C. Preparation of HCV CKS-5'ENV
- Using the methods detailed above, a 489 base pair DNA fragment from the
putative envebpe region of HCV was gen~rated by PCR. This fragment represents
the HCV amino acids 114-276 and was cloned into the CKS expression vector
1 5 pJ0202 using EcoRI-BamHI restriction sites. The resulting clone, designated
pHCV-103 (SEQ.ID.NO. 26 and 27), expresses the HCV CKS-5'ENV antigen under
control of the lac promoter. The HCV CKS-5'ENV antigen consists of 239 amino
acids of CKS, 7 amino acids contributed by linker DNA sequences, 163 amino acidsfrom the HCV envelope region (amino acids 114-276), and 16 additional arnino
2 0 æids contributed by îinker DNA sequences. Figure 38 presents a schematic
representation of the pHCV-103 antigen. SEQ.ID.NO. 26 and 27 presents the DNA
and amino acid sequence of the HCV CKS-5'ENV recombinant antigen produced by
pWCV-103. Figure 37 presents me expression of pHCV-103 proteins in E.coli.
Lane 1 contained the E.coli Iysate containing pHCV-103 expressing the HCV CKS-
25 5'ENV antigen (amino acids 114-276) prior to induction and lanes ~ and 6 after 2
and 4 hours induction, respectively. These results show that the pHCV-103 fusionprotein has an apparent mobility corresponding to a molecular mass of 47,000
daltons. This compares acceptably to the predicted molecular mass of 46,091
daltons.
3 0 D. Preparation of HCV CKS-3'ENV
Using the methods detailed above, a 621 base pair DNA fragment form the
putative envelope region of HCV was generated by PCR. This fragment represents
HCV amino acids 263469 and was cloned into the CKS expression vector pJ0202
using EcoRI restriction sites. The resulting clone, designated pHCV-101
36 (SEQ.ID.NO. 17), expresses the HCV CKS-3'ENV antigen under control of the lacpromoter. The HCV CKS-3'ENV antigen consists of 239 amino acids of CKS, 7
amino acids contributed by linker DNA sequences, 207 amino acids from the HCV

WO93/04088 ~ i ~ j PCr/US92/07188


envelope region (amino acids 263-469), and 15 additional amino acids contributedby linker DNA sequences. Figure 39 presents a schematic representation of the
pHCV-101 antigen. SEQ.ID.NO. 28 and 29 presents the DNA and amino acW sequence
of the HCV CKS-3'ENV recombinant antigen produced by pHCV-101. Figure 37
5 presents the expression of pHCV-101 prnteins in ~ i Lane 7 contained the ~
Iysate containing pHCV-101 expressing the HCV CKS-3'ENV antigen ~amino acids
263-469) prior to induction and lanes 8 and 9 after 2 and 4 hours induction,
respectively. These resulting show that the pHCV-101 fusion protein has an
apparent mobility corresponding to a molecular mass of 47,000 daltons. This
1 0 compares acceptably to the predicted molecular mass of 51,181 daltons.
E. Preparation of HCV CKS-NS2
Using the methods detailed above, a 636 base pair DNA fragment from the
putative NS2 region of HCV was generated by PCR. This fragment represents the
HCV amino acids 994-1205 and was cloned into the CKS expression vector pJ0201
1 5 using EcoRI restriction sites. The resulting clone, designated pHCV-102, expresses
the HCV CKS-NS2 antigen under control of the lac promoter. The HCV CKS-NS2
antigen consists of 239 amino acids of CKS, 7 amino acids contributed by linker
DNA sequences, 212 amino acids from the HCV NS2 region (amino acids 994-
1205), and 16 additional amino acids contributed by linker DNA sequences. Figure40 presents a schematic representation of the pHCV-102 antigen. SEQ.Il).NO. 30
and 31 presents the DNA and amino acid sequence of the HCV CKS-Næ recombinant
antigen produced by pHCV-102. Figure 41 presents the expression of pHCV^102
proteins in E.coli. Lane 1 contained the ~ Iysate containing pHCV-102
expressing the HCV CKS-NS2 antigen (amino acids g94-1205) prior to induction
2 5 and lanes 2 and 3 after 2 and 4 hours induction, respectively. These results show
that the pHCV-102 fusion protein has an apparent mobili~ corresponding to a
molecular mass of ~3,000 daltons. This compares acceptably to the predicted
molecular mass of 51,213 daltons.
F. PreDaration of HCV CKS-NS1
3 0 Using the methods detailed above, a 654 base pair DNA fragment from the
putative NS1 region of HCV was generated by PCR. This fragment represents HCV
amino acids 617-834 and was cloned into the CKS expression vector pJ0200 using
EcoRI-BamHI restriction sites. The resulting clone, designated pHCV-107,
expresses the HCV CKS-NS1 antigen under control of the lac promoter. The HCV
CKS-NS1 antigen consists of 239 amino acids of CKS, 10 amino acids contributed by
iinker DNA sequences, and 218 amino acids from the HCV NS1 region (amino acids
617-834). Figure 42 presents a schematic representation of the pHCV-107

WO g3/04088 2 ~ ~ 3 ~ ~ ~i PCI`/USg2/07188 ~ ~`
34
antigen. SEQ.ID.NO. 32 and 33 presents the DNA and amino acid sequence of the HCV
CK~NS1 recombinant anbgen produced by pHCV-107.
G. Preparation of HCV CKS-ENV
Using the methods detailed above, a 1068 base pair DNA fragment from the
5 putative envebpe region of HCV was generated by PCR. This fragment represents
HCV amino acids #114469 and was cloned into the CKS expression vector pJ0202
using EcoRI restriction sites. The resulting clone, designated pHCV-104, expresses
the HCV CKS-ENV antigen under control of the lac promoter. The HCV CKS-ENV
anbgen consists of 239 amino acids of CKS, 7 amino acids contributed by linker
1 0 DNA sequences, 356 amino acids from the HCV envelope region (amino acids 114-
469), and 15 additional amino acids contributed by linker DNA sequences. Figure
43 presents a schematic representation of the pHCV-104 antigen. SEQ.ID.NO. 34
and 35 presents the DNA and amino acid sequence of the HCV CKS-ENV recombinant
antigen produced by pHCV-104.
EXAMPLE 10. HCY (~KS-NS1S1
A. Construction of the HCV CKS-NS1 S1 Expression Vector
Eight individual oligonucleotides representing amino acids 365-579 of the
HCV genome were ligated together and cloned as a 645 base pair EcoRI/BamHI
2 0 fragment into the CKS fusion vector pJO200. The amino acid sequence of this
antigen is designated as pHCV-77 (SEQ. ID. NO. 1). The resultant fusion protein
HCV CKS-NS1S1 consists of 239 amino acids of CKS, seven amino acids contributed
by linked DNA sequences, and ~15 amino acids from the NS1 region of the HCV
gerlome.
25 B. Production and Characterization of the Recombinant Antiaen HCV-NS1S1
pHCV-77 was transformed into E.coli K-12 strain XL-1 (recA1, endA1,
gyrA96, thi-1, hsdR17, SupE44, relA1, lac/f1, p10AB, lacl1ADM15, TN10)
cells. Expression analysis and characterization of the recombinant protein was done
using polyacrylamide gel electrophoresis as described in Example 1. The apparent3 0 molecular weight of the pHCV-77 antigen was the same as the expected molecular
weight of 50,228 as visualized on a coumassie stained gel. The immunoreactivity as
determined by Western blot analysis using human sera indicated that this
recombinant antigen was indeed immunoreactive. FIGURE 47A presents the
expression of pHCV-77 in E. coli. FIGURE 47B presents an immunoblot of the
3 ~ pHvV-77 antigen expressed in E. coli. Lane 1 contained the E. çoli Iysate containing
pHCV-77 expressing the HCV CKS-NS1S1 antigen prior to induction and Lanes 2
and 3 are 2 and 4 hours post-induction, respectfully.

. ~

W0 93/04088 ~ 2 ~ PCI/US92/0~188


EXAMPLE 11. HCV CK~NSlS2
A. Construction of the HCV CKS-NS1S2 Expression Vector
Six individual oligonucleotides representing amino acids 565-731 of the
5 HCV genome was ligated together and cloned as a 501 base pair EcoRI/BamHI
fragment into the CKS fusion vector p~O200. The complete amino acid sequence of
this antigen is designated as pHCV-65 (SEQ. ID. NO. 2). The resultant fusion
protein HCV CKS-NSlS2 oonsists of 239 amino acids of CKS, eight amino acids
contributed by linker DNA sequences, and 167 amino acids from the NS1 region of
1 0 the HCV genome.
B. Production and Characterization of the Recombinant Antioen HCV-NS1S2
pHCV-65 was transformed into E.coli K-12 strain XL-1 (recA1, endA1,
gyrA96, thi-1, hsdR17, SupE44, relA1, lac/f1, p10AB, laclqAMD15, TN10) cells.
Expression analysis and characterization of the recombinant protein was done using
1~ polyacrylamide gel electrophoresis as described in Example 1. The apparent
molecular weight of the pHCV-66 antigen was the same as the expected molecular
weight ot 46,223 as visualized on a coumassie stained gel. The immunoreactivity as
determined by Western blot analyis using human sera indicated that this
recombinant antigen was indeed immunoreactive. FIGURE 48A presents the
2 0 expression of pHCV-65 in E. coli. FIGURE 48B presents an immunobiot of the
pHCV-65 antigen expressed in E. coli. Lane 1 contained the E. coli Iysate containing
pHCV-65 expressing the HCV CKS-NS1S2 antigen prior to induction and Lanes 2
and 3 are 2 and 4 hours post-induc~ion, respectively.

EXAMPLE 12. CKS-NS1S3
A. Construction of the HCV CKS-NS1 S3 Expression Vector
Six individual oligonucleotides representing amino acids 717-847 of the
HCV genome were ligated together and cloned as a 393 base pair EcoRltBamHI
fragment into the CKS fusion vector pJO200. The complete amino acid sequence of
3 0 this antigen is designated as pHCV-78 (SEQ. ID. NO. 3). The resultant fusion
protein HCV CKS-NS1S3 consists of 239 amino acids of CKS, eight amino acids
contributed by linker DNA sequences, and 131 amino acids from the NS1 region of
the HCV genome.
B. Production and C~haracterization of the Recombiant Antiaen HCV-NS1S3
pHCV-78 was transformed into E.coli K-12 strain XL-1 (recA1, endA1,
gyrA96, thi-1, hsdR17, SupE44, relA1, lactf1, p10AB, laclqADM15, TN10) cells.
Expression analysis and characterization of the recombinant protein was done using

WO 93/04088 ~ i ~ PCl'/US92/07188


polyacryiamide gel electrophoresis as described in Example 1. Analysis of the
coumassie stained gel indicated very low levels of expression of the protein with an
expected molecular weight of 42,1141. Western blot analysis also failed to show
any immunoreactivity and we are continuing to identify human sera that is specific
to this region of NS1.

EXAMPLE 13. CKS-NS1S1-NS1S2
A. Construction of the HCV CKS-NS1S1-NSlS2 Expression Vector
The construction of pHCV-80 (NS1S1-NS1S2) involved using the
SACI/BamHI insert from pHCV-65 and ligating that into the Sacl/BamHI vector
backbone of pHCV-77. The resultant HCV gene represents amino acids 365-731 of
the HCV genome. This resulted in a 1101 base pair EcoRI/BamHI fragment of HCV
cloned into the CKS fusion vector pJO20~ The complete amino acid sequence of this
antigen is designated as pHCV-80 (SEQ. ID. NO. 4). The resultant fusion protein
1 5 HCV CKS NS1S1-NS1S2 consists of 239 amino acids of CKS, seven amino acids
contributed by linker DNA sequences, and 367 amino acids from the NS1 region of
~e HCV genorne.
B. Production and Characterization of the Recombinant Antiaen HCV-NS1S1:~1S1S2
pHCV-80 was transformed into E.coli K-12 strain XL-1 (recA1, endA1,
gyrA96, thi-1, hsdR17, SupE44, relA1, lac/fl, p10AB, laclqADM15, TN10) cells.
Expression analysis and characterization of the recombinant protein was done using
polyac~ylamide gel electrophoresis as described in Example 1. The apparent
molecular weight of the pHCV-80 antigen was the same as the expected molecular
weight of 68,454 as visualized on a coumassie stained gel. The immunoreactivity as
2 5 determined by Western blot analysis using human sera indicated that this
recombinant antigen was very immunoreactive. FIGURE 49A presents the
expression of pHCV-80 in E. coli. FIGURE 49B presents an immunoblot of pHCV-
80 antigen expressed in E. coli. Lane 1 contained the E. coli Iysate containing pHCV-
80 expressing the HCV CKS-NS1S1-NS1S2 antigen prior to induction and Lanes 2
and 3 are 2 and 4 hours post-induction, respectively.

E)(AMPLE 14. HCV CKS-FULL LENGTH NS1
A. Construction of the HCV CKS-full lenath NS1 Expression Vector
The construction of pHCV-92 (SEQ. ID. NO. 5) full length NS1) involved
using the Xhol/BamHI insert from pHCV-78 (SEQ. ID. NO. 3) and ligating that intothe XhoVBamHI vector backbone of pHCV-80 (SEQ. ID. NO. 4) The resultant HCV
gene represents amino acids 36~-847 of the HCV genorne. This resulted in a 1449

! WO 93/04088 ~ i i J ~ ~ 6 Pcr/usg2!o7188


base pair EcoRI/BamHI fragment of HCV cloned into CKS fusion vector pJO200. The
complete amino acid sequence of this antigen is designated as pHCV-92 (SEQ. ID. NO.
5). The resultant fusion protein HCV CKS-full length NS1 consists of 239 amino
acids of CKS, seven amino acids contributed by linker DNA sequences, and 483
5 amino acids from the NS1 region of the HCV genome.
B. Production and Characterization of the Recombinant Antigen pHCV-92
pHCV-92 was transformed into E.coli K-12 strain XL-1 ~recA1, endA1,
gyrA96, thi-1, hsdR17, SupE44, relA1, lac/f1, p10AB, laclqADM15, TN10) cells.
Expression analysis and characterization of the recombinant protein was done using
10 polyacrylameide gel electrophoresis as described in Example 1. The expressionlevels as seen by counassie stained gel were virtually undectable and the Western
blot indicated no immunoreactivity. We are still in the process of identifying sera
that will recognize this region of HCV N~1.

The present invention thus provides unique recombinant antigens
representing dis~inct antigenic regions of the HCV genome which can be used as
reagents for ~e detection and/or confirmation of antibodies and antigens in testsamples from individuals exposed to HCV. The NS1 protein is considered to be a
non-structural membrane glycoprotein and to be able to elicit a protective immune
2 ~ response of the host against iethal viral infection.
The recombinant antigens, either alone or in combination, can be used in the
assay formats provided herein and exemplified in the Examples. It also is
contemplated that these recombinant antigens can be used to develop specific
inhibitors of viral replication and used for therapeutic purposes, such as for
2 5 vaccines. Other applications and modifications of the use of these antigens and the
specific embodiments of this inventions as set forth herein, will be apparent tothose skilied in the art. Accordingly, the invention is intended to be limited only in
accordance with the appended claims.
, .

PCrNS 9 2 / 0 7 1 8 8
BO/US 23DEC~992

3~

SEQUENCE LISTING
~1) GENERAL INFORMATION:
(i) APPLICANT: DEV~RE, S.
DESAI, S.
DAILEY, S.
(ii) TITLE OF INVENTION: HCV SYNTHETIC PEPTIDE FROM NSl REGION
(iii) NUMBER OF SEQUENCES: 35
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: ABBOTT LABORATORIES
(B) STREET: ONE ABBOTT PARK ROAD
(C) CITY: ABBOTT PARK
(D) STATE: ILLINOIS
(E) COUNTRY: U.S.
~F-) ZIP: 60065-3500
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
tD) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
- (C~ CLASSIFICATION:
~viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: POREMBSKI, PRISCILLA E.
(B) REGISTR~TION NU~BER: 33,207
(C) REF~RENCE/DOCKET NUMBER: d834PC.02
(ix) TELECOMMUNICATION INFORMATION: -
(A) TELEPHONE: 708-937-6365
(8~ TELEFAX: 708-937-9556

(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: ~63 amino acids
(B) TYPE: amino acid
(C) STRP~JDEDNESS: single
(D) TOPOLOGY: linear
(ii) NOLECULE TYPE: pep~ide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu

'}~ ~ r 92G PCTIlJS 92/0718 8
12O/IJS 23 DC 1992

3g

Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

VA1 Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Vial Ala

Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn
al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Aisp Asn Leu Ala Gln Arg Gln Val Gly ~et Thr Thr Leu Ala Val
115 120 - 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Vial
130 135 140
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val ('-ly Asp
165 170 175
isn Phe L~u Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
18~ 1~5 190
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu G:lu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
sp Pro Ser Thr Asn Ser Thr Met Val Gly Asn Trp Ala Lys Val Leu
245 250 255
al Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr His Val Thr
260 265 270
Gly Gly Ser Ala Gly His Thr Val Ser Gly Phe Val Ser Leu Leu Ala
275 280 285
Pro Gly Ala Lys Gln Asn Val Gln Leu I le Asn Thr Asn Gly Ser Trp
2gO 295 300

PCTlllS 9 2/ 07 1 8 8
B0/ljS 23 DEC lm



Hi~ Leu Asn Ser Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn Thr Gly
~05 310 315 320
Trp Leu Ala Gly Leu Phe Tyr His His Lys Phe Asn Ser Ser Gly Cy5
325 330 335
Pro Glu Arg Leu Ala Ser Cys Arg Pro Leu Thr Asp Phe A~p Gln Gly
340 345 350
Trp Gly Gln Ile Ser Tyr Ala Asn Gly Ser Gly Pro Asp Gln Arg Pro
355 360 365
Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys
370 375 380
Ser Val Cys Gly Pro Val Tyr.Cys Phe~Thr Pro Ser Pro Val Val Val
385 390 395 400
Gly Ihr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Glu Asn
4.05 410 415
Asp Thr Asp Val Phe ~al Leu Asn Asn Thr Arg Pro Pro Leu Gly Asn
420 ~25 430
Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys Val Cys
g35 440 g45
Gly Ala Pro Pro Cys Val Ile Gly Gly Ala Gly A~n Asn Thr Leu
450 ~55 460

(2) INFORMATION FOR SEQ ID No~2:
ti) SEQU~NCE CHARACTERISTICS.
(A) LENGIH: 414 amino acids
(B) TYPE: amino acid
(C) STRANDXDNESS: single
(D~ TOPOLOGY: linear
(ii) ~GLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu

Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
g5
Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

`~ PCTIUS 92/07l8a
~ O / 1~ S 2 3 DEC 1992

41

Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
lu Val ~'al Glu Lys Cys Ala Phe S~r A~p Asp Thr Val Ile Val Asn
al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val
115 120 1~5
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Al~ Val Lys Val
130 135 lgO
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr I le
145 150 155 160
ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly A~p
165 170 175
sn Phe Leu Arg His Leu Gly Ile Iyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
lgS 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Yal Ala Gln Glu Val Pro Gly Thr Gly Val A p Thr Pro Glu Asp Leu
225 230 23~ 24Q
sp Pro Ser Thr Asn Ser Met Gly Ala Pro Pro Cys Val Ile Gly Gly
245 250 255
la Gly Asn Asn Thr Leu His Cy~ Pro Thr Asp CyY Phe Arg Lys His
260 2~5 270
Pro Asp Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro
275 2~0 285
Arg Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Thr Pro Cys Thr
290 295 300
Ile Asn Thr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu
305 310 315 . 320
is Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp
325 330 335
,eu Glu Asp Arg Asp Arg Ser Glu Leu S,er Pro Leu Leu Leu Thr Thr
340 34S 350

- hLi~f~2~ PCTI~JS 92/071 8 8
K0/US 23DEClg~
42

Thr Gln Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu
355 360 365
Ser Thr Gly Leu Ile His Leu Gly Gln Asn Ile Val Asp Val Gln Tyr
370 375 380
Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu
385 390 395 g00
Tyr Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val
405 gl0

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 378 ami~o acids
(B) TYPE: amino ~cid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide

- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met S~r Phe Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro
1 5 10 15
Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro ~et Ile Val His Val

Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr

Asp His Glu A3p Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val

Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu

Val Val Glu Lys Cy9 Ala Phe Ser Asp Asp Thr Val Ile Val Aqn Val

Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala
100 105 110
Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val Pro
115 120 125
Ile ~is Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val
130 135 140
Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile Pro

PCT/US 92/0718 8
`2~ 0/US 23 DEC 199

43

145 150 155 160
Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp Asn
165 170 175
Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg
180 185 190
Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu Hi~ Ile Glu Met Leu
195 200 205
Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala V~l
210 215 220
Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu A~p
225 230 235 2~0
Pro Ser Thr Asn Ser Thr Met Glu Tyr Val V~l Leu Leu Phe Leu L~u
245 250 255
Leu Ala Asp Ala Arg Val Cy9 Ser Cy9 Leu Trp Met Met Leu Leu Ile
260 265 270
Ser Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Ile Leu Asn Ala Ala
275 280 285
Ser Leu Ala Gly Thr His Gly Leu Val Ser Phe Leu Val Phe Phe Cys
290 295 300
Phe Ala Trp Tyr Leu Lys Gly Lys Trp Val Pro Gly Al~ Val Tyr Thr
305 310 315 320
Phe Tyr Gly Met Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Gln
325 330 335
Arg Ala Tyr Al~ Leu A~p Thr G;u Val Ala Al~ Ser Cy~ Gly Gly Val
340 3~5 3S0
Val Leu Val Gly Leu Met Ala Leu Thr Leu Ser Pro Tyr Tyr Lys Arg
355 360 365
Tyr Ile Ser Trp Cys Leu Trp Trp Leu Gln
370 375

~2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 622 amino acids
(B) TYPE: amino a-cid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide

- PCTllJS 9 2 / 0 7 1 8
0/~S 23 DEC 1992

44

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
ro Gly Lyq Pro Leu Val Asp Ile Asn Gly Lyq Pro Met Ile Val His

Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cys Met Thr Arg Ala Asp His Gln S~r Gly Thr Glu Arg Leu Ala
~ 75 80
lu Val Val Glu Lys Cys Ala Phe Ser A3p Asp Thr Val Ile Val A~n
~l Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 1~0
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
ro ~rp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly A~p
165 170 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Ar~ Ala Gly Phe Ile
180 1~5 190
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 ~ 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
qp Pro Ser Thr Asn Ser Thr Met Val Gly Asn Trp Ala Lys Val Leu
245 250 255
al Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr His Val Thr
260 265 270
ly Gly Ser Ala Gly His Thr Val Ser Gly Phe Val Ser Leu Leu Ala

PCT/lJS 9z/O718.
- - ~O/lJS 23 OEC 1992


275 280 285
Pro Gly Ala Lys Gln Asn Val Gln Leu Ile Asn Thr Asn Gly Ser Trp
290 295 300
His Leu A~n Ser Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn Thr Gly
305 310 315 320
Trp LQU Ala Gly Leu Phe T~r His His Lys Phe A~n Ser Ser Gly Cys
325 330 335
Pro Glu Arg Leu Ala Ser Cys Arg Pro Leu Thr Asp Phe Asp Gln Gly
3gO 3g5 350
Trp Gly Gln Ile Ser Tyr Ala Asn Gly Ser Gly Pro Asp Gln Arg Pro
355 360 365
Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys
370 375 380
Ser Val C-ys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val
385 390 395 400
Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Glu Asn
gO5 410 415
Asp Thr Asp Val Phe Val Leu Asn A~n Thr Arg Pro Pro Leu Gly Asn
420 425 430
Trp Phe Gly Cys Thr Trp Met A~n Ser Thr Gly Phe Thr Lys Vnl Cys
435 440 445
Gly Ala Pro Pro Cys Val Ile Gly Pro Pro Cys ~al Ile Gly Gly Ala
~50 455 g60
Gly Asn A~n Thr L~u Hi~ Cys Pro Thr Asp Cys Phe Arg Ly~ His Pro
465 470 ~75 ~80
Asp Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg
485 490 495
Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile
500 505 510
Asn Tyr Thr Ile Phe Ly~ Ile Arg Met Tyr Val Gly Gly Val Glu His
515 520 525
Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu
530 535 540
Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Thr Thr Thr
5g5 550 555 560
Gln Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser
565 570 575

` i- PCTIUS ~/0718 8 ~
3~b ~O/US 23 aEC 1992

46

Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu
580 585 590
Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr
595 600 605
Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Xaa
610 615 620

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ?38 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Ser Phe Val Val Ile Ile Pro Ala Arg ~yr Ala Ser Thr Arg Leu
1 5 10 15
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

V~l Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr Asp His Glu A~p Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val cy9 Met Thr Arg Ala Asp Hi~ Gln Ser Gly Thr Glu Arg Leu Ala

Glu V~l Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn

Val Gln Gly A~p Glu Pro ~et Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 1~0
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Th~ Val Gly A~p

PCT/US 9 2 / 0 7 1 8 8 .
b ~0/liS 23 DEC ~992


4 7

165 170 175
Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
Arg Asg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
Asp Pro Ser Thr Asn Ser Thr Met Val Gly Asn Trp Ala Lys Val Leu
245 250 255
Val Val Leu Leu Leu Phe Alà Gly Val Asp Ala Glu Thr His Val Thr
260 265 270
Gly Gly Ser Ala Gly His Thr Val Ser Gly Phe Val Ser Leu Leu Ala
275 28~ 285
Pro Gly Al~ Lys Gln Asn Val Gln Leu Ile Asn Thr Asn Gly Ser Trp
290 295 300
His Leu Asn Ser Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn Thr Gly
305 310 315 . 320
Trp Leu Ala Gly Leu Phe Tyr His His Lys Phe As~ Ser Ser Gly Cys
325 330 335
Pro Glu Arg Leu Ala Ser ~ys Arg Pro Leu Thr Asp Phe Asp Gln Gly
. 340 345 350
Trp Gly Gln Ile Ser Tyr Ala Asn Gly Ser Gly Pro Asp Gln Arg Pro
355 360 365
Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys
370 375 380
Ser Val ~yg Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val
385 390 395 400
Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Glu Asn
gO5 410 415
Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro Pro Leu Gly Asn
420 q25 g30
Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys Val Cys
435 440 445
Gly Ala Pro Pro Cys Val Ile Gly Pro Pro Cys Val Ile Gly Gly Ala
450 455 460

'

- PCTIUS 9 2/ 07 1 8 8
O/ IJ S 2 3 OEC 1992

48
I


Gly Asn Asn Thr Leu His Cys Pro Thr Asp Cys Phe Arg Lys Hi~ Pro
465 470 475 480
sp Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg
485 490 495
y.s Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Iyr Pro Cys Thr Ile
500 505 S10
Asn Tyr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu His
515 520 525
Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg qr8 A-~p Leu
530 535 5~0
Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Thr Thr rhr
545 550 555 560
ln Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser
565 570 575
hr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu
580 585 590
Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr
S9S 600 605
Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys
610 615 620
Leu Trp Met Met Leu Leu Ile Ser Gln Ala Glu Ala Ala I~eu Glu Asn
625 630 635 640
eu Val Ile Leu Asn Ala Ala Ser Leu Ala Gly 'rhr His Gly Leu Val
645 650 655
er Phe Leu Val Phe Phe Cys Phe Ala Trp Iyr Leu Lyc Gly Lys Trp
660 665 670
Val Pro Gly Ala Val l~rr Thr Phe ~r Gly Met Trp Pro Leu Leu Leu
675 ~80 685
Leu Lau Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Val
690 695 700
A1A Ala Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr
705 710 715 720
eu Ser Pro Tyr Tyr Lys Arg Tyr I le Ser Trp Cys Leu Trp Trp Leu
725 730 735
ln Xaa

- PCTIUS 9 2/ ~ 7 1 8 8
~LL,~ /l,S- 23 o~c lm

4 9

(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~481 base pairs
(B) TYPE: nucleic acid
(C~ STRANDEDNESS: 9 ingle
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 130..1317

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:~:
GAATTAATTC CCATTAATGT GAGTTAGCTC ACTCATTAGG CACCCCAGGC m ACACTTT 60
AT~TICCGGC TCGTATTTTG TGTG~AATTG TGAGCGGATA ACAA~TGGGC ATCCAGTAAG 120
GAG~TTTAA ATG AGT TTT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG . 168
Met Ser Phe Va} Val Ile Ile Pro Ala Arg Tyr Ala Ser
1 ' 5 10
ACG CGT CT~ CCC GGT AAA CCA TTG G~T GAT ATT AAC GGC AAA CCC ATG 216
Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met
15 20 25
ATT GTT CAT GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC 264
Ile Val His Val Lou Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile
30 -.35 g0 45
ATC GTG GCA ACC GAT CAT GA~ GAT GTT GCC CGC GCC GTT GAA GCC GCT 312
lle V~l Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala
50 55 60
GGC GGT GAA GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA 360
Gly Gly Glu V~l Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu
65 70 75
C~T CTG GCG GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG 408
Arg Leu Ala Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val
80 85 90
ATC GTT AAT GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT 456
Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile
95 100 105 -
CGT CAG GTT GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT 504
Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr
110 115 120 125
CTG GCG GTG CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG . 552
.

,~:
,,,

PCT/US 9 2 / 0 7 1 8 8
Y~ 0/liS- 23 DEC 1992


Leu Ala VA1 Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala
130 135 lgO
GTG AAA GTG GTT CTC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC 600
VA1 Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg
145 150 155
GCC ACC ATT CCT TGG GAT CGT GAT CGT TTT GCA GAA GGC CTT GAA ACC 648
Ala Thr Ile Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr
160 165 170
GTT GGC GAT AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA 696
Val Gly A~p Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Al~
175 180 185
GGC TTT ATC CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC 744
Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His
190 195 200 205
ATC GAA ATG TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC 792
Ile Glu Met Leu Glu Gln Leu Arg Va' Leu Trp Tyr Gly Glu Lys Ile
210 215 220
CAT GTT GCT GTT GCT CAG GAA GTT CGT GGC ACA GGT GTG GAT ACC CCT 840
Hi~ Val Ala V 1 Ala Gln Glu Val Pro Gly Thr Gly Yal Asp Thr Pro
225 230 235
GAA GAT CTC GAC CCG TCG ACG AAT TCC ATG TCT ACC AAC CCG AAA CCG 888
Glu Asp Leu Asp Pro Ser Thr Asn Ser Met Ser Thr Asn Pro Lys Pro
- 240 245 250
CAC AAA AAA AAC AAA CGT AAC ACC AAC CGT CGT CCG CAG GAC GTT AAA 936
Gln Lyq Ly~ Asn Lys Arg A~n Thr A n Arg Arg Pro Gln A9P Val Lys
255 260 26S
TTC CCG GGT GGT GGT C~G ATC GTT GGT GGT GTT TAC CTG C~G CCG CGT 984
Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg
270 275 280 2a5
CGT GGT CCG CGT CTG GGT GTT CGT GCT ACG CGT AAA ACC TCT GAA CGT 1032
Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg
290 295 300
TCT CAG CCG CGT GGG CGT CGT CAG CCG ATC CCG AAA GCT CGT CGT CCG 1080
Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro
305 310 315
GAA GGT CGT ACC TGG GCT CAG CCG GGT TAC CCG TGG CCG CTG TAC GGT 112 ?3
Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly
320 325 330
AAC GAA GGT TGC GGT TGG GCT GGT TGG CTG CTG TCT CCG CGT GGA TCT 1176
A~n Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser
335 340 345

~ 1 L J .j ~ ti PCTIUS 9 2 / 0 7 1 8 8


51

CGT CCG TCT TGG GGT CCG ACC GAC CCG CGT CGT CGT TCT CGT AAC CTT 1224
Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu
350 355 360 365
GGT AAA GTT ATC GAT ACC CTG ACC TGC GGT TTC GCT GAC CTG ATG GGT 1272
Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly
370 375 380
TAC ATA CCG CTG GTT GGA GCT CCG CTG GGT GGT GCT GCT CGT GCT 1317
Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala
385 390 395
TAACCCATGG ATCCTCTAGA CTGC~GGCAT GCTAAGTAAG TAGATCTTGA GCGCGTTCGC 1377
GCTGAAATGC GCTAATTTCA CTTCACGACA CTTCAGCCAA TTTTGGGAGG AGTGTCGTAC 1437
CGTTACGATT TTCCTCAATT TTTCTTTTCA ACAATTGATC TCATTCAGGT GACATCTTTT 1~97
ATATTGGCGC TCATTATGAA AGCAGTAGCT TTTATGAGGG TAATCTGAAT GGAACAGCTG 1557
CGTGCCGAAT TAAGCCATTT ACTGGGCGAA AAACTCAGTC GTATTGAGTG CGTCAATGAA 1617
AAAGCGGATA CGGCGTTGTG GGCTTTGTAT GACAGCCAGG GAAACCCAAT GCCGTTAATG 1677
GCAAGAAGCT TAGCCCGCCT AATGAGCGGG CITTTTTTTC GACGCGAGGC TGGATGGCCT 1737
TCCCCATTAT GATTCT$CTC GCTTCCGGCG GCATCGGGAT GCCCGCGTTG CAGGCCATGC 1797
TGTCCAGGCA GGTAGATGAC G~CCATCAGG GACAGCTTCA AGGATCGCTC GCGGCTCTTA 1857
CCAGCCTAAC TTCGATCACT GGACCGCTGA TCGTCACGGC GATTTA~GCC GCCTCGGCGA 1917
GCAC~TGGAA CGGGTTGGCA TGGATTGTAG GCGCCGCCCT ATACCTTGTC TGCCTCCCCG 1977
CGTTGCGTCG CGGTGCATGG AGCCGGGCCA CCTCGACCTG AATGGAAGCC GGCGGCACCT 2037
CGCTAACGGA TTCACCACTC CAAGAATTGG AGCCAATCAA TTCTTGCGGA GAACTGTGAA 2097
TGCGCAA~CC AACCCTTGGC AGAACATATC CATCGCGTCC GCCATCTCCA GCAGCCGCAC 2157
GCGGCGCA~C TCGGGCAGCG TTGGGTCCTG GCCACGGGTG CCCATGATCG TGCTCCTGTC 2217
GTTGAGGACC CGGCTAGGCT GGCGGGGTTG CCTTACTGGT TAGCAGAATG AATCACCGAT 2277
ACGCGAGCGA ACGTGAAGCG ACTGCTGCTG CAAAACGTCT GCGACCTGAG CAACAACATG 2337
AATGGTCTTC GGTTTCCGTG TTTCGTAAAG TCTGGAAACG CGGAAGTCAG CGCCCTGCAC 2397
CATTATGTTC CGGATCTGCA TCGCAGGATG CTGCTGGCTA CCCTGTGGAA CACCTACATC 2457
~GTATTAACG AAGCGCTTCT TCCGCTTCCT CGCTCACl'GA CTCGCTGCGC TCGGTCGTTC 2517
GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG 2577
GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA 2637

2 ~ PCTIJS 9 2 / 0 7 1 8 8
~0/luS 23DEC1992
52

AGGCCGCGTT GCTGGCG m TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC 2697
GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC 2757
CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG 2817
CCTTTCTCCC TTCGGGAAGC GTGGCGC m CTCAATGCTC ACGCTGTAGG TATCTCAGTT 2877
CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC 2937
GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC 2997
CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG 3057
AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTA m GGTATCTGCG 3117
CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA 3177
CCACCGCTGG TAGCGGTGGT TTTTTTG m GCAAGCAGCA GATTACGCGC AGAAAAAAAG 3237
GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT 3297
CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA - 3357
ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT 3gl7
ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG 3477
TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA 3537
GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCG AGA m ATCA GCAATAAACC 3597
. .
AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC m ATCCGCC TCCATCCAGT 3657
CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG 3717
TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA 3777
GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAA~GCGG 3837
TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GA~GTAAGTT GGCCGCAGTG TTATCACTCA 3897
TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG 3957
TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT 4017
CTTGCCCGGC GTCAACACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA 4077
TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA 4137
GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG 4197
TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC 4257

8~us 23DEC1992


GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT 4317
ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC 4377
C~CGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT 4437
TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT TCAA 4481

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 396 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: prOteiQ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
~0 55 60
Va~ Cy Met Thr Arg Ala Asp Hi3 Gln Ser Gly Thr Glu Arg Leu Ala
lu Val Val Glu Lys Cy9 Ala Phe Ser A~p Asp Thr Val Ilç Val Asn
al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 ' 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
Val Leu Asp Ala Glu Gly Tyr-Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile

- PCTllJS 92/0718 8
K0/US 23 DEC 1992


180 185 190
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly T~r Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
sp Pro Ser Thr Asn Ser Met Ser Thr Asn Pro Lys Pro Gl~ Lys Lys
245 250 255
sn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly
260 265 270
Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro
275 280 285
Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro
290 295 30~
Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Axg
305 . 310 315 320
hr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly A~n Glu Gly
325 330 335
ys Gly TTP Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser
340 345 350
Trp Gly Pro m r Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly ~ys Val
355 360 36~
Ile Asp Thr Leu Thr Cy~ Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro
370 375 380
Leu Val Gly Ala Pro Leu Gly Gly Ala Ala AIg Ala
385 390 395

(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5600 base pairs
(B~ TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE IYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 130..2472

PcTNs 9 2 / 0 7 1 8 8
O/US 23 D~ 1992




(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GAATTAATTC CCATTAATGT GAGTTAGCTC ACTCATTAGG CACCCCAGGC TTTACAC m 60
ATGTTCCGGC TCGTATTTTG TGTGGAATTG TGAGCGGATA ACAATTGGGC ATCCAGTAAG 120
GAGGTTTAA ATG AGT TTT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG 168
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser
5 10
ACG CGT CTG CCC GGT AAA CCA TTG GTT GAT ATT AAC GGC AAA CCC ATG 216
Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met
15 20 25
ATT GTT CAT GTT CTT GAA CGC GCG CGT GAA TSA GGT GCC GAG CGC ATC 264
Ile Val His Val ~eu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile
30 35 40 45
ATC GTG GCA ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT 312
Ile V~l Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala
50 55 60
GGC G~T GAA GTA TGT ATG AC~ CGC GCC GAT CAT CAG TCA GGA ACA GAA . 360
Gly Gly Glu V~l Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu
65 ' 70 75
CGT CTG GCG GAA GTT GTC GAA AAA TGC GCA TT~C AGC GAC GAC ACG GTG 408
Arg Lou Ala Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val
80 a5 90
ATC GTT AAT GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT 456
Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile
95 100 105
CGT CAG GTT GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT 504
Arg Gln Val Ala Asp Asn Lou Ala Gln Arg Gln Val Gly Met Ala m r
110 115 120 125
CTG GCG GTG CCA ATC CAC AAT GCG GAA GAA GCG m AAC CCG AAT GCG 552
Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala
130 135 140
GTG AAA GTG GTT CTC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC 600
Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg
14S 150 155
GCC ACC ATT CCT TGG GAT CGT GAT CGT m GCA GAA GGC CTT GAA ACC 648
Ala Thr Ile Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr
160 165 170
GTT GGC GAT AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA 696
Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala
175 180 185


.

.. . . . . .... ... .. .. . .. .. . .. . .. . ..

P~IUS 9 2 / 0 7 1 8
~US 23D~Cl992

56

GGC TTT ATC CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC 744
Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His
190 195 200 205
ATC GAA ATG TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC 792
Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile
210 215 220
CAT GTT GCT GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT 840
Hi~ Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val A~p Thr Pro
225 230 235
GAA GAT CTC GAC CCG TCG ACG AAT TCC ATG GCT GTT GAC TTT ATC CCG 888
Glu Asp Leu Asp Pro Ser Thr Asn Ser Met Ala Val Asp Phe Ile Pro
240 245 _ 250
GTT GAA AAT CTC GAG ACT ACT ATG CGT TCT CCG GTT TTC ACT GAC AAC 936
Val Glu Asn Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr A~p Asn
255 260 265
TCT TCT CCG CCG GTT GTT CCG CAG TCT TTC CAG GTT GCT CAC C~G CAT 984
Ser Ser Pro Pro Val Val Pro Gln Ser Phe Gln Val Ala His Leu His
270 275 280 285
GCT CCG ACT GGT TCT GGT AAA TCT ACT AAA GTT CCA GCT GCT TAC GCT 1032
Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala
290 295 300
GCT CAG GGT TAC AAA GTT CTG GrT CTG AAC CCG TCT GTT GCT GCT ACT 1080
Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr
305 310 315
CTG GGT TTC GGC GCC TAC ATG TCT AAA GCT CAC GGT ATC GAC CCG AAC 1128
~ Leu Gly Phe Gly Ala Tyr Met Ser Lys Al~ His Gly Ile Aqp Pro A~n
J 320 325 330
A~T CGT ACT GGT GTA CGT ACT ATC ACT ACT GGT TCT CCG ATC ACT TAC 1176
Ile Ary Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr
335 340 345
TCT A.T TAC GGT AAA TTC CTG GCT GAC GGT GGT TGC TCT GGT GGT GCT 1224
Ser Thr Tyr Gly Lys Phs Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala
350 355 360 365
TAC GAT ATC ATC ATC TGC GAC GAA TGC CAC TCT ACT GAC GCT ACT TCT 1272
Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Sex
370 375 380
ATC CTG GGT ATC GGT ACC GTT CTG GAC CAG GCT GAA ACT GCA GGT GCT 1320
Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala
385 390 ~95
CGT CTG GTT GTT C~G GCT ACT GCT ACT CCG CCG G5T TCT GTT ACT GTT 1368
Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val` Thr Val

- PCTNS 92/07188
2~ ~O/~s 23 nEC ~992


400 405 410
CCG CAC CCG AAC ATC GAA GAA GTT GCT CTG TCG ACT ACT GGT GAA ATC 1416
Pro Hi~ Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile
415 420 425
CCG TTC TAs- GGT AAA GCT ATC CCG CTC GAG GTT ATC AAA GGT GGT CGT 14 6 4
Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Arg
430 435 440 445
CAC CTG ATT TTC TGC CAC TCT AAA AAA AAA TGC GAC GAA CTG GCT GCT 1512
~is Leu I le Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala
450 455 460
AAG CTT GTT GCT CTG GGT ATC AAC GCT GTT GCT TAC TAC CGT GGT Cl~ 15 6 0Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala Tyr l~r Arg Gly Leu
465 470 ~ 475
GAC GTT TCT GTT ATC CCG ACT TCT GGT GAC GTT GTT GTT GTG GCC ACT i 6 0 8
Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val VA1 Val Val Ala Thr
480 485 490
GAC GCT CTG ATG ACT GGT TAC ACT GGT GAC TTC GAC TCT GTT ATC GAT 16 5 6A~p Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp
495 500 505
TGC AAC ACT TGC AAT TCG TCG ACC GGT TGC Gl~ GTT ATC GTT GGT CGT 17 0 4Cys Asn Thr Cys Asn Ser Ser Thr Gly Cys Val Val Ile Val Gly Arg
510 515 520 525
GTT GlT C:TG TCT GGT AAA CCG BCC Aq~ ATC CCG GAC CGT GAA Gl~ CTG 17 52
~al Val Leu Sex Gly Lys Pro Ala Ile Ile Pro Asp Ar~ Glu Val L~u
, 530 535 540
TAC CGT GAG TTC GAC GAA ATG GAA GAA 5Y~C TCT CAG CAC CTG CCG TAC 18 0 0Tyr Arg Blu Phe Asp Glu Met Glu Glu Cy~ Ser Gln His Leu Pro Tyr
545 550 555
ATC GAA C~G GGT ATG ATG CTG BCT GAA CAG TTC AAA CAG AAA GCT CT& 1848
Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu
560 56~ 570
GGT CTB CTG CAG ACC GCT TCT CGT CAG GCT GAA GTT ATC GCT CCG GCT 1896
Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala Glu Val Ile Ala Pro Ala
575 580 585
GTT CAG ACC AAC TGG CAG AAA CTC GAG ACC TTC TGG GCT AAA CAC ATG 19 4 4Val Gln Thr Asn Trp Gln Lys Leu Glu Thr Phe Trp Ala Lys His Met
590 595 600 605
TGG AAC TTC ATC TCT GGT ATC CAG TAC CTG GCT GGT CTG TCT ACC CTG l 9 9 2
Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu
610 615 620
CCG GGT AAC CCG GCT ATC GCA AGC TTG ATG GCT TTC ACC GCT GCT GTT 2 0 4 0

PCTIUS 92/0718~
12b 1~0/l~S 23 DEC 1992

58
Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala Val
625 630 635
ACC ICT CCG CTG ACC ACC TCT CAG ACC CTG CTG TTC AAC ATT CTG GGT 2088
m r Ser Pro Leu Thr Thr Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly
640 645 650
GGT TGG GTT GCT GCT CAG CTG GCT GCT CCG GGT GCT GCT ACC GCT TTC 2136
Gly Trp Val Ala Ala Gln Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe
655 660 665
GTT GGT GCT GGT CTG GCT GGT GCT GCT ATC GGT TCT GTA GGC CTG GGT 2184
Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu Gly
670 675 680 685
AAA GTT CTG ATC GAC ATT CTG GCT GGT TAC GGT GCT GGT GTT GCT GGA 2 2 3 2
Lys Val Leu Ile Asp Ile Leu Ala Gly Tyr Gly Ala Gly V~l Ala Gly
690 695 700
GCT CTG GTT GCT TTC AAA ATC ATG TCT GGT GAA GTT CCG TCT ACC GAA 2 2 80Ala Leu V~l Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu
705 710 715
GAT CTG GTT AAC CTG CTG CCG GCT ATC CTG TCT CCG GGT GCT CTG GTT 2 3 2 8
Asp L u Val Asn Leu Leu Pro Ala Ile L~u Ser Pro Gly Ala Leu 'Jal
720 725 730
~TT G~T GTT GTT TGC GCT GCT ATC CTG CGT CGT CAC GTT GGC CCG GGT 2 3 7 6
Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly
735 740 7~5
GAA G~T GCT GTT CAG TGG ATG AAC CGT CTG ATC GCT TTC GCT TCT CGT 2424
Glu Gly Ala V~l Gln Trp Met A~n Ary Leu Ile Ala Phe Ala Ser Arg
750 755 76~ 765
GGT AAC CAC GTT TCT CCA TGG GAT CCT CTA GAC TGC AGG CAT GCT AAG 2472
Gly A~n ~is Val S~r Pro Trp A~p Pro Leu Asp Cys Arg Hi~ Ala Lys
770 775 780
TAAGTAGATC lTGAtCBCGT TCGCGCTGAA ATGCGCTAAT TTCACTTCAC GACA~`TTCAG 2532
CCAATTTT~;;G GAGGAGTGTC GTACCGTTAC GAT m CCTC AAT m TCTT TTCAACAATT 2 5 9 2
GATCTCATTC AGGTGACATC T m ATATTG GCGCTCATTA TGAAAGCAGT AGCTTTTATG 2652
AGGGTAATCT GAATGGAACA GCTGCGTGCC GAATTAAGCC A m ACTGGG CGAAAAACTC 2712
AGTCGTATTG AGTGCGTCAA TGAAAAAGCG GATACGGCGT TGTGGGCTTT GTATGACAGC 2 7 7 2
CAGGGAAACC CAATGCCGTT AATGGCAAGA AGCTTAGCCC GCCTAAT~AG CGGGCTTTTT 2832
TTTCGACGCG AGGCTGGATG GCCTTCCCCA TTATGATTCT TCTCGCTTCC GGCGGCATCG 2892
GGATGCCCGC GTTGCAGGCC ATGCTGTCCA GGCAGGTAGA TGACGACCAT CAGGGACAGC 2952

` - , PCTIUS 92/0718~.
i 1 v 8 ~b K0/ IJ S 2 3 DEC l992

ss
TTCAAGGATC GCTCGCGGCT CTTACCAGCC TAACTTCGAT CACTGGACCG CTGATCGTCA 3012
CGGCGA m A TGCCGCCTCG GCGAGCACAT GGAACGGGTT GGCATGGATT GTAGGCGCCG 3072
CCCTATACCT TGTCTGCCTC CCCGCGTTGC GTCGCGGTGC ATGGAGCCGG GCCACCTCGA 3132
CCTGAATGGA AGCCGGCGGC ACCTCGCTAA CGGATTCACC ACTCCAAGAA TTGGAGCCAA 3192
TCAATTCTTG CGGAGAACTG TGAATGCGCA AACCAACCCT TGGCAGAACA TATCCATCGC 3252
GTCCGCCATC TCCAGCAGCC GCACGCGGCG CATCTCGGGC AGCGTTGGGT CCTGGCCACG 3312
GGTGCGCATG ATCGTGCTCC TGTCGTTGAG GACCCGGCTA GGCTGGCGGG GTTGCCTTAC 3372
TGGTTAGCAG AATGAATCAC CGATACGCGA GCGAACGTGA AGCGACTGCT GCTGCAAAAC 3432
GTCTGCGACC TGAGCAACAA CATGAATGGT CTTCGGTTTC CGTG m CGT AAAGTCTGGA 3492
AACGCGGAAG TCAGCGCCCT GCACCATTAT GTTCCGGATC TGCATCGCAG GATGCTGCTG 3552
GCTACCCTGT GGA.~CACCTA CATCTGTATT AACGAAGCGC TTCTTCCGCT TCCTCGCTCA 3612
CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG 3672
TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC 3732
AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTT m CCAT AGGCTCCGCC 3792
CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC 3852
TATAAAGATA CCAGGCG m CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC 3912
TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG C m CTCAAT 3972
GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC 4032
ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA 4092
ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG 4152
CGAGGTATGT AGGCGGTGCT ACAGAGTTCT T~AAGTGGTG GCCTAACTAC GGCTACACTA g212
GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG 4272
GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC 4332
AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATC m TCTACGGGGT 4392
CTGACGCTCA GTGGAACGA~ AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA 4g52
GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAG m TAAATCAATC TAAAGTATAT 4512
ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA 4572
TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC 4632

,~

PC~IJS 92/07188
6 ~O/US 23 0~C 1992



GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CA~TGATACC GCGAGACCCA CGCTCACCGG 4692
CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG ~752
CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT 4812
CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT 4872
CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT 4932
CCCCCATGTT ~-TGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA 4992
AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA 5052
TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT 5112
AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAC ACGGGATAAT ACCGCGCCAC 5172
ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA 5232
GGATCTTACC GCTGTIG~GA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT 5292
CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG 5352
CAAAAAAGOG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT 5412
ATTATTGAAG CA m ATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT 5~72
AGAAAAATAA ACAAAT~GGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT 5532
AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC 5592
GTCITCAA 5600

(2) INFORMATION FOR SEQ ID NO:9:
(~) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~81 amino acids
~) TYPE: amino acid
(D) TOPOLOGY: linear
~ii) MOLECVLE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Ser Phe VA1 Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 lS
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

` - , PCTIUS 92/0718 8
iJ~b BOJIS ~3 OEC 1992

61

Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala

Glu Val Val Glu Lys Cys Ala Phe Ser Asp Acp Thr Val Ile Val Asn

Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 - 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
Val Lèu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
Pro TSp A~p Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
Arq Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 ~05
Leu Glu Gln Leu Arg V~l Leu Trp Tyr Gly Glu Lys Ile Hi~ Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Ihr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
Asp Pro S~r Thr Asn Ser Met Ala Val A~p Phe Ile Pro Val Glu Asn
245 250 255
Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro
260 265 270
Pro Val Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr
275 ~80 285
Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly
290 295 300
Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe
305 310 315 320
Gly Ala Tyr Met Ser Lys Ala His Gly Ile A.~p Pro Asn Ile Arg Thr
325 330 ~335

PCTIUS 92/07188
O/US 23 OEC 1992

62

Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr
3gO 345 350
Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile
355 360 36~
Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly
370 375 380
Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val
385 390 395 ~00
al Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro
~05 410 ~15
n Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr
420 425 g30
Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Arg His Leu Ile
435 ~gO 445
Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val
450 455 460
Al~ Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser
465 470 475 480
al Ile Pro mr Ser Gly A~p Val Val Val Val Ala Thr Asp Ala Leu
485 g90 495
Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr
' 500 505 510
Cys Asn Ser Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu
~15 520 525
Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu
530 535 540
Phe Asp alu Met Glu Glu Cy8 Ser Gln Hi~ Leu Pro Tyr Ile Glu Gln
545 550 555 560
ly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu
565 570 575
ln Thr Ala Ser Arg Gln Ala Glu Val Ile Ala Pro Ala Val Gln Thr
580 585 590
Asn Trp Gln Lys Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe
595 600 605
Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn
610 615 620

PCTIUS 92/07188
;~,113!J~ O/l~S 23 DEC t

63
Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro
625 630 635 640
e~ Thr Thr Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val
6~5 650 655
la Ala Gln Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala
660 665 670
Gly Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu Gly Lys Val Leu
675 680 685
lle A~p Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu VA1
690 695 700
Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu A~p Leu Val
705 710 ~15 720
sn 'eu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val
725 730 735
al Cys Al~ Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala
740 745 750
Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His
755 760 765
Val Ser Pro Trp Asp Pro Leu Asp Cys Arg His Ala Lys
770 775 780

(~) INFOKMATION FOR SEQ ID NO:10:
(i~ SEQU~NCE CHARACTFRISTICS:
(A) LENGTH: 1548 base pair~
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: ~ingle
(D~ TOPOLOGY: circular
(ii) MO~CULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1548

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
ATG AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CG~ CTG 4 8
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
CCC GGT AAA CCA TTG GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 9 6
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His


- PCTIUS 92/07188
~ i l J ~ ~ ii BO/ ~ S 2 3 OEC 1~2

6 4

GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GTG GCA 144
Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 g5
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192
Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
50 55 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
Val Cys Mot Thr Arg Ala Asp His Gln Ser Gly Thr Glu Ar~ Leu Ala
65 70 75 80
GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288
Glu Val Val Glu Lys Cys Ala Phe Ser Asp A~p Thr Val Ile Val Asn
85 90 95
GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
GCT GAT AA~' CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
A1A A~p A~l Leu Ala Gln Arq Gln Val Gly Met Ala Thr Leu Ala Val
llS 120 1~5
CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG 432
Pro Il- Ni~ Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
GTT CTC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
V~l L~u Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
CCT Tr~G ~AT CGT GAT.CGT m GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro Txp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC m ATC 576
Asn Phe L-u Arg Hia Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
'. 180 185 190
, . .
CGT ~GT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 ~05
TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 672
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile Hiq Val Ala
210 215 220
GTT GCT CAG GAA GTT CCT GGC.ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
GAC CCG TCG ACG AAT TCC CCA TGG ACC CAC TAC GTT CCG GAA TCT GAC 768
Asp Pro Ser Thr Asn Ser Pro Trp Thr His Tyr Val Pro Glu Ser Asp


, ~ .

` - PCTIUS 92/071 8 8 .
~ l L J ~ ''li ~ 0 / ~ S 23DECl~


245 250 255
GCT GCT GCT CGA GTT ACC GCT ATC CTG TCT TCT CTG ACC GTT ACC CAG 816
Ala Ala Ala Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln
260 265 270
CTT CTG CGT CGT CTG CAC CAG TGG ATC TCT TCT GAA TGC ACC ACC CCG 864
Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro
275 280 - 285
TGC TCT GGT TCT TGG CTG CGT GAC ATC TGG GAC TGG ATC TGC GAA GTT 912
Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu VA1
290 295 300
CTG TCT GAC TTC AAA ACC TGG CTG AAA GCT AAA CTG ATG CCG CAG CTG 960
Leu Ser A~p Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln ~eu
305 310 ~15 320
CCG GGT ATC CCG TTC GTT TCT TGC CAG CGT GGT TAC AAA GGT GTT TGG 1008
Pro Gly Ile Pro Phe V~l Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp
325 330 335
CGT GTT GAC GGT ATC ATG CAC ACC CGT TGC CA TGC GGT GCT GAA ATC ~ 1056
~rg V~l Asp Gly Ile Met His Thr Arg Cy8 Hig Cy~ Gly Ala Glu Ile
340 345 350
ACC GGT CAC GTT AAA AAC GGT ACC ATG CGT ATC GTT GGT CCG CGT ACC 1104
Thr Gly His Val Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr
355 360 365
TGC CGT AAC ATG TGG TCT GGC ACC TTC CCG ATC AAC GCT TAC ACC ACC 1152
Cys Arg Asn Met Trp Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr
370 375 380
GGT CCG TGC ACC CCG CTG CCG GCT CCG AAC TAC ACC TTC GCT CTG TGG . 1200
~ Gly Pro Cy~ Thr Pro Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp
3~ 385 390 395 ~00
CGT GTT 5C~ GCT GAA GAA ~AC GTT GAA ATC CGT CAG GTT GGT GAC TTC 12g8
Arg V~l ~ ff Ala Glu Glu Tyr Val C-lu Ile Arg Gln V~1 Gly Asp Phe
~- qO5 410 415
CAC TAC ~TT ACC GGT ATG ACC ACC GAC AAC CTG AAA TGC CCG TGC CAG 1296
His Tyr Val Thr Cly Met Thr Thr Asp Asn Leu Lys Cy~ Pro Cya Gln
420 425 430
GTT ~CG TCT CCG GAG TTC TTC ACC GAA CTG GAC GGT GTT CGT CTG CAC 1344
Va1 PrO Ser Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His
435 440 445
CGT TIC GCT CCG CCG TGC AAA CCG CTG CTG CGT GAA GAA GTT TCT TTC 1392
Arg Phe Ala Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe
450 455 460
C5T GTT GGT CTG CAC GAA TAC CCG GTT GGT TCT CAG CTG CCG TGC GAA 1440

PCTNS 92/0718 8
KO/US 2 3 ~EC 1992

66

Arg Val Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro Cy9 Glu
465 470 475 480
CCG GAA CCG GAC GTT GCT GTT CTG ACC TCT ATG CTG ACC GAC CCG TCT 1488
Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser
g85 490 495
CAC ATC ACC GCT GAA GCT GCT GGT CGT CGA CTG GAT CCT CTA GAC TGC 1536
His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu Asp Pro Leu Asp Cys
500 505 510
AGG CAT GCT AAG 1548
Arg His Ala Lys
515

(2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 516 amino acids
(B) TYPE: amino acid
lD) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N~:ll:
Met Ser Phe Val Val Ile Il~ Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val Hi~

V~l Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr Asp His Glu Asp VA1 Ala Arg Ala Yal Glu Ala Ala Gly Gly Glu

~ .
Val Cys ~ ~hr Arg Al~ Asp His 21n Ser Gly Thr Glu Arg Leu Ala
65 ~ 70 75 ~ 80
Glu Val Val Glu Ly~ Cys Ala Ph~ Ser A~p Asp Thr Val Ile Val Asn
85 90 95
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile

&TIIJS 92fO7188
,~ S 2 3 OEC 1992

67

lqS 150 155 160
ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
rg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
sp Pro Ser Thr Asn Ser Pro Trp Thr His Tyr Val Pro Glu Ser Asp
245 250 255
l~ Ala Al~ Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln
260 265 270
eu Leu Arg Arg ~-eu Hi~ Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro
275 280 285
;Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val
2~0 295 300
Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala T-yc Leu Met Pro Gln Leu
305 310 315 320
ro Gly Ile Pro Phe Val S~r Cy_ Gln Arg Gly Tyr Lys Gly Val Trp
3~5 330 335
r~ Val A~p Gly Ile ~e~ His Thr Arg Cyq His Cyq Gly Ala Glu Ile
340 345 350
hr Gly ~ ~ ~1 Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr
3Ss 360 3~5
Cys Arg As~ Met Trp Ser Gly Thr Phe PrG Ile Asn Ala Tyr Thr Thr
370 375 380
Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp
385 390 395 gO0
rg Val Ser Ala Glu Glu Iyr Val Glu Ile Arg Gln Val Gly Asp Phe
405 410 415 .
is Tyr Val Thr Gly Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln
420 425 430
A1 Pro Ser Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His
g35 g40 445

PCT/IJS 9 2/ 0718 8
JJ ~l3 l~O/lJS 23 DEC l992

68

Arg Phe Al~ Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe
g50 455 460
Arg Val Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro cys Glu
465 g70 475 480
Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr A~p Pro Ser
g85 490 495
His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu Asp Pro Leu Asp Cys
500 S05 510
Arg His Ala Lys
515

(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1623 b~se pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
- (B) LOCATION: 1.... 1623

(xi) SEQUENCE DESC~IPTION: SEQ ID NO:12:
ATG A~T TTT GIG GTC ATT ATT ecc GCG CGC TAC GCG TCG ACG CGT CTG 48
Mst Ser Ph~ Val Val Ile Ile Pro Ala Arg Tyr Al~ Ser Thr Arg Leu
1 5 10 15
CCC CGT Ah~ CCA T~G GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 96
Pro Gly Ly~ Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His
20 25 30
GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GTG GCA 144
Val Leu Glu ~rg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 45
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192
Thr Asp His Glu Aqp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
50 5S 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
V~l Cys Met Thr Arg Ala Asp Hiq Gln Ser Gly Thr Glu Arg Leu Al~
65 70 75 80
GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GT~ AAT 288

PCT/U~ ~2/0718 8
26 R0/US 23 D~ 1992

69
Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val A~n

GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG 432
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
GTT CTC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
CCT TGG GAT CGT GAT CGT TTT GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro Trp A~p Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 . 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 576
A n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC TGG CAG CCA A~T CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
TTA GAG CAG CTT CGT GTT CIG TGG TAC GGC GAA AAA ATC CAT GTT GCT 672
Leu Glu Gln Leu Ar~ Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Ala Gln Glu V~l Pro Gly Thr Gly Val Aqp Thr Pro Glu Asp Leu
225 230 235 240
GAC CCG TCG ACG AAT TCT ATG CGT CGA CTG GCT CGT GGT TCT CCG CCG 768
Asp Pro Ser Thr Asn Ser Met Arg Arg Leu Ala Arg Gly Ser Pro Pro
245 250 255
TCT GTT GCT TCT TCT TCT GCT TCT CAA CTG TCT GCT CCG TCT CTG AAA 816
Ser Val Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys
260 265 270
GCT ACC TGC ACC GCT AAC CAC GAC TCT CCG GAC GCT GAA CTG ATC GAA 864
Ala Thr Cys Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu
275 280 285
GCT AAC CTG CTG TGG CGT CAG GAA ATG GGT GGT AAC ATC ACC CGT GTT 912
Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val
290 295 300

`~` i~i PC~lUS 92/07 1 8 8
R0/IJS 23 DEC l9g.



GAA TCT GAA AAC AAA GTT GTT ATC CTG GAC TCT TTC GAC CCG CTG GTT 960
Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Asp Pro Leu Val
305 310 315 320
GCT GAA GAA GAC GAA CGT GAG ATC TCT GTT CCG GCT ~-AA ATC CTG CGT 1008
Ala Glu Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg
325 330 335
AAA TCT CGT CGT TTC GCT CAG GCT CTG CCG GTT TGG GCT CGT CCG GAC 1056
Lys Ser Arg Arg Phe Ala Gln Ala Leu Pro Val Trp Ala Arg Pro Asp
340 345 350
TAC AAC CCG CCG CTG GTT GAA ACC TGG AAA AAA CCG GAC TAC GAA CCG 1104
Tyr Asn Pro Pro Leu Val Glu Thr Trp Lys Lys Pro A~p Tyr Glu Pro
355 360 365
CCG GTT GTT CAC GGT TGC CCG CTG CCG CCG ~CG AAA TCT CCG CCG GTT 1152
Pro Val Val His Gly Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro Val
370 375 380
CCG CCG CCG CGT AAA AAA CGT ACC GTT GTT CTG ACC GAA TCT ACC CTG 1200
Pro Pro Pro Arg Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu
385 390 395 400
TC~ ACC GCT CTG 5CT GAA CTG GCT ACC CGT TCT TTC GGT TCT TCT TCT 1248
Ser Thr Ala Leu Ala Glu Leu A1A Thr Arg Ser Phe Gly Ser Ser Ser
405 410 415
ACC TCG GGT ATC ACC GGT GAC AAC ACC ACC ACC TCT TCT GAA CCG GCT 1296
Thr Ser Gly Ile Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala
42~ 425 g30
CCG TCT GGT TGC CCG CCG GAC TCT GAC GCT GAA ICT TAC TCT ~CT ATG 13g4
Pro Ser Gly Cys Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser Met
435 440 445
CCG CCG CTG GAA GGT GAA CCG GGT GAC CCG GAT CTG TCT GAC GGT TCT 1392
Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro A8p Leu Ser Asp Gly Ser
450 455 ~60
TGG TCT ACC GTT TCT TCT GAA GCT AAC GCT GAA GAC GTT GTT TGC TGC 1440
Trp Ser Thr Val Ser Ser Glu Ala Asn Ala Glu Asp Val Val Cys Cys
465 470 475 480
TCT ATG TCT TAC TCT TGG ACC GGT GCT CTG GTT ACT CCG TGC GCT GCT 1488
Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala
485 490 495
GAA GAA CAG AAA CTG CCG ATC AAC GCT CTG TCT AAC TCT CTG CTG. CGT 1536
Glu Glu Gln Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg
S00 505 510
CAC CAC AAC CTG GTT TAC TCT ACC ACC TCT CGT TCT GCT TGC CAG CGT 1584
His His Asn Leu Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln Arg
515 520 525

PCT/US 92/07188
O/ljS 2 3 DEC 1992




CAG AAA AAA GTT ACC TTC GAC CGT CTG CAA GTT CTA GAC 1623
Gln Ly~ Lys Val Thr Phe A~p Arg Leu Gln Val Leu Asp
530 535 540

(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 541 amino acids
(B~ TYPE: amino acid
(D) TOPOLOGY: linear
(,i) MOLECULE TYPE: pro~ein

(xi) SEQUENCE DESCRIPTION:. SEQ ID NO:13:
et Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
ro Gly Lys Pro Lau ~al Asp Ile Asn Gly Lys Pro Met Ile Val His

Val Leu Glu Ar~ Ala Arg Glu Ssr Gly Ala Glu Arg Ile Ile Val Ala

mr Asp Hi3 Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cy~ Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
lu Val Val Glu Lys Cys Al~ Phe Ser Asp Asp Thr Val Ile Val A~n
~l Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp AJn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 1~5
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
Val Leu A~p Ala Glu Gly Tyr-~la Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
ro Trp A~p Arg A~p Arg Phe Ala Glu Gly Leu Glu Thr Val Gly. Asp
165 170 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
rg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met

PC~IUS 92/07188
2~ 120/US 23 OEC ~992

72

l9S 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu L~s Ile His Val Ala
210 2~5 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
sp Pro Ser Thr Asn Ser Met Arg Arg Leu Ala Arg Gly Ser Pro Pro
245 250 255
er Val Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys
260 265 270
Ala Thr Cys Thr Ala Asn Hiq Asp Ser Pro A~p Ala Glu Leu Ile Glu
275 280 285
Al s A~n Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val
290 295 300
Glu Ser Glu Asns Lys Val Val Ile Leu Asp Ser Phe A~p Pro Leu Val
305 310 315 320
las Glu Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg
325 330 335
ys Ser Arg Arg Phe Alas Gl~s Ala Leu Pro Val Trp Al~ Arg Pro Asp
340 345 350
Tyr A~s Pro Pro Leu Val Glu Thr Trp Ly~ Lys Pro Asp Tyr Glu Pro
355 360 365
Pro Val Val Hi~ Gly Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro V~l
370 , 375 3ao
Pro Pro Pro Asrg Ly3 Ly~ Arg Thr Val Vasl Leu Thsr Glu Ser Thr Leu
385 390 395 400
er I~Sr Ala Leu Ala Glu Leu Ala Thr Arg Ser Phe Gly Ser Ser Ser
405 410 415
hr Ser Gly Ile Thr Gly Asp Asn Thr l~.Sr Thr Ser Ser Glu Pro Ala
420 425 430
Pro Ser Gly Cys Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser Met
435 440 4gS
Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser
g50 455 460
Trp Ser Thr Val Ser Ser Glu Ala Asn Ala Glu Asp Val Val Cys Cys
465 470 475 ~80
Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala
g85 g90 ~9~

"~.TlUS ~ Z l O 7 1 8 8
i ,j ~ v/ ~ S 2 3 OEC 1992




lu Glu Gln Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg
500 505 510
His His Asn Leu Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln Arg
S15 520 525
Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Yal Leu Asp
530 535 540

(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1488 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single ~-
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1488

~xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ATG AGT TTT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG ~8
Met S~r Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
CCC GGT AAA CCA T~G GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 96
Pro Gly Lys Pro Leu Val Acp Ile Asn Gly Ly~ Pro Met Ile Val His
20 25 30
GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GT~ GCA 14~ -Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 ~5
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192
Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
50 55 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
Val Cys Met Thr Arg Ala Asp ~is Gln Ser Gly Thr Glu Arg Leu Ala
65 ~0 75 80
GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288
Glu Val Val Glu Lys Cys Ala-Phe Ser Asp Asp Thr Val Ile Val Asn
85 30 95
GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
VA1 Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110

PCT/US 9210718 8
12 0/ U S - 2 3 OEC- 1992



GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG 432
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
GTT CTC GAC GCT GAA GSG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
V~l Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
CCT TGG GAT CGT GAT CGT TTT GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 . 170 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 5?6
Asn Phe Lou Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
TTA GAG CAG CTT CGT GTT CTG TGG TAC G -C GAA AAA ATC CAT GTT GCT . 672
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Ly~ Ile His Val Ala
21~ 215 220
GTT 5CT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
YA l Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 23~ 240
GAC CCG TCG ACG AAT -TCT CTA GAC TCC CAC TAC CAG GAC Gl~ CTG AAA 768
Asp Pro Ser Thr Asn Ser Leu Asp Ser His Tyr Gln Asp Val Leu Lys
-~ 245 250 255
GAA GTT AAA ~CT GCT GCT TCT AbA GTT AAA GCT AAC CTG CTG TCT GTT 816
Glu Val Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val
260 265 270
.
GAA GAA GCA TGC TCT CT& ACC CCG CCG CAC TCT GCT AAA TCT AAA TTC 864
Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Ly~ Ser Lys Phe
275 280 285
GGT TAC GGT GCT AAA GAC GTT CGT TGC CAC GCT CGT AAA GCT GTT ACC 912
Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Thr
290 295 300
CAC ATC AAC TCT GTT TGG AAA-GAT CTG CTG GAA GAC AAC GTT ACC CCG 960
His Ile A~n Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr Pro
305 310 315 320
ATC GAC ACC ACC ATC ATG GCT AAA AAC GAA GTT TTC TGC GTT CAG CCG ~008
Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro
.
'

PCTIUs 92/07188
w~ 2~i ~0/lJS 23 OC l992


325 330 335
GAA AAA GGT GGT CGT AAA CCG GCT CGT CTG ATC GTT TTC CCG GAC CTG 1056
Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile V~l Phe Pro Asp Leu
340 345 350
GGT GTT CGT GTT TGC GAA AAA ATG GCT CTG TAC GAC GTT GTT ACC AAA 1104
Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr Lys
355 360 365
CTG CCG CTG GCT GTT ATG GGT TCT TCT TAC GGT TTC CAG TAC TCT CCG 1152
Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gln Tyr Ser Pro
370 375 380
GGT CAG CGT GTT GAG TTC CTG GTT CAG GCT TGG AAA TCT AAA AAA ACC 1200
Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Lys Ser Lys Lys Thr
385 390 ~95 400
CCG ATG GGT TTC TCT TAC GAC ACC CGT TGC TTC GAC TCT ACC GTT ACC 1248
Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr
g05 . 410 415
GAA TCT GAC ATT CGT ACC GAA GAA GCT ATC TAC CAG TGC TGC GAC CTG 1296
Glu Ser A~p Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp Leu
420 425 430
GAC CCG CAG GCT CGT GTT GCT ATC AAA TCT CTG A~C GAA CGT CTG TAC 134g
A~p Pro Gln Ala Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr
435 440 ~5
GTT GGT GGT CCG CTG ACC AAC TCT CGG GGT G~A A~C TGC GGT TAC CGT 1392
Val Gly Gly Pro Leu ThF Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg
450 455 460
CGT TGC CGT GCT TCT GGT GTT CTG ACC ACC TCT TGC GGT AAC ACC CTG - 1440
~ Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cy~ Gly Asn Thr Leu
F 465 470 ~75 480
ACC IGC ~AC ATC AAA GCT CGT GCT GCT TGC CGT GCT GCT GGT CTG CAG 1488
Thr Cy8 Tyr Ile Lys Ala Arg Ala Ala Cy Arg Ala Ala Gly Leu Gln
485 g90 ~95

(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS: -
(A) LENGTH: 496 amino acids
(B~ TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

PCT/US 9 2 / 0 7 1 8 E
O/ ~ S 2 3 OEC Ig92


Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His
al Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val A3n
~5 90 95
al Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala A p Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro A~n Ala Val Lys Val
130 135 lgO
Val Leu Asp Ala Glu Gly Iyr Ala Leu ~yr Phe Ser Arg Ala Thr Ile
145 i50 155 160
ro TSp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 17~ 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 1~0
Arg Arg ~yr V~l Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 2~0
sp Pro Ser Thr Asn Ser Leu Asp Ser His Tyr Gln Asp Val Leu Lys
245 250 255
lu Val Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val
260 265 270
Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe
275 280 285
Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Thr

PCT/US 92/0718
~`2~ ~O/US 23 DEC 1992


290 295 300
His Ile Asn Ser Val Trp Lys Asp L~u Leu Glu Asp Asn Val Thr Pro
305 310 315 320
Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro
325 3~0 335
Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu
340 345 350
Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr Ly~
355 360 365
Leu Pro Leu Ala Vai Met Gly Ser Ser l~r Gly Phe Gln ryr Ser Pro '-
370 375 380
Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Ly~ Ser Ly~ Lys Thr
385 390 395 400
Pro Met Gly Phe Ser Tyr A~p Thr Arg Cys Phe Asp Ser Thr Val Thr
405 410 gl5
Glu Ser Asp Ile Arg ~r Glu Glu Ala Ile l~r Glsl Cys Cys Asp Leu
~20 425 430
Asp Pro Gln Al~ Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu ryr
435 440 4g~
Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg
450 ~55 460
Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu
465 ~ 470 475 480
Thr Cys Tyr Ile Lys Ala Ars~ Ala Ala Cys Arg Ala Ala Gly Lou Gln
485 490 495

(2) INFO~5ATION FOR SEQ ID NO: 16:
( i ) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 1161 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
~ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
( A ~ NAME/KEY: CDS
(B) LOCATION: 1..1161

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

PCTIUS 9 2 / 0 7 1 8 8
;'.L.l~ 6 B0/1~S 23 DEC ~992

78

ATG AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 4 8
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
5 10 15
CCC GGT AAA CCA rrG GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 9 6
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His
20 25 30
GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GTG GCA 14 4
Val Leu Glu Arg Ala Arg Glu Ser Gly Alzl Glu Arg Ile Ile Val Ala
35 40 45
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 19 2
Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
50 55 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 2 4 0
Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
65 70 75 80
GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC AC~: GTG ATC GTT AAT 2 8 8
Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val ~le V~l A~n
85 90 95
GTG CAG GGT GAT GAA CCG Aq G ATC CCT GCG ACA ATC ATT CGT CAG GTT 3 3 6
Val Gln Gly Asp Glu Pro Met Ile Pro Al~ Thr Ile Ile Arg Gln Val
100 105 110
GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 3 8 4
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG m AAC CCG AAT GCG GTG AAA Gl~ 4 3 2
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro A~;n Al~ Val Ly~ Val
130 135 140
GTT CTC GAC GCT GAA GGG TAT GrA CTG TAC TTC TCT CGC GCC ACC ATT 480
Val Leu A~p Ala Glu Gly Tyr Ala Leu Iyr Phe Ser Arg Ala Thr Ile
145 150 155 160
CCT TGG GAT CGT GAT CGT m GCA GAA GGC crr GAA ACC GTT GGC GAT 5 2 8
Pro Trp Asp Arg A~p Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 57 6
Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 6 2 4
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTr GCT 6 7 2
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala

PCTIUS 9 2/ 07 1 8 a
26 ~O/US 23 DEC 1992

7 9

210 215 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Al~ Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu A~p Leu
225 230 235 240
GAC CCG TCG ACG AAT TGC ATG CTG CAG GAC TGC ACC ATG CTG GTT TGC 768
Asp Pro Ser Thr Asn Cys Met Leu Gln Asp Cys Thr Met Leu Val Cys
245 250 255
GGT GAC GAC CTG GTT GTT ATC TGC GAA TCT GCT GGT GTT CAG GAA GAC 816
Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Val Gln Glu Asp
260 265 270
GCT GCT TCT CTG CGT GCT TTC ACC GAA GCT ATG ACC CGT TAC TCT GCT 864
Al~ Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala
275 2a0 ~ 285
CCC CCG GGT GAC CCG CCG CAG CCG GAA TAC GAC CTG GAA CTG ATC ACC 912
Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr
290 295 300
TCT ~GC TCT TCT AAC GTT TCT GTT GCT CAC GAC GGT GCT GGT AAA CGT 960
Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys Arg
305 310 315 320
GTT TAC TAC CTG ACC CGT GAC CCG ACC ACC CCG CIG GCT CGT GCT GCT 1008
V~l Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala
325 330 335
TGG GAA ACC GCT CGT CAC ACC CCG GTA AAC TCT TGG CTG GGT AAC ATC 1056
Trp Glu Thr Al~ Arg His Thr Pro V~l Asn Ser Trp Leu Gly Asn Ile
340 345 35Q
ATC ATG TTC GCT CCG ACC CTG TGG GCC CGT ATG ATC CTG ATG ACC CAC 1104
- Ile Met Phe Al~ Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His
J 355 36~ 365
TTC TTC TCT GTT CTG ATC GCT CGT GAC CAG CTG GAA CAG GCT CTG GAC 1152
Phe Phe S-r Val Leu Ile Ala Arg Asp Gln Leu Glu Gln Ala Leu Asp
370 375 380
TGC GAG ATC 1161
Cys Glu Ile
385

~2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 387 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

PCTIUS 92/ 07 1 8 8
J~ O~S 2 3 DE~ 1992



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Ser Phe Val Val Ile Ile Pro Ala Ar~ Tyr Ala Ser Thr Arg Leu
1 5 10 15
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cys Met Thr Arg Ala Asp His Gln Ser ~ly Thr Glu Arg Leu Ala

Glu V~l Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val A~n
. 90 95
Val Gln Gly Asp Glu Pro ~et Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
Pro Ilo His A~n Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
~ 130 135 140
Val Leu Asp Ala Glu Gly Tyr Ala Lou Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
Pro Trp Asp Arg A~p Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
- ~0 185 190
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Me~
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
V~l Al~ Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
A~p Pro Ser Thr A~n Cys Met Leu Gln Asp Cys Thr Met Leu Val Cys
245 250 255
Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Val Gln Glu Asp
260 265 270

P~ U~ ~ ~ / U 7 1 8 8
2G l~O/~S 23 DEC ~992

81

Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Aln
275 280 285
Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr
290 2~5 300
Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys Ars~
305 310 315 320
al Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala
325 330 335
rp Glu Thr Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile
340 345 350
Ile Met Phe Ala Pro Thr Leu Trp Ala Axg Met Ile Leu Met Thr Hi~
355 360 365
Phe Phe Ser Val Leu Ile Ala Arg Asp Gln Leu Glu Gln Ala Leu Asp
370 375 380
Cys Glu Ile
385

(2~ INFORMI~TION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1179 base pairs
(~) TYPE: nucleic acid
( C ~ STR~NDEDNESS: g ingle
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA ~genomic)
(ix) FEATURE:
~A) NAME/~ CDS
( El ) LOCATION: 1. .1179

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
ATG AGT m GTG GTC Al~ Al~ CCC GCG CGC TAC GCG TCG ACG CGT CTG 48
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
5 10 15
CCC GGT AAA CCA TTG GTT GAT Al~ A~C GGC AAA CCC ATG ATT GTT CAT 96
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His
20 25 30
GTT CTT GAA CGC GCG CGT GAA . TCA GGT GCC GAG CGC ATC ATC GTG GCA 1 ~ 4
Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 . ~5
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192

i PCT/US 92/071a8
b ~O/~i 23 OEC 1992.


82

Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
So 55 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
65 70 75 80
GAA GTT GTC GAA AAA T~C GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288
Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn
85 90 95
GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG 432
P,ro Il- Hi~ Asn Al~ Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
GTT CTC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
Val L-u Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
CCT TGG GAT CGT GAT CGT TTT GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro ~rp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC TTC CTG CGT CAT CTT GGT ~TT TAT GGC TAC CGT GCA GGC m ATC 576
Asn Pho Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC IGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arq Arg Tyr V 1 Asn TSp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
TTA GAG C~C CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 672
Leu Glu Gln L~u Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
2I0 215 ~ 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
GAC CCG TCG ACG AAT TCC ATG GAG ATC TAC GGT GCT TGC TAC TCT ATC 768
Asp Pro Ser Thr Asn Ser Met Glu Ile Tyr Gly Ala Cys ~yr Ser Ile
24S 250 255
GAA CCG CTG GAC CTG CCG CCG ATC ATT CAG CGT CTG CAC GGT CTG TCT 816
Glu Pro Leu Asp Leu Pro Pro Ile Ile Gln Arg Leu His GIy Leu Ser
260 265 270


.

- - PCTIUS 9 2/ 07 1 8 8
;lia~ O/US ~3 DE~ 1992


GCT TTC TCT CTG CAC TCT TAC TCC CCG GGT GAA ATC AAC CGT GTT GCT 86i
Ala Phe S-r Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala
275 280 285
GCT TBC CTG CGT AAA CTG.GGT GTT CCG CCG CTG CGT GCT TGG CGT CAC 912
Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp Arg His
290 295 300
CGT GCT CGT TCT GTT CGT GCT CGT CTG CTG GCT CGT GGT GGC CGT GCT 960
Arg Ala Arg Ser Val Arg Ala Arg Leu Leu Ala Arg Gly Gly Arg Ala
305 310 315 320
GC~ ATC T&C GGT AAA TAC CTG TTC AAC TGG GCT GTT CGT ACC AAA CTG 1008
Ala Ile Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Arg m r Lys Leu
325 330 335
AAA CTG ACC CCG ATC GCT GCT GCT GGT CAG GTG GAC CTG TCT GGT TGG 1056
Lys Leu m r Pro Ile Ala Ala Ala Gly Gln Leu Asp Leu Ser Gly Trp
340 345 350
TTC ACC GC. GGT TAC TCT GGT GGT GAC ATC TAC CAC TCT GTT TCT CAC 1104
Ph Thr Al~ Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val Ser His
355 360 365
GCT CGT-CC6 OGT TGG ATC TGG TTC TGC CTG CTG CTG CTG GCT GCT GGT ' 1152
Al~ Arg Pro Arg Trp Ile Trp Phe Cys Leu Leu Leu Leu Ala Ala Gly
370 375 380
GTT CGT AT~ TAC CTG CTG CCG AAC CGT 117g
Val Gly Ile Tyr Leu Leu Pro Asn Arg
385 390
:
l2) INFO~MATION FOR SEQ ID NO:19:
.
(i) SEQUENCE CHAR~CTERISTICS:
~A) LENGTH: 3g3 amino acids
~B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Met Ser Phe Val Vai Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val- His

- Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr Asp His Glu Aqp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu


.. . _ . ... .. . . . . .. ..

PCTIUS 92/071 8
: l~J~2~ V/~- 23 DEC 1992

84


Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala

lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn
al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
la Asp A~n Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 ~25
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys V~l
130 135 140
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
1~0 185 190
rg Arg Tyr Val A~n Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val A1A Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 .230 235 240
sp Pro Ser Thr Asn Ser Met Glu Ile Tyr Gly Ala Cy8 Tyr Ser Ile
245 250 255
lu Pro L~u A~p L~u Pro Pro Ile Ile Gln Arg Leu Hi3 Gly Leu Ser
~60 265 270
Ala Phe Ssr Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala
275 280 285
Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp Arg His
-290 295 300
Arg Ala Arg Ser Val Arg Ala Arg Leu Leu Ala Arg Gly Gly Arg Ala
305 310 315 320
la Ile Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr Lys Leu
325 330 335
ys Leu Thr Pro Ile Ala Ala Ala Gly Gln Leu Asp Leu Ser Gly Trp
340 345 350

PCT/US 92/0718
_ .L ~ 0/ U S 2 3 o~c 1992



Phe Thr Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser V~l Ser Hi~
355 360 365
Ala Arg Pro Arg Trp Ile Trp Phe Cys Leu Leu Leu Leu Ala Ala Gly
370 375 380
V~l Gly Ile Tyr Leu Leu Pro Asn Arg
385 390

(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1791 b~se pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
~A3 N~ME~KEY: CDS
(B) LOCATION: 1..1791

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
ATG AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 48
Met Ser Ph~ Val Val Ile Ile Pro Ala Arg Tyr Al~ Ser Thr Arg Leu
5 10 1~
CCC GG~ AAA CCA TTG GTT GAT A~T AAC GGC AAA CCC ATG ATT GTT C~T 96
Pro Gly Lys Pro L~u Val A~p Ile A3n Gly Ly~ Pxo M~t Ile Val His
20 25 30
GTT CTT GA~ CGC ~CG rGT GAA TCA GGT GCC GAG C~C ATC ATC GTG GCA 144
Val Leu Glu Arg Ala Arg G1U Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 45
ACC GAT CAT GAG G~T GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192
Thr Asp His Glu Asp Val Ala Arg Al~ Val Glu Ala Ala Gly Gly Glu
50 55 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
65 70 75 80
GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288
Glu Val Val Glu Ly-~ Cys Ala Phe Ser Asp A~p Thr Val Ile Val Asn
85 90 95
GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 3 3 6
Val Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110

PCTIUS 92/0718 8
w~32~j ~O/~S 23 DEC l992

86

GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
Al~ Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG 432
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 . 135 140
GTT CTC GAC GCT GAA GnG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
1~5 150 ~55 160
CCT TGG GAT CGT GAT CGT TTT GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC T~C CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 576
A n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 62g
Arg Arg ~yr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile ~-lu Met
195 200 205
TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 672
Leu Glu Gln ~eu Arg Val Leu T~ qyr Gly Glu Lys Ile His Val Ala
210 215 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
GAC CCG TCG ~CG AAT TCC ATG GAC GCT CAC TTC CTG TCT CAG GCG CCG 768
Asp Pro S~r Thr Asn Ser Met Asp Ala Hi~ Phe Leu Ser Gln Ala Pro
245 250 255
CCG CCG TCT TGG GAT CAG ~TG TGG AAA TGC CTG ATC CGT CTG AAA CCG 816
Pro Pro S~r Trp A~p Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro
260 265 270
ACC CTG CAC GGC CCG ACC CCG CTG CTG TAC CGT CTG GGT GCT GTT CAG 86g
Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln
275 280 285
AAC GAA ATC ACC CTG ACC CAC CCG GTT ACC AAA TAC ATC ATG ACC TGC 912
Asn Glu Ile Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys
2~0 295 300
ATG TCT GCT GAT CTA GAA GTT GTT ACC TCT ACC TGG GTT CTG GTT GGT 960
Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly
305 310 315 320
GGT GTT CrG GCT GCT CTG GCT GCT TAC TGC CTG TCG ACC GGT TGC GTT 1008
Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val

PCrlUS 92/0718 8
0/US 2 3 DE~ 1992


8 7

325 330 335
GTT ATC GTT GGT CGT GTT GTT CTG TCT GGT AAA CCG GCC ATT ATC CCG 1056
Val Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro
340 3g5 350
GAC CGT GAA GTT CTG TAC CGT GAG TTC GAC GAA ATG GAA GAA TGC TCT 1104
Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser
355 360 365
CAG CAC CTG CCG TAC ATC GAA CAG GGT ATG ATG CTG GCT GAA CAG TTC 1152
Gln Hi~ Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe
370 375 380
AAA CAG AAA GCT CTG GGT CTG CTG CAG ACC GCT TCT CGT CAG GCT GAA 1200
Ly~ Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala Glu
385 390 . 395 400
GTT ATC GCT CCG GCT GTT CAG ACC AAC TGG CAG AAA CTC GAG ACC TTC 12~R
Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Lys Leu Glu Thr Phe
405 410 41S
TGG GCT AAA CAC ATG TGG AAC Tl~- ATC TCT GGT ATC C~G TAC CTG GCT 1296
Trp Ala Ly~ His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala
420 425 g30
GGT CTG TCT ACC CTG CCG GGT AAC CCG GCT ATC ~CA AGC TTG ATG GCT 1344
Gly Leu Ser Thr Leu Pro Gly A~n Pro Ala Ile Ala Ser Leu Met Ala
435 443 445
TTC ACC GCT GCT GTT ACC TCT CCG CTG ACC ACC TCT CAG ACC CTG CTG 1392
Phe Thr A1~ Ala Val Thr Ser Pro Leu Ihr Thr Ser Gln ~hr Leu Leu
450 g55 g60
TTC AAC ATT CTG GGT GGT TGG GTT GCT GCT CAG CTG GCT GCT CCG GGT 1440
Phe Asn Ile Leu Gly 51y Trp Val Ala Ala Gln Leu Ala Al~ Pro Gly
465 470 47~ 480
GCT GCT ACC GCT TTC GTT GGT GCT GGT CTG GCT GGT GCT GCT ATC G5T 1488
Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly
485 490 g95
TCT GTA GGC CTG GGT AAA GTT CTG ATC GAC ATT CTG GCT GGT TAC GGT 1536
Ser Val Gly Leu Gly Lys Val Leu Ile Asp Ile Leu Ala Gly Tyr Gly
500 505 510
GCT GGT GTT GCT GGA GCT CTG GTT GCT TTC AAA ATC ATG TCT GGT GAA 1584
Ala ~ly Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu
515 520 525
GTT CCG TCT ACC GAA GAT CTG GTT AAC CTG CTG CCG GCT ATC CTG TCT 1632
Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser
530 535 540
CCG GGT GCT CTG GTT GTT GGT GTT GTT TGC GCT GCT ATC CTG CGT CGT 1680

PCTIUS 92/07188 .
32~i BO/~S 23 ~EC t992

88
Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg
545 550 555 560
CAC GTT GGC CCG GGT GAA GGT GCT GT~ CAG TGG AIG AAC CGT CTG ATC 1728
His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile
565 570 575
GCT TTC GCT TCT CGT GGT AAC CAC GTT TCT CCA TGG GAT CCT CTA GAC 1776
Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Trp Asp Pro Leu Asp
580 585 590
TGC AGG CAT GCT AAG 1791
Cys Arg His Ala Lys
595

(2) INFORMATION FOR SEQ ID NO:21-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 597 amino acids
(B) TYPE: amino acid
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
- 1 5 10 15
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His
~5 30
Val Leu Glu Arg Ala Arg ~lu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr A3p Hi~ Glu A~p Val Ala Arg Ala ~al Glu Ala Ala Gly Gly Glu

Val Cy~ M t Thr Arg Ala Asp His Gln Ser Gly Thr Glu Ar~ Leu Ala
` 70 75 ~0
Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn

Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg ~ln Val Gly Met Ala Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140

,,

aJ~i PCTAJS 92/071~ 8
8,0/~S 23DECt9~2


Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
lgS 150 155 160
ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180. 185 190
rg Arg ~yr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly V~l Asp Thr Pro Glu Asp Leu
225 230 . 235 240
sp Pro Ser Thr Asn Ser Met Asp Ala His Phe Leu Ser Gln Al~ Pro
2~5 250 255
ro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Il-e Arg Leu Ly~ Pro
260 265 270
hr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln
275 280 285
Asn Glu Il~ Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys
290 295 300
Met Ser Ala Asp Leu Glu V~l Val Thr 5er Thr Trp Val Leu Val Gly
305 310 315 3~0
ly Val Leu Al~ Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val
325 330 335
al Ile V~l Gly Arg V~l Val Leu Ser Gly Lys Pro Ala Ile Ile Pro
340 34S 350
sp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser
355 360 365
Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe
3~0 315 380
Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala Glu
385 390 395 400
al Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Lys Leu Glu Thr Phe
405 410 415
rp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala
420 g25 430
ly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala

PCT/US 92/0718 8
;i ij 1~ 0/ U S 2 3 0~c 199;!


435 440 445
Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Ser Gln Thr Leu Leu
450 gS5 460
Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala Pro Gly
465 470 a,75 480 *
la Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly
~85 g90 495
er Val Gly Leu Gly Lys Val Leu Ile A~p Ile Leu Ala Gly Tyr Gly
500 505 510
Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lyc~ Ile Met Ser Gly Glu
515 520 525
Val Pro Ser Thr Glu A~p Leu Val A~n Leu Leu Pro Ala Ile Leu Ser
530 535 540
Pro Gly Ala Leu Val Val Gly Val Val Cy~ Ala Ala Ile Leu Arg Arg
545 550 555 560
i~ Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile
565 570 575
la Phe Ala Ser Arg Gly Asn His Val Ser Pro Trp Asp Pro Leu A~p
580 585 590
C~rs Arg His Ala Lys
595
2~ INFORMATION FOR SEQ ID NO-22:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENG~: 1797 ba~e pairs
(B) TYPE: nucleic acid
( C ) S'rRANDEDNESS: s inS~le
(D~ TOPS)LC)GY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix~ FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.. 1797

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
A~G AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 48
Met Ser Phe Val Val Ile Ile Pro Ala Arg ~r Ala Ser Thr Arg Leu
1 5 10 15
CCC GGT AAA CCA TTG GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 96

- PCTIUS 9 2 / 0 7 1 8 8
r~ 0/ 1~ S 2 3 DEC 1992

91
ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GTG GCA 144
V~l Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 g5
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192
Thr Asp Hi3 Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
50 55 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
65 70 75 80
GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288
Glu Val Val Glu Lys Cys Ala Phe Ser Asp *9p Thr Val Ile Val Asn
85 90 95
GIG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 ' 110
GCT GAT AAC CTC GCT CAG CGT CAG GTG G~T ATG GCG ACT CTG GCG GTG 384
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG g32
Pro Ile His Asn Ala Glu Glu Ala Phe A~n Pro Asn Ala Val Lys Val
- 130 135 lgO
GTT CTC GAC &CT GAA G G TAT GCA CT~ TAC TTC TCT CGC GCC ACC ATT 480
~al Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 15g 155 160
CCT TGG GAT CGT GAT CG. TTT GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro ~rp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 576
Asn Phe Lou Arg His Leu Gly Ile Tyr Gly ~yr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 672
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240

~`, . P~CTIUS 9 2 1 0 7 1 8 8
U/~TS 23 0EC ~992


GAC CCG TCG ACG AAT TCC ATG GAC GCT CAC TTC CTG TCT CAG ACC AAA 768
Asp Pro Ser Thr Asn Ser Met Asp Ala His Phe Leu Ser Gln Thr Lys
245 250 255
CAG TCT GGT GAA AAC CTT CCG TAC CTG GTT GCT TAC CAG GCT ACC GTT 816
Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val
260 265 270
TGC GCT CGT GCT CAG GCC CCG ACC CCG CTG CTG TAC CGT CTG GGT GCT 864
Cys Ala Arg Ala Gln Ala Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala
275 280 285
GTT CAG AAC GAA ATC ACC CTG ACC CAC CCG GTT ACC AAA TAC ATC ATG 912
Val Gln Asn Glu Ile Thr Leu Thr His Pro Val Thr Ly~ Tyr Ile Met
290 295 300
ACC TGC ATG TCT GCT GAT CTA GAA GTT GTT ACC TCT ACC TGG GTT CTG 960
Thr Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu
305 310 315 320
GTT GGT GGT GTT CTG GCT GCT CTG GCT GCT TAC TGC CTG TCG ACC GGT 1008
Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly
325 330 335
TGC GTT GTT ATC GTT GGT CGT GTT GTT CTG TCT GGT AAA CCG GCC ATT 1056
Cy~ Val Val Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala Ile
340 3~5 350
ATC CCG GAC CGT GAA GTT CTG TAC CGT GAG TTC GAC GAA ATG GAA GAA 1104
- Ile Pro A9p Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu
355 360 365
TGC TCT CAG CAC CTG CCG TAC ATC GAA CAG GGT ATG ATG CTG GCT GAA 1152
Cys Ser Gln Hi~ Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu
370 375 3ao
CAG TTC AAA CAG AAA GCT CTG GGT CTG CTG CAG ACC GCT ~CT CGT CAG 1200
Gln Phe Lys Gln Lys Ala Leu Gly Leu ~eu Gln Thr Ala Ser Arg Gln
385 390 395 400
GCT GAA GTT ATC GCT CCG GCT GTT CAG ACC AAC TGG CAG AAA CTC GAG 1248
Ala Glu VA1 Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Ly~ Leu Glu
gO5 glO 415
ACC TTC TGG GCT AAA CAC ATG TGG AAC TTC ATC TCT GGT ATC CAG TAC 1296
Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr
420 425 430
CTG GCT GGT CTG TCT ACC CTG CCG GGT AAC CCG GCT ATC GCA AGC TTG 1344
Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu
435 440 gg5
ATG ~-CT TTC ACC GCT GCT GTT ACC TCT CCG CTG ACC ACC TCT CAG ACC 1392
Met Ala Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Ser Gln Thr
~ g50 455 460
"~

PCT/US 92/0718 8
i J ~ / ~ S 23 DEC 1~




CTG CTG TTC AAC A~T CTG GGT GGT TGG GTT GCT GCT CA~ CTG GCT GCT1440
Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala
465 470 g75 480
CCG GGT GCT GCT ACC GCT TTC GTT GGT GCT GGT CTG GCT GGT GCT GCT1488
Pro Gly Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala
485 490 g95
ATC GGT T~T GTA GGC CTG GGT AAA GTT CTG ATC GAC ATT CTG GCT GGT1536
Ile Gly Ser Val Gly Leu Gly Lys Val Leu Ile Asp Ile Leu Ala Gly
500 505 510
TAC GGT GCT GGT GTT GCT GGA GCT CTG GTT GCT TTC AAA ATC ATG TCT1584
Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser
515 520 525
GG~ GAA GTT CCG TCT ACC GAA GAT CTG GTT AAC CTG CTG CCG GCT ATC1632
Gly Glu Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile
530 535 540
CTG TCT CCG GGT GCT CTG GTT GTT GGT GTT GTT TGC GCT GCT ATC CTG1680
Leu Ser Pro Gly Ala Leu V~l Val Gly Val Val Cys Ala Ala Ile Leu
545 550 555 560
CGT CGT CAC GTT GGC CCG GGT GAA GGT GCT GTT CAG TGG ATG AAC CGT. 1728
Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg
565 570 575
- C$G ATC GCT TTC GCT TCT CGT GGT AAC CAC GTT TCT CCA TGG GAT CCT 1776
Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val 5er Pro Trp Asp Pro
580 585 590
CTA GAC TGC AGG CAT GCT AAG 1797
~eu A~p Cys Arg His Ala Lys
~ 595
J
(2) INFO~MATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 599 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu

Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

' ~

PCTIUS 92/07188
'J~6 E~O~-S 23 DEC 199

9 4
al Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
4~
Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cys Met Thr Arg Ala Asp His Gln Ser ¢ly Thr Glu Arg Leu Ala

Glu Val Val Glu Lys Cys Ala Phe Ser A p Asp Thr Val Ile Val Asn
~5 90 95
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln V~l
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Mht Ala Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 lgO
Val Leu A~p Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Acp
165 170 175
As~ Phe Leu Ar~ His Leu Gly Ile ~yr Gly Tyr Arg Ala Gly Phe Ile
- ~80 185 lgO
Arg Arg Tyr Val Asn Trp.Gln Pro Ser Pro Leu Glu Hi Ile Glu ~et
195 2G0 205
Lsu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile ~i9 Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu A~p Leu
225 230 235 240
Asp Pro Sar Thr A~n Ser Met Asp Ala His Phe Leu Ser Gln Thr Ly~
245 250 255
Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val
260 265 270
ys Ala Arg Ala Gln Ala Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala
275 280 285
Val Gln Asn Glu Ile Thr Leu -Thr H1s Pro Val Thr Lys Tyr Ile Met
290 295 300
Thr Cy5 Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu
305 310 315 320

` - `. PCTIUS 't 2 / 0 7 1 8 8
b R0/~S 23 DEC 1992


Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly
325 330 335
Cys Val Val Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala Ile
340 345 350
Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu
355 360 365
Cy~ Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu
370 375 380
Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg Gln
385 390 395 400
Ala Glu Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Lya Leu Glu
405 . 410 ~ 415
Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr
420 425 430
Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu
435 440 445
Met Ala Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Ser Gln Thr
450 455 ~60
Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala
465 470 475 ~80
Pro Gly Ala Ala Thr Al~ Phe Val Gly Ala Gly Leu Ala Gly Ala Ala
485 490 495
Ile Gly Ser Val Gly Leu Gly Lya Val Leu Ile Asp Ile Leu Ala Gly
500 505 510
Tyr Gly Al~ Gly V~l Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser
515 520 525
Gly Glu V~l Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile
530 535 540
Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu
545 550 S55 560
Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg
565 570 575
Leu Ile Ala Phe Al~ Ser Arg Gly Asn His Val Ser Pro Trp Asp Pro
580 585 590
Leu Asp Cys Arg His Ala Lys
595

- - PCTIUS 92/07188 .
2~ R0/lJS 23 DEC 1992
96

~2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1251 ba~e pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1251

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ATG AGT TTT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 48
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
CCC GGT AAA CCA TTG GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 96
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly3 Pro Met Ile Val His
20 ~5 30
GTT CTT GAA CGC GCG CGT GAA TCA G-T GCC GAG CGC ATC ATC GTG GCA 144
Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 45
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192
Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
50 55 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
V~l Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
65 70 75 80
GAA GTT GTC GAA AAA TCC GCA TTC AGC GAC GAC ACG GTG ~TC GTT AAT 288
Glu Val V~l Glu Ly3 Cy8 Ala Phe Ser Asp Asp Thr Val Ile Val Asn
85 90 95
G~G CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG 432
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 . 140
GTT CTC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile

` - PCT/US 92/0718 8
L;J~ 2~ ~0/~S 23 DEC l992

97
1~5 150 155 160
CCT TGG GAT CGT GAT CGT TTT GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 576
A~n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC T~G CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 672
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
GTT GCT CAG GAA GTT CCT G C ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Al~ Gln Glu Val Pro Gly m r Gly V~l Asp m r Pro Glu Asp Leu
225 230 235 . 240
GAC CCG TCG ACT CGA ATT CGA GCT CGG TAC CCT GAG ACA ATC ACG CTT 768
A~p Pro Ser Thr Arg Ile Arg Ala Arg Tyr Pro Glu Thr Ile Thr Leu
2~5 250 255
CCC CAG GAT GCT GTC TCC CGC ACC CAG CGT CGG GGC AGG ACT GGC AGG 816
Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg m r Gly Arg
260 265 270
GGG AAG CCA GGC ATC TAC AGA TTT GT~ GCA CCG GGG GAG CGC CCT TCC 864
Gly Lys Pro Gly Ile Tyr Arg Phe Val Al~ Pro Gly Glu Arg Pro Ser
2~5 280 285
GGC ATG TTC GAC TCG TCC GTC CTC TGC GAG TGC TAT GAC GCG GGC TGG 912
Gly ~et Phe Asp Ser Ser Val Leu Cy~ Glu Cys Tyr Asp Ala Gly Trp
290 295 300
CCT TGG TAT GAG CTC ACA CCC GCC GAG ACC ACA GTT AGG CTA CGA GCG 960
Pro Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala
305 310 ~ 315 320
TAC ATG AAC ACC CCG GGA CTC CCC GTG TGC CAA GAC CAT CTT GAA TTT 1008
Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe
325 330 335
TGG GAG GGC GTC TTC ACG GGT CTC ACC CAT ATA GAC GCC CAC TTT CTA 1056
Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu
340 345 350
TCC CAG ACA AAG CAG AGT GGG GAA AAC CTT CCT TAC CTG GTA GCG TAC 1104
: Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr
~: 355 360 365
CAA GCC ACC GTG TGC GCT AGA GCT CAA GCC CCT CCC CCA TCG TGG GAC 1152

PCTIIJS 9 2 / 0 7 1 8 8
2r, ~O/US 23 DEC ~g92

98

Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp
370 375 380
CAG ATG TGG AAG TGC TTG ATC CGC CTC AAG CCT ACC CTT CAT GGG CCG 1200
Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro
385 390 395 400
ACC CCC CTG CTA TAC AGA CTG GGC GGG GGA TCC TCT AGA CTG CAG GCA 1248
Thr Pro Leu Leu Tyr Arg Leu Gly Gly Gly Ser Ser Arg Leu Gln Ala
g05 410 415
~GC 1251
Cys

(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 417 amino acids
(B) TYPE: amino acid
(D) TOPOL~GY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SE~ ID NO:25:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

~al Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

r Asp Hi~ Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cy~ Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn
al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr.Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pxo Asn Ala Val Lys Val
130 135 140
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile

PCUUS 9 2/ 07 1 8 8
r~ l l 3 ~ b l~ O / U S 2 3 DEC ~

99
145 150 155 160
ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
rg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
lg5 200 205
Leu Glu Gln Leu Arg Vai Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
sp Pro Ser Thr Arg Ile Arg Ala Arg Tyr Pro Glu Thr Ile Thr Leu
245 250 255
ro G1~ Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg
260 265 2~0
ly Lys Pro Gly Ile Tyr Arg Phe V~l Ala Pro Gly Glu Arg Pro Ser
275 . 280 285
Gly het Phe Asp Ser Ser Val Leu Cys Glu Cy9 Tyr Asp Ala Gly Trp
290 295 300
Pro Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala
305 310 315 320
yr Met Asn Thr Pro Gly Leu Pro Val Cy~ Gln Asp Hi~ Leu Glu Phe
325 330 335
~p Glu Gly Val Phe Thr G~y Leu Thr His Ile Asp Ala His Phe Leu
340 345 ~50
er Gln Thr Ly3 Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr
355 360 365
Gln Al~ Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp
370 375 380
Gln Met Trp Lys Cy5 Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro
385 390 395 400
hr Pro Leu Leu Tyr Arg Leu Gly Gly Gly Ser Ser Arg Leu Gln Ala
40~ 410 415
ys
2) ~NFORMATION FOR SEQ ID NO:26:

- PCTIUS 92/07188~ ~ ~ J ~ 2 ~ BO/ U S 2 3 OEC 1992
100
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1275 base pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
~D) TOPOLOGY:.circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1275

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
ATG AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 48
Met Ser Phe Val Val Ile Ile Pro.Ala Arg Iyr Ala Ser Thr Arg Leu
1 5 10 15
CCC GGT AAA CCA TTG GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 96
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly~ Pro Met Ile Val Hi~
20 25 30
GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GTG GCA 144
Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 ~5
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192
Tbr Asp His Glu Asp Val Ala Arg Ala Val Glu Al~ Ala Gly Gly Glu
- 50 55 6
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
V~l Cys Met Thr Arg Al~ Asp His Gln Ser Gly Thr Glu Arg Leu Ala
65 70 75 80
GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288
Glu Val Yal Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn
85 90 95
GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
Val Gln Gly Asp Glu Pro:Met Ile Pro Ala Thr Ile Ile Ar~ Gln Val
100 105 110
GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ~TC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG 432
Pro Ile His Asn Ala Glu Glu Ala Phe A~n Pro Asn Ala Val Lys Val
130 135 lg0
GTT CIC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala ~hr Ile
145 150 155 160

P~TIUS 9 2 / 0 7
X ~ 1~0/ ~ S 2 3 OEC 1992
101

CCT TGG GAT CGT GAT CGT m GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC m ATC 576
A~n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 lB5 190
CGT CGT TAC GTC AAC T~G CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
TTA GAG CAG CTT CGT GTT CT~ TGG TAC GGC GAA AAA ATC CAT GTT GCT 672
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
V~l Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
CAC CCG TCG ACT CGA ATT CGT AGG TCG CGC AAT TTG GGT AAG GTC ATC 768
Asp Pro Ser Thr Arg Ile Arg Arg Ser Arg Asn Leu Gly Lys Val Ile
245 250 255
GAC ACC CTC ACG TGC GGC TTC GCC GAC CTC ATG GGG TAT ATT CCG CTC 816
Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu
260 265 270
GrC GGC GCC CCT CTT GGA GGC GCT GCC AGG GCC CTG GGC CAT GGC GTC 86g
Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Gly His Gly Val
275 280 285
CGG Gl~ CTG GAA GAC GGC GTG AAC TAT GCG ACA GGG AAT Cl~ CCT GGT 912
ArS,~ Val Leu Glu Asp Gly V~l Asn Tyr Ala Thr Gly Asn Leu Pro Gly
2gO 295 300
TGC TCT TTC TCT ATC I~C CTT CTG GCC CTG CTC TCT TGC CTG ACC GTG 960
Cys Ser Ph~ Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val
305 310 315 320
CC~C GCA TCA GCC TAC CAA GTA CGC AAC TCC TCG GGC CTT TAC CAT GTC 1008
Pro Ala Ser Al~ Tyr Gln Val Arg Asn Ser Ser Gly Leu Tyr His Val
325 330 335
ACC AAT GAT TGC CCC AAC TCG AGT ATT GTG TAC GAG ACG GCC GAT GCC 1056
mr Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr Glu Thr Ala Asp Ala
340 3g5 ` 350
ATC CTG CAC ACT CCG GGG TGC GTC CCT TGC GTT CGT GAG GGC AAC GCC 1104
Ile Leu His m r Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ala
355 360 365
TCG AGA TGT TGG GTG GCG GTG GCC CCC ACA GTG GCC ACC AGG GAT GGA 1152
Ser Arg Cys Trp Val Ala Val Ala Pro Thr Val Ala Thr Arg Asp Gly
370 375 380

.~

- PCT/US 92/0718 8
0/ ~ S 2 3 OEC ~992

1 02

AAA CTC CCC GCA ACG CAG CTT CGA CGT CAC ATT GAT CTG CrT GTC GGG 12 0 0
Lys Leu Pro Ala Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly
385 390 395 d,00
AGC GCC ACC CTC TGT TCG GCC CTC TAC TTA AGG AGC TCG GTA CCC GGG 1248
Ser Ala Thr Leu Cys Ser Ala Leu Tyr Leu Arg Ser Ser Val Pro Gly
405 410 415
GAT CCT CTA GAC TGC AGG CAT GCT AAG 127 5
Asp Pro Leu Asp Cys Arg His Ala Lys
420 425

(2) INFORMATION FOR SEQ ID NO : 27:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~25 amino aclds
(B) IrYPE: amino acid
( D ) TOPOLOGY: l inear
( ii ) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
. 15
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His
;25 30
~al Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
,35 40 45
Thr Asp His Glu Asp Val Ala Ar~7 Ala Val Glu Ala Ala (~ly Gly Glu

Val Cys M0t Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala

Glu Val Val Glu Lys Cy9 Ala Phe Ser A~p Asp Thr Val Ile Val Asn
~0 95
Val Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160

- - PCTIUS 't2/07188 .
26 BO/I~S 23 OEC t992

103

Pro Trp Asp Arg Aisp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
Asn Phe Leu Arg His Leu Gly Ile Tyr Gly ryr Arg Ala Gly Phe Ile
180 185 190
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
Asp Pro Ser Thr Arg Ile Arg Arg Ser Arg A~sn Leu Gly Lys Val Ile
245 250 255
Asp m r Leu Thr Cys Gly Phe Ala Asp Leu Met Gly l~r Ile Pro Leu
260 265 270
~1 Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Gly His Gly Val
2'75 280 285
Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly
290 295 300
Cy~ Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val
305 310 315 320
Pro Ala Ser Ala Tyr Gln Val Arg Asn Ser Ser Gly Leu l~r Hls Val
:; 325 330 335
Thr Asn Asp Cys Pro Asn Ser Ser Ile Yal Tyr Glu Thr Ala Asp Ala
340 345 350
Ile Leu His m r Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ala
355 360 365
'Ser Arg Cys Trp Val Ala Val Ala Pro Thr Val Ala Thr AL-g Asp Gly
370 375 380
Lyis Leu Pro Ala Thr Gln Leu Arg Arg Xis Ile Asp Leu Leu Val Gly
3~5 390 395 400
Ser Ala Thr Leu Cys Ser Ala Leu Tyr Leu Arg Ser Ser Val Pro Gly
405 410 415
:~ Asp Pro Leu Asp Cy5 Arg His Ala Lys
420 425

(2) INFORMATION FOR SEQ ID NO:28:

` - PCTIUS 92/07188
~2~ l~O/~iS 23 DEC 1992

104
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1401 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(~) LOCATION: 1..1401

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
ATG AGT TTT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 48
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
CCC GGT AAA CCA TTG GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 96
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His
20 25 30
- GIT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GTG GCA 144
Val Leu Glu Arg AlA Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 45
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192
Thr A~p Ni~ Glu Asp Val Ala Arg Al~ Val Glu Ala Ala Gly Gly Glu
- -50 55 60
, ~,
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
65 70 75 80
GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288
Glu Val Val Glu Lys Cys Ala Phe Ser Asp A~p Thr Val Ile Val Asn
85 90 95
GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG 432
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Ual
. 130 135 }40
GTT CTC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
,.~;,,
,

PCTIUS 9 21 07 1 8 8
~L~ 6 ~0/1,S 23 DE~ 1992

105

CCT TGG GAT CGT GAT CGT TTT GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 576
Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 672
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Al~
210 215 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
GAC CCG TCG ACT CGA ATT CTG CTT GTC GGG AGC GCC ACC CTC TGC TCG 768
Asp Pro Ser Thr Arg Ile Leu Leu Val Gly Ser Ala Thr Leu Cys Ser
245 250 255
GCC CTC TAT GTG GGG GAC TTG TGC GGG TCT GTC TTT CTT GTC GGT CAA 816
Ala Leu ~yr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Gly Gln
260 265 270
CTG TTC ACT TTC TCC CCC AGG CAG CAC T~G ACA ACG CAA GAC TGC AAC 864
Leu Phe m r Phe Ser Pro Arg Gl~ His Trp Thr Thr Gln Asp Cys Asn
275 280 285
TGT TCT ATC TAC CCC GGC CAC GTA ACG ~GT CAC CGC ATG GCA TGG GAT 912
Cy~ Ser Ile Tyr Pro Gly Hi~ Val Thr Gly His Arg Met Ala Trp Asp
290 295 300
ATG ATG ATG AAC TGG TCC CCT ACG ACA GCG CTG GTA GTA GCT CAG CIG 960
Met Met M~t Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ala Gln Leu
305 310 315 320
CTC AGG GTC CCG CAA GCC ATC TTG GAC ATG ATC GCT GGT GCC CAC TGG 1008
Leu Arg Val Pro Gln Ala Ile Leu A~p Met Ile Al~ Gly Ala His Trp
325 330 335
GGA GTC CTA GCG GGC ATA GCG TAT TTC TCC ATG GTG GGG AAC TGG GCG 1056
Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val ~-ly Asn Trp Ala
3gO 345 350
AAG GTC CTG GTA GTG CTG CTG.CTA TTT GCC GGC GTT GAC GCG GAA ACC 1104
Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr
355 360 365
CAC GTC ACC GGG GGA AGT GCC GGC CAC ATT ACG GCT GGG CTT GTT CGT 1152
His Val Thr Gly Gly Ser Ala Gly His Ile Thr Ala Gly Le~ Val Arg
370 375 380

` PCT/IJS 92/0718 8
2G B,O~ ~,S 2 3 OEC l992
106

CTC CTT TCA CCA GGC GCC AAG CAG AAC ATC CAA CTG ATC AAC ACC AAC 1200
Leu Leu Ser Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn
385 390 395 400
GGC AGT TGG CAC ATC AAT AGC ACG GCC TTG AAC TGC AAT GAA AGC CTT 124 a
Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu
405 410 415
AAC ACC GGC TGG TTA GCA GGG CTC TTC TAT CAC CAC AAA TTC AAC TCT 1296
Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr His His Lys Phe Asn Ser
420 425 430
TCA GGC TGT CCT GAG AGG GTT GCC AGC TGC CGT CGC CTT ACC GAT TTT 1344
Ser Gly Cy9 Pro Glu Arg Val Ala Ser Cys Arg Arg Leu Thr Asp Phe
435 ~40 445
GAC CAG GGC TGG GAA TTC GAG CTC GGT ACC CGG GGA TCC TCT AGA CTG 1392
Asp Gln Gly Trp Glu Phe Glu Leu Gly Thr Arg Gly Ser Ser Arg Leu
450 455 460
CAG GCA TGC 1~01
Gln Ala Cys
465

2) INFORMATION FOR SEQ ID NO:29:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids
(P) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Met Ser Ph~ V~l Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
Pro Gly ~y~ Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

m r Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala

Glu Val Val Glu Ly~ Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn


,: .

- , ~ PCT/US 9 2 / 0 7 1 8 8
1~ 0 / ~ S 2 3 DEC 1992

1 07

Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
VA1 Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu Hi~ Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gl~ Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 ~30 235 240
sp Pro Ser Thr Arg Ile Leu Leu Val Gly Ser Ala Thr Leu Cys Ser
24~ 250 255
Ala Leu Tyr V~l Gly A3p Leu Cys Gly Ser Val Phe Leu Val Gly Gln
~ 260 265 270
L~u Phe Thr Phe Ser Pro Arg Gln His Trp Thr Thr Gln Asp Cys Asn
275 280 285
Cyq Ser Ils Tyr Pro Gly Hi~ Val Thr Gly His Arg Met Ala Trp A~p
290 295 300
Met ~et Met A~n Trp Ser Pro Thr Thr Ala Leu Val Val Ala Gln Leu
305 10 315 320
eu Arg Val Pro Gln Ala Ile Leu Asp Met Ile Ala Gly Ala His Trp
325 330 335
ly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val Gly Asn Trp Ala
340 345 350
Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val A~p Ala Glu Thr
355 360 365
His Val Thr Gly Gly Ser Ala Gly His Ile Thr Ala Gly Leu Val Arg
370 375 380

PCTlUS 92/0718 8
6 ~O/~ 23 DE~

108

Leu Leu Ser Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn
385 390 395 400
Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu
405 410 415
Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr His His Lys Phe Asn Ser
420 425 430
Ser Gly Cys Pro Glu Arg Val Ala Ser Cys Arg Arg Leu Thr Asp Phe
435 440 445
Asp Gln Gly Trp Glu Phe Glu Leu Gly Thr Arg Gly Ser Ser Arg Leu
450 455 460
Gln Ala Cys
465

(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1422 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
- (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1422

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
ATG AGT TTT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 48
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
CCC GGT AAA CCA TTG GTT GAT ATT AAC GGC:AAA CCC ATG ATT GTT CAT ~6
Pro Gly Lys Pro Leu V~l A~p Ile A~n Gly Lys Pro Met Ile Val Hi-~
20 25 30
GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GTG GCA 144
Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 45
ACC GAT CAT GAG GAT G~T GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA . 192
Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
50 55 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 240
Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala


- PCT/US 92/0718 8
~Q~ 2 3 DEC ~992

os

GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288
Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn
85 90 95
GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
Ala A p Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG ~ 32
Pro Ile ~is Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
GTT CTC GAC GCT GAA G G TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Ars1 Ala Thr Ile
1~5 150 155 160
CCT T~G GAT CGT GAT CGT m GCA GAA GGC CTT GAA ACC GTT GGC GAT 528
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 576
A~n Phe Lell Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr V~l A3n Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 . 200 205
TTA GAG CAG CTT CGT G~T CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 6?2
Leu Glu Gln Leu Arg V~l Leu Trp Tyr Gly Glu Ly~ Ile His Val Ala
210 215 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Ala Gln Glu Val Pro Gly Thr Gly Val A~3p Thr Pro Glu Asp Leu
225 230 235 2~0
GAC CCG TCG ACC GAA TTC GGT GAC ATC ATC AAC GGC TTG CCC GTC TCC 768
Asp Pro Ser Thr Glu Phe Gly Asp Ile Ile Asn Gly Leu Pro Val Ser
245 250 255
GCC CGT AGG GGC CAG GAG ATA CTG CTC GGA CCA GCC GAC GGA ATG GTC 816
Al~ hrg Arg Gly Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Val
260 265 270
TCC AAG GGG Tt; G AGG 1~ CTG GCG CCC ATC ACG GCG TAC GCC CAG CAG 864
Ser Lys Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln
275 280 285
ACA AGG GGC CTC CTA GGG TGT ATA ATC ACC AGC CTG ACT GGC CGG GAC 912
Thr Arg Gly Leu Leu Gly Cys lle Ile Thr Ser Leu Thr Gly Arg A~p



~_3 PCTIUS 9 2 / 0 71 8 8
.BO/l~ 23 DEC 1992

1 1 0

290 295 300
AAA AAC CAA GCG GAG GGT GAG GTC CAG ATT GTG TCA ACT GCT GCC CAA 960
1ys Asn Gln Ala Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln
305 310 315 320
ACT TTC CTG GCA ACG TGC ATC AAT GGG GTA TGC TGG ACT GTC TAC CAT 1008
Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His
325 330 335
GGG GCC GGA ACG AGG ACC CTC GCA TCA CCC AAG GGT CCT GTT ATC CAG 1056
Gly Ala Gly Thr Arg Thr Leu Ala S~r Pro Lys Gly Pro V~l Ile Gln
340 345 350
ATG TAT ACC AAT GTA GAC CAA GAC CTT GTG GGC I~G CCC GCT CCT CAA 1104
Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln
355 360 ~ 365
GGT GCC CGC TCA TTG ACA CCC TGC ACC TGC GGC TCC TCG GAC CTT TAC 1152
Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr
370 375 380
CTG GTT ACG AGG CAC GCC GAT GTC ATT CCC GTG CGC CGG CGG GGT GAT 1200
Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp
385 390 395 400
AGC AGG GSC AGC CTG CTT TCG CCC CGG CCC ATT TCT TAT TTG AAA GGC 1248
Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly
405 410 gl5
TCC TCG GGG GGT CCG CTG ~G 5GC CCC GCG GGA CAC GCC GTG GGC ATA 1296
Sor Ser Gly Gly Pro L~u Leu Cys Pro Al~ Gly Hi~ Ala Val Gly Ile
~20 425 g30
~IC AGG GCC GCG GIY~ TGT ACC C5T GGA GIY~ GCT AAG GCG GTG GAC l~T 1344
Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe
435 440 445
GTC CCC G~G GAG AAC CTC GA~ ACA ACC ATG AAT TCG AGC TCG GTA CCC 1392
Val Pro Val Glu Asn Leu Glu Thr Thr Met Asn Ser Ser Ser Val PI~O
450 455 460
GGG GAT CCT CTA GAC TGC AGt; CAT GCT AAG 1422
Gly Asp Pro Leu Asp Cy4 Arg His Ala Lys
465 470

(2) INFORMATION FOR SEQ ID NO: 31:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 474 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLEC'JLE TYPE: protein

- PCTIUS 92/0718 8
~ J ~ 0 ~ ~ S 23 OEC ~2

1 1 1


(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
~et S~r Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His

Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu

Val Cyq Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
~0
1U VA1 Val Glu Lys Cys Ala Phe Ser Asp Asp Thr V~l Ile Val Asn
. g5
al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gln ~al Gly Met Ala Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
~45 lS0 155 160
ro Irp Asp Arg A3p Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
sn Phe Lau Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 1~5 190
Arg Arg ~yr Val Asn Trp Gln Pro Ser Pro Leu Glu Hi~ Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
sp Pro Ser Thr Glu Phe Gly Asp Ile Ile Asn Gly Leu Pro Val Ser
245 250 255
la Arg Arg Gly Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Met V~l
260 265 270

- PCTIUS 92/07188
!3~ ~O/US 23 DEC 1992
1 1 2

Ser Lys Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln
275 28~ 285
Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp
290 295 300
Lys Asn Gln Ala Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln
305 310 315 320
hr Phe Leu Ala Thr Cy~ Ile Asn Gly Val Cy3 Trp Thr Val Tyr His
325 330 335
ly Ala Gly Thr Arg Thr Leu Ala Ser Pro Lys Gly Pro Val Ile Gln
340 345 350
Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln
355 360 365
Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp L~u Tyr
370 375 380
Leu Val m r Arg His Ala Asp Val Ile Pro Val Arg Arg ~rg Gly Asp
385 390 395 400
er Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly
405 ~10 415
er Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile
420 425 ~30
Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe
- 435 440 4~65
V~l Pro V~l Glu Asn Leu Glu Thr Thr Met Ac~ Ser Ser Ser Val Pro
450 455 460
Gly A~p Pro L~u Asp Cys Arg His Ala Ly
465 470

(2) INFORMATION FOR SEQ ID NO:32.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTB: 1401 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAMEtKEY: CDS
~B) LOCATION: l..lg01

JS 9 2 / 07 1 8 8
~Ll`~9~ O/~ S 23 DEC l992

1 1 3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
ATG AGT TTT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 4 8
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
CCC GGT AAA CCA TTG GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 9 6
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His
20 25 30
GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GTG GCA 14 4
Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
35 40 45
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 19 2
Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
S0 55 . 60
GTA TGT ATG ACG CGC GCC GAT CAT CAG TCA GGA ACA GAA CGT CTG GCG 2 4 0
V~l Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
65 70 75 80
GAA GTT GTC GAA AAA T~C GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 2 8 8
Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn
- 85 90 95
G$G CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 3 3 6
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
GCT GAT AA~: CTC GCT CAG CGT CAG GTG GGT ATG ACG ACT CTG GCG GTG 3 8 g
Al~ A~p Asn Leu Ala Gln Arg Gln Val Gly M~t Thr Thr Leu Ala V~l
115 120 125
CCA ATC CAC AAT GCG GAA GAA GCG m AAC CCG ~T GCG GTG AAA GTG 432
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro A~n Ala Val Lys Val
130 135 140
GTT CTC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 4 8 0
Val Leu Asp Ala Glu Gly q~r Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
CCT TGG GAT CGT GAT CGT 1~ GCA GAA GGC CTT GAA ACC GTT GGC GAT 5 2 8
Pro Trp A~p Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC 1~1 C CTG CGT CAT CTT GGT ATT TAT S~GC TAC CGT GCA GGC TTT ATC 57 6
Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 lgO
CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
lg5 200 205
TTA GAG CAG CTT CGT Gl~ CTG TGG TAC GGC GAA AAA ATC CAT Gl~ GCT 67 2

~ T/US ~ 2 / 0 7 1 8 8
y~f~ K~ S 2 3 OEC 1992


114

Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
GAC CCG TCG ACG AAT TCC ACC ATG GGG CAT TAT CCT TGT ACC ATC AAC 768
Asp Pro Ser Thr Asn Ser Thr Met Gly His Tyr Pro Cys Thr Ile Asn
245 250 255
TAC ACC CTG TTC AAA GTC AGG ATG TAC GTG GGA GGG GTC GAG CAC AGG 816
Tyr Thr Leu Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg
260 265 270
CTG GAA GTT GCT TGC AAC TGG ACG CGG GGC GAA CGT TGT GAT CTG GAC 864
Leu Glu Val Ala Cys Asn Trp Thr Arg Gly Glu Ars Cys Asp Leu Asp
275 280 285
GAC AGG GAC AGG TCC GAG CTC AGC CCG CTG CTG CTG TCC ACC ACT CAG 912
AJP Arg A~p Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln
290 295 300
TGG CAG GTC CTT CCG TGT TCC TTC ACG ACC TTG CCA GCC TTG ACC ACC . 960
Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Thr Thr
305 310 315 320
G~C CTC ATC CAC CTC CAC CAG AAC ATC GTG GAC GTG CAA TAC TTG TAC 1008
Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr
325 330 335
G~G GTG GGG TCA AGC ATT GTG TCC TG5 GCC ATC AAG TGG GAG TAC GTC 1056
Gly Val Gly Ser Ser Ile Val Ser Irp Ala Ile Lys Trp Glu Tyr Val
340 345 350 . -~
ATC CTC TTG TTT CTC CTG CTT GCA GAC GCG CGC ATC TGC TCC TGC TTG 1104
Ile 1eu Lou Phs Leu Leu Lau Ala Asp Ala Arg Ile Cy~ Ser Cys Leu
355 360 365
TGG ATG ATG TTA CTC ATA TCC CAA GCG GAG GCA GCC TTG GAA AAC CTT 1152
Trp Met ~et Leu Leu Ile Ser Gln Ala Glu Ala Ala Leu Glu Asn Leu
370 375 380
GTG TTA CTC AAT GCG GCG TCT CTG GCC GGG ACG CAC GGT CTT GTG TCC 1200
Val Leu Leu Asn Ala Ala Ser Leu Ala Gly Thr His Gly Leu Val Ser
385 390 395 400
TTC CTC GTG m TTC TGC TTT GCA TGG TAT CTG AAG GGT AAG TGG GTG 1248
~: Phe Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Lys Gly Lys Trp Val
; gOS 410 415
CCC GGA GTG GCC TAC GCC TTC TAC GGG ATG TGG CCT TTC CTC CTG CTC 1296
Pro Gly Val Ala Tyr Ala Phe Tyr Gly Met Trp Pro Phe Leu Leu Leu
420 425 430

PCT/US 9 2/ 07 1 8 8
326 BO/~S 23 OEC Im

1 15

CTG TTA GCG TTG CCC CAA CGG GCA TAC GCG CTG GAC ACG GAG ATG GCC 1344
Lau Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Met Ala
435 g40 445
GCG TCG TGT GGC GGC GTT GTT CTT GTC GGG TTA ATG GCG CTG ACT CTG 1392
Ala Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr Leu
450 455 460
TCA CCA TAT 14Ql
Ser Pro Tyr
465

(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amIno acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu
1 5 10 15
ro Gly Lys Pro Leu V~l Asp Ile Asn Gly Lys Pro Met Ile Val His

Val Leu Glu Axg Ala Ar~ Glu Ser Gly Ala Glu Ars Ile Ile Val Ala

Thr Asp His Glu A~p Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
5~ 60
Val Cys Met m r Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn
B5 90 35
al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Al~ Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val
115 120 12~
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160

PCT/US 92/07188
0/l,S 230ECl992
b

1 1 6
Pro Trp A~p Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
~n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
Arg Arg Tyr Val A!3n Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
l9S 200 20S
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
sp Pro Ser Thr Asn Ser Thr Met Gly His Tyr Pro Cys Thr Ile Asn
2gS 250 ~ 255
yr Thr Leu Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg
260 265 270
Leu Glu Val Ala Cys As~ Trp Thr Arg Gly Glu Arg Cys Asp Leu A7sp
275 280 285
Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln
29~ 295 30~
Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Thr Thr
30~ ' 310 315 320
ly L~u Ile His Leu Hi~ Gln A~n Ile Val Asp Val Gln 1~7r Leu l~rr
325 330 335
ly Val Gly Ser Ser Ile Val Ser Trp Ala Ile Lys Trp Glu l~r Val
3~0 3~5 350
Ile Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Ile Cys Ser Cy~ Leu
355 360 ' 365
Trp Met Mot Leu Leu Ile Ser Gln Ala Glu Ala Ala Leu Glu Asn Leu
370 375 380
Val Leu Leu Asn Ala Ala Ser Leu Ala Gly Thr ~lis Gly Leu Val Ser
385 390 395 gO0
he Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Lys Gly Lys Trp Val
gOS 410 glS
ro Gly Val Ala Tyr Ala Phe Tyr Gly Met Trp Pro Phe Leu Leu Leu
420 ' 425 430
Leu Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Met Ala
435 440 445
Ala Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr Leu

PCTIUS 9 2/ 07 1 8 ~ .
t ~ O/ ~ S 2 3 DEC 199~

1 1 7

450 455 460
Ser Pro Tyr
465

(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1851 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1851

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3~:
ATG AGT ITT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG . 48
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu

CGC GGT AAA CCA TTG GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 96
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly~ Pro Met Ile Val His
20 25 30
GTT-CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GTG GCA 144
Yal Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala
3~ 40 45
ACC GAT CAT GAG GAT GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192
Thr Asp His Glu Asp V~l Ala Arg Ala Val Glu Ala Ala Gly Gly Glu
50 55 50
GTA TGT ATG ACG CGC GCC GAT CAT C~G TCA GGA ACA GAA CGT CTG GCG 240
Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala
6~ 70 75 80
GAA GTT 5TC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288
Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn
85 90 95
GTG CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336
Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
10~ 105 110
GCT GAT AAC CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384
Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val
115 120 125

2 ~ lllS 9 2 / 0 7 1 8 8
~0/l~S 23DEC199

118

CCA ATC CAC AAT GCG GAA GAA GCG m AAC CCG AAT GCG GTG AAA GTG 432
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 lgO
GTT CTC GAC GCT GAA GGG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160
C'-T TGG GAT CGT GAT CGT TTT GCA GAA GGC CTI GAA ACC GTT GGC GAT 528
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 576
A~n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 672
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile Hi3 Val Ala
210 215 220
GTT GCT CAG GAA GTT CCT GGC ACA GGT GTG GAT ACC CCT GAA GAT CTC 720
~al Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu
225 230 235 240
GAC CCG TCG ACT CGA ATT CGT AGG TCG CGC AAT $TG GGT AAG GTC ATC 7 68
Asp Pro Ser Thr Arg Ile Arg Arg 5er Arg Asn Leu Gly Ly3 Val Ile
245 250 255
GAT ACC CTC ACG TGC GGC TTC GCC GAC CTC ATG GGG TAC Al~r CCG CTC 816
A~p Thr Leu Thr Cys Gly Phe Ala A~p Leu Met Gly l~r Ile Pro Leu
260 265 270
Gl~ GGC GCC CCT CTT GGA GGC GCT GCC AGG GCC CTG GCG CAT GGC GTC 864
Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val
275 280 285
CGG GTT csa GAA GAC GGC GTG AAC TAT GCA ACA GGG AAC CrT CCC GGT 912
Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Prs Gly
290 295 300
TGC TCT TTC TCT ATC TTC CTT CTG GCC CTG CTC TCT TGC CTG ACT GTG 960
Cy~ Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val
305 310 315 320
CCC GCG TCA TCC TAC CAA GTA CGC AAC TCC TCG GGC CTT TAT CP.T GTC 1008
Pro hla Ser Ser Tyr Gln Val Arg Asn Ser Ser Gly Leu Tyr His Val
325 330 335
ACC AAT GAT TGC CCC AAC TCG AGC ATT GTG TAC GAG ACG GCC GAT ACC 1056
Thr Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr Glu Thr Ala Asp Thr
340 345 350~

~ O/ ~TS 2 3 OEC ~


1 1 9

ATC CTA CAC TCT CCG GGG TGC GTC CCT TGC GTT CGC GAG GGC AAC ACC 1104
Ile Leu His Ser Pro Gly Cy~ Val Pro Cys Val Arg Glu Gly A~n Thr
355 360 365
TCG AAA TGT TGG GTG GCG GTG GCC CCC ACA GTG GCC ACC AGG GAC GGC 1152
Ser Lys Cy8 Trp Val Ala Val Ala Pro Thr Val Ala Thr Arg Asp Gly
370 375 380
AAA CTC CCC TCA ACG CAG CTT CGA CGT CAC ATC GAT CTG CTC GTC GGG 1200
Lys Leu Pro Ser Thr Gln Leu Arg Arg His Il~ Asp Leu Leu Val Gly
385 390 395 400
AGC GCC ACC CTC TGC TCG GCC CTC TAT GTG GGG GAC TTG TGC GGG TCT 1248
Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser
405 glO ~15
GTC TTT CTT GTC AGT CAA CTG TTC ACC TTC TCC CCT AGG CGC CAT TGG 1296
Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp
420 425 430
ACA ACG CAA GAC TGC ~C TGT TCT ATC TAC CCC GGC CAT ATA ACG GGT 1344
Thr m r Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly
435 440 445
CAC CGC ATG GCA TGG GAT ATG ATG ATG AAC TGG TCC CCT ACA ACG GCG 1392
His Arg M~t Ala Trp Asp Met Met M~t Asn Trp Ser Pro Thr m r Ala
450 455 460
CTG GTA G~A GCT CAG CTG CTC AGG GTC CCA CAA GCC ATC ~TG GAC ATG 1440
Leu Val V~l Ala Gln Leu Leu Arg Val Pro Gln Ala Ile Leu Asp Met
465 470 g75 480
ATC GCA GGT GCC CAC TGG GGA GTC CTA GCG GGC ATA GCG TAT TTC TCC 1488
Ile Al~ Gly Ala His Irp Gly V~l Leu Ala Gly Ile Al~ Tyr Phe Ser
~85 g90 ~95
ATG GTG GGG AAC T¢G GCG AAG GTC CTG GTA GTG CTG ~TG CTG m TCC 1536
Met V~l Gly Asn Trp Al~ Ly~ Val Leu Val Val Leu Leu Lel~ Phe Ser
~00 505 510
GGC GTC GAT GCG GCA ACC TAC ACC ACC GGG GGG AGC GTT GCT AGG ACC 158g
Gly Val Asp Ala Ala Thr Tyr Thr Thr Gly Gly Ser Val Ala Arg Thr
515 520 525
ACG CAT GGA TTC TCC AGC TTA TTC AGT CAA GGC GCC AAG CAG AAC ATC 1632
Thr His Gly Phe Ser Ser Leu Phe Ser Gln Gly Ala Lys Gln Asn Ile
~30 535 540
CAG CTG ATT AAC ACC AAC GGC AGT TGG CAC ATC AAT CGC ACG GCC TTG 1680
Gln Leu Ile Asn Thr A~n Gly Ser Trp His Ile Asn Arg Thr Ala Leu
5g5 550 555 560
AAC TGT AAT GCG AGC CTC GAC ACT GGC TGG GTA GCG GGG CTC TTC TAT 1728
Asn Cy~ Asn Ala Ser Leu Asp Thr Gly Trp Val Ala Gly Leu Phe Tyr

- i i J ~ ~ S PCTNS 9 2 / 0 71 8 l

120

565 570 575
TAC CAC AAA TTC AAC TCT TCA GGC TGC CCT GAG AGG ATG GCC AGC TGT 177 6
Tyr His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser C ys
580 585 590
AGA CCC CTT GCC GAT m GAC CAG GGC IY~G GAA TTC GAG CTC GGT ACC 18 2 4
Arg Pro Leu Ala Asp Phe Asp Gln Gly Trp Glu Phe Glu Leu Gly Thr
595 600 605
CGG GGA TCC TCT AGA CTG CAG GCA TGC 18 51
Arg Gly Ser Ser Arg Leu Gln Ala Cys
610 615

( 2 ) INFORMATION FOR SEQ ID NO:35:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 617 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu

Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Mst Ile Val His

VA1 LeU Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala

Thr Asp Hi~ Glu Asp Val Ala Arg A1A Val Glu Ala Al~ Gly Gly Glu
_~ 50 55 60
Val Cyi~ M~t Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala

Glu Val V~l Glu Lys Cys Ala Phe Ser Asp A~p Thr Val Ile Val Asn

Val Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val
100 105 110
Ala Asp Asn Leu Ala Gln Arg Gl,rl Val Gly Met Ala Thr Leu Ala Val
115 120 125
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val
130 135 140
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile
145 150 155 160

9~6 1~0/1/S 2 3 DEC 1992

121
Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp
165 170 175
sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile
180 185 190
Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met
195 200 205
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala
210 215 220
Val Ala Gln Glu Val Pro Gly Thr Gly Val A~p Thr Pro Glu Aap Leu
2~5 230 235 2~0
sp Pro Ser Thr Arg Ile Arg Arg Ser Arg Asn Leu Gly Lys Val Ile
245 250 255
sp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu
260 265 270
Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val
275 280 285
Arg Val L~u Glu Asp Gly Val Asn Tyr Ala Thr Gly A~n Leu Pro Gly
290 295 300
Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val
305 319 315 320
ro Ala Ser ser Tyr Gln Val Arg Asn Ser Ser Gly Leu Tyr His Val
325 330 335
hr As~ Asp Cys Pro A~n Ser Ser Ile Val Tyr Glu Thr Al~ A~p Thr
3~0 345 350
Ile Leu Hia Ser Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Thr
355 360 365
Ser Lys cy9 Trp Val Ala Val Ala Pro Thr Val Ala Thr Arg Asp Gly
370 375 ~80
Lys Leu Pro Ser Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly
385 390 395 400
er A~a Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser
405 410 415.
al Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp
420 g25 430
hr Thr Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly
g35 440 445

26 PCTIJS 92/07188
1~0/ ~; S 2 3 DEC t992
1 2l~

His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala
450 455 460
Leu Val Val Ala Gln Leu Leu Arg Val Pro Gln Ala Ile Leu Asp Met
465 470 475 480
le Ala Gly Ala His Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser
4~5 490 495
et Val Gly Asn Trp Ala Lys Val Leu Val Val Leu Leu Leu Phe Ser
500 505 SlO
Gly Val Asp Ala Ala Thr Tyr Thr Thr Gly Gly Ser Val Ala Arg Thr
SlS 520 525
m r His Gly Phe Ser Ser Leu Phe Ser Gln Gly Ala Lys Gln Asn Ile
530 535 540
Gln Leu Ile Asn Thr A~n Gly Ser Trp His Ile Asn Arg Thr Ala Leu
545 550 555 560
sn Cys Asn Ala Ser Leu Aap Thr Gly Trp Val Ala Gly Leu Phe Tyr
565 570 575
yr His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys
580 585 590
Arg Pro Leu Ala Asp Phe Asp Gln Gly Trp Glu Phe Glu Leu Gly Thr
595 600 605
Arg Gly Ser Ssr Arg Leu Gln Ala Cys
610 615