Note: Descriptions are shown in the official language in which they were submitted.
wogs/29234 r~
2 1 88258
Human gamma 3 GA~A-A receptor subunit and stably ~c-transfected cell l~nes
. .
This invention concerns t: loning of a novel cDNA
5 sequence encoding a particular subuni. ~he hulnan GABAA receptor. In
addition, the invention relates to a stable cell line capable f ~
said cDNA and to the use of the cell line in a screening technique for the
design and ~1u~ .,l of subtype-specific P ~
Gamma-amino butyric acid (GABA) is a major inhibitory
10 n~ . in the central nervous system. It mediates fast synaptic
inhihit.i--n by opening the chloride channF-l intrinsic to the GABAA
receptor. This receptor ~ ; `F.~ a ~I~ltimP~ri~. protein of m~ r size
230-270 kDa with specific binding sites for a variety of dru~s including
b~..,.u~ 7~ .c, ks~ c and ~-carbolines, in additi . to sites for the
agonist ligand GABA (for reviews see StPphp~ncnn~ Biochem. J., 1988, 249.
21; Olsen and Tobin, Faseb J., 1990, _, 14~9; and Sieghart, Tren~s in
PharmQcol. Sci., 1989, lQ, 407).
MnlF~C~ r biûlogical studies tipmnnetr~tp~ that the receptor is
composed of several distinct types of subunit, which are divided into four
classes (a, ~, ~, and ~) based on their sequence ~imil~ritiP.c To date, six
types of a (.~I`hnfiPl(i et al., N~tture (Lo7t~70n), 1987, 328, 221; Levitan et al.,
Nature ~London), 1988, ~, 76; Ymer et al., EMB0 J., 1989, ~, 1665;
Pritchett & Seeberg, J. Neurochem., 1990, 54, 802; Luddens et al., Nature
(I,ondon), 1990, 346, 648; and Rhrpqtrh ~ticl~y et al., Neuron, 1989, 3, 745),
three types of ~ (Ymer et al., EMBO J., 1989, 8, 1665), two types of y
(Ymer et al., EMB0 J., 1990, ~, 32G1; and Shivers et al., Neuro7l, 1989, 3,
327) and one ~ subunit (Shivers et al., Neuron, 1989, 3, 327) have been
i~lPntifiPA
The .liffPrPntl~l rlic~rihlltlr~n of many of the subunits has
been ~ by in situ hyhri~liq~tinn (Sequier et al., Proc. Natl,
wossns234 21~8258 P~' 9'~
- 2 -
Acad. Sci. USA, 1988, ~5, 7815; Malherbe et al., J. Neurosci., 1990, 10~
2330; and Shivers et al., Neuron, 1989, ~ 327) and this has pPrmitt~d it to
be speculated which subunits, by their co-lnr~lic~t~nn could thPr,rpti/~lly . .
exist in the same receptor complex.
Various cnmhin ~h ~m c of subunits have been co~ r~ l pd
into cells to identify synthetic rnnnhin~tinn.c of subunits whose
rh~rnl~rrl~gy parallels that of bona fide GABAA receptors in uiuo
~Pritchett et al., Science, 1989, 245. 1389; Malherbe et al., J. Neurosci.,
1990, 10, 2330; Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802; and
Luddens et al., Nature (Zondo1~), 1990, ~, 648). This approach has
revealed that, in addition to an a and ,~ subunit, either Yl or Y2 (Pritchett
et al., Nature (London,~, 1989, 338, 582; Ymer et al., EMB0 J., 1990, 9,
32~il; and Malherbe et al., J. Neurosci., 1990, 1~, 2330) or y3 ~Herb et al.,
Proc. Natl. Acad. Sci. ~JSA, 1992, 89, 1433; Knoflach et al., ~EBS Lett.,
1991, ~, 191; and Wilson-Shaw et al., ~EBS Lett., 1991, 284. 211) is also
generally required to confer b~, . 7 ~ i . . P se~siiivi~y, and that the
bPn7.n~ 7Prinr rh~rm~rnln~y of the expressed receptor is largely
dependent on the identity of the a and y subunits present. Receptors
rnnts~ininE a o subunit (i.e. a,~o) do not appear to bind bPn7n~ 7prinpc
(Shivers et al., Neuron, 1989, 3, 327). Cnmhin~t~nc of subunits have been
identified which exhibit the ph slrm ~rl-lgir~l profile of a BZ 1 type receptor
(al~1y2) and a BZ2 type receptor (a2~1y2 or a3ply2, Pritchett et al.,
Nature (London), 1989, ~, 582), as well as two GABAA receptors with a
novel ph~rm~rnlngy, as,~2~2 (eritchett and Seeberg, J. Neurochem., 1990,
54, 1802) and a6~2~2 (Luddens et al., Nature (London), 1990, ~i, 648).
Although the ph~r~ rnlngy of these expressed receptors appears similar
to that of those i~lPntifie~ in brain tissue by rs~-linli~nd binding, it has
nnnPthPlPcR not been shown that these receptor subunit rnmhin~tinnc
exist in viuo.
_ ~ 218~258 `
A r~!mhin ~ tinn of subunits rnmrri cin E the human y3 GABAA
receptor subunit has not hitherto been possible due to the non-availability
of the human y3 cDNA. T_is has consequently limited the use of cell lines
in screening for subtype-specific m~lir~mPnt~, it being impossible to study
5 the ph~rm~rnlngir~l profile of subunit rnTnhinsltinn~ rn nrriqine the y3
subunit.
We have now ascertained the cDNA sequence of the y3
subunit of the human GABAA receptor. This nucleotide sequence,
together with the deduced amino acid sequence corresponding thereto, is
10 depicted in SEQ.ID.NO.7 of the accompanying Sequence Listing.
The present invention accordingly provides, in a first aspect,
a DNA molecule encoding the y3 subunit of the human GABAA receptor
comprising all or a portion of the sequence depicted in Figure 2, or a
modified human sequence.
The sequencing of the novel cDNA molecule in accordance
with the invention can conveniently be carried out by the standard
procedure described in accompanying Example 1; or may be Ar~nrnrlichrd
by alternative molecular cloning techniques which are well known in the
art, such as those described by Maniatis et al. in Molecular Cloning, A
20 I,aboratory Manual, Cold Spring ~arbor Press, New York, 2nd edition,
1989.
In another aspect, the invention provides a reromhin~nt
expression vector comprising the nucleotide sequence of the GABAA
receptor y3 subunit together with ~ itinn~l sequences capable of
25 directing the synthesis of the said GABAA receptor y3 subunit in cultures
of stably co-tr~n ~fe~te :1 eukaryotic cells.
The term "expression vectors" as used herein refers to DNA
sequences that are required for the transcription of cloned copies of
rrrnmhin~nt DNA sequences or genes and the tr:ln~l:3tlnn of their mRNAs
AMENCF.D S~EET
.. . , .... . .. ... _ .. .. .. _
woss/2s234 ~ 1 88258 ,~ . s. ~
in an i-~ ,uru~liaL~ host. Such vectors can be used to express eukaryotic
genes in a variety of hosts such as bacteria, blue-green algae, yeast ce~ls,
insect cells, plant cells and animal cells. Specifically designed vectors
allow the shuttling of DNA between bacteria-yeast, bacteria-plant or
5 bacteria-animal cells. An appropriately cv s~ru~Led t~l'.,Ul~ - vector
should contain: an origin of replication for ~ - c ~ aLiuu in host
cells, selective markers, a limited number of useful rPe~rt~r~n enzyme
sites, a high copy number, and strong promoters. A promoter is defined as
a DNA sequence that directs RNA polymerase to bind to DNA and to
10 initiate RNA synthesis. A strong promoter is one vhich causes mRNAs to
be initiated at high frequency. Expression vectors may include, but are
not limited to, cloning vectors, modified cloning vectors, specifically
designed plasmids or viruses.
The term "cloning vector" as used herein refers to a DNA
15 molecule, usually a smaU plasmid or b~rtnrirplt~ DNA capable of se~f-
replication in a host nr~niem, and used to introduce a fragment of foreign
DNA into a host celL The foreign DNA combined with the vector DNA
rr,ncfitlltPc a rernmhin~nt DNA molecule which is derived from
rPcnmhin slnt technology. Cloning vectors may include plasmids,
20 bacteriophages, viruses and cosmids.
The rPrnmhinslnt ~:~,Ult:~S;ull vector in a~u~da~ with the
invention may be prepared by inserting the nucleotide sequence of the
GABAA ~3 subunit into a suitable precursor expression vector ~hereinafter
referred to as the ",Ull,~ Ul vector") using conventional rprnmh;n~nt~ DNA
25 mPtho~nlngy known from the art. The precursor vector may be obtained
cnmmPrri~lly, or constructed by standard techniques from known
expression vectors. The precursor vector suitably contains a selection
marker, typically an antibiotic resistance gene, such as the neomycin or
simpirillin l~,~ e gene. The precursor vector preferably contains a
30 neomycin l~s~ldu~,e gene, adjacent the SV40 early splicing and
Tl271 2 ~ 2~8
-5-
polyadenylation region; an ampicillin resistance gene; and an origin of
r~li~t1nn, e.g. pBR322 ori. The vector also preferably contains an
inducible promoter, such as MMTV-LTR (inducible with (1~Y~lm~tllA.con~)
or metallothionin (inducible with zinc), so that transcription can be
5 controlled in the cell line of this invention. This reduces or avoids any
problem of toxicity in the cells because of the chloride channel intrinsic to
the GABAA receptor.
One suitable precursor vector is pMAMneo, available from
Clontech Laboratories Inc. (Lee et al., N~ture, 1981, 294. 228; and Sardet
et al., Cell, 1989, 56, 271). Alternatively the precursor vector pMSGneo
can be constructed from the vectors pMSG and pSV2neo.
The r~ om~in~nt expression vector of the present invention i9
then produced by cloning the GABAA receptor 1'3 subunit cDNA into the
above precursor vector. The receptor subunit cDNA is subcloned from the
15 vector in which it is harboured, and ligated into a restriction enzyme site,
e.g. the Hindm site, in the polylinker of the precursor vector, for example
pMAMneo or pMSGneo, by standard cloning methodology known from the
art, and in particular by techniques analogous to those described herein.
Before this subcloning, it is often advantageous, in order to improve
20 expression, to modify the end of the ~3 subunit cDNA with ~ititm~l 5
untranslated sequences, for example by modifying the 5' end of the ~3
subunit DNA by addition of 5~ lln~.r~n~l~t.oll region sequences from the a
subunit DNA.
AMENI'ED SHEET
woss/2s234 2' 3~rj8 ~.,. s
According to a further aspect of the present invention, there
is provided a stably CO-I, J. .~ d eukaryotic cell Iine capable of
PYrrPCCin ~ a GABAA receptor, which receptor ~ c at least one
alpha, one beta and the y3 subunit.
This is achieved by co-tr~n cfPctln ~ cells with three e~JL
vectors, each ~ e cDNAs encoding for an a, ,~ or y3 GABAA
receptor subunit. In a further aspect, therefore, the present invention
provides a process for the preparation of a eukaryotic cell Line capable of
expressing a GABAA receptor, which ~o~rricPc stably co-tr~n cfPctin E a
eukaryotic host cell with at least three expression vectors, one such vector
h s~rhollrin ~ the cDNA sequence encoding for an alpha, another such vector
harbouring the cDNA sequence encoding for a beta, and a third such
vector harbouring the cDNA sequence encoding for the y3 GABAA receptor
subunit. The stable cell-line which is Psf~hlichP(l expresses an a~y3
GABAA receptor. Each receptor thereby P~rPCCP~ a unique
rnmhin ~tinn of , ~ and y3 subunits, wi~l be referred to hereinafter as a
GABAAreceptor"subunit c~lmhin~tinn'~, Ph ~rm~r~ and
electrophysiological data conf~nn that the rP~mhin~nt a~y3 receptor
expressed by the cells of the present invention has the properties expected
of a native GABAA receptor.
Expression of the GABAA receptor may be ~cc~-mplichPd by a
variety of di~ferent promoter-~ systems in a v riety of different
host cells. The eukaryotic host cells suitably include yeast, insect and
mslmm~ n cells. Pr~erably the eukaryotic cells which can provide the
host for the ~ of the receptor are m~mms~ n cells. Suitable host
cells include rodent fibroblast lines, for example mouse Ltk-, Chinese
hamster ovary (CH0) and baby hamster kidney (BHK); HeLa; and
HEK293 cells. It is necessary to incorporate at least one a, one ,~ and the
wo g5ng234 ~ 1 8 ~ ~ 5 ~
y3 subunit into the cell line in order to produce the required receptor.
Within this linnit~ti~n, the choice of receptor subunit cnnnhin~tinn is made
according to the t,ype of activity or 3el~_Lvi~y which is being screened for.
For example, bpn7n~ 7prinpq ((lP4;~ d BZ) represent one class of
5 drugs which act upon the GABAA receptor. The presence of an al subunit
is specific for a class of 1~ P.4 having the rh~rn~ y
(lP.Ci~n ~tPd BZl; whereas 2 to as define different ph~rm ~rnln~jrsll
profiles, broadly ~lPCiF~tPd as BZ2. The type of ~ subunit is not critical in
defining the class of bn~ J~l; s ~l); ~P, although a ~ subunit is required.
10 The y3 subunit is also innptlrt~nt in defining BZ scle_Lvi~y. It is lil~ely
that .lil~ tiu . between a subunit s~le~,~ivi~,y is conferred by the y3
subunit.
In order to employ this invention most ~ ly for
screening purposes, it is llrPfPr~hl^ to build up a library of cell lines, each
15 with a different rmnhin~ti~n of subunits. Typically a library of 5 or 6 cell
line types is convenient for this purpose. Preferred subunit rnmhin~tinnc
include: a2,~1y3 and a3~1y3, and in particular as~3y3. These may be used
with cell lines cnnt:3inin~ other subunit rnmhin~tinn~ such as all31y2;
al,B2y2; a2~1yl; a2,~1y2; a3~1Y2; ~4~1Y2; a5~1Y2; a6~1Y2; and all3ly2L
As stated above, for each cell line of the present invention,
three such vectors will be necessary, one ~rnnt~ininE an a subunit, one
r~nt:lininF a ~ subunit, and the third rnnt~ininF the ~3 subunit.
Cells are then co-tr~ncfP~tPd with the desired cnmhin~tinn of
three expression vectors. There are several commonly used techniques for
tr~n cfpcti nn of eukaryotic cells in vitro. Calcium phosphate ~ n
of DNA is most commonly used (Bachetti et al., Proc. N~tl. Acad . Sci.
USA, 1977, 74, 1590-1594; Maitland et ~1., Cell, 1977, 14, 133-141), and
represents a favoured tPrhniqllP in the context of the present invention.
wo ss/2s234 2 ~ 8 8 2 5 8
- 8 -
A small ~ la~ of the host cells takes up the rPrnmhin~nt
DNA. In a small percentage of those, the DNA will integrate into the host
cell chromosome. Because the neomycin . ~ gene will have been
i .. ~.. ,. I ~d into these host ce s, they can be selected by isolating the
5 individual clones which will grow in the presence of neomycin. Each suc'n
clone is then tested to identify those which will produce the receptor. This
is achieved by inducing the production, for example with llPY~mPthslennp
and then detecting the presence of receptor by means of r~flinliF~n~
binding.
In a further aspect, the present invention provides protein
prep~r~tinn e of GABAA receptor subunit rnmhin sl* nn e especially human
GABAA receptor subunit rnmhin~tinnc, comprising the human y3 GABAA
receptor subunit derived from cultures of stably ll aul;~L~ d eukaryotic
cells. The invention also provides rrPr~r~tinn A of mPmhr~n Pq n ~ E
15 subunit cnmhin~*nnc of the GABAA receptor, especially human GABAA
receptor subunit rnmhin:ltinne ~ e the human y3 GABAA receptor
subunit derived from cultures of stably tr~nqfPct~d eukaryotic cells. In an
especiaUy preferred Pmho~imPnt, the invent;on provides cell mPmhrs~nPA
rnnt:linine a human GABAA receptor ~...u.i~l;..e of an a~y3 subunit
20 rnmhin~tinn isolated from stably ~ r~ mouse Ltk- fibroblast cells,
most especially an as~3y3 subunit r~nhin~tinn
The cell line, and the mPmhr~nP preparations therefrom,
according to the present invention have utility in screening and design of
drugs which act upon the GABAA receptor, for example b~ ",.l ;~ ,~l.; . .PA,
25 ba~ u~a~t:s, ,B-carbolines and neurosteroids The present invention
s~rrnrAin ely provides the use of the cell line described above, and
m~mhr~nP pr~pPr~tinne derived therefrom, in screening for and designing
mPtlir~mrntA which act upon the GABAA receptor. Of particular interest
in this context are molecules capable of interacting selectively with
~ woss/2s234 ~ ~ 882~;8 r ~
GABAA receptors made up of varying subunit f~nmhin ~ti rn q As will be
readily apparent, the cell line in ~ .1u ~ f- with the present invention,
and the mPmhr~nP prPr~r~tinns derived thPrPfrnm provide ideal systems
for the study of structure, ph~r~ ~ ,,y and function of the various
5 GABAA receptor subtypes.
The following non-limiting FY~mI'lPC illustrate the present
invention.
EXAMPLE I
ISOLATION AND SEQUENCING OF cDNAS ENCODING THE
HUMAN GABAA RECEPTOR y3 SUBUNIT
a) cDNA libraries
cDNAs were cloned from human foetal brain cDNA libraries.
All cDNA libraries were cu~ u~l~d in the lambdaZAP vector, and were
purchased from Stratagene (San Diego, C~lifnrni~). For screening, the
cDNA libraries were plated according to the ~ u~ ,. r~ instructions,
at 40,000 pfu per 137 mm plate. Filter lifts .~ere taken using Hybond N
filters ~AmPrch~m) according to the m~nllf~rrl-rer's ~ u.,l.iu..s.
b) Isolation of cDNA encodin~ human ~3 subunit,
A rat y3 cDNA probe was first ~n~r~t~d by PCR using
25 nli~nnllnlPoti~o primers derived from the rat y3 sequence f~Knoflach et fll,
FEBS Lett., 1991, 293,191):
5'ATTCAAGCTTACCATGGCTGCAAAGCTGCTGCTTCTCTGCCTGTTCT
CGGG3'(bp 177-217,with 13basesonthe5'endf lluil~ aHind m
restriction Cite) SEQ. ID. NO.: 1, and
- ~=~
Wog5129234 2 ~ ~8258 ]~,IJ~ 1
- - 10 -
5'GGAATTGTTTAACGTGATCATCACGGGTG3' (bp 1330-1358, antisense)
SEQ. ID. NO.: 2. PCR ~vas performed as described, for example, by
Whiting et al in Proc Natl. Acad. Sc~. USA, 1990, ~, 9966, using rat ,.
brain cDNA as a template. A 1250bp PCR product was obtained which
when digested with Hind m was cut into 2 pieces of 900bp and 350bp in
size. The 900bp fragment was subcloned into the Hind m site of
pRlll~srrillt SK-(str~t~nf~) and its identity cnn~rm~d by DNA
~q..~ using standard terhnigll~c and t_e ~ u~ II enzyme
(United States R~rl..-...;. ,~lc).
A human foetal brain cDNA Iibrary was screened using 32p
labelled rat y3 900bp DNA as described above. A single cDNA clone was
obtained. Sequence analysis was p~rfnrmf~r~, using an Applied Biosystems
373A DNA se4uG..~G~ and dye l. . ~ chemistry according to the
~z"~..r~ 2' iUal~lu-~..iOl12. This cDNAlackedboth the 5' and 3' ends of
15 the coding region. These were sllhseqll~ntly obtained by anchored PCR.
For the 3' end, a sense nli~ -rl~ derivedfrom sequence near the 3'
end of the truncated cDNA clone
(5'CCAGATTCCTCAAGATGATTCCTGAGCGAATAAG3', incorporating an
EcoRI site) SEQ. ID. NO.: 3 was used in conjunction with an
20 oligonucleotide "anchor" primer derived from the T7 primer sequence of
the pRll~crri~t vector
(5'AGCGCGCGTAATACGACTCACTATAGGGCGAA3') SEQ. ID. NO.: 4 in
a PCR reaction with human foetal brain cDNA library as template. A
500bp PCR product was obtained and cllhrlnnrtl into EcoRl cut
25 pF~ r-~rnl)t SK-. Sequencing, as above, rr.,nfirm~d that it contained the 3'
end of the human y3 coding region, together with 13 lbp of 3~ llntr~ncl~tRd
region sequence. The missing 5' ~e4..~.l~s of the y3 cDNA were obtained
using human brain "5' RACE Ready cDNA", obtained from CLONTECH -
(part no. 7302-1), using the antisense primers
5~GCTTTTTATCATATGCTCTTAGCAAC3' SEQ. ID. NO: 5 and
, . . . _ . .... .. , = .
T1271 ~ 2~
.
- 11
5'CAAGACCCACATATGG~GATGGAGA3' SEQ. ID. NO.: 6, derived
from the very 5' end of the truncated r3 cDNA clone. The anchored PCR
was performed according to manufacturers' instructions, and a 200bp PCR
product obtained which was subcloned into the p-CR-Script vector
(Stratagene),againaccordingtothem~ (" ~ r~ instructions. DNA
sequencing confirm~d that the 200bp PCR product contained the missing
5' coding region of the human y3 cDNA, together with 25bp of 5'
ntr~n.c1s3t.~d region.
The complete nucleotide sequence of the cDNA encoding the
human r3 subunit, together with the deduced amino acid sequence
corresponding thereto is shown in SEQ. ID. NO.: 7.
EXAMPLE ~
PREPARATION OF STABLY TRANSFECTED CELLS
EXPRESSING a6133r3 SUBUNIT COMBINATION OF THE ~UMAN
GABAA RECEPTOR
Human as (see Tntf~rn~ti~nsll patent sp~ ifi-~tl~n no. WO
92/22~52), ,B3 (Wagstaff et al, Genomics, lg91, 11, 1071) and y3 cDNAs
were subcloned into the eukaryotic ~p~ iiUIl vector pMSGneo (the
preparation of which is described in WO 92/22(~52) using standard
techniques (cf. Maniatis et al., in Molecular Clo~ing, A l,aboratory
Manual, Cold Spring Harbor Press, New York, 2nd Edition, 1989) and a
25 stable cell line expressing human as~3y3 GABA-A receptor ~c~hlich~d
according to the methodology described in Example l of WO 92/22652.
A~ !~.3 ~IEE,
wo 9sl2s~34 2 l 8 8 2 5 8
- 12-
EXAMPLE 3
ClIARACT~RT~ATTON OF STABLY TRANSFECTED CELLS
EXPRESSING as~3y3 SUBUNIT COMBINATION OF TiIE HUMAN
6 GABAA RECEPTOR
Expression of r~acr~hin ~nt s,~3Y3 hurnan GABAA receptors
is ~ ' ' ' by r~linln~ir~l binding. Tr~ncfartPd cells which had been
induced by culture in ~a~nl ~th ~c~ma crnt~ininE medium for 3-5 days
(according to math n~l -' r ~y described in Example 2 of WO 92l22652) were
harvested and cell mamhr~n Pc prepared (again according to m~th r ~nl rgy
described in Example 2 of WO 92/22652). ~t..rAti.~n binding curves
(Figure 3) were obtained by inrllh~tinF cell mannhr~n~c with various
.~.. l ~ ~I:.. c of 3H Rol5-1788 (obtained from New England Nuclear, Du15 Pont (U.K.) Ltd., Stevenage), with non-specific binding measured in the
presence of 10~1M n....;~ .. (obtained from Sigma Chemical
Company, Poole, UE~). All binding assays were p~ 1 in triplicate in
an assay volume of 0.5ml, with an inCllh~tirn time of 90min at 4C.
Tnrllh~tinnq were iarmin~tPd by filtration through GF/B filters ~randel,
20 Gathersberg, MD) on a Tomtech cell harvester, followed by three washes
in ice-cold assay buffer. After drying, filter-retained radioactivity was
measured by liquid srintill~tinn counting.
A ce~l line prepared as described in Example 2 expressed
approximately 80fmol [3H]Rol5-1788 binding sites/mg protein following a
25 5-day induction of receptor ''~ 4' '`" The ~,UleS~u.~ of human as, ~3
and y3 mRNA transcripts was rrnfirnn~ by isolation of mRNA, cDNA
synthesis and PCR using subunit specific oligonucleotide primers in a
conventional manner.
W0 9~/29234 j
- 13 -
Scatchard anRlysis of S~tl~rRti-m binding curves for
[3HlRol5-1788 was p~ d for mPmhr~R~n~ pr~pRrRtirn~ from two cell
lines ~ g the as,B3y3 subunit f'A~nhinRtii n according to the present
invention, giving the following KD values (mean i SEM~: 0.32iO.06nM and
5 O.G3iO.llnM.
woss/2s234 2 1 8 ~258 ,~
- 14-
SEQUENCE LISTING
(1) GENERAL INFORMATION:
6 (i) APPLICANT:
(A) NAME: Merck Sharp & Dohrae Limited
~B) STREET: T~ings Park
(C) CITY: Harlow
(D) STATE: Essex
(E) COUNTRY: England
(F~ POSTAL CODE (ZIP): CM20 2QR
(ii) TmE OF I~tENTION: Nucleic Acids
(iii) NUMBER OF SEQUENCES: 8
(iv) COMPUTER RT.~AnAT~T.T1~ FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC rnmr;l~ihlp
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Versior~ #1.25 (EPO)
(2) INFORMATTON ~OR SEQ ID NO: 1:
26
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: ~1 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: sir gle
a)) TOPGLOGY: linear
Vl/O 95/29234 ~ 1 8 8 2 ~ ~ r~
- 15-
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
ATTC,.AGCTT ACC~TGGCTG CMAGCTGCT GCTTCTCTGC CTGTTCTCGG C 51
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARAl;l~ llCS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 3inear
16
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DES5RIPTION: SEQ ID NO: 2:
~CMTTGTTT AACGTGATCA TCACGGGTG 29
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARA( ~ CS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(I)) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
woss/2s234 21 8 8 2 5 8 P~
- 16 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CCAUTTCCT CAAUTUTT CCTCACCGM TAAC 34
(2) INFOR~IATION FOR SEQ ID NO: ~:
(i) SEQUENCE C~A :l~;~lSllCS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
AGCCCGCGTA ATACCACTC~ CTATAGGGCG M 32
20 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
~) TYPE: nucleic acid
26 (C) STRANDEDNESS: single
(D) TOPGLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:~
GCTTTTTATC ATATGCTCTT AGCAAC 26
wo gs/29234 2 1 8 8 2 5 8 r~~
.
- 17-
-~ (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARA(;~ lCS:
(A) LENGTH: 27 base pairs
~) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUEN5E DESCRIPTION: SEQ ID NO: 6:
C~ACACCCAC ATATCGTTTG ATCCACA 27
(2) INFOP~MATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1565 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
- (A) NAMEIKEY: CDS
(B) LOCATION: 33..143G
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
=
WO 9929D4 2 1 ~ 8 2 ~ 8
- 18 -
TCAATTCGTG AGATGGCCAG CTCCACGGU CC ATG GCC CCG MG CTG CTG CTC 53
Met Al~ Pro Lys Leu Leu Leu
CTC CTC TGC CTG TTC TCG GGC TTG CAC GCG CGG TCC AGA MG GTG GAA 101
LeU Leu Cy Leu Ph~ ser Gly L~u His Al- Arg 8er Ars Ly~ V~ CIU
10 15 20
CAC GAT GM TAT G~A GAT TU TCA TU MC CAA AAG TGC GTC TTC CCT 149
0 Clu Asp Clu Tyr GIU Asp Ser ser S~r Asn Gln Lys Trp Val LeU Aln
25 30 35
CCA MM TCC CAA G~C ACC GAC CTC ACT CtT ATT CTC MC AAC TTC CTA 197
Pro Lys s~r Cln Asp Thr Asp V l Thr Leu lle Leu Asn Lys LeU Leu
40 45 S0 SS
AGA GAG TAT GAT MA MG CTG AGG CCA CAT ATT GGA ATA AAA CCG ACC 245
Arg Glu Tyr Asp Lys Lys LeU Arg Pro Asp lle Gly lle Lys Pro Thr
60 65 70
GTA ATT GAC GTT GAC ATT TAT GTT MC AGC ATT GGT CCT GTG TCA Ta 293
V~l lle Asp Val Asp lle Tyr Val ~sn Ser lle Gly Pro Vol Ser ser
75 80 BS
ATA MC ATG GAA TAC UM ATT GAC ATA TTT TTT GCT UG ACC TGG ACA 341
lle Asn Me~ Glu Tyr Gln lle Asp lle Phe Phe Ala G~n Thr Trp Thr
90 9S 100
CAT AGT CGC CTT CCA TTC AAC AGC AU ATG MA ATT CTT ACT CTG AAC 3B9
Asp Ser Aro Leu Arg Ph~ Asn S~r Thr M~t Lys l~e L~u Thr Leu Asn
105 110 115
AGC MC ATG GTG GGG TTA ATC TGG ATC CCA GAC ACC ATC TTC CGC MT 437
ser Asn Met Va~ G~y Leu l~e Trp l~e Pro Asp Thr l~e Phe Arg Asn
lZ0 125 130 135
TCT MA ACC GCA GAG GCT CAC TGG ATC ACC ACA CCC AAT CAG CTC CTC 4BS
ser Lys Thr Ala G~u Al~ H~s Trp lle Thr Thr Pro Asn Gln Leu Leu
140 145 150
CGG ATT TGG MT GAC GGG AAA ATC CTT TAC ACT TTG AGG CTC ACC ATC 533
Arg lle Trp Asn Asp Gly Lys lle Leu Tyr Thr Leu Arg Leu Thr lle
155 160 165
. . .
~ WO 9~Q9234 2 1 8 ~ 2 5 8 P~
MT GCT GAG TGC CAC CTG UG CTC CAC MC TTC CCC ATG GAC GAA UC 581
A~n Al- Glu Cy~ Gln Leu G~n Leu Hi~ Asn Phe Pro Met Asp Glu Nis
170 1h 180
5TCc TGC CCG CTG ATT TTC TCC AGC TAT CGC TAT CCC MM C~A CM ATG 629
Ser Cys Pro Leu l~e Ph~ Ser Ser Tyr Gly Tyr Pro Lys Glu Glu Met
185 190 195
ATT TAT AGA TGG AGA MM MT TCA GTG GAG GCA GCT GAC CAG MM TCA 677
0lle Tyr Arg Trp Arg Ly~ Asn Ser Vnl Glu Ala Al- Asp Cln Ly Ser
200 205 210 215
TGG CGG CTT TAT CAC TTT GAC TTC ATG GGC CTC AGA MC ACC ACA GM 7Z5
Trp Arg Leu Tyr Gln Phe Asp Ph~ M~t Gly Leu Arg A~n Thr Thr Glu
15 220 ZS 230
ATC GTG ACA ACG TCT GCA GGT GAT TAT GTT GTC ATG ACT ATA TAT TTT m
lle Val Thr Thr Ser ~1- Gly Asp Tyr V-l V-l Met Thr 11- Tyr Ph~
235 240 245
GAA TTG AGT AGA AGA ATG GGA TAC TTC ACC ATT CAG AC~ TAC ATT CCC 821
Glu Leu S~r Arg Arg Met Gly Tyr Phe Thr l~e Gln Thr Tyr lle Pro
ZSO 255 260
25TGT ATA CTG ACT GTG CTT TTA TCC TGG GTG TU TTT TGG ATC AAA A~A 869
Cy~ ~e Leu Thr Yal V~l Leu ser Trp Val Ser Phe Trp lle Ly5 Lys
Z65 270 275
GAT GCT ACG CCA GCA AGA ACA GCA TTA GGC ATC ACC A~G GTG CTG ACC 917
30Asp Al- Thr Pro Al- Arg Thr Al~ Leu Gly lle Thr ~ - V~l Leu Thr
280 285 Z90 Z95
ATG ACC ACC CTC AGC ACC ATC GCC AGG MG TCC TTG CCA CGC GTG TCC 965
Met Thr Thr Leu Ser Thr lle Alfl Arg Lys Ser Leu Pro Arg V-l Ser
35 ~oo 305 310
TAC OTG ACC GCC ATG GAC CTT TTT GTG ACT GTG TGC TTC CTG TTT GTC 1013
Tyr V-l Thr Ala Met Asp Leu Phe Val Thr Val Cys Phe Leu Phe Val
315 3Z0 325
TTC GCC GCG CTG ATG GAG TAT GCC ACC CTC MC TAC TAT TCC AGC TGT 1061
Phe Al- Ala Leu Met GIU Tyr AL- Thr Leu Asn Tyr Tyr Ser Ser Cys
330 335 340
wossns234 2 ~ 8 ~258 r~
- 20 -
AGA AAA CCA ACC ACC ACG AAA MG ACA ACA TCC TTA CTA CAT CCA CAT t109
Aro ~ys Pro Thr Thr Thr Lys Lyi Thr Thr S~r Leu Leu hi~ Pro Asp
345 350 355
TCC TCA AGA TGC ATT CCT GAC CGA ATA ACC CTA CAA LCC CCT TCC MC 1157
Ser S-r Arg Trp lle Pro Glu Ar~ lle S~r Leu C~n Al~ Pro ser Asn
360 365 370 375
TAT TCC CTC CTC GAC ATC AGG CQ~ CCA CCA CCT CCG ATG ATC ACT TTA 1205
0 Tyr s~r Leu Leu Asp Het Ars Pro Pro Pro Pro Ala Met lle Thr Leu
3aO 3a5 390
MC MT TCC CTT TAC TCC CAC GAA TTT GAA GAT ACC TGT GTC TAT GAG ~253
A~n Asn S~r Va~ Tyr Trp Cln Clu Phe Clu Asp Thr Cys V~ Tyr CIU
395 400 405
TCT CTG GAT GCC AAA GAC TCT CAC ACC TTC TT~ TCC TGC TAT GAA CAA 1301
Cyc Leu Asp Gly Ly~ Asp Cyi Gln Ser Ph~ Phe Cys Cyr. Tyr GIU Glu
410 415 420
TGT A~A TCA GGA TCC TCC ACC AAA CCC CCT ATT CAC ATA GAC ATC TTC 1349
Cy~ Lys Ser Gly Ser Trp Ars LYs G~y Ars lle Uis lle Asp lle Leu
425 U0 435
GAC CTG GAC TCG TAC TCC CGG GTC TTT TTC CCC ACG TCC TTC CTG CTC 1397
Clu Leu ASp Ser Tyr ser Arc V~l~ Phe Phe Pro Thr Ser Phe Leu Leu
440 445 450 455
TTT AAC CTC CTC TAC TGG GTT GGA TAC CTG TAT CTC TAAGTGTTGC 1443
Ph~ Asn Leu Vl~ Tyr Trp Val G~y Tyr Leu Tyr Leu
460 465
TCAGAGTGAA GAGTGMGAG CATTTGGTAC ACACTTGACC TTCTGTCGTC CCCAGACC~G 1503
TAGTGACCM TCGGGAGTAG C4AGGAAGGA CACTGCCCAG TGTATCTTGT TATAMTGAC 1563
CG 1565
(2) INFOR~ATION FOR SEQ ID NO: 8: .
(i) SEQUENCE CHARA~ lCS:
(A) LE~GTH: 467 amino aci~s
wo 95~2~234 2 ~ 8 8 2 ~ 8 r~
- 21 -
~) TYPE: an~ino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ m NO: 8:
Met Ala Pro Lys Leu Leu Leu Leu Leu Cys L~u Phe Ser Sly Leu Hi~
5 10 15
Ala Arg Ser Arg Lys VAI Glu Glu Asp Glu Tyr G~u Asp Ser Ser Ser
20 25 30
Asn Gln Lys Trp V~l Leu Al- Pro Lys Ser Gln Asp Thr Asp V~l Thr
35 40 45
L~u lle Leu Asn Lys Leu Leu Ars Glu Tyr Asp Lys Lys Leu Arg Pro
50 55 60
Asp 1~ Gly l~e Lys Pro Thr Val lle Asp Val ~sp lle Tyr Val Asn
65 70 75 80
ser lle Cly Pro Vl~l 5~r Ser lle Asn Met Olu Tyr Cln ILe Asp 11
85 90 95
Phe Phe Al~ Gln Thr Trp Thr Asp Ser Arg Leu Arg Phe Asn Ser Thr
100 105 110
Mee Lys lle Leu Thr Leu Asn Ser Asri Met V~l C~y Leu lle Trp lle
115 120 125
Pro Asp Thr lle Phe Arg Asn ser Lys Thr Al~ Clu Ala His Trp lle
130 135 140
Thr Thr Pro Asn Cln Leu Leu Ars 1 le Trp Asn Asp Gly Lys I le Leu
145 tS0 155 160
Tyr Thr Leu Arg Leu Thr lle Asn Al~ Clu Cys Cln Leu Cln Leu His
165 170 175
Asn Phe Pro Met Asp Clu His Ser Cys Pro Leu lle Phe Ser Ser Tyr
180 185 190
W0 95/29234 2 1 8 8 2 5 8 ~ . ~ 1 ~
-22 -
Gly Tyr Pro Ly~ C~u Clu Met lle Tyr Arg Trp Arg Ly~ A~n Ser Val
195 Z00 205
Glu Al- Al- Asp Gln Ly~ Ser Trp Arg Leu Tyr Gln Ph~ Asp Phe Ret
2~0 215 220
Gly Leu Arg A~n Thr Thr Glu 11~ Val Thr Thr Ser AIA Gly Asp Tyr
225 230 235 240
0 Val V~l Met Thr lle Tyr Phe Glu L~U Ser Arg Arg Met Gly Tyr Phe
245 250 255
Thr lle Gln Thr Tyr lle Pro Cys 11~ Leu Thr V l V-l Leu Ser Trp
260 265 270
Val Ser Phe Trp lle Ly~ Lys A~p Ala Thr Pro Al- Arg Thr Al- Leu
275 280 285
Cly lle Thr Thr Vol Leu Thr Met Thr Thr Leu ser Thr lle Alo Ar~
290 295 300
Lys Ser Leu Pro Arg V-l Ser Tyr V~l Thr Al- Met Asp L~u Phe Val
305 310 ~tS 320
Thr V l Cys Phe Leu Phe V-l Phe Al- Alo Leu Met Glu Tyr Al- Thr
325 330 335
Leu Asn Tyr Tyr Ser Ser Cys Arg Lys Pro Thr Thr Thr Lys Lys Thr
340 345 350
Thr Ser Leu Leu Ni~ Pro A~p Ser Ser Arg Trp lle Pro Glu Arg lle
355 360 365
Ser Leu Cln Al- Pro ser A~n Tyr Ser Leu Leu Asp Met Arg Pro Pro
370 375 380
Pro Pro A~a Met lle Thr Leu A~n Asn Scr Y-l Tyr Trp Gln Glu Phe
385 390 395 400
0 GIU Asp Thr Cy~ Val Tyr Glu Cy~ Leu Asp Gly Ly~ Asp Cys Gln Ser
405 4~0 415
he Phe CYs Cys Tyr Glu Glu Cys Lys Ser Gly Ser Trp Arg Lys Giy
420 425 430
46
~ W0 95129234 2 1 ~ 8 2 5 8
-
- 23 -
Ar9 lle ~i- Ile ~sp lle Leu Glu Leu Asp Ser Tyr Ser Ar~ V~l Phe
435 440 445
Phe Prr, Thr Ser Phe Leu Leu Phe Asn Leu V~ Tyr Trp Y~Z~ Gly Iyr
450 455 ~,60
Leu Tyr Leu
465
