Note: Descriptions are shown in the official language in which they were submitted.
2~8~
I'itle of the lnvention
Protein Partner Screening Assays and Uses Thereof
Cross-References to Related Applications
This application is a continuation-in7art of U.S. Application No.
07/915,745 filed July 21, 1992, which is a continuation of U.S. Application
No. 07/815,880, filed January 7, 1992, which is a continuation of U.S.
Application No. 07/510,254 filed April 19, 1990.
F'ield of the Invenfion
This invention is in the field of molecular biology and is directed to a
method of identifying a peptide capable of associating with another peptide in
a heterodimeric complex. The invention is also directed to a method of
identifying inhibitors of such heterodimeric complex formation.
BackgrQund of the Invention
Many regulatory proteins are heterodimers, that is, they are composed
of two different peptide chains which interact to generate the native protein.
Among such regulatory proteins are DNA binding proteins which are
capable of binding to specific DNA sequences and thereby regulating
transcription of DNA into RNA. The dimerization of such proteins is
necessary in order for these proteins to exhibit such binding specificity. A
large number of transcriptional regulatory proteins have been identified: Myc,
Fos, Jun, Ebp, Fra-1, Jun-B, Spl, H2TF-1/NF-KB-like protein, PRDI, TDF,
GLI, Evi-1, the glucocorticoid receptor, the estrogen receptor, the
progesterone receptor, the thyroid hormone receptor (c-erbA) and ZIF/268,
OTF-1(OCT1), OTF-2(0CT2) and PIT-1; the yeast proteins GCN4, GAL4,
HAPl, ADR1, SWI5, ARGRII and LAC9, mating type factors MATa1,
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MATa2 and MATal; the Neurospora proteins cys-3 and possibly cpc-l; and
the Drosophila protein bsg 25D, kruppel, snail, hunchback, serendipity, and
suppressor of hairy wing, antennapedia, ultrabithorax, paired, fushi tarazu,
cut, and engMiled. Eukaryotic transcriptional regulatory proteins, and the
methods used to characterize such proteins, have been recently reviewed
(Pabo, C.O. et al., Ann. Rev. Biochem. 61:1053-1095 (1992); Johnson, P. F.
et al., Ann. Rev. Biochem. 58:799-839 (1989)).
Members of the mammalian transcriptional regulatory protein families
Jun/Fos and ATF/CREB only bind to DNA as dimers. The proteins in these
families are "leucine zipper" proteins which contain a region rich in basic
amino acids followed by a stretch of about 35 amino acids which contains 4-S
leucine residues separated from each other by 6 amino acids (the "leucine
zipper" region). Collectively, the combination of a basic region and the
leucine zipper region is termed the bZlP domain.
Generally, it is the basic region which has been found to be
predominantly involved in contacting DNA whereas the zipper region mediates
the dimerization. Many dimeric combinations are possible, however, the
particular nature of the zipper specifies which partnerships are permissible
(Abel, T. et al., Nature 341:24-25 (1989)).
Another large family of proteins contains the DNA
binding/dimerization motif known as the basic helix-loop-helix motif (bHLH)
(Jones, N., Cell 61:9-11 (1990)). A bHLH protein generally contains a basic
N-terminus followed by a helix-loop-helix structure; two short amphipathic
helices containing hydrophobic residues at every third or fourth position. The
sequence of the basic region characteristically reveals no indication of an
amphipathic helix. The intervening loop region usually contains one or more
helix-breaking residues.
The bHLH motif was first detected in two proteins, E12 and E47, that
bind to a specific "E box" DNA enhancer sequence found in immunoglobulin
enhancers (Murre C. et al.~ Cell 56:777-783 (1989)). E motifs generally are
double stranded variants of the 5'-CAGGTGGC-3' consensus sequence. For
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example, the ~E1 motif is GTCAAGATGGC [Seq. ID NO. 1], ~4E2 motif is
AC~CAGCTGGC[SEQID NO. 2], ~E3 is GTCATGTGGC [Seq. ID NO. 3~,
,uEisTGCAGGTGT (Murre, C. et al., Cell 56:777-783 (1989)). Like many
transcriptional factors, peptides containing the bHLH motif often dimerize with
each other, either as a homodimer which contains two identical peptides or as
a heterodimer which contains two different peptides. Examples of
heterodimeric complex of two bHLH proteins binding DNA with a greater
efficiency than homodimeric complexes of either peptide in the heterodimer
are known (Murre C. et al., Cell 56:777-783 (1989); Murre, C e~ al., Cell
58:537-544 (1989)).
Identification of partners which direct protein-DNA binding and
compounds which inhibit such activity by inhibiting such protein partner
interaction could be very useful. For example, identification of partners of themyc protein and inhibitors of myc-partner interactions could provide a means
for treating diseases in which expression and activity of myc is a factor in
promoting cell growth or in maintaining the cell in a transformed state.
Myc is a bHLH protein and the bHLH domain of c-myc is encoded in
c-myc amino acids 255-410. The sequence homology between the proteins
expressed by the three n~yc genes (human N-myc 393437, human c-myc 34~
401, and human ~myc 289-338) and other genes which contain a bHLH
domain have been compared (Murre C. et al., Cell 56;777-783 (1989)).
Proteins such as myc which contain the bHl,H motif also possess the
ability to dimerize with other bHLH motif proteins. Such interactions among
bHLH proteins may play a critical role in their function andtor regulation.
ldentification of these protein partners would be useful not only in
understanding how these proteins function, but also in developing or
identifying inhibitors of these proteins. For example, identification of myc-
partners would make it possible to identify inhibitors of myc-partner
interactions. By inhibiting such interactions, inhibition and/or control of myc-induced cell growth may be achieved.
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To date, no myc inhibitors have been identified. The identification of
such inhibitors has suffered for lack of a simple, inexpensive and reliable
screening assay which could rapidly identify potential inhibitors and active
derivatives thereof. Thus a need still exists for rapid, economical screening
S assays which identify specific inhibitors of oncogene activity.
Sl~mnza~y of the Invention
~ecognizing the potential importance of inhibitors of oncoproteins in
the therapeutic treatment of many forms of cancer, and cognizant of the lack
of a simple assay system in which such inhibitors might be identified, the
inventors have investigated the use of chimeric oncogene constructs in in vitro
assays in prokaryotic hosts as a model system for identifying agents which
alter oncogene expression.
These efforts have culminated in the development of a simple,
inexpensive assay which can be used to identify protein partners in general,
and partners of transcriptional regulatory proteins in particular.
The methods of the invention are especially useful for the identification
of partners which influence transcriptional regulatory proteins, and especially
oncoprotein activity.
The method of the invention further provides a method of identifying,
isolating and characteri~ing inhibitors of such partner formation and especiallyinhibitors of oncoprotein activity.
The invention further provides a quick, reliable and accurate method
for objectively classifying compounds, including human pharmaceuticals, as
inhibitors of oncogene activity.
The invention further provides a method of identifying protein partners
by their ability to disrupt ~cl induced repression of phage promoters in
bacterial hosts which express fusion proteins containing the cl DNA binding
domain and a dimerization domain from a protein of interest. Proteins
identified by this method are partners of the protein from which the
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dimerization domain was obtained. Protein partners thus identified are already
in a cloned form, amenable to further characterization.
Brief Description of the Drawings
Figure 1 shows the DNA sequence (Seq. ID No. 4) and protein
sequence (Seq. ID No. S) of human c-myc exon 3 and the sites used to
synthesize the HLH/LZ and HLH fragments of c-myc.
Description of the Preferred Embodiments
In the description that follows, a number of terms used in recombinant
DNA technology are extensively utiliæd. In order to provide a clearer and
more consistent understanding of the specification and claims, including the
scope to be given such terms, the following definitions are provided in
alphabetical order.
Bioactive Compound. The term "bioactive compound" is intended to
refer to any compound which induces a measurable response in the assays of
the invention.
Clonin~ vehicle. A "cloning vehicle" is any molecular entity which is
capable of providing a nucleic acid sequence to a host cell for cloning
purposes. Examples of cloning vehicles include plasmids or phage genomes.
A plasmid which can replicate autonomously in the host cell is especially
desired. Alternatively, a nucleic acid molecule which can insert into the host
cell's chromosomal DNA is especially useful.
Cloning vehicles are often characterized by one or a small number of
endonuclease recognition sites at which such DNA sequences may be cut in
a determinable fashion without loss of an essential biological function of the
vehicle, and into which DNA may be spliced in order to bring about its
replication and cloning.
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The cloning vehicle may further contain a marker suitable for use in
the identification of cells transforrned with the cloning vehicle. Markers, for
example, are tetracycline resistance or ampicillin resistance. The word
"vector" is sometimes used for "cloning vehicle. "
Compound. The term "compound" is intended to refer to a chemical
entity, whether in the solid, liquid, or gaseous phase. The term should be read
to include synthetic compounds, natural products and macromolecular entities
such as polypeptides, polynucleotides, or lipids, and also small entities such
as neurotransmitters, ligands, horrnones or elemental compounds.
Dimeric Protein. The term "dimeric protein" is intended to refer to a
protein which contains two polypeptide chains that associate with one another,
but which are not bound to one another by an amino acid linkage. Association
of the polypeptide chains may be due to, for example, hydrogen bonding,
ionic interactions, hydrophobic interactions, disulfide bonds, and the like.
Dimerization Domain. The term "dimerization domain" is intended to
refer to that portion of each polypeptide chain of a dimeric protein which is
necessary for the polypeptide chains to associate with one another. The
dimerization domains of a dimeric protein, which may be identical or
different, are referred to herein as complimentary to each other.
Expression. Expression is the process by which the information
encoded within a gene is transcribed and translated into protein.
A nucleic acid molecu1e, such as a DNA or gene is said to be "capable
of expressing" a polypeptide if the molecule contains the sequences which
code for the polypeptide and the expression control sequences which, in the
appropriate host environment, provide the ability to transcribe, process and
translate the genetic information contained in the DNA into a protein product,
and if such expression control sequences are operably-linked to the nucleotide
sequence which encodes the polypeptide.
Expression vehicle. An "expression vehicle" is a vehicle or vector
similar to a cloning vehicle but is especially designed to provide sequences
capable of expressing the cloned gene after transformation into a host.
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In an expression vehicle, the gene to be cloned is operably-linked to
certain control sequences such as promoter sequences.
Expression control sequences will vary depending on whether the
vector is designed to express the operably-linked gene in a prokaryotic or
eukaryotic host and may additionally contain transcriptional host specific
elements such as operator elements, upstream activator regions, enhancer
elements, termination sequences, tissue-specificity elements, andlor
translational initiation and termination sites.
Functional Derivative. A "functional derivative" of a fusion protein is
a protein which possesses an ability to dimerize with a partner protein, and/or
an ability to bind to a desired DNA target, that is substantially similar to theability of the fusion protein constructs of the invention to dimerize. By
"substantially similar" is meant that the above-described biological activities
are qualitatively similar to the fusion proteins of the invention but
quantitatively different. For example, a functional derivative of a fusion
protein might recognize the same target as the fusion protein, or form
heterodimers with the same partner protein, but not with the same affinity.
As used herein, for example, a peptide is said to be a "functional
derivative" when it contains the amino acid sequence of the fusion protein plus
additional chemical moieties not usually a part of a fusion protein. Such
moieties may improve the derivative's solubility, absorption, biological half-
life, etc. The moieties may alternatively decrease the toxicity of the
derivative, or eliminate or attenuate any undesirable side effect of the
derivative, etc. Moieties capable of mediating such effects are disclosed in
Remington's Pha~7naceuhcal Sciences (1980). Procedures for coupling such
moieties to a molecule are well known in the art.
A functional derivative of a fusion protein may or may not contain
post-translational modifications such as covalently linked carbohydrate,
depending on the necessity of such modifications for the performance of the
methods of the invention.
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The term "functional derivative" is intended to encompass functional
"fragments, " "variants, " "analogues, u or "chemical derivatives" of a molecule.
Fusion protein. As used herein, "fusion protein" is a hybrid protein
which has been constructed to contain domains from two different proteins.
The term "fusion protein gene" is meant to refer to a DNA sequence
which codes for a fusion protein, including, where appropriate, the transcrip-
tional and translational regulatory elements thereof.
Heterodimer. The term "heterodimer" or "heterodimeric protein" is
intended to refer to a protein which contains two different polypeptide chains
that associate with one another, but which are not bound to one another by an
amino acid linkage.
Homodimer. The term "homodimer" or "homodimeric protein" is
intended to refer to a protein which contains two identical polypeptide chains
that associate with one another, but which are not bound to one another by an
amino acid linkage. This term may be modified to refer only to a particular
portion of a dimeric protein. For instance, a DNA binding domain
homodimer is intended to refer to any dimeric protein containing identical
DNA binding domains on its separate polypeptide chains.
Host. By "host" is meant any organism that is the recipient of a
cloning or expression vehicle as deflned herein. Appropriate hosts for use in
the method of the invention include, but are not limited to, bacteria, yeast, and
mammalian cells.
Marker Gene. The term "marker gene" is intended to refer to a gene
whose expression in a host cell produces a readily observable, assayable, or
selectable phenotype. Examples of marker genes which may be useful in the
method of the invention include, but are not limited to, lacZ, aada (which
confers spectinomycin and streptomycin resistance), and ble-1 (which confers
bleomycin and phleomycin resistance).
Operably-linked. As used herein, two macromolecular elements are
operably-linked when the two macromolecular elements are physically
9 2~
arranged such that factors which influence the activity of the first element
cause the first element to induce an effect on the second element.
Promoter. A "promoter" is a DNA sequence located proximal to
the start of transcription at the 5' end of the transcribed sequence, at which
RNA polymerase binds or initiates transcription. The promoter may contain
multiple regulatory elements that interact in modulating transcription of the
operably-linked gene.
Protein Partner. The term "protein partner" is intended to refer to a
polypeptide chain capable of associating with a heterologous polypeptide chain
to form a heterodimeric protein. The two polypeptide chains of a
heterodimeric protein are herein referred to as "partners" of one another. A
polypeptide chain of a homodimeric protein may act as a partner in a
heterodimeric protein.
Response. The term "response" is intended to refer to a change in any
parameter which can be used to measure and describe the effect of a
compound on the activity of a protein. The response may be revealed as a
physical change (such as a change in phenotype) or a molecular change (such
as a change in a reaction rate or affinity constant). Detection of the response
may be performed by any means appropriate.
Variant. A "variant" of a fusion protein is a protein which
contains an amino acid sequence that is substantially similar to, but not
identical to, the amino acid sequence of a fusion protein constructed from
naturally-occurring domains, that is, domains containing the native with the
amino acid sequence.
By a "substantially similar" amino acid sequence is meant an amino
acid sequence that is highly homologous to, but not identical to, the amino
acid sequence found in a fusion protein. Highly homologous amino acid
sequences include sequences of 80% or more homology, and possibly lower
homology, especially if the homology is concentrated in domains of interest.
Transcription regulatory proteins, which normally function as dimeric
proteins, have been found to possess discrete dimerization domains and DNA
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bincling domains. The inventors have used these findings to develop the
method of the invention for identifying a partner of a dimeric protein. This
method involves construction of chimeric peptides with (1) known
complementary dimerization domains and (2) DNA binding domains which,
when present in homodimer form, are capable of conferring a detectable
phenotype upon a host cell (preferably a bacterial host cell, such as E. coli).
This detectable phenotype is a marker other than resistance to phage
infection, such as infection by lambda phage. It has been discovered that this
phenotype may be detected by methods which do not depend upon phage
resistance, or phage-induced cell Iysis.
In the host cell, the chimeric peptides form DNA binding domain
homodimers by association of the known complementary dimerization
domains. Protein partners capable of associating with the chimeric peptides
to form heterodimeric proteins will interfere with formation of the chimeric
peptides into DNA binding domain homodimers. By monitoring the
homodimer-conferred phenotype in the host cell, formation of interfering
heterodimers may be detected and protein partners thus identified.
This method of the invention is generally useful to identify partners for
any homodimer or heterodimer. For a homodimer, a single chimeric peptide
containing the dimerization domain of the homodimer is used. For a
heterodimer, two separate chimeric peptides are used; each containing one of
the complementary dimerization domains of the heterodimer. The chimeric
peptides also contain a DNA binding domain that confers a detectable
phenotype in homodimer form.
DNA binding domains useful in construction of chimeric peptides of
the invention may be obtained from proteins where they have been identified.
For example, DNA binding domains may be obtained from bacteriophage
repressors, such as bacteriophage lambda (~) repressor. In particular, the
lambda repressor protein cI is useful as a source of a DNA binding domain.
cl represses lambda gene expression in its homodimeric form (Lambda 11,
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Hendrix, R.W. et al., eds., Cold Spring Harbor Laboratory, New York,
(1983).
Other DNA binding domains may be identified by a variety of
techniques known in the art and previously used to identify such domains (see
Pabo, C.O. etal., Ann. Rev. Biochem. 61:1053-1095 (1992); Johnson, P. E.
et al., Annu. Rev. Biochem. ~8:799-839 (1989) for a review of such domains).
DNA binding proteins, and DNA binding domains in such proteins, are
identified and purified by their affinity for DNA. For example, DNA binding
may be revealed in filter hybridization experiments in which the protein
10 (usually labelled to facilitate detection) is allowed to bind to DNA immobilized
on a filter or, vice versa, in which the DNA binding site (usually labelled) is
bound to a filter upon which the protein has been immobilized. ~he sequence
specificity and affinity of such binding is revealed with DNA protection assays
and gel retardation assays. Purification of such proteins may be performed
15 utilizing sequence-specific DNA affinity chromatography techniques, that is,
column chromatography with a resin derivatized with the DNA to which the
domain binds. Proteolytic degradation of DNA binding proteins may be used
to reveal the domain which retains the DNA binding ability.
Dimeric proteins for which protein partners are desired to be identified
20 sene as the source of dimerization domains useful in the construction of
chimeric peptides of the invention. Dimerization domains may be currently
known dimerization domains or those recognized by their homology to known
dimerization domains. Other dimerization domains may be predicted by
analysis of the three-dimensional structure of a protein using the amino acid
25 sequence and computer analysis techniques commonly known in the art, for
example, the Chou-Fasman algorithm. Such techniques allow for the
identification of helical domains and other areas of interest, for example,
hydrophobic or hydrophilic domains, in the peptide structure.
One class of known dimerization domains are the HLH domains, which
30 share a common helix-loop-helix amino acid structure. The bHLH region of
the c-myc protein is one such dimerization domain. This domain is
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cornplementary to itself and is therefore useful in the construction of chimericpeptides that form homodimers.
An HLH dimerization domain in a protein can be identified by
comparison of an amino acid sequence with that of ten known HLH
S dimerization domains (amino acids 336-393 in E12, 33~393 in E47, 55~613
in daughterless, 357-407 in ~st, 393-437 in human N-myc, 289-338 in
human ~myc, 346~01 in human c-myc, 108-164 in MyoD, and genes of the
achaete-scute locus: 101-167 of T4, 26-9S of T5 (Murre, ~. et al., Goll
56:777-783 (1989)). The HLH dimerization domain contains two amphipathic
helices separated by an intervening loop. The first helix contains 12 amino
acids and the second helix contains 13 amino acids. Certain amino acids
appear to be conserved in the HLH format, especially the hydrophobic
residues which are present in the helices. Comparisons of the two sequences
named above shows that there are five virtually identical hydrophilic residues
within the 5' end of the homologous region and a set of mainly hydrophobic
residues located in two short segments that are separated form one another by
a sequence that generally contains prolines or clustered glycines.
Another class of known dimerization domains are the leucine zipper
domains. This domain is typically about 35 amino acids long and contains a
repeating heptad array of leucine residues and an exceedingly high density of
oppositely charged amino acids (acidics and basics) juxtaposed in a manner
suitable for intrahelical ion pairing. It is thought that the leucines extendingfrom the helix of one polypeptide interdigitate with those of the analogous
helix of a second peptide (the partner) and form the interlock termed the
leucine zipper.
The DNA binding domain and the dimerization domain are engineered
into the fusion protein in a manner which does not destroy the function of
either domain; that is, the DNA binding domain, when properly dimerized,
can recognize the DNA element to which it naturally binds and the
dimerization domain retains the ability to dimerize with its partners. One of
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skill in the art, by running control assays, will be able to establish that the
fusion protein functions in the proper manner.
The DNA sequence encoding the fusion protein may be chemically
constructed or constructed by recombinant means known in the art. Methods
of chemically synthesizing DNA are well known in the art (Oligonucleot~de
Synthesis, A Practical Approach, M.J. Gail, ed., IRL Press, Washington,
D.C., 1094; Synthesis and Applicahons of DNA and RNA, S.A. Narang, ed.,
Academic Press, San Diego, CA, 1987). Because the genetic code is
degenerate, more than one codon may be used to construct the DN~ sequence
encoding a particular amino acid (Watson, J.D., In: Molecular Biology of the
Gene, 3rd edition, W.A. Benjamin, Inc., Menlo Park, CA, 1977, pp. 35~-
357).
To express the recombinant fusion constructs of the invention,
transcriptional and translational signals recognizable by the host are necessary.
A cloned fusion protein gene, obtained through the methods described above,
and preferably in a double-stranded form, may be operably-linked to sequences
controlling transcriptional expression in an expression vector, and introduced,
for example by transformation, into a host cell to produce the recombinant
fusion proteins, or functional derivatives thereof, for use in the methods of the
invention.
Transcriptional initiation regulatory signals can be selected which allow
for repression or activation of the expression of the gene encoding the fusion
protein, so that expression of the fusion construct can be modulated, if
desired. Of interest are regulatory signals which are temperature-sensitive so
that by varying the temperature, expression can be repressed or initiated, or
are subject to chemical regulation, for example, by a metabolite or a substrate
added to the growth medium. Alternatively, the fusion construct may be
constitutively expressed in the host cell.
It is necessary to express the proteins in a host wherein the ability of
the protein to retain its biological function is not hindered. Expression of
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proteins in bacterial hosts is preferably achieved using prokaryotic regulatory
signals.
Expression vectors typically contain discrete DNA elements such as,
for example, (a) an origin of replication which allows for autonomous
replication of the vector, or elements which promote insertion of the vector
into the host's chromosome in a stable manner, and (b) specific genes which
are capable of providing phenotypic selection in transformed cells. Many
appropriate expression vector systems are commercially available which are
useful in the methods of the invention.
Once the vector or DNA sequence containing the construct(s) is
prepared for expression, the DNA construct(s) is introduced into an
appropriate host cell by any of a variety of suitable means, for example by
transformation. After the introduction of the vector, recipient cells are grown
in a selective medium, which selects for the growth of vector-containing cells.
Expression of the cloned gene sequence(s) results in the production of the
fusion protein.
If the fusion protein DNA encoding sequence and an operably-linked
promoter is introduced into a recipient host cell as a non-replicating DNA (or
RNA) molecule, which may either be a linear molecule or, more preferably,
a closed covalent circular molecule which is incapable of autonomous replica-
tion, the expression of the fusion protein may occur through the transient
expression of the introduced sequence.
Genetically stable transformants may be constructed with vector
systems, or transformation systems, whereby the fusion protein DNAis
integrated into the host chromosome. Such integration may occur de noYo
within the cell or be assisted by tMnsformation with a vector which
functionally inserts itself into the host chromosome, for example, with
bacteriophage, transposons or other DNA elements which promote integration
of DNA sequences in chromosomes.
Cells which have been transformed with the fusion protein DNA
vectors of the invention are selected by also introducing one or more markers
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which allow for selection of host cells which contain the vector. Markers
incorporated in the vector may provide, for example, biocide resistance, e.g.,
resistance to antibiotics, or the like.
The transformed host cell can be fermented according to means known
in the art to achieve optimal cell growth, and also to achieve optimal
expression of the cloned fusion protein sequence fragments. Optimal
expression of the fusion protein is expression which provides no more than the
same moles of fusion protein subunit as the moles of the partner protein which
are being expressed. However, variations in this amount are acceptable if they
do not prevent the partner from forming heterodimers with the fusion protein,
thereby interfering with fusion protein homodimer activity.
Any protein that possesses a binding domain which can form a
heterodimer with the fusion protein will impair or prevent the formation of
fusion protein homodimers. Such proteins can thus be identified by their
ability to interfere with the phenotype conferred by the fusion protein
homodimer.
In one embodiment the bacterial host, which is expressing a fusion
protein as described above, is transformed with a ~ expression library capable
of expressing cloned eukaryotic genes. Those cells transformed with a
eukaryotic gene expressing a protein which is a partner of the fusion protein
can then be detected due to loss of the phenotype conferred by the fusion
partner homodimer.
~gtl 1 packaging systems for the creation of expression libraries from
mRNA, which are useful in the methods of the invention, are known in the art
and may be obtained commercially (for example, through Promega
Corporation, Madison, Wisconsin). Further, custom genomic expression
libraries may also be obtained commercially. Using the commercial kits, an
oligo(dT)-primed cDNA library in ~gtll may be generated with the use of
cytoplasmic poly(A)-containing mRNA from any desired mammalian source.
To induce expression of the cloned proteins contained therein, 10 mM IPTG
(isopropyl-thiogalactoside) may be added.
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A particular advantage of the method of the invention for the
identification of protein partners is that, where approximately equal amounts
of the fusion protein(s) and the protein partner are present in the host cell, the
partner which is identified will have a higher affinity for the fusion protein(s)
than the fusion protein(s) has to itself. If the disrupted dimeriza~ion is normally
associated with a biological activity, such a protein partner is highly likely to
be an important regulator of that biological activity. Further, the partner
which is identified is already in a cloned, expressing form which may be
utilized to obtain larger quantities of the protein for its isolation and further
characterization by protein and molecular biology techniques known in the art.
Utilizing the above techniques, a chimeric peptide containing the bHLH
dimerization region of c-myc and the DNA binding domain of cI was
constructed (see Example 1). In the appropriate host cell, this chimeric
peptide formed homodimers and repressed expression of the l~acZ gènè under
the control of a lambda PL promoter and repressed phage Iysis (see Example
2). Introduction of a partner protein into the host cell interfered with
homodimer formation and de-repressed exp~ession of the lacZ gene (see
Example 3). The inventors used this method to screen a cDNA expression
library and discovered a specific partner protein which associates with c-myc
in ViYo (see Example 3).
Compounds which inhibit the ability of protein partners to form
interfering heterodimers, but which do not interfere with homodimer
formation, may be identified by screening for the ability of a compound to
reverse the interfering effect of the heterodimers and restore the homodimer-
conferred phenotype.
For example, for partners identified by de-repression of the lacZ gene
as described above (see also Example X), compounds which prevent or
otherwise interfere with heterodimer formation of the protein partners can be
identified by screening for the ability of such compounds to restore repression
of the lacZ gene and cause partner-containing cells to remain white when
grown on X-gal plates. A compound which is found to restore lacZ gene
,~ .
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repression in this example would be a compound which (a) prevents the fusion
protein from associating with the partner peptide which is also being expressed
in tEle host, (b) does not prevent homodimer formation and (c) does not inhibit
cell growth.
The methods of the invention can be used to screen compounds in their
pure form, at a variety of concentrations, and also in their impure form. The
methods of the invention can also be used to identify the presence of such
inhibitors in crude extracts, and to follow the purification of the inhibitors
therefrom. The methods of the invention are also useful in the evaluation of
the stability of the inhibitors identified as above, to evaluate the efficacy ofvarious preparations.
Analogs of such compounds which are more permeable across bacterial
host cell membranes may also be used. For example, dibutyryl derivatives
often display an enhanced permeability.
Partners, and compounds which inhibit the association of such partners,
of any type of transcriptional regulation protein which associates into dimers
may be identified by the bacterial methods of the invention. The methods of
the invention can also be used to identify partners, and compounds which
interfere with such partners, of membrane-localized and/or cytoplasmically-
localiæd proteins which associate into dimers.
It may be desired to fur~her characterize the partner proteins of c-myc
which are identified by the methods of the invention in a eukaryotic expression
system. Such characterization may be performed according to the methods
described in the inventor's copending U.S. patent application entitled "C-Myc
Screening Assays," Serial 07/785,567 filed Oct. 30, I99I and incorporated
herein by reference.
The following examples further describe the materials and methods
used in carrying out the invention. The examples are not intended to limit the
invention in any manner.
-18- 2~ 7
Examples
E~cample I
Constr~ction of cl/c-myc Fusion Proteins
Chimeric genes capable of expressing fusion pr~teins containing the
DNA binding domain of the lambda repressor d and either 1) the c-myc basic
helix-loop-helix (bHLH) dimerization domain or 2) the c-myc bHLH and
leucine zipper (LZ) dimerization domains were constructed.
The promoterloperator region used consists of the ,B-lactamase
promoter, lac operator and Shine-Delgarno (S.D.) sequence. The sequence
is as follows:
GGA TCC TCT AAA TAC ATT CAA ATA AGT ATC CGC TCA TGA
BamHI - 3 5
CAC AAT AAC GGT AAC CAG AAT TGT GAG CGC TCA CAA TTT TG
-10 BstEII
ATC GAT AGC AAA CTC GAG ATG...... [Seq. ID No. Ç]
ClaI S.D. XhoI +l cI
The N-terminal 336 bp (112 amino acids) of d, which contains the
DNA binding domain of this protein, was incorporated into this construct.
This portion was amplified for cloning using polymerase chain reaction with
primers adding ,~oI and X~al sites on the 5' and 3' ends, respectively. The
promoter/operator and cI DNA were cloned into pUC18 digested with BamHI
and XbaI to generate pUC3d.
The sequence around the XbaI site is as follows:
5 CAG GCA GGG TCT AGA . . . [ SEQ ID NO . 7 ]
Gln Ala Gly XbaI
cI coding seq.
The bHLH/LZ and bHLH fragments of c-myc were generated by PCR
using a human c-myc cDNA as a template. The bHLH/LZ fragment used was
a 258 bp fragment synthesized with primers starting at sites #2 and #9 (Figure
. .
,, ~ :
. , - . . :
,
.:
-l9-
1) with Xbal and SalI sites added at the S' and 3' ends, respectively. The -bHLH fragment used is a 165 bp fragment with Xbal and PstI sites added on
the 5' and 3' ends, respectively. The boundaries of bHLH are at sites marked
#2 and #10 (Figure 1). The primer used at site #10 included a termination
codon, as does that used at site #9. Insertion of the c-myc sequences into
pU3cl was at the restriction sites corresponding to those added by the
indicated PCR primers. The resulting constructs containing c-myc bHLH/LZ
and bHLH were referred to as pU3.29 and pU3.210, respectively. As a result
of the cloning procedure used, an X~7al site (TCT AGA) encoding amino acids
Serine and Arginine was incorporated in-between the cl and c-myc sequences.
The chimeric cl/c-myc gene constructs in pUC18 were subcloned into
pACYC177 (Chang, A.C.Y. et al., J. Bacteriol. 134: 1141-1156 (1978)) as
follows. Both chimeric genes were excised from pUC18 by digestion with
Hindlll, fill-in of the Hindlll overlap with Klenow, and subsequent BamHI
digestion. The chimeric gene fragments were then cloned into pACYC177
digested with Bgn (filled in with Klenow) and BamHI. The resulting
constructs were designated pYC188 which contains cI-bHLH/LZ and pYC192
which contains cI-bHLH. These pYC-constmcts confer kanamycin resistance
upon transformed E. coli host cells and are normally maintained in low copy
number (5-20 copies/cell).
Example 2
Assaying transformed bacteria for the phenotype
conferred by the cl/c-myc fusion protein in homodimer form
The DNA binding domain of the d protein must be present in dimer
form to function as a repressor of lambda transcription/infection. Native d
protein is unable to form dimers at physiological levels and is therefore
functionally inactive. In contrast, fusion proteins containing a functional DNA
binding domain from cI and a functional dimeriza~ion domain from c-myc
should be able to form functional homodimer repressors. To detect the
- 2~8~8~7
-20-
repressor phenotype in bacterial cells transformed with the cl/c-myc fusion
constructs described in Example 1, two different assays were used.
In the "dot plaque assay" (DPA), transformed E. coli cells were tested
for susceptibility to lambda phage infection. These cells were predicted to be
S resistant to infection if the cl/c-myc fusion protein was adequately expressed
and formed functional homodimers. Cell strains carrying fusion protein
constructs were grown in L-broth media containing 30 ~g/ml Kanamycin, 10
mM MgSO4, and 0.2% maltose at 37C. 0.25-0.5 ml of culture at an OD60"
of 1.0 to 2.0 was added to 3mls of 48C top agar, mixed by vortexing and
plated on pre-warmed L-broth/Kanamycin plates. The top agar was allowed
to solidify for 2-3 min. at room temperature and then 5~41 aliquots of lambda
phage KH54 (provided by J. Hu and R. Sauer of the Massachussetts Institute
of Technology) of titer 5x101-5x106 plaque forming units (pfu) were dotted
onto the top agar. Lambda phage KH4i434 (provided by J. Hu and R. Sauer
of the Massachussetts Institute of Technology), which carries the immunity
region of phage 434 and is therefore not affected by lambda cI, was also
dotted on as a control. Phage aliquots were allowed to dry and then the plates
were incubated overnight at 37C.
In this assay, the titer of phage required to create a clear spot is used
as a measure of phage resistance. Bacteria that express native cI protein
(which is unable to form dimers) from pACYC177 (Chang, A.C.Y. et al., J.
Bacteriol. 134: 1141-1156 (1978)) are not resistant and clearing can be seen
at < 102 pfu. In contrast, bacterial strains containing pYC188 and expressing
the cl-bHLH/LZ fusion protein are resistant up to 105-106 pfu and those
containing pYC192 and expressing the cl-bHLH are resistant up to 107 pfu.
This resistance demonstrates the ability of the cl/c-myc fusion proteins to
dimerize and effectively repress phage transcription/infection.
In the second assay, referred to herein as the X-gal assay, cells
transformed with the cl/c-myc fusion construct pYC192 also contained a
chimeric lacZ gene under the control of the lambda PL promoter. In these
host cells, expression of functional cl/c-myc fusion protein would be expected
.
..
2 ~
-21-
to repress lacZ expression via repression of the lambda PL promoter.
Expression of the lacZ gene is easily detectable by growth of cells on X-gal
corltaining media. Cells expressing lacZ become blue on this media while
nonexpressing or poorly expressing cells become white or pale blue,
respectively.
As expected, those cells transformed with pYC192 grew as white or
pale blue colonies due to repression of the PL-IacZ gene while nontransformed
cells grew as blue colonies due to expression of the PL-lacZ gene.
Exan~ple 3
Screening a cDNA expression library for protein partners
able to form heterodimers with the cl/c-myc fusion protein
Interference with dimerization by direct protein-protein interaction
between the dimerization domain of the chimeric repressor and a cDNA-
encoded protein is the basis for the screening system of the invention. Upon
dimerization of a repressor monomer with a heterologous protein partner,
which is not part of a cl fusion, the repressor chimera will be inactivated, as
it is unable to bind DNA as a monomer.
The dot plaque assay (DPA) and X-gal assay, described in Example 2,
were used in the screening system. Bacteria expressing cl/c-myc fusion
proteins and exhibiting the homodimer conferred repression phenotype (either
phage resistance or repression of PL-IacZ expression) were used.
Screening with the Dot Plaque Assay
For the DPA, E. coli strain Y1090 (available from Promega Corpora-
tion, Madison, Wisconsin) expressing the chimeric repressor cllc-myc, which
are resistant to infection by )~gtll, were used. Using this bacterial stMin,
~gtl 1 phage cDNA libraries, expressing cDNA encoded proteins as C-terminal
fusions with lacZ, were screened. Only those phage containing a cDNA
.
2081807
-22-
encoding a protein partner were predicted to form plaques due to interference
with cl/c-myc repressor/homodimer formation.
~gtll libraries were screened as follows. Bacterial strain Y1090
containing pYC192 and expressing cl-mycbHLH was grown in L-broth media
containing Kanamycin, Mg2+, and maltose essentially the same as for the
DPA described in Example 2. 0.6 mls of culture were exposed to lx106-
5X106 pfu of i~gt11 library phage in liquid for 20 min. at 30C and then mixed
with 7 mls of top agar and poured on 150 mm L-broth/Kanamycin plates.
Plates were incubated overnight at 42C. Four cDNA libraries were screened:
one from HeLa cells, one from T cell line EL4, one from the pre-B cell line
38B9, and one from primary tonsil cells which are almost exclusively B cells.
These libraries were respectively obtained from T. Kadesch at the University
of Pennsylvania, K. Georgopolas of Massachussetts General Hospital, D.
Weaver of the Dana Farber Cancer Institute (DFCI), and T. Tedder of DFCI.
The lambda cDNA libraries were also plated onto a bacterial strain
expressing a chimeric repressor from plasmid PJH370 (Hu, l.C. etal.,
Science 250:1400-1403 (199~)) containing the d DNA binding domain and the
leucine zipper dimeriMtion domain of the yeast transcription factor GCN4.
For initial screenings, this strain acted as a comparison control to provide a
baseline number of false positive plaques to be expected from the libraries
resulting from phage mutations rendering them insensitive to the d repressor.
This strain also acted as a control for subsequent screening of put~tive positive
plaques to determine if interference was due to a specific interaction with the
cI/c-myc fusion protein or if the interference was of a more general nature,
affecting the GCN4 dimeriMtion domain as well.
For all libraries screened, essentially equal numbers of plaques were
observed with the strain containing cI-GCN4 vs. the strain containing pYC192
(cl-myc), indicating that these plaques were likely to be false positives. The
number of plaques obtained varied from 5 to approximately 250, depending
on the library used. Ninety phage which formed plaques on the strain
containing pYCl92 were plaque purified and subsequently screened on the cl-
., ' ~ "'
2~g~
-23-
GCN4 containing strain. All these phage again formed pla~ues, indicating that
they did not specifically interact with the cl/c-myc fusion protein.
In light of these results, a subsequent experiment was performed to
determine if a known protein partner could be detected with this screeening
procedure. In this experiment a bacterial strain expressing a cl/c-myc fusion
protein was challenged with a ~gtl 1 phage expressing a Max DNA. Max is
a bHLH/LZ protein known to interact with c-myc. The challenged cells
exhibited full resistance to the Max ~gtl 1 phage.
In contrast to these results, DPA screening for a predicted protein
partner introduced before phage infection was succesful. In this experiment,
a pUC18 plasmid capable of expressing a protein containing the bHLH/LZ
domains of c-myc, but not the DNA binding domain of d, was introduced into
a bacterial strain which already contained a pACYC177 plasmid capable of
expressing a cl/c-myc fusion protein. The protein containing the bHLH/LZ
domains was predicted to function as a partner to the cI/c-myc fusion protein
and interfere with the repression of phage infection. As predicted, cells
expressing cl/c-myc and the bHLH/LZ protein were approximately 100-fold
less resistant to phage infection than cells expressing cl/c-myc only, as
measured by the DPA.
These results indicate that the DPA can be used to screen for protein
partners, but that the protein partner must be expressed in the bacteria before
it is challenged with phage. Simultaneous introduction of the protein partner
gene with the challenging phage, as occurs in the direct screen, probably does
not work because the phage is effectively repressed before the protein partner
gene is given a chance to express and interfere with the cl/c-myc fusion
protein repressor.
Screening with the X-Gal Assay
As described above, in cells with an active cltc-myc repressor the PL-
lacZ gene is turned off resulting in the generation of white colonies on X-gal
,
2~8180~
-24-
indicator plates. Interference with repressor dimerization is predicted to yieldblue colonies as the lacZ gene would be expressed (de-repressed).
Screening using the X-gal assay was performed as follows. The strain
Y1090 was transformed with the lacZ target plasmid pNNPL387 and pYC188
which expresses cl-bHLH/LZ. The plasmid pNNPL387 was constructed by
inserting a PCR generated DNA fragment containing the left promoter of
phage lambda upstream of lacZ in pNN387 (provided by S. Elledge, Baylor
University, See Elledge, S.J. et al., Genes & Develop. 3: 185-1g7 (1989)).
These cells, when plated on L-broth/Kanamycin/Chloramphenicol/X-gal, form
white to pale blue colonies. This strain, referred to as 10B18, was made
competent for electroporation (see Current Protocols in Molecular Biology,sec.
1.8.4, Wiley Interscience, ed. by Ausubel et al. (1987)) and transformed with
a plasmid-based cDNA library made from human peripheral blood
Iymphocytes which had been transformed with Epstein-Barr Virus (provided
by S. Elledge, See Elledge, S.J. et al., Proc. Natl. Acad. Sci. USA 88: 1731-
1735 (1991)). There were about 107 recombinants in this once-amplified
library.
10B18 was electropoMted on two separate occasions with 500 ng of
library DNA. Cells were allowed to recover from electroporation for 45 min.
at 37C in SOC media and then plated on M9/0.2 % mannitol with
Chloramphenicol (20~g/ml), Kanamycin (30~g/ml), IPrG (2mM), Ampicillin
(50~g/ml), and X-gal (0.004%). The electroporations yielded 2.8x106 and
5.6x105 tsansformants, of which approximately 500 and 29, respectively, were
blue. A total of 322 blue colonies were picked and restreaked to isolate single
colonies. From these, 97 blue clonal colonies were isolated and plasmid
DNAs were prepared. Plasmid DNA from each clone was then retransformed
into 10B18 and plated as above. Only one clone consistently produced blue
colonies.
This clone was shown to be Tecific for c-myc by comparing the
phenotypes it produced in different repressor chimera backgrounds. Bacterial
strains similar to 10B18 which contain different cl-dimerization domain fusion
2~81~7
-25-
constructs were used. These strains express d fusions with the c-myc bHLH
domain (lOB19), the transcription factor E2/SbHLH domain (lOBE2/5), or
thyroid hormone receptor ,B (lOB,B). The positive clone isolated in the originalscreen produced blue colonies only in 10B18 and 10B19, where dimerization
S was mediated by a c-myc domain. Strains 10BE2/S and 10B,6' remained white on X-gal plates after transformation with this clone.
The high number of false positives obtained during the initial rounds
of screening could be due to the instability of the plasmid containing the
chimeric repressor gene in the screening strain. Alternatively, blue colonies
could result from an increase in the copy number of the PL-lacZ containing
plasmid or increased expression of the PL-IacZ gene which titrates out
repressor dimers. Whatever the cause, repeated passages through the 10B18
strain was effective in screening out false positives.
Example 4
Identification of compounds which prevent
c-myc partnerfor~na~ion
To identify compounds which inhibit c-myc partner heterodimerization
without interfering with c-myc homodimerization, cells identified according to
the method described in Example 3 which contain the cllc-myc fusion protein
and a partner protein are used along with cells containing only the cl/c-myc
fusion protein as described in Example 2. These cells are further exposed to
experimental compounds W, X, Y, and Z and the effect of such compounds
on the homodimer/heterodimer dependent phenotype is determined.
Typical results from such an experiment are shown in Table 1.
.
.
~Q8~07
-26-
Table 1. Identification of C-myc-protein Partner Inhibitors
Compound ¦ Protein Partner ¦ Assay Phenotype
none no plaques/white
_ + plaques/blue
W no plaques/white
+ plaques/blue
X ~ plaques/blue
+ plaques/blue
Y no plaques/white
_ + no plaques/white
The results of the above table indicate that, in the absence of the
partner protein, compound W had no effect on the ability of the cIlc-myc
protein to form homodimers and exhibit the corresponding phenotype.
Compound W also had no effect on the ability of the partner to form
heterodimers with the myc fusion protein and reverse the homodimer-
conferred phenotype. Therefore, compound W will not be a compound of
interest.
Compound X interfered with homodimer formation and therefore will
not be a compound of interest.
Compound Y is an inhibitor of heterodimer formation. Compound Y
did not interfere with homodimer formation but did interfere with hete.rodimer
formation. Therefore, compound Y is a compound of interest as it may
disrupt c-myc action in vivo.
All references cited herein are fully incorpoMted by reference. Having
now fully described the invention, it will be understood by those with skill in
the art that the scope may be performed within a wide and equivalent range
of conditions, paMmeters and the like, without affecting the spirit or scope of
the invention or any embodiment thereof.
,
.
:.
-27- 2 0 ~ 7
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Kinqston, Robert E.
Bunker, Christopher
(ii) TITLE OF INVENTION: Protein Partner Screening Assays and
Uses Thereof
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sterne, Ressler, Goldstein and Fox
(B) STREET: 1225 Connecticut Avenue
(C) CITY: Washington
(D) STATE: D.C.
(E) COUNTRY: U.S.A.
(F) ZIP: 20036
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) CONPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NVMBER: US (to be assigned)
(B) FILING DATE: (herewith)
(C) CLASSIFICATION:
~viii) ATTORN~Y/AGENT INFORMATION:
(A) NAME: Cimbala, Michelle A.
(B) REGISTRATION NUMBER: 33,851
(C) REFERENCE/DOCRET NUMBER: 0609.3630004
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 833-7533
(B) TELEFAX: (202) 833-8716
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQVENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GTCAAGATGG C 11
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(c) STRANDEDNESS: double
.
.
2 ~
-28-
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
AGCAGCTGGC 10
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 ba~e pair#
(8) TYPE: nucleic ac~d
(C) STRaNDEDNESS- double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GTCATGTGGC 10
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1419 ba~e pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
AGGAGGAACA AGAAGATGAG GAAGA~ATCG ATGTTGTTTC TGTGGA~AAG AGGCAGGCTC 60
CTGGCAaAAG GTCAGAGTCT GGATCACCTT CTGCTGGAGG CCACAGGAaA CCTCCTCACA 120
GCCCACTGGT CCTCAAGAGG TGCCACGTCT CCACACATCA GCACAACTAC GCAGCGCCTC 180
CCTCCACTCG GAAGGACTAT CCTGCTGCCA AGAGGGTCAA GTTGGACAGT GTCAGAGTCC 240
TGAG~CAGAT CAGCAACAAC CGAAAATGCA CCAGCCCCAG GTCCTCGGAC ACCGAGGAGA 300
ATGTCAAGAG GCGAACACAC AACGTCTTGG AGCGCCAGAG GAGGAACGAG CTAaAACGGA 360
GCTTTTTTGC CCTGCGTGAC CAGATCCCGG AGTTGGAAaA CAATGA~AAG GCCCCCAAGG 420
TAGTTATCCT TAAAAAAGCC ACAGCATACA TCCTGTCCGT CCAAGCAGAG GAGCA~AAGC 480
TCATTTCTGA AGAGGACTTG TTGCGGA~AC GACGAGAACA GTTGAaACAC A~ACTTGAAC 540
AGCTACGGAA CTCTTGTGCG TAAGGAAAAG TAAGGAaaAC GATTCCTTCT AACAGAaATG 600
TCCTGAGCAA TCACCTATGA ACTTGTTTCA AATGCATGAT CAAATGCAAC CTCACAACCT 660
TGGCTGAGTC TTGAGACTGA AAGATTTAGC CATAATGTAA ACTGCCTCAA ATTGGACTTT 720
.
- 2~818~7
-29-
GGGCATAAAA GAACTTTTTT ATGCTTACCA TCTTTTTTTT TTCTTTAACA GATTTGTATT 780
TAAGAATTGT TTTTAAAAAA TTTTAAGATT TACACAATGT TTCTCTGTAA ATATTGCCAT 840
TA~ATGTAAA TAACTTTAAT AAAACGTTTA TAGCAGTTAC ACAGAATTTC AATCCTAGTA 900
TATAGTACCT AGTATTATAG GTACTATAAA CCCTAATTTT TTTTATTTAA GTACATTTTG 960
CTTTTTAAAG TTGATTTTTT TCTATTGTTT TTAGAAAAAA TAAAATAACT GGCAAATATA 1020
TCATTGAGCC AAATCTTAAG TTGTGAATGT TTTGTTTCGT TTCTTCCCCC TCCCAACCAC 1080
CACCATCCCT GTTTGTTTTC ATCAATTGCC CCTTCAGAGG GTGGTCTTAA GAAAGGCAAG 1140
AGTTTTCCTC TGTTGAAATG GGTCTGGGGG CCTTAAGGTC TTTA~GTTCT TGGAGGTTCT 1200
AAGATGCTTC CTGGAGACTA TGATAACAGC CGAAGTTGAC AGTTAGAAGG AATGGCAGAA 1260
GGCAGGTGAG AAGGTGAGAG GTAGGCAAAG GAGATACAAG AGGTCAAAGG TAGCAGTTAA 1320
GTACACAAAG AGGCATAAGG ACTGGGGAGT TGGGAGGAAG GTGAGGAAGA AACTCCTGTT 1380
ACTTTAGTTA ACCAGTGCCA GTCCCCTGCT CACTCCAAA 1419
~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:.186 amino acid~
~B) TYPE: amino acid
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Glu Glu Gln Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys
1 5 10 15
Arg Gln Ala Pro Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly
20 25 30
Gly His Ser Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His
35 40 45
Val Ser Thr His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys
Asp Tyr Pro Ala Ala Lys Arg Val Ly~ Lsu Asp Ser Val Arg Val Leu
Arg Gln Ile Ser Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp
85 90 95
Thr Glu Glu Asn Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln
100 105 110
Arg Arg Asn Glu Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile
115 120 125
Pro Glu Leu Glu Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys
130 135 140
`
2~18~7
-30-
Lys Ala Thr Ala Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu
145 150 155 160
Ile Ser Glu Glu Asp Leu Leu Arg Ly~ Ar3 Arg Glu Gln Leu Lys His
Lys Leu Glu Gln Leu Arg Asn Ser Cys Ala
180 185
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 101 base pairs
(B) TYPE: nucleic acid
tc) STRaNDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GGATCCTCTA AATACATTCA AATAAGTATC CGCTCATGAG ACAATAACGG TAACCAGAAT 60
TGTGAGCGCT CACAATTTTG ATCGATAGGA AACTCGAGAT G 101
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 ba~e pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA -
(xi) SEOUENCE DESCRIPTION: SEQ ID NO:7:
QGGCAGGGT CTAGA 15
- ~ :
,
~ .
