Note: Descriptions are shown in the official language in which they were submitted.
~0 9G/01899 1~ r ~1~ /4
219431~1
~IF,THOD FOR IDENTIFYING NUCl,F,lC ACIDS I~NCODIl~IG
c-fos PROMOTE,R ACTIVATlNG PROTEINS
The present invention relates to materials and
5 methods for identifying signal trAneducing molecules which
activate the human c-fos proto-oncogene promoter and
antagonists of such molecules.
BACKGROUND O~ THF, INVF:I~TION
Cell activation as a result of mutation or over-
expression of signalling mnl~cules, such as the proto-oncogenes
Ha-ras, c-fos, c-myc, and c-jun, has been implicated in the
aberrant growth of cells that forms the basis of neoplasia. See,
15 DeFeo, etaL,Proc. Natl. Acad. Sci..78, 3328-3332 (1981); Miller,
et al., Cell, 36, 51-60 (1984); Kelekar, et al., Mol. Cell. Biol.. 6. 7-
14 (1986); and Vogt, et al., Adv. Cancer Res.. 55, 1-35 (1990).
Induction of cfos occurs in response to the
activation of growth-relate.d signalling pathways following
20 serum stim~ tion of mouse 3T3 cells, or in response to
o~G.c~ ,;,sion of the normal and transforming versions of
Ha-ras, IGs~e~liYGly. It has also been shown that COI~lilulive
e~pression of c-fos occurs in certain human tumor lines. These
findings suggest that the aberrant growth characteristic of the
25 neoplastic phenotype can involve the constitutive activation of
signal transduction pathways participating in c-fos
proto-oncogene induction. See, Greenberg. et al., Nature, ~,
433-438 (1984); Stacey, et al., Mol. Cell. Biol.. ~, 523-527
(1987); and O'Hara, et al., Mol. Cell. Biol.. 7, 2941-2946 (1987).
By using c-fos promoter-driven reporter genes,
specific enhancers in the c-fos proto-oncogene promoter have
been identified which respond to activated signal transduction
pathways. These enhancers include a tyrosine kinase
responsive SCM, raf~ ollsivG direct repeats, a protein kinase
WO 96/01899 ~ ~ P~.l/u,.... ~
2~ 3~
-2-
C-responsive AP-1 site, and a ras-responsive semm response
element. See, Fujii, e~ al., Mol. Cell. Biol.. 2, 2493-249g (1989);
Hayes, c~al.,Proc. N~tl. A(~l Sci.. USA.~L, 1272-1276 (1987);
Jamal, et al., Nature, 344, 463466 (1990); Gutman, et aL,~,
C-~.11 BjQI.. I1, 5381-5387 (1991); and Fisch, et al., J~ol. Cell. Biol
9, 1327-1331 (1989).
Contingent replication systems employing
l...nsc~ ional activation of the SV40 T antigen gene to identify
enhancers and stably interacting transcription factors are
10 known. See, Vasavada, er a1., Ind. J. Biochrm Bio~hys.. ~. 488-
494 (1988); Vasavada, et al., (',ene, ~, 29-40 (1987~;
Vasavada, et al., Proc. Natl. Acad. Sci.. 88, 10686-10690 (1991);
and Rusconi, et ~1.,~,~, 211-221 (1990).
Because of the i~ .-hncc of signalling mnlt~cnll~s in
15 the control of cellular proliferation, there is a need for methods
to identify mnlecllles involved in growth-related signaling
systems which can in turn be used to identify biological targets
for antitumor drug discovery. There is also a ne,-d for methods
of identifying agents that can interfere with such growth-
20 related signaling systems to restore normal growth whenabnormal cell proliferation is occurring.
SUMMARY OF THE INV~NT~ON
The present invention fills the foregoing needs by
providing materials and methods for identifying signal
transduction mnlect-l~s and antagonists thereof. More
specifically, this invention provides m~nm~ n cell lines, the
cells of which comprise
~a) a l~co.. ~ nt vector comprising an inducible. or
tissue specific promoter operatively linked to a nucleic acid
encoding polyomavirus large T antigen; and
(b) a recomhin~nt expression vector comprising a
polyomavirus origin of replication and a nucleic acid suspected
35 to encode an activating protein of said promoter.
~O 96101899 ~ 1 g ~L 3 6 1 PCTIUS95/07874
Preferably the promoter is the human c-fos
promoter and the activating protein is a human c-fos promoter
activating protein.
The present invention further provides a method for
5 identifying a nucleic acid encoding a promoter activating
protein, comprising:
(a) culturing a m~mm~lion cell line, the cells of which
comprise:
(i~ a recombinant vector comprising an inducible
or tissue specific promoter operatively linked to the
coding region of the polyomavirus large T antigen gene;
and
(ii) a recombinant expression vector comprising a
polyomavirus origin of replication and a nucleic acid
suspected to encode an activating protein of said
promoter,
under conditions in which such nucleic acids are expressed; and
(b) measuring the levels of replicated vectors in the
cells after a period of incubation sufficient to permit vector
replication;
whereby a nucleic acid encoding a human promoter activating
protein is identified by mc&sui,;.--elll of increased levels of
vectors in the cells.
Preferably the promoter is a human c-fos promoter
and the activating protein is a human c-fos promoter activating
protein.
A preferred recombinant vector comprising a human
c-fos promoter for use in the present invention is the plasmid
PfLAG-8.
A preferred recombinant expression vector
comprising a polyomavirus origin of replication is the plasmid
La2.
The present invention also provides a human c-f os
promoter activating proteins having the amino acid sequences
defined in the Sequence Listings SEQ ID NO:l and SEQ ID NO:3, or
WO 9C101899 T~ 4
2 ~ 6 t
-4-
an antigenic fragments thereof, and nucleic acids encoding such
protein or fragments.
In another embodiment, the present invention
provides mS~mm~ n cell lines, the cells of which comprise:
(a) a first recombinant expression vector ~ i"g a
reporter gene operatively linked to a human c-fos promoter;
and
(b) a second recombinant expression vector comprising
a nucleic acid encoding a human c-fos promoter activating
1 0 protein.
The present invention also provides a me~hod for
identifying an antagonist of a human c-fos promoter activating
protein, comprising-
(a) providing a ms)mm~ n cell line, the cells of which
1 5 comprise:
(i) a first recombinant expression vector
c~ ;llg a reporter gene operatively lin~:ed to a human
c-fos promoter; and
(ii) a second recombinant expressi(m vector
comprising a nucleic acid encoding a human c-fos
promoter activating protein;
(b) cont~ting the cell line of step (a) with a sample
suspected to contain an antagonist of the human c-fos promoter
activating protein; and
(c) measuring the level of exprcssion of the reporter
gene;
whereby an antagonist of the human c-fos promoter activating
protein in the sarnple is identified by ,..easu-c~ t of a
reduced level of expression of the reporter gene.
Preferably the second recombinant expression
vector encodes CROC-l protein, CROC-4 protein or
o~2-macroglobulin receptor-associated protein.
~10 g6/01899 ~ /4
3 6 ~
-s-
DETAILED DESCRIPTION
All references cited herein are hereby
incorporated in their entirety by reference.
The following terms are herein denoted by the
indicated abbrevintions: long terminal repeat (LTR);
Dulbecco's modified Eagle's medium (DMEM); serum
response element (SRE); chloramphenicol acetyltransferase
~ (CAT).
All nucleic acid sequences disclosed follow the
normal 5' to 3' convention, as read from left to right.
Standard single-letter abbreviations are used for the
nncl~.otitle bases in the seqllences (37 C.F.R. 1.822).
The term "antagonist" is defined herein as a
15 substance that blocks or inhibits the effects of a human c-fos
promoter actvating protein, such as the CROC-l protein or
c~2-macroglobulin receptor-associated protein.
The term "reporter gene" as used herein means
either a DNA molecule isolated from genomic DNA, which
20 may or may not contain introns, or a comrlP.ml~nt~ry DNA
(cDNA) prepared using messenger RNA as a template. In
either case, the DNA encodes an expression product that is
readily measurable, e.g., by enzymatic activity, enzyme-
linked immunosorbent assay (ELISA) or ra~ imml-noassay
25 (RIA). Preferred reporter genes for use in the present
invention include the E. co~i Lac-Z gene from pCH110
(Stratagene #27-4508-01). The expression level of this gene
can be measured by a sensitive fluorescent substrate assay.
Also preferred is the CAT reporter gene described below,
30 although many others well known in the art could be used
instead.
The term "recombinant expression vector" means
a vector prepared using recombinanl techniques said vector
comprising an inserted nucleic acid encoding a protein such
35 that said vector is capable of expressing the protein upon
wo g6,0l899 2 1 g ~ 3 6 1 ~ P~IUS95/078'~4 1~
-6~
transfection or transformation into a suitable host cell.
Preferred is a vector comprising a nucleic acid encoding a
promoter activating protein. Also preferred is a vector
comprising a reporter gene operatively linked to a human c-
fos promoter.
Cells which have been "stably transformed" have
rec---'- DNA incorporated into their genomic DNA. Such
stably incol~ol~tcd DNA is retained by the transi'ormed cells
because it is introduced into the cells with a selection marker,
such as G418 resistance, which forces retention when the cells
are grown in selection medium. The present invention employs
transiently transfected m!lmmS li51n cell lines, however stably
t~ ro-lllcd m~mm~liAn cell lines comprising a c-fos promoter
regulated large T antigen can also be used.
The inducible or tissue specific promoters of the
present invention are non-hous~keeping promoters, i.e., they
are regulated and are not transcriptionally active under normal
conditions, except to the extent that low basal levels of
constitutive expression may occur.
As defined herein, "inducible plol~lot~l~" are
,,lllolc~ the l ~Ins~,liL~lion activity of which is activated or
enhanced in response to changes in the cellular environment
that results in a cellular response, such as stress, hormonal
~timnl~tion or differentiation. Induction occurs via activation of
a sigr!~lling cascade resulting in the enhanced binding and
activity of transcription factors at the promoter site. Molecules
involYed in such induction include promoter activating proteins
as descr;bed herein. Inducible promoters include the c-fos and
c-myc ~)..l - Another inducible promoter is the multidrug
30 resistance gene promoter described in J. Biol. Chem,. ~,
15347-15350 (1993).
The term "tissue specific promoter" means a
promoter which is active only within a subset of cell types, such
as promoters which are active only in prostate cells. See. Young,
ef al.,Biochem.. 31, 818-824 (1992); and Riegman, et al., ~QL
01899 ~ 14
Endocrinol..~, (No. 12) 1921-lg30 (1991). Other tissue specific
promoters include promoters of late histone genes and
n(IIIW~ of muscle regulatory elements. See, Genes Dev.. ~,
849-859 (1990); Mol. Cell. Biol..,~. 515-522 (1989); and Mol.
5 Cell. Biol.. 2, 2191-2201 (1989).
Promoters that can be used in this invention include
but are not limited to the promoters of the proto-oncog. ~s c-
fos and c-myc . See, Miller, et al., supra; and Kelekar, et al.,
s~pra. Both of these promoters regulate expression i~Z v~vo of
10 genes the ù~ c~ o;~ion of which can lead to aberrant cell
growth. Most preferred is the c-fos promoter.
The term "aberrant cell growth" is herein defined as
the abnormal or uncontrolled cell proliferation characteristic of
neoplasms .
As used hereiD, the term "promoter activating
protein" is defined as a protein which causes transcriptional
activation of one of the above-mentioned promoters. Preferably
the promoter activating protein is a human c-fos promoter
activating protein. Most preferred is an activating protein
20 having an amino acid sequence subsl lnti?lly identical to that of
the a2-macroglobulin receptor-associated protein. Also most
preferred is an activating protein having an amino acid
sequence 5~hs~?n~ 1y identical to that of the CROC-4 protein or
the CROC-l protein, the S~IU~ C~ of which are defined by SEQ
25 ID NO:3 and SEQ ID NO:1, respectively. Substantial identity of
amino acid ~ uellccs means that the sequence of another c-fos
promoter activating protein compared to the sequence defined
by either SEQ ID NO:1 or
SEQ ID NO:3 is identical or differs by one or more amino acid
30 alterations (deletions, additions, substitutions) that do not
subst~ntiSllly impair transcription activating activity as
described herein. For example, there may be allelic or
interspecies variants of the sequences defined by either
S~Q ID NO: 1 or SEQ ID NO:3.
W0 ~6/0~89g ~ /Q I4 ~
gl ~lA~~ i
l~ld~Vl
FullL~ o~c. it is well within the skill of the art.
e.g., by chernical synthesis or by the use of modi~led
polymerase chain reaction (PCR) primers or site-directed
m~-t~gPn~cic to modify DNA encoding a c-fos promoter
5 activating protein having the sequence defined by either SEQ
ID NO:] or SEO 11~ NO-3, to produce single or multiple base
substitutions which do not suhst~n~i~lly impair the activity
of c-fos promoter activating proteins produced therefrom.
Such c-~- vdtiYely modified variants are within the scope
10 of this invention.
Sequence identity, is det~ incd by optimizing
residue matches, if necessary, and by introducing gaps as
required. This changes when considering conservative
subsfitnlione as matches. Conservative substitutions typically
15 include substitutions within the ~ollowing groups: glycine,
alanine; valine, icol -, leucine; aspartic acid, glutamic acid;
asparagine, ~lut~minP; serine, threonine; Iysine, arginine; and
phenylalanine, tyrosine. Homologous amino acid sequences are
typically intended to include natural allelic and intc.~ ie;s
20 variations in each respective protein sequence. Typical
homologous proteins or peptides will have from 25-100%
homology (if gaps can be introduced), to 50-100% homology (if
conservative subQ~it~ltiong are included) with the amino acid
sequence of the CROC-1 protein or CROC-4 protein. Homology
25 measures will be at least about 50%, and typically at least 60%
or more.
The present invention also comprises "antigenic
fragments" of a human c-fos promoter activating protein. It is
well known in the art that antigenic determinants ~epitopes)
30 generally contain at least about 5 amino acid residues. Ohno et
al., Proc. Natl Acad. Sci. USA. 82, Zg45 (1985). The antigenic
fragments of the invention comprise from about 5 to about 100,
and preferably about 5 to about 50, amino acid residues.
Whether a ~iven polypeptide falls within the scope of this
~f096/01899 ~ 361 ~ vr~4
g
invention can readily be det~-rmin~d by routine
e ~ nt~tion using the methods described below.
Such antigenic fragm~rlte can be made by
proteolysis of the whole human c-fos promoter activating
5 protein or by chemical or recomhin~lnt DNA synthesis. The
antigenic fragml~n~s can be used to elicit production of
antibodies, preferably in a mammal, by standard methods. The
antibodies thus produced can be used to assay for or purify the
activating protein, using standard in munoassay or
10 immunoadsorption methods.
The present invention utilizes a recombinant vector
co-llplisi,lg the polyomavirus T antigen gene and extends the
system of contingent replication to identify proteins the
production of which leads to transcriptional activation of gene
15 promoters. In contrast to the SV40 T antigen gene used by
Vasavada, et al., supra, the replicating and transforming
properties of the polyoma T antigen gene can be separated.
Separation of the replirating and transforming
properties is ~rComrlich~d by inserting a stop codon in the large
20 T intron in a region overlapping the central coding se4u~,..ccs
for middle T antigen. This separability of functions is important
in the case of the c-fos promoter, where prevention of middle T
expression eliminates the possibility of transcriptional
activation of the promoter via the rniddle T-activated c-src- and
25 phosphatidylinositol 3-kinase-associated signalling systems
(identified in Talmage, et al., Clell, ~i!, 55-65 (1989)) due to low
level, basal transcription from the promoter.
Use of the polyomavirus system enables the
extension of contingent replication to several well-characteri~ed
30 murine systems. In contrast, the SV40 T system used by
Vasavada, et al., supra, is limited primarily to simian (monkey)
~ systems. In addition, the present system does not appear to
suffer the high frequency of truncated or rearranged inserts
(approximately 25 percent) previously reported for the SV40 T
W096101899 v r~ a,..~ /1"4--
21~3~1
-10-
antigen-based system. Alteration of inserts occurs at a
frequency of less than 2 percent in present system.
A preferred embodiment includes the incorporation
of multiple enhancers from the promoter upstream of the
polyomavirus large T antigen gene to achieve sufficient
sensitivity of the promoter to permit large T induction in
response to low level expression of a cDNA-encoded signalling
molecule. Large T induction in turn results in plasmid
replication. Co-transfection with a cDNA library as described
below allows the percentage of cDNAs encoding signalling
proteins to be enriched within the library population. through
such large T-induced plasmid replication. The resulting
enrichment permits successive screening of increasingly srnaller
groups of library plasmids within a cDNA library, resulting in
the i~l~ntific~tica of single library plasmids encoding biologically
active mnh~cl~les which activate the promoter.
The self-~mplifi~ l~ion process of the present
invention provides additional sensitivity towards the detect;on
of cDNAs encoding signalling molecules. Initial plasmid
replication, in response to induction, leads to enhanced
expression of active signalling mr~lPc~ s due to greater gene
copy number. This increase in signalling molecules results in
greater :~mp~ific~til: of large T antigen expression, which in
turn leads to greater plasmid replication.
Preferred vectors of the present invention include
novel plasmids, denoted PfLAG-8 and Lo~2, as described below.
The present invention further provides a method for
identifying cDNAs encoding proteins which can activate a
promoter, preferably a human promoter, and more preferably
the human c-fos promoter. More preferred are the cDNAs,
denoted CROC-I and CROC-4, which encode c-fos promoter
activating proteins. For example CROC-I encodes a specific c-fos
promoter activating protein, denoted CROC-I protein, having the
amino acid sequence shown in SEQ ID NO:l. Similarly, CROC-4
encodes a specific c-fos promoter activating protein, denoted
~0 96/01899 PCT/IJS95/U7874
21~4~61
CROC-4 protein, having the amino acid sequence shown in SEQ
ID NO:3. Most preferred are the nnrleotidP se~ ncts shown in
SEQ ID NO:I and SEQ ID NO:3.
The present invention also provides cl~NAs encoding
5 c-fos promoter activating proteins which are conservative
mutants of the proteins encoded by CROC-l or CROC-4. Such
mutants possess the binding and c-fos promoter activating
functions of the proteins encoded by CROC-1 and CROC-4,
respectively .
In addition, the presenl invention provides
compounds which are antagonists of the protein encoded by
CROC-I or CROC-4. These antagonists include proteins which are
deletional, substitutional or ~ ditionnl mutants of the CROC-l
protein or CROC-4 protein, and which bind to, but do not
15 activate, the human c-fos promoter.
It is recognized that, because of the dtge~ of
the genetic code, there are many functionally equivalent nucleic
acid sequences that can encode c-fos promoter activating
proteins and c-fos promoter activating protein antagonists as
20 defined herein. Such functionally equivalent sequences, which
can }eadily be prepared using known methods such as chemical
synthesis, PCR employing modified primers, and site-directed
mn~gen~oeiC~ are within the scope of this invention.
As used herein, the term "recombinant vector"
25 includes both rec~-mhin~nt plasmids such as those mPn~ ned
herein and rec~mhin~n~ retroviral vectors, which can also be
engineered as described by Geller et al., Proc. Natl. Acad. Sci.
USA, 87, 1149(1990).
The foregoing recombinant vectors can be used to
30 transfect any m~mm~ n cell capable of undergoing
transfection and permitting vector replication, as herein
defined. Although cells from fresh tissue explants (primary
cells) could in principle be used, the use of ~st~hlichcd cell lines
is preferred. Many such cell lines are available including, e.g.,
35 NIH 3T3 mouse (ATCC# CRL1658),L-M(TK-) mouse (ATCC# CCL
WO Y61V189~ P~ /4
21 9~
-12-
1.3) and BALB/c 3T3 Clone A31 mouse (ATCC# CCL 163~ cell
lines.
The choice of a cell or cell line for use in the
methods of the present invention will be dictated by the known
5 or ~Pt~rminol~lP specifiritipc- of the vectors used. For example,
the murine cell lines are preferred for use with vectors
CO~ illg a recnmhinqnt vector containing the polyomavirus
large T antigen gene under the control of a regulated promoter,
such as the human c-fos promoter; and a mqmmalian
10 recombinant expression vector co~ lising a polyomavirus
origin of replication and a nucleic acid suspected ~o encode a
human promoter activating protein, such as a retroviral
expression vector ctmrrieing a retroviral LTR capable of
expressing the nucleic acid.
Although cells for use in the present invention were
transiently transfected, stably-transformed cells can also be
used. Stable transformation of a mqmmqliqn cell line can be
~cr mrlich~ d by using standard methods to co-b-ansfect the
cells with one of the above-mPntion~d recombinant vectors and
20 with a second vector which confers resistance to a selection
agent such as an antibiotic.
To identify nucleic acids encoding human c-fos
promoter activating proteins using the methods of this
invention, cells are co-l~"src~t~d with a lecol-lbillallt vector
25 comprising a human c-fos promoter operatively linked to
pol~ul~vi-us large T antigen gene, and a cDNA library
incol~u.~t~d into a n.qmmqliqn recombinant expression vector
comprising a polyomavirus origin of replication. The cells are
then incubated under conditions in which vectors containing
30 cDNA encoding a human c-fos promoter activating protein will
stimulate increased vector replication. The cells are then
harvested, the plasmids extracted and unreplicated vectors
selectively digested with Dpnl. Replicated plasmids are
recovered by transforming competent bacteria with the Dpnl
35 digest.
96tO1899 ~ 3 ~ 14
Typical incubations are carried out for 2 days at
37~C in a h-~mitlifif~d C02 incubator, although the choice of
e ~ "l'O~c will be apparent to those skilled in the art and will
depend, e.g., upon the nature of the cells, the medium used and
the type of culture container. Tr~llh~tinn is c~ntimled for a
period of time sufficient to permit development of a strong
replicative response. The optimal time is determined by
routine experimentation but will typically be in the range of
about 24 to 72 hours.
0 A s~hs~:mti~lly increased level of vector replication
and recovery after Dpnl digestion will be detected for those
vectors comprising nucleic acids encoding human c-fos
promoter activating proteins as compared to background
resulting from replication of vectors lacking such nucleic acids.
A su~hst~nti~l increase in vector replication and
recovery is typically an increase of at least about ~-fold,
preferably about 8-fold, and most preferably about 20-fold,
above the level measured in the complete absence of a plasmid
comprising a nucleic acid encoding a human c-fos promoter
activating protein. The degree of increase will be primarily
dep~ndent upon the level of background replication.
SUbst~nti~lly the same procedures are used for
identifying nucleic acids encoding other human promoter
activating proteins, by utilizing vectors comprising the promoter
operatively linked to a nucleic acid encoding polyomavirus large
T antigen.
In screening human c-fos promoter activating
protein antagonists using the methods of this invention, cells
are provided which are sim--lt~r~ously transfected with a first
rec--mhin~nt expression vector comprising a reporter gene
opel~Li~ly linked to a human c-fos promoter and a second
vector comprising a nucleic acid encoding a human c-fos
promoter activating protein. Preferred reporter genes are the
fos-CAT reporter gene described below or a fos-lac Z reporter
WO961018~9 21~436L ~ 4 ~
-14-
gene. The cells are planted in a culture medium appropriate to
the kind of cells used.
The cells are then incubated in the absence (control)
or presence of varying quantities of samples containing
5 suspected antagonists under conr~ nQ in which the gene
encoding the human c-fos promoter activating protein is
expressed. Under such conditions, and in the absence of an
antagonist, stimnl:ltion of the human c-fos promoter will occur,
resulting in reporter gene expression. The samples can be, e.g.
10 aqueous or water-miscible solutions in which isolated
cnmpounf~ have been dissolved, or individual or pooled
fractions from purification steps such as chromatographic or
electrophoretic fractions.
Typical incubations are carried out at about 37~C in
15 a hnmidifi~od C02 incubator, although the choice of conditions
will be apparent to those skilled in the art and will depend, e.g.,
upon the nature of the cells, the medium used and the type of
culture container.
ub~tion is continued for a period of time
20 sufficient to permit signific~nt reporter gene induction, at which
time the level of expression of the reporter gene is measured by
an appropriate assay. The optimal time for making the
measurement is det~rmi -d by routine experimentation but
will typically be in the range of about 24 to 72 hours,
25 preferably about 48 hours.
The highest levels of reporter gene expression will
be measured in the control (antagonist free) cultures. V~here a
culture contains a human c-fos promoter activating protein
antagonist, a reduction in the level of reporter gene expression
30 will be measured, the degree of which will be a direct function
of the quantity of antagonist added to the medium. Antagonists
present in the samples added to some of the cultures will be
identified by rn~Curing a ~uhst~nti~lly decreased level of
reporter gene expression, compared to the level measured in
35 the control cultures.
~1096101899 r.,~
~@3~ ~
A subsrAnti~lly decreased level of reporter gene
expression is defined as a decrease of at least about 50%, and
preferably at least about 70%, of the level measured in the
complete absence of an arltagonist of a human c-fos promoter
activating protein. Of course, the degree of decrease may be
intl~ ced by the quantity of antagonist present in the sample
compared to the quantity of human c-fos promoter activating
protein used and the efficiency of the antagonist.
Decreased levels of reporter gene expression due to
general toxicity of samples can be ~co~lntPd for by transfecting
a second constitutively expressed reporter gene, such as lac-Z
driven by a ~-actin promoter and normalizing c-fos reporter
gene activity to lac-Z expression.
The following non-limiting Examples will serve to
illustrate the present invention.
FxAMpl F~~
Materials and Gener~ql Me~hods-
Unless otherwise specified, percentages given below
for solids in solid mixtures, liquids in liquid mixtures, and solids
in liquids are on a wt/wt, vol/vol and wt/vol basis,
l~o}Jccli~cly~ Sterile conditions are r~qin~qinPd during cell
culture.
Standard recornbinant methods were used
throughout, such as those described in Sambrook, e~ al.
"Molecular Cloning. A Laboratory Manual, 2 ed.", Cold Spring
Harbor Laboratory Press (1989~.
Dpnl is a known restriction endonuclease isolated
from Diplococcus pneumoniae and is commercially available
from ICN Bi~nlPAicqlc~ Sigma Chemical Company or New England
BioLabs, Inc.
The restriction endonl~clPq~es Asel, ~amHI, Bglll,
BstXI, Clal, Fspl, ~incll, Narl, Notl, Sacll, Sall, Scal, X~aI and
wo s6/ols~s ;~ PCSIUS9~107874 --
-16-
Xhol are known and are co~ ially available, e.g. from Sigm~
Chemical Company.
The restriction endonucleases BamHI, BssHII, BsfXI,
HlncII, Sall, Scal and Xbal are known and are commercially
available, e.g. from ICN Biom~ al~.
The restriction en~onl~r!~cps AfllII, Asel, BamHI,
~gllI, Bs~HII, BstXI, Clal, FspI, Hincn, Nael, Narl, NorI, SaoII,
Sall, ScaI, XbaI and Xhol are known and are cornrnercially
available, e.g. from New England BioLabs, Inc.
The restriction endonuclease Saltl is known and is
cul~ c~ially available, e.g. from Boehringer h~nnh~im
The enzyme mung bean nuclease is known and is
commercially available from New England Biolabs, Inc or Sigma
Chemical Company.
The synthetic polylinker used in preparing the
vector La2 was obtained from New England Biolabs, Inc. and
has the sequence shown in SEQ ID NO:2. The NcoI linker
d(pAGCCATGGCT) is known and is cormmercially available fiom
New England Biolabs, Inc. (catalog $t 1150).
The vector pUCI9 (ATCC 37254, GenBank Accession
#: X02514~ is colll~ ;ially available from New England Biolabs,
Inc or ICN Bi-mPdic~llg The nucelotide sequence and restriction
sites of pUCl9 are described by Yanisch-Perron, et al., in Gene,
33, 103-119 (1985).
The following DNA, utilized in preparing the
plasmids of the present invention, is publicly available:
Polyollu.~,ilu6 DNA strain A2 (ATCC # 45017); a~1d human
genomic c-fos (ATCC # 41û42).
In addition, the DNA sequence of polyomavirus strain A2 is
reported in DNA Tumor Viruses. ed. Tooze, J. (1980) (Cold
Spring Harbor Press), pp. 834-838.
Construction of the retroviral vector pMV7 is
described by Kirschmeier, ~t al., PNA, 1., 219-225 (1988~,
starting from plasmids pPyori and pMV (ATCC# 37190). The
vector pMV7 is well known in the art and has been freely and
~o 96,0l89g ~ 1 9 4 3 6 1 ~ 14
.
-17-
widely distributed in many laboratories. In addition,
l~,hu~ cs similar to pM'V7 which could be used instead in
this invention are readily available, such as pV-mos (ATCC#
4 1 037).
The fos-CAT reporter gene construct described
below was prepared using the commercially available
pCAT-basic vector (Promega catalog # E1041).
Mouse monoclonal antibodies directed against the
hemagglutinin epitope and fluorescein-conjugated rabbit anti-
mouse IgG are commercially available from Boehringer
Mannheim.
For cDNA library screening a unidirectional cDNA
library was made from human brain poly A RNA (Clontech, Palo
Alto, CA) using the GIBCO (Grand Island, NY) Su~ l cloning
kit, and inserted into the SalllNotl sites in plasmid L~2.
Separation and visualization of nucleic acids was
carried out as described in Sambrook, et al., supra, by
electrophoresis on agarose gels and visualization with ethidium
bromide. All nucleotide sequencing was performed using the
dideoxy-mediated chain termination method described in
Sanger, et al., Proc. Natl. Acad. Sci. IJSA.l~, 5463-5467 (1977).
To obtain the sequences of CROC-I and CROC-4, DNA sequencing
was performed on both strands.
Co-transfection of cells with PfLAG and La2
containing a cDNA encoding a biologically active ~ign~lling
molecule causes activation of the c-fos promoter, resulting in
the production of large T antigen. The production of large T
antigen stim~ tes intracellular replication of plasmids
c~nt~ining the polyull~a\~ s origin of replication. Plasmids are
recovered from tl e transfected cell cultures by "Hirt extraction"
using the methods described in Hirt, J. Mol Biol.. 26, 365-369
(1967). Unreplicated plasmids are selectively destroyed by
restriction with DpnI. Replicated plasmids are then recovered
by transformation into competent bacteria.
WO Y6101899 2 L 9 ~ 3 5 1 . ~ 4 ~
-18-
Early passage NIH 3T3 mouse fibroblasts (ATCC#
CRL 1658) and Rat 2 fibroblasts (ATCC# CRL 17641 were grown
in DMEM ~u~ with l0'3'c bovine calf serum and 50
~lg/ml gentamycin sulfate.
The DHIOB E. coli used in the present invention are
commercially available from GIBCO.
Construction of Plagmi-l~
Two basic plasmids were constructed for use in the
prescnt invention. The first (denoted PfLAG) comprised a
human promoter-regulated polyomavirus large T antigen gene
which served as a source of large T antigen upon activation of
the promoter, and was based on the human c-fos promoter. The
second plasmid (denoted La2) was a retroviral cDNA expression
vector co~~ining the polyomavirus origin of replication.
The retroviral cDNA vector La2 was prepared as
follows. Polyomavirus DNA strain A2 was digested with
BamHIlNarI and the resulting 750 bp fragment was ligated into
the BamHllNarI sites in pUCl9 to give a plasmid denoted pOri.
The retrovira] vector pMV7 was digested with F spl/AflllI and
the resulting 4 kb band conlAining the two Moloney murine
sarcoma virus LTRs was ligated into the HincIIlAfllII fragment
of pOri, to give a plasmid denoted pMV7-2. A neomycin
resistance gene present between the two Moloney murine
sarcoma virus LTRs in pMV7-2 was removed by Saul/ClaI
digestion and replaced by a synthetic polylinker (described
above) to give the plasmid pMV7-3. To enable blue-white
screening, the polylinker in pUCl9 was replaced with a NcoI
linker, then the 360 bp lac Z region was removed by AseI/lVarl
digestion, blunt ended Witll mung bean nuclease, and ligated
into the pMV7-3 polylinker. The resultant plasmid, denoted
La2, was 4.5 kb and contained unique Sall and Norl sites at the
5' and 3' ends, ~ ,uc~Li~ly~ of ehe ~ac Z gene. A translaeional
start codon, followed by a DNA sequence encoding a histidine
~V0 961018gg P~ 14
21~43~1
-19-
hexamer, was inserted 5' to the cDNA insertion site to insure
expression of cDNA-encoded protein from truncated cDNA
inserts lacking start codons, and to aid in ~ubse~lLFrt protein
purification.
The PfLAG plasmid was prepared via the following
procedure. The polyomavirus large T anligen under the control
of the human c-fos promoter was introduced by digesting the
5.9 kb BamHI fragment of pcfos-l, disclosed by Curran, et al.,
Mol. Cell. Biol.. ~, 914-921(1983), with Nael to remove the
1 0 entire coding region of the c-fos gene and inserting the 2.8 kb
Bs~XII~incll band from polyomavirus, encoding the polyoma T
antigen. Middle and small T expression was t~limin~ d by
inserting a stop codon in the ScaI site located at position 605 of
the polyomavirus DNA sequence reported in Tooze, s~pra. The
15 resulting construct was denoted PfLAG-1 (for
promoterfO5/large T antigen).
A third vector, denoted HEL, was prepared for use in
identifying the intracellular locations of CROC-I. The histidine
hexamer coding sequl~nres of Lo~2 were removed by BglII/Sall
20 digestion and replaced witll coding scqucnccs for the nine
amino acid influenza virus HAI epitope described in Field, e~ aL,
Mol. Cell. Biol.. 8, 2159-2165 (1988). The SV40 origin of
replir~ti~ n was then inserted at the unique Xbal site between
the polyoma origin of replication and the 5' LTR, to give HEL.
2~ A fourth vector was prepared for use in confirming
the ability of suspected human c-fos promoter activating
proteins to stimulate the c-fos promoter. The fos-CAT reporter
gene described by Desch~mrs. et al., in ~cience, 233, 1174-1177
(1985), was prepared by inserting the human c-fos promoter
30 from the -735 (Bam~ll site) to +42 (~ael site) in front of the
bacterial CAT gene in the pCAT basic vector (Promega).
W0961018~9 ~1943~1 r~ 4 --
-20-
Deterrninin~ Enh~ncer Re,luh. .~..ts for P~LAG-de,pendent
Contingent Rep]ication:
For purposes of the present invention, the human
5 c-fos promoter in PfLAG must remain llans~ ionally s;lent in
quiescent cells, but be sensitive enough to respond to the low
level expression of active, cDNA-encoded signalling molecules
by producing sufficient T antigen to cause plasmid replication.
The sensitivity and level of gene induction from the promoter
10 can be increased by the incorporation of additional enhancer
elements into the promoter. Multiple enhancer elements were
incorporated into PfLAG-I by isolating an approximately 500 bp
or more Xhol/BssHIl(blunt-ended) fragment containing the c-
fos enhancer elements, and ligating the enhancer region into
15 the XhoI/Sacll (blunt-ended) site of the previous PfLA&.
To determine the number of enh~r~,-rc required to
display contingent replication, a series of PfLAGs, containing 1,2,
4 and 8 enhancer regions, were col~ The following
experiments were then conducted to define the enhancer
20 l~quilcll.~,ots for PfLAG-dependent contingent replication.
Muramatsu, et al., Mol. Cell. Biol.. 2, 831-836 (1989)
have shown that expression of the catalytic domain of protein
kinase C induces the c-fos promoter. The nucleotide and
deduced amino acid sequence of rat protein kinase C-~ I are
25 described in Housey, et al., Cell, 52, 343-354 (1988). The
catalytic domain of rat protein kinase C-~l was incorporated
into La2 to m~ke a construct, denoted pMvpkc~
Co-transfection of a pMVPkC~ /Lc~2 mixture with a Pfl.AG
containing 1, 2, 4 or 8 enhancer regions would therefore provide
30 a means of testing the sensitivity of each PfLAG.
A threshold sensitivity of detecting about one
plasmid out of forty for cDNA screening was used. Therefore a
1:40 (wt/wt) ratio of pMypkc~llLo~2 for co-transfection with
each of the PfLAGs into NIH 3T3 cells was utilized in the
35 procedure described below. Cells were incubated for forty-eight
VOg6/01899 r~J~
~9~3~
-21 -
(48) hours following transfection. The plasmids were extracted
and e~r -~, following ~pnl digestion, for elevated plasmid
recovery indicative of contingent replication. The results
obtained under these conditions are presented in Table 1.
5 These data show that eight enhancer regions (PfLAG-8) were
required for significant activation of plasmid replication,
permitting an eight-fold increase in plasmid recovery over
background resulting from co-transfection of PfLAG-8 with
vector alone. Induction with PfLAG-8 ranged from 6-fold to
10 greater than 20-fold increases in plasmid recovery, dep~ntling
primarily on the level of background.
Table 1. Human c-fos enhancer requirement to activate
polyomavirus large T antigen-activated contingent
15replicatior.~
Plasmid No. of Co-transfecte~ Total
Construct Enhancer Plasmid Number of
Re~ions Colonies
PfLAG-I 1 pMV7-Z 8
PfLAG-I I pMV7PkC~31 5
LoL2
PfLAG-2 2 pMV7-Z 3 3
PfLAG-2 2 pMV7PkC~l~l 2 2
Lo~2
PfLAG-4 4 pMV7-Z 109
PfLAG-4 4 pMV7PkC~ 101
Lo~2
PfLAG-8 8 pMV7-Z 363
PfLAG-8 8 pMV7PkC~31 1915
Lor2
- - pMV7PkC~,B1 7
L~2
2 llg PfLAG is co-transfected with 18 ~,lg of either
pMV7-Z or a 1:40 (wt/wt) ratio of pMV7PkC~,BI/Loc2.
The results presented are the average of two
experiments.
WO96/01899 2Ig~36~ .t/~/4--
-22-
The effect of the concentration of plasmids encoding
a promoter actvating protein on the recovery of pMVPkC~p l
within a total population of plasmids is de~-~minPd by varying
the concentration of pMVPkC~l in a pMVPkC~l/La2 mixture
5 prior to co-transfection with PfLAG-8. Because Lc~2 has a
modified lac Z gene derived from pUCl9, bacteria transformed
with L~2 will turn blue, whereas bacteria transformed with
pMVPkC~ will remain white, when plated on agar plates
c~n~aining ampicillin, X-gal, and IPTG. The percentage of
10 pMVPkC~l is d~t- ...;.fd by expressing the number of white
colonies as a percentage of totnl colonies formed after bacterial
transformation of Dpnl-digested Hirt extracts.
Lxperiments were conducted by co-transfecting
PfLAG-8 with the pMV7PkCI~l/La2 mixtures beginning at a
1:80 ratio (wtlwt), then diluting down to a 1400 ratio, using the
methods described below. Competent DHlO13 E. coli were
transformed with Dpn I-digested Hirt extracts and plated on
agar cont:~ining ~lmpicillin X-gal, and IPTG. The percent of
pMV7PkC~I in the recovered colonies was determined by the
2G number of white colonies over the total colonies.
To insure that the white colonies resulted from
transformation of pMVPkC~ l, plasmids were recovered and
restriction mapped. All white colonies showed the correct
pMVPkC~I restriction pattern. The results presented in Table
25 2 show that although the number of recovered colonies is
reduced to background levels at high pMV7Pk~ dilution, the
actual percentage of pMV7Pk~ colonies increases; i~lica~inE~
that a minimum of 400 library colonies can be transfected with
PfLAG-8 to enrich a cDNA library population for cDNA encoding
30 signal transducing molecules. Initial cDNA library screening
was therefore performed with plasmid pools comprised of four
hundred or more plasmids in order to acquire a library
population enriched in cDNAs encoding activators of the c-fos
promoter.
~,~O gC/01899 P~
~9~3$l
-23-
Table 2. Conc~ .tion dependence of pMV7PkC~ ~1 on
plasmid r covery,~
Ratio of co- Number of Percent Colonies
transfected Colonies per Dish
pMV7PkC~1 Blue V~Thite pM~T7PkC~
/Lc~2
1:8~ 206 4 I.g 210
0 146 7 4.o 153
1:2'0 134 6 4.3 150
I :3' 0 97 24 19.8 1 21
1:4()0 90 24 21.1 1 14
~Co-transfection with PfLAG-8 and Lc~2 alone gave a
background of 102 colonies/dish in this experiment.
Cell Culture and Transfection:
For transfections. 8 x 105 3T3 cells were planted in
10 growth medium in 100 mm dishes and allowed to attach
overnight. The following day, transfections were performed by
the method of Wigler, et a/., Cell, I l, 223-232 (1977), using
calcium ph~5rhatP. After a 4-hour exposure to the calcium
phosphate precipitate, cells were washed twice with phosphate
15 buffered saline, re-fed with DMIEM supplemented with 0.5%
bovine calf serum, and incubated at 37~C for 40-48 hours. Cells
were harvested and the plasmids were extracted by the
procedure of Hirt, supra. The extracted plasmids were digested
with DpnI for a minimum of 24 hours. Dpnl digests were
20 phenol extracted and ethanol precipitated. DNA was
res~lcrenrlPd in 20 IlL TF, (I mM EDTA +10 mM Tris, pH8.0), and
transformed into c~ l DHIOB bacteria (GIBCO).
Co-transfections were performed via the above
procedure at a cDNA/PfLAG ratio of 9:1 (wt/wt), using 20 llg
25 DNA per dish.
wo g6/01899 2 ~ 9 ~ 3 ~ ,Ol4
-24-
cDNA Library Screenin~ usin~ Contin~ent Replication
h human brain cDNA library was co-transefected
with PfLAG-S into NIH 3T3 cells via the methods described
5 above. Plasmid pools, comprised of approximately 30-40
plasmids, were co-transfected with PfLAG-8 and examined for a
minimum 5-fold increase in plasmid recovery. Plasmids from
active pools were recovered and subdivided into secondary
pools of four plasmids each. and similarly examined for
10 activation of contingent replication. Plasmids from each active
secondary pool were then examined individually for contingent
replication. From approximately 1400 plasmids screened
initially, two plasmids, denoted CROC-I and CROC-2 (for
cl~nfingPnt leplication of cDNA), consistently gave elevated
15 plasmid recovery when co-transfected with PfLAG-8. The
nucleotide sequence for CROC-I is shown in SEQ ID NO:l.
A third plasmid, denoted CROC-4, was identified by
further plasmid screening. Plasmid CROC-4 also co~ ff~ntly
gave elevated plasmid recovery when co-transfected with
20 P~LAG-8. The nucelotide sequence for CROC-4is shown in
SEQ ID NO:3.
Confirmation of c-fos Promoter Activation usin~ a fo~s-CAT
Reporter Gen~-
Certain extraneous factors could also cause theelevated plasmid recovery observed in the contingent
replication assay. For example, incomplete bactelial
methylation of the Dpnl sites, which will confer Dpr~l resistance.
30 or differences in transfection or transformation efficiency. To
eliminate these p~c~ihilifi~s, each of CROC-I,CROC-2 and CROC-4
was co-l~ sf~ d with a fos-CAT reporter gene and tested for
elevation of CAT activity as follows. Rat 2 cells were
co-transfected with 18 ~,lg Lo~-e~pressed cDNA (i.e., CROC I,
CROC-2 or CROC-4~ + 2 ,ugfos-CAT for 4 h, then refed with D~IEM
~0 96/01899 2 ~ ~ ~ 3 6 1 PCTIUS9~107874
-25-
+ 0.5% calf serum. Cells were harvested 72 hours after
transfection and CAT assays performed via the procedure of
Gorman, etal.,Mol. Cell. Biol..~, 1044-1051 (1982).
CAT activity was cignificln~ly induced by CROC-l,
5 CROC-2 and CROC-4, indicative of c-fos promoter activation. The
extenl of activation was approximately 50% of the activation
caused by co-transfection with pMVPkC~ . In contrast, vector
alone did not induce snhst:mfi~l CAT activity, nor did randomly
chosen cDNA library plasmids isolated from the same plasmid
10 pools as CROCs 1, 2 and 4, but which did not activate c~-ntinge
replication. These results confirm that the elevated plasmid
recovery observed upon co-transfection of CROCs 1, 2 or 4 with
PfLAC-8 was due to activation of the c-fos promoter in PfLAG-
mediated contingent replication.
An~ysis of c-fos Activatin~ Proteins:
Sequencing revealed that CROC-2 encodes the
recently i~lel-tified a2-macroglobulin receptor-associated
20 protein (AMRAP) disclosed in Strickland, et al., J. Biol. Chem..
266, 13364-13369 (1991). The insert is nearly full length and
extends from the start codon, which is in frame with the
internal vector start codon, to the poly A tail.
A 347 base pair sequence corresponding to
25 nucleotides 555-897 of CROC-4 has been submit~d to GenBank
(Accession # Z40809) as an expression sequence tag.
CROC-I cDNA encodes a 19 kd protein with an acidic
amino terminal half and a basic carboxy terminus, as shown in
SEQ ID NO:I. The protein includes a kinase target domain which
30 contains phosphorylation sites for a variety of kinases involved
in signal transduction. Specifically, the kinase target region is
comprised of adjacent proximal potential phosphorylation sites
for: (a) tyrosine kinases (RXXXEXXXY motif, amino acids 81-89),
Cooper, et al., J. Biol. Chem.. 259, 7835-7841 (1984); casein
35 kinase 2 (TIYE motif, amino acids 82-85), Kuenzel, et al., J. Biol.
WO 96/01899 ~ 4
-26-
Çhem., ~, 9136-9140 (1987); cAMP-dependent protein
kinases (RIYS motif, amino acids 87-90) Glass, et al., J. Biol.
~h~m,.. ~L. 2987-2993 (1986) and Kichim-tc. et al., J. Biol.
Chem., ~1, 12492-12499 (1985); and pro~ein kinase C (SLK
5 motif, amino acids 90-92), Kishimoto, ef al., supra.
The kinase target domain of the CROC-I protein is a
twelve amino acid stretch located at the start of ~he basic
domain. The known transactivating ability of acidic domains in
general, combined with the potential of basic dormains to bind
DNA, suggests that CROC-l could function as a transcriptional
activator whose activity is regulated by phosphorylation of the
kinase target domain. Phosphorylation would cause a further
increase in the acidity of the region, thereby enh:~ncing its
potential for trPnc~riptictn~l activation, as well as cause a change
15 in the structural conformation of the protein.
The length and tissue distribution of CROC-I mRNA
was determined by Northern analysis of poly A-containing RNA,
isolated from various human tissues, using the 1.8 kb Sall/~otl
insert of CROC-1 as a probe. CROC-1 mRNA was approximately
20 2.3 kb in length, about 0.5 kb longer than our cDl~A insert, and
present in all tissues eY~min~d, with the highest levels being
expressed in brain, skeletal muscle, and kidney. In CU~ I.DVU,
the 1.5 kb CROC-2 mRNA was present in all tissues ç~ rnin~tl
but with the highest levels being expressed in heart, placenta,
25 and kidncy. No evidence was found for additional transcripts,
as a result of alternative splicing or multiple sets of
transcription-termination-polyadenylation signals, as reported
for CROC-2 by Strickland, e~ al., s~pra.
Intracellular localization of the CROC- I protein was
30 detprminpd by cloning CROC-1 in HEL and electroporating the
resultant plasmid into COS-7 cells (ATCC# CRL 1651).
Incorporation of CROC-I nucleic acid into the MEL vector enables
the in frame fusion of the hemagglutinin epitope to the CROC-I
protein. The intracellular location of CROC-I prolein was then
3~ determined by immunofluorescence microscopy osing mouse
~0 96/01899 - PC~/IJS9~/07874
~9~3~1L
-27-
monoclonal antibody directed against the hemagglutinin
epitope. Elc~l..,po.dtion of CROC-I in HEL resulted in intense
nuclear fluorescence. In cw~trast, elc~,tlopo,.,tion of HEL alone
resulted in general cytoplas [nic fluorescence, ind~ ting that
5 nuclear loc~li7~tion is an inherent property of the CROC-I
protein .
The present invention encompasses modifications
and variations which will be evident to those skilled in the art.
The specific embodiments described herein are representative
10 examples only, the scope of the present invention being defined
by the claims.
wo g6,0l899 2 1 9 ~ 3 6 1 . ~ 14
;;
-28-
SEQUENOE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Schering Corporation
Patent Depart" ,el ,l K-6-1 (1990)
(B) STREET: 2000 Galloping Hill Road
(C) CITY: Kenilworth
1 0 (D) STATE: New Jersey
(E) COUNTRY: U.S.A.
(F) POSTAL CODE ~ZIP): 07033-0530
(G) TELEPHONE: 908-298-5150
(H) TELEFAX: 908-298-5388
(I) TELEX:
(ii) TITLE OF INVENTION: Method for Identifying Nucleic Acids
Encoding c-fos Promoter Activating Proteins
~iii) NUMBER OFSEQUENCES: 3
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: Apple Macintosh
(C) OPERATING SYSTEM: Macintosh 7.1
(D) SOFTWARE: Microsoft Word 5.1 a
(v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US95/
(B) FILING DATE: -June-1995
(vi) PRIOR APPUCATION DATA
(A) APPLICATION NUMBER: US 081272,412
(B) FILING DATE: 8-JUL-1994
~ 096l0l899 219 4 ~ 6 1 r~ ,4
-29-
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1930 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) SEOUENCE DESCRIPTION: SEQ ID NO:I:
15 ATG GAT CTC AGG CCT AGA TCT C'AT CAC CAT CAC CAT CAT TGG TGC CAG 48
Met Asp Leu Arg Pro Arg Ser His His His His His His Trp Cys Gln
TGT GCT GGT CGA CCC ACG caT CCG GAT GGC AGC CAC CAC GGG CTC GGG 96
20 cys Ala Gly Arg Pro Thr Arg Pro Asp Gly Ser His His Gly Leu Gly
20 25 30
AGT AAA AGT CCC TCG CAA TTT CGA CTG TTG GAA GAA CTC GAA GAA GGC 144
Ser Lys Ser Pro Ser Gln Phe Arg Leu Leu Glu Glu Leu Glu Glu Gly
35 40 45
CAG AAA GGA GTA GGA GAT GGC ACA GTT AGC TaG GGT CTA GAA GAT GAC 132
Gln lys Gly Val Gly Asp Gly Thr Val Ser Trp Gly Leu Glu Asp Asp
GAA GAC ATG ACA CTT ACA AGA TGG ACA GGG ATG ATA ATT GGG CCT CCA 240
Glu Asp Met Thr Leu Thr Arg Trp Thr Gly Met Ile Ile Gly Pro Pro
AGA ACA ATT TAT GAA AAC CGA ATA TAC AGC CTT AAA ATA GAA TGT GGA 288
Ara Thr Ile TYr Glu Asn Ara Ile Tvr Ser Leu Lvs Ile Glu Cys Gly
CCT AAA TAC CCA GAA GCA CCC CCC TTT GTA AGA TTT GTA ACA AAA ATT 336
40 Pro Lys Tyr Pro Glu Ala Pro Pro Phe Val Arg Phe Val Thr Lys Ile
100 105 llO
AAT ATG AAT GGA GTA AAT AGT TCT AAT GGA GTG GTG GAC CCA AGA GCC 384
Asn Met Asn Gly Val Asn Ser Ser Asn Gly Val Val Asp Pro Arg Ala
115 120 125
ATA TCA GTG CTA GCA AAA TGG CAG AAT TCA TAT AGC ATC AAA GTT GTC 432
Ile Ser Val Leu Ala Lys Trp Gln Asn Ser Tyr Ser Ile Lys Val Val
WO 96/OlD99 ~ ' r l/L /D/.4
21 ~
-30-
130 135 140
CTG CAA GAG CTT CGG CGC CTA ATG ATG TCT AAA GAA AAT ATG AAA CTC 480
Leu Gln Glu Leu Arg Arg Leu Met Met Slr Lys Glu Asn Met Lys Leu
145 150 155 160
CCT CAG CCG CCC GAA GGA CAG TGT TAC AGC AAT TAA TCA AAA AGA AAA 528
Pro Gln Pro Pro Glu Gly Gln Cy~ Tyr Ser Asn
165 170
ACC ACA GGC CCT TCC CCT TCC CCC CAA TTC GAT TTA ATC AGT CTT CAT 576
TTT CCA CAG TAG TAA ATT TTC TAG ATA CGT CTT GTA GAC CTC AAA CTA 6a4
CCG GAA AGG AAG CTC CCA TTC AAA GGA AAT TTA TCT TAA GAT ACT GTA 672
AAT GAT ACT AAT TTT TTG TCC ATT TGA AAT ATA TAA GTT GTG CTA TAA 72U
CAA ATC ATC CTG TCA AGT GTA ACC ACT GTC CAC GTA GTT GAA CTT CTG 768
GGA TCA AGA AAG TCT ATT TAA ATT GAT TCC CAT CAT AAC TGG TC~7 GGC 816
ACA TCT AAC TCA ACT GTG AAA AGA CAC ATC ACA CAA TCA CCT TGC TGC 864
TGA TTA CAC GCC CTG GGG TCT CTG CCT TCT CCC TTT ACC CTC CCG CCT 912
CCC ACC CTC CCT GCA ACA ACA GCC CTC TAG CCT GGG GGG CTT GTT AGA 960
GTA GAT GTG AAG GTT TCA GGT CGC AGC CTG TGG GAC TAC TGC TAG GTG 1008
TGT GGG GTG m CGC CTG CAC CCC TGG TTC CTT TAA GTC TTA AGT GAT 1056
GCC CCT TCC AAA CCA TCA TCC TGT CCC CAC GCT CCT CCA CTC CCG CCC 1104
TTG GCC GAA CCA TAG ATT GTA ACC CCT CCA CTC CCC TCT GAG ATT GGC 1152
TTC GGT GAG GAA TTC AGG GCT TTC CCC ATA TCT TCT CTC CCC CCA CCT 1200
TTA TCG AGG GGT GCT GCT TTT TCT CCC TCC TCC TCA AGT TCC TTT TTG 1248
CAC CGT CAC CAC CCA ACA CCT TCC ATG ACA CTT CCT TGC TTT GGC CAG 1296
AAG CCA TCA GGT AAG GTT GGA AAG AGC CTC TGA CCT CCC TTG TTT AGT 1344
TTT GGA ACC ATA CTC ACT CAC TCT CCA CCA GCC TGG GAA ATG AAT ATT 1392
GGG TCC TCA GCC CTG CCA CCC TCT GCT GTC ATC AGC TGA TGC ATT GTT 1440
TTT AGC TCA GGT TTT GAT AAG GTG AAA AGA ATA GTC ACC AGG GTT ACT 1488
CAG ACC TGC CAG CTC TCG GAG TCC TTG GTG GTT GAA CTT GGA GAA AGA 1536
CCG CAT GAA GAT ACT TGT AAG CAC ACA TGA TCC CTC TGA ATT GTT TTA 1584
CTT TCC TGT AAC TGC m TGC TTT TAA AAA TTG AAG AAG TTT TAA ACA 1632
GGG CTT TCA TTT GGT CAT CCT TGC AAT CCA TTG GGG TCT AGT TTG GAA 1680
~0~6~ 21~ 1 r~l/u. ~4
-31-
TCT GAC AAC TGG AAC AAA A~G AAC CTT GAA TCC GGT GCA TGC CTT GGT 1728
TTT GGT GCT GCT GCT GCT TCC CAA GAT CCT CAG CAG GGA TTA AGA AGG 1776
AAC CCG GTG TGC ACA GCA GAT CCC CGA AAT TGG TGG GCT TGA CCT CCT 1324
GGC AAA TTG CTG CGT CTT TCC ACT TGC TGT TCA GGA CCA CTA AAT GCG 1872
10 AAA TGT GGA TGC ATA CCG AAA TAA AAG CAA TTC ATT GTG TAC TAA AAA 1920
AAA AAA AAA A 1930
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B~ TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) SEQUENCE DESCRIPI'ION: SEQ ID NO:2:
CAC GTG AAT TCA AGA TCT CTG CAG AAG CTl' TCC GGA CCG GGC CGC GTA 48
GCA CGC GTA ATA ATT ATC GAT 6~
W 096~1899 ~ 1 9 4 ~ 6 ~ /4 -
-32-
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 926 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: iinear
(xi) SEQUENCE DESCRlPTiON: SEQ ID NO:3:
GTCGACCCAC ~o~..u~,- CTCACAGAAG CCTGGAGCTG GGCATCCAAa ~ Ar~A~ 60
CTCATTTCTT il~G.~l~ ATCGTAGCTG GCCACCTATG 4U~ ,A ATGTA~AAAG 120
- GGCAGCTCTC TGGC ATG TTC CTG ACT GAG GAT CTC ATA ACA TTT AAC TTa 170
Met Phe Leu Thr Glu Asp Leu Ile Thr Phe Asn Leu
5 10
AGG AAC TTC CTC CTT TTC CAG CTT TGG GAG TCA AGC TTC TCA CCT GGG 218
Arg Asn Phe Leu Leu Phe Gln Leu Trp Glu Ser Ser Phe Ser Pro aly
~5 15 20 25
GCG GGT GGG TTC TGC ACC ACC CTC CCA CCC TCC TTC CTC CGT GTG GAC 266
Ala Gly Gly Phe Cys Thr Thr Leu Pro Pro Ser Phe Leu Arg Val Asp
GAT AGA GCC ACA TCC AGC ACC ACG GAC AGC TCC CGG GCG CCT TCA TCT 314
A~p Arg Ala Thr Ser Ser Thr Thr Asp Ser Ser Arg Ala Pro Ser Ser
35 CCT CGT CCT CCA GGC AGC ACA AGC CAT TGT GGA ATC TCC ACC AGG TGT 362
Pro Arg Pro Pro Gly Ser Thr Ser His Cy~ Cly Ile Ser Thr Arg Cys
ACA GAA CGG TCC CTC TGC GTC CTG CCA CTC AGC. ACC TCT CAA GTC CCC 410
Thr Glu Arg Cy~ Leu cy5 Val Leu Pro Leu Arg Thr Ser Gln Val Pro
~30 85 90
GAT GTG ATG GCT CCT CAG CAT GAT CAG GAG AAA TTC CAT GAT CT~ GCT 458
Asp Val Met Ala Pro Gln Hi8 A~p Gln Glu Lys Phe His A~p Leu Ala
gs loo loS
TAT TCC TGT CTT GGG AAG TCC TTC TCC ATC TCT AAC CAA GAT CTA TAT 506
Tyr Ser CYs Leu Gly Lys Ser Phe Ser Met Ser Asn Gln Asp Leu ~yr
~ 096l018g9 ~ 1 9 ~ 3 ~ I r~
-33-
110 115 120
GGC TAT AGC ACC AGC TCT TTG GCT CTT GGC TTG CCA TGG CTA AGT TGG 554
Gly Tyr Ser Thr Ser Ser Leu Ala Leu Gly Leu Ala Trp Leu Ser Trp
'~ 5 12~ 130 135 140
GAG ACC AAA AAG AAG AAT GTA CTT CAT CTG GTT GGG CTG GAT TCC CTC 602
Glu Thr Lys Lys Lys As~ Val Leu ~is Leu Val Gly Leu Asp Ser Leu
145 150 155
TGATAAGCCT TCCCAGTTGA CTGAAAGATG AGGCTAGGCT CTAGCAAGTT GAAGTCAAAC 662
CAGCTCCTTC AAGAAGCTTT GAGCAGAATG AAGTGGGGAG GACCCAGCTT CCAGCCCAGG 722
AAGCCCACTG TACCTGGAGC CATCTGGGAT AAGACTTTGA CCCATGACTC CCATATCCAC 782
AGCCTGTCCA TCCTAGCCCA TCCCAG~TTA l~Ul~l~'l~A TTTGAGCTGG GATTCCCACA ~342
20 TCCTCTGAGT TGGAAGTCCC ATCTCAAGTC TTCAATAAAG ACTCTTGAAT ATTGAAAAAA 902
~ A~AAAA AbArr~r~rr-rr CGC 925
