Peptides and compositions which modulate apoptosis

The present invention is directed to novel peptides and compositions capable of modulating apoptosis in cells, and to methods of modulating apoptosis employing the novel peptides and compositions of the invention. In one aspect, the invention is directed to a novel peptide designated the "GD domain," which is essential both to Bak's interaction with Bcl-x.sub.L, and to Bak's cell killing function. Methods of identifying agonists or antagonists of GD domain function are provided. The GD domain is responsible for mediating key protein/protein interactions of significance to the actions of multiple cell death regulatory molecules.

FIELD OF THE INVENTION 
The present invention relates generally to the field of cell physiology, 
and more particularly, to programmed cell death, or apoptosis. The novel 
peptides and compositions of the invention are useful for modulating 
apoptosis in cells. 
BACKGROUND OF THE INVENTION 
The phenomenon of programmed cell death, or "apoptosis," is known to be 
involved in and important to the normal course of a wide variety of 
developmental processes, including immune and nervous system maturation. 
Apoptosis also plays a role in adult tissues having high cell turnover 
rates (Ellis, R. E., et al., Annu. Rev. Cell. Biol. 7: 663-698 (1991); 
Oppenheim, R. W., Annu. Rev. Neurosci. 14: 453-501 (1991); Cohen, J. J., 
et al. Annu. Rev. Immunol. 10: 267-293 (1992); Raff, M. C., Nature 356: 
397-400 (1992)). A number of different physiological signals normally 
activate programmed cell death in these contexts, but non-physiological 
insults, such as irradiation and exposure to drugs which damage DNA, also 
can trigger apoptosis (Eastman, A., Cancer Cells 2: 275-280 (1990); Dive, 
C., et al., Br. J. Cancer 64: 192-196 (1991); Lennon, S. V., et al., Cell 
Prolif. 24: 203-214 (1991)). 
In addition to its role in development, apoptosis has been implicated as an 
important cellular safeguard against tumorigenesis (Williams, G. T., Cell 
65: 1097-1098 (1991); Lane, D. P., Nature 362: 786-787 (1993)). Under 
certain conditions, cells die by apoptosis in response to high-level or 
deregulated expression of oncogenes (Askew, D., et al., Oncogene 6: 
1915-1922 (1991); Evan, G. I., et al., Cell 69: 119-128 (1992); Rao, L., 
et al., Proc. Natl. Acad. Sci. USA 89: 7742-7746 (1992); Smeyne, R. J., et 
al., Nature 363: 166-169 (1993); Tanaka, S., et al., Cell 77: 829-839 
(1994); Wu, X., et al., Proc. Natl. Acad. Sci. USA 91: 3602-3606 (1994)). 
Suppression of the apoptotic program, by a variety of genetic lesions, may 
contribute to the development and progression of malignancies. This is 
well illustrated by the frequent mutation of the p53 tumor suppressor gene 
in human tumors (Levine, A. J., et al., Nature 351: 453-456 (1991)). 
Wild-type p53 is required for efficient induction of apoptosis following 
DNA damage (Clarke, A. R., et al., Nature 362: 849-852 (1993); Lowe, S. 
W., et al., Cell 74: 957-967 (1993); Lowe, S. W., et al., Nature 362: 
847-849 (1993)) and cell death induced by constitutive expression of 
certain oncogenes (Debbas, M., et al., Genes & Dev. 7: 546-554 (1993); 
Hermeking, H., et al., Science 265: 2091-2093 (1994); Tanaka, S., et al., 
Cell 77: 829-839 (1994); Wu, X., et al., Natl. Acad. Sci. USA 91: 
3602-3606 (1994)). The cytotoxicity of many commonly used chemotherapeutic 
agents is mediated by wild-type p53 (Lowe, S. W., et al., Cell 74: 957-967 
(1993); Fisher, D. E., Cell 78: 539-542 (1994)). Thus, loss of p53 
function may contribute to the clinically significant problem of drug 
resistant tumor cells emerging following chemotherapy regimens. 
The expression product of the bcl-2 oncogene functions as a potent 
suppressor of apoptotic cell death (McDonnell, T. J., et al., Cell 57: 
79-88 (1989); Hockenbery, D., et al., Nature 348: 334-336 (1990)). 
Constitutive Bcl-2 expression can suppress apoptosis triggered by diverse 
stimuli, including growth factor withdrawal, oncogene expression, DNA 
damage, and oxidative stress (Vaux, D. L., et al., Nature 335: 440-442 
(1988); Sentman, C. L., et al., Cell 67: 879-888 (1991); Strasser, A., et 
al., Cell 67: 889-899 (1991); Fanidi, A., et al., Nature 359: 554-556 
(1992); Hockenbery, D. M., et al., Cell 75: 241-251 (1993)). There is also 
conservation of Bcl-2 function across species. For example, the ced-9 gene 
of the nematode C. elegans appears to be a structural and functional 
homolog of bcl-2 (Hengartner, M. O., et al., Cell 76: 665-676 (1994)) and 
bcl-2 can complement ced-9 mutations in transgenic animals (Vaux, D. L., 
et al., Science 258: 1955-1957 (1991)). These observations suggest that 
Bcl-2 is intimately connected with an evolutionarily conserved cell death 
program. 
It is known that bcl-2 is a member of a family of related genes, at least 
some of which also modulate apoptosis. Of these, bcl-x bears the highest 
degree of homology to bcl-2, and is differentially spliced to produce a 
long form, termed bcl-x.sub.L, and a shorter form, bcl-x.sub.S, related 
genes, at least some of which also modulate harboring an internal deletion 
(Boise, L. H., et al., Cell 74: 597-608 (1993)). Bcl-x.sub.L functions to 
suppress apoptosis, whereas the deleted form, Bcl-x.sub.S, inhibits the 
protection against cell death provided by Bcl-2 expression. A second Bcl-2 
homolog, Bax, forms heterodimers with Bcl-2 (Oltvai, Z. N., et al., Cell 
74: 609-619 (1993)) and has been shown to counteract Bcl-2 and accelerate 
apoptosis. Mutational analysis of Bcl-2 has suggested that the interaction 
with Bax is required for Bcl-2 to function as an inhibitor of cell death 
(Yin, X. -M., et al., Nature 369: 321-323 (1994)). 
The isolation and characterization of a bci-2 related gene, termed bak, is 
described in co-pending U.S. application Ser. No. 08/321,071, filed 11 
Oct. 1994, which is a continuation-in-part of U.S. application Ser. No. 
08/287,427, filed 9 Aug. 1994, now abandoned (bak is referred to therein 
as bcl-y), the disclosures of which are incorporated herein by reference. 
Ectopic Bak expression accelerates the death of an IL-3 dependent cell 
line upon cytokine withdrawal, and opposes the protection against 
apoptosis afforded by Bcl-2. In addition, enforced expression of Bak is 
sufficient to induce apoptosis of serum deprived fibroblasts, raising the 
possibility that Bak directly activates, or is itself a component of, the 
cell death machinery. 
The known cellular Bcl-2 related genes, where analyzed, have distinct 
patterns of expression and thus may function in different tissues. While 
Bcl-2 expression appears to be required for maintenance of the mature 
immune system, it is desirable to identify other genes which may govern 
apoptotic cell death in other lineages. In addition, the identification of 
particular regions or domains of the proteins encoded by such genes may 
provide a basis for understanding their structural and functional 
characteristics and allow the development of valuable diagnostics and 
therapeutics. For example, the identification of agents capable of 
restoring or inducing apoptosis in tumor cells (in which loss of p53 tumor 
suppressor gene function may be implicated in tumorigenesis and in 
clinically significant drug resistance) would be of significant 
therapeutic value, particularly where such restoration or induction was 
independent of p53 function. Similarly, the development of agents capable 
of counteracting the anti-apoptotic function of oncogenes such as as 
bcl-2, the activation of which is implicated in tumorigenesis (e.g., 
lymphoma) and in chemotherapeutic drug resistance, would be of great 
potential value. 
SUMMARY OF THE INVENTION 
The present invention is directed to a novel protein domain of general 
significance to the actions of multiple cell death regulatory molecules, 
which has been identified and mapped to a short subsequence in the central 
portion of the Bak molecule. This heretofore unrecognized protein domain, 
which the inventor has designated the "GD domain," is essential both to 
Bak'sinteraction with Bcl-x.sub.L, and to Bak's cell killing function. 
Truncated Bak species encompassing the GD domain are themselves sufficient 
to bind to Bcl-x.sub.L and to kill cells in transfection assays. 
The GD domain has been identified in two other Bcl-2 binding proteins that 
function to induce apoptosis: Bax and Bip1a. As with Bak, mutation of the 
homologous GD domain elements in Bax and Bip1a diminishes cell killing and 
protein binding function. Thus, the GD domain is responsible for mediating 
key protein/protein interactions of significance to the actions of 
multiple cell death regulatory molecules. 
In one aspect, then, the invention is directed to purified and isolated 
peptides comprising the GD domain and to molecules that mimic its 
structure and/or function, useful for inducing or modulating the apoptotic 
state of a cell. Chemical compounds that disrupt the function of the GD 
domain have utility as apoptosis-modulating agents. Accordingly, in 
another aspect, the invention is directed to agents capable of disrupting 
GD domain function. Such agents include, but are not limited to, molecules 
that bind to the GD domain, molecules that interfere with the interaction 
of the GD domain with other protein(s), and molecules comprising the GD 
domain which is altered in some manner. The invention provides methods to 
identify molecules that modulate apoptosis by disrupting the function of 
the GD domain, which accordingly comprise additional contemplated 
embodiments. 
In additional aspects, the present invention relates to products and 
processes involved in the cloning, preparation and expression of peptides 
comprising the GD domain; antibodies with specificity to the GD domain; 
and nucleotide sequences encoding the GD domain or portions thereof. 
Peptides comprising the GD domain are useful for producing antibodies 
thereto. Such antibodies are useful for detecting and isolating proteins 
comprising the GD domain in biological specimens including, for example, 
cells from all human tissues including heart tissue, lung tissue, tumor 
cells, brain tissue, placenta, liver, skeletal muscle, kidney, and 
pancreas, as well as for modulating the apoptotic activity of proteins 
comprising the GD domain in and from such biological specimens, and 
constitute additional aspects of the invention. 
In yet another aspect, the invention provides for expression vectors 
containing genetic sequences, hosts transformed with such expression 
vectors, and methods for producing the recombinant GD domain peptides of 
the invention. 
The present invention is further directed to methods for inducing or 
suppressing apoptosis in the cells and/or tissues of individuals suffering 
from degenerative disorders characterized by inappropriate cell 
proliferation or inappropriate cell death, respectively. Degenerative 
disorders characterized by inappropriate cell proliferation include, for 
example, inflammatory conditions, cancer, including lymphomas, such as 
prostate hyperplasia, genotypic tumors, etc. Degenerative disorders 
characterized by inappropriate cell death include, for example, autoimmune 
diseases, acquired immunodeficiency disease (AIDS), cell death due to 
radiation therapy or chemotherapy, neurodegenerative diseases, such as 
Alzheimer's disease and Parkinson's disease, etc. 
The present invention also relates to methods for detecting the presence of 
the GD domain peptide, as well as methods directed to the diagnosis of 
degenerative disorders, which disorders are associated with an increased 
or decreased level of expression of proteins comprising the GD domain, as 
compared to the expected level of expression of such proteins in the 
normal cell population. 
The present invention relates to the therapeutic use of peptides comprising 
the GD domain. 
The present invention also relates to methods for modulating the apoptotic 
state of a cell by administering peptides comprising the GD domain 
peptide, or mutants thereof, to an individual suffering from a 
degenerative disorder characterized by inappropriate cell proliferation or 
inappropriate cell death, in order to stabilize inappropriate cell 
proliferation (i.e., induce apoptosis) or stabilize inappropriate cell 
death (i.e., suppress apoptosis), respectively, and/or in either case to 
restore normal cell behavior. 
In another aspect, the present invention is related to the surprising 
discovery that the Bak GD domain is involved in and sufficient for 
homodimerization and heterodimerization of Bak. Nonlimiting examples of 
Bak GD domain dimerization include Bak (homodimerization), Bax 
(heterodimerization with a different killer protein) and Bcl-x.sub.L 
(heterodimerization with a survival protein). Further, it has unexpectedly 
been discovered that the non-essential regions of the Bak protein in this 
aspect include the two domains in the carboxyl terminal half of the 
protein that show the highest degree of homology to other Bcl-2 family 
members (Bcl-2 homology domains I and II). Thus, peptides comprising the 
GD domain are capable of mediating interactions not only with Bcl-x.sub.L, 
but also with Bak and Bax. 
These and other objects and aspects of the invention will be apparent to 
those of skill from the description which follows.

DETAILED DESCRIPTION OF THE INVENTION 
Technical and scientific terms used herein have the meanings commonly 
understood by one of ordinary skill in the art to which the present 
invention pertains, unless otherwise defined. Reference is made herein to 
various methodologies known to those of skill in the art. Publications and 
other materials setting forth such known methodologies to which reference 
is made are incorporated herein by reference in their entireties as though 
set forth in full. Standard reference works setting forth the general 
principles of recombinant DNA technology include Sambrook, J., et al., 
Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor 
Laboratory Press, Planview, N.Y. (1989); McPherson, M. J., Ed., Directed 
Mutagenesis: A Practical Approach, IRL Press, Oxford (1991); Jones, J., 
Amino Acid and Peptide Synthesis, Oxford Science Publications, Oxford 
(1992); Austen, B. M. and Westwood, O. M. R., Protein Targeting and 
Secretion, IRL Press, Oxford (1991). Any suitable materials and/or methods 
known to those of skill can be utilized in carrying out the present 
invention; however, preferred materials and/or methods are described. 
Materials, reagents and the like to which reference is made in the 
following description and examples are obtainable from commercial sources, 
unless otherwise noted. 
A previously unrecognized domain within the Bak molecule that appears to be 
both necessary and sufficient for the known biological activities of Bak 
has now been identified. This domain, designated herein as the "GD 
domain," is sufficient to mediate cell killing function and physical 
interaction with Bcl-xL. Sequences homologous to the Bak GD domain have 
also been identified within Bax and Bip1a and shown to be similarly 
required for the cell killing and Bcl-x.sub.L binding activities of these 
proteins. These observations suggest that Bak, Bax and Bip1a modulate or 
regulate apoptosis through a similar mechanism that, in each case, 
involves their respective GD domains. As those of skill familiar with the 
present invention will appreciate, sequences comprising the GD domain are 
useful in modulating apoptosis in cells. Similarly, compounds and 
compositions which are capable of binding to the GD domain are useful as 
agents for the modulation of apoptotic activity in cells. 
As used herein, the term "GD domain" refers to a protein domain first 
identified in Bak, demonstrated herein to be essential for the interaction 
of Bak with Bcl-x.sub.L and for Bak's cell killing function, and to 
peptides and/or molecules capable of mimicking its structure and/or 
function. In a preferred embodiment, the present invention comprises a 
peptide having the following amino acid sequence: 
EQU GDDINRRYDSEFQ [SEQ ID NO:1] 
corresponding to amino acid residues 82-94 of Bak, as well as functional 
equivalents thereof. By "functional equivalent" is meant a peptide 
possessing a biological activity or immunological characteristic 
substantially similar to that of the GD domain, and is intended to include 
"fragments", "variants", "analogs", "homologs", or "chemical derivatives" 
possessing such activity or characteristic. Functional equivalents of the 
GD domain, then, may not share an identical amino acid sequence, and 
conservative or non-conservative amino acid substitutions of conventional 
or unconventional amino acids are possible. 
Reference herein to "conservative" amino acid substitution is intended to 
mean the interchangeability of amino acid residues having similar side 
chains. For example, glycine, alanine, valine, leucine and isoleucine make 
up a group of amino acids having aliphatic side chains; serine and 
threonine are amino acids having aliphatic-hydroxyl side chains; 
asparagine and glutamine are amino acids having amide-containing side 
chains; phenylalanine, tyrosine and tryptophan are amino acids having 
aromatic side chains; lysine, arginine and histidine are amino acids 
having basic side chains; and cysteine and methionine are amino acids 
having sulfur-containing side chains. Interchanging one amino acid from a 
given group with another amino acid from that same group would be 
considered a conservative substitution. Preferred conservative 
substitution groups include asparagine-glutamine, alanine-valine, 
lysine-arginine, phenylalanine-tyrosine and valine-leucine-isoleucine. 
In a preferred embodiment of the invention, there is provided a peptide 
having the following amino acid sequence: 
EQU PSSTMGQVGRQLAIIGDDINRRYDSEFQ [SEQ ID NO:2] 
corresponding to amino acid residues 67-94 of Bak, uniquely required for 
Bak cell killing function. 
In another preferred embodiment, there is provided a peptide having the 
following amino acid sequence: 
EQU QVGRQLAIIGDDINRRYDSEFQTMLQHLQPT [SEQ ID NO:3] 
corresponding to amino acid residues 73-103 of Bak, sufficient for the cell 
killing function of Bak. 
The present data indicate that the biological activity of the GD domain and 
its functional derivatives will be affected by the sub-cellular 
localization of these compositions. Accordingly, in another preferred 
embodiment of the invention, the GD domain peptides of the invention will 
have fused to their C-terminal end an appropriate hydrophobic tail, which 
may comprise amino acids 187-211 of Bak. Other suitable means of effecting 
sub-cellular localization, including the selection of suitable hydrophobic 
tails, such as amino acids 172-192 of Bax, amino acids 213-233 of 
Bcl-x.sub.L, amino acids 220-240 of Bcl-2, and hydrophobic tails 
introduced through protein lipidation (Casey, T. J., Science, 268: 221-225 
(1995)) such as prenylation and acylation (e.g., myristylation, 
palmitylation) may be employed by those of skill using known methods. 
The GD domain disclosed herein is uniquely involved in both cell killing 
and Bcl-x.sub.L binding activity of Bak. Moreover, other Bcl-2 interacting 
proteins having functional properties resembling those of Bak are 
demonstrated herein to contain amino acid regions having sequences bearing 
homology to sequences within the GD domain of Bak. These proteins include 
Bax and Bip1a which, like Bak, interact with Bcl-2, and both of these 
proteins contain amino acid regions bearing homology to sequences within 
the GD domain of Bak. In Bax, this region comprises amino acids 59-73, 
which bears homology to amino acids 74-88 within the GD domain of Bak. The 
protein Bip1a similarly contains an amino acid region comprising amino 
acids 57-71 bearing homology to the same sequences (amino acids 74-88) 
within the Bak GD domain. Deletion of the Bax and Bip1a GD domain regions 
identified above impaired their cell killing activity and prevented 
binding to Bcl-x.sub.L. Bip1a lacks sequences homologous to the two highly 
conserved regions, designated Domain I and Domain II (also referred to in 
the literature as "Bcl-2 Homology domains" or "BH domains" I and II or 
"BH1" and "BH2"). It has been suggested that these two conserved regions, 
and especially Domain I, are instrumental in dictating homo- and 
heterodimerization in Bcl-2, Bax, and other Bcl-2 family members. 
Accordingly, the GD domain constitutes a key element involved in the 
biological activity of proteins such as Bak, Bax and Bip1a, not 
necessarily shared with Bcl-2 family members, which activity is 
independent of BH domains I and II. This suggests that the GD domain 
defines a distinct family of proteins, including Bak, Bax and Bip1a. 
Accordingly, in an additional preferred embodiment, there is provided a 
peptide comprising the following amino acids:ps ti LSECLKRIGDELDSN [SEQ ID 
NO:4] 
corresponding to amino acids 59-73 of Bax. In another preferred embodiment, 
a peptide is provided which comprises amino acid sequence: 
EQU LKRIGDELD [SEQ ID NO:5] 
corresponding to amino acids 63-71 of Bax. In another preferred embodiment, 
a peptide is provided which comprises amino acid sequence: 
EQU QDASTKKLSECLKRIGDELDSNMELQ [SEQ ID NO:6] 
corresponding to amino acids 52-77 of Bax. In another preferred embodiment, 
a peptide is provided which comprises amino acid sequence: 
EQU LALRLACIGDEMDVS [SEQ ID NO:7] 
corresponding to amino acids 57-71 of Bip1a. In another preferred 
embodiment, there is provided a peptide comprising the following amino 
acid sequence: 
EQU IGDEM [SEQ ID NO:8] 
corresponding to amino acids 64-68 of Bip1a. In another preferred 
embodiment, there is provided a peptide comprising the following amino 
acid sequence: 
EQU CMEGSDALALRLACIGDEMDVSLRAPRL [SEQ ID NO:9] 
corresponding to amino acids 50-77 of Bip1a. In another preferred 
embodiment, there is provided a peptide comprising the following amino 
acid sequence: 
EQU VGRQLAIIGDDINRR [SEQ ID NO:10] 
corresponding to amino acids 74-88 of Bak. 
A surprising aspect of the present invention is the discovery that the GD 
domain alone is sufficient for homodimerization of Bak, as well as for 
heterodimerization of Bak with Bax and Bcl-x.sub.L, and that the highly 
conserved Bcl-2 family Domains I and II are not necessary for this 
dimerization. This indicates that the GD domain is capable of modulating 
the function of proteins including Bak, Bax and Bcl-x.sub.L directly 
through dimerization, and thus may also modulate the function of other 
proteins including Bcl-2. 
The functional importance of the GD domain, then, is likely to be related 
to its ability to mediate one or more protein/protein interactions with 
other Bcl-2 family members, or with other as yet unidentified cellular 
protein(s). It is possible that survival proteins like Bcl-2 and 
Bcl-x.sub.L suppress apoptosis by binding and inactivating proteins that 
actively promote cell death, such as Bak, Bax and Bip1a, through their GD 
domains. In support of this view, the interaction with Bax appears to be 
required for Bcl-2 to suppress apoptosis (Yin et al., Nature 369: 321-323 
(1994)). A second possibility is that Bak, Bax, and Bip1a induce cell 
death bybinding (via their GD domains) and inactivating proteins, 
including Bcl-2 and Bcl-x.sub.L, that actively promote cell survival. It 
is also possible that Bak, Bax and Bip1a bind one or more additional 
cellular proteins and that this interaction mediates cell death function. 
The present inventor does not intend to be bound by a particular theory; 
however, regardless of its mechanism(s) of action, the GD domain in Bak, 
Bax and Bip1a is of central importance for mediating these protein/protein 
interactions. 
Agents capable of modulating GD domain mediated protein/protein 
interactions may include peptides comprising the GD domain, as well as 
mutants of the GD domain or of proteins comprising the GD domain. A 
"mutant" as used herein refers to a peptide having an amino acid sequence 
which differs from that of the naturally occurring peptide or protein by 
at least one amino acid. Mutants may have the same biological and 
immunological activity as the naturally occurring GD domain peptide or the 
naturally occurring protein. However, the biological or immunological 
activity of mutants may differ or be lacking. For example, a GD domain 
mutant may lack the biological activity which characterizes naturally 
occurring GD domain peptide, but may be useful as an antigen for raising 
antibodies against the GD domain or for the detection or purification of 
antibodies against the GD domain, or as an agonist (competitive or 
non-competitive), antagonist, or partial agonist of the function of the 
naturally occurring GD domain peptide. 
Modulation of GD domain mediated protein/protein interactions may be 
effected by agonists or antagonists of GD domain peptides as well. 
Screening of peptide libraries, compound libraries and other information 
banks to identify agonists or antagonists of the function of proteins 
comprising the GD domain is accomplished with assays for detecting the 
ability of potential agonists or antagonists to inhibit or augment GD 
domain binding, e.g., GD domain homodimerization or heterodimerization. 
For example, high through-put screening assays may be used to identify 
compounds that modulate the protein binding function of the GD domain. 
Such screening assays facilitate the identification of compounds that 
accelerate or inhibit apoptosis by influencing protein/protein 
interactions mediated by the GD domain. For example, an in vitro screen 
for compounds that disrupt the Bak GD domain interaction with 
GST-Bcl-x.sub.L comprises multiwell plates coated with GST-Bcl-x.sub.L 
which are incubated with a labeled GD domain peptide probe in the presence 
of one or more compounds to be tested. Molecules that specifically disrupt 
the interaction could, in principle, bind to either the GD domain "ligand" 
or to the as yet undefined "receptor" domain in Bcl-x.sub.L. Either class 
of compound would be a candidate apoptosis-modulating agent. 
Thus, the invention provides a method of screening for an agent capable of 
modulating apoptosis which comprises coating a multiwell plate with 
GST-Bcl-x.sub.L and incubating the coated multiwell plate with a labeled 
GD domain peptide probe in the presence of an agent which it is desired to 
test, wherein disruption of GD domain interaction with GST-Bcl-x.sub.L 
indicates that said agent is capable of modulating apoptosis. Agents 
identified by this method are also contemplated embodiments of the 
invention. 
Suitable labels include a detectable label such as an enzyme, radioactive 
isotope, fluorescent compound, chemiluminescent compound, or 
bioluminescent compound. Those of ordinary skill in the art will know of 
other suitable labels or will be able to ascertain such using routine 
experimentation. Furthermore, the binding of these labels to the peptides 
is accomplished using standard techniques known in the art. 
A high speed screen for agents that bind directly to the GD domain may 
employ immobilized or "tagged" combinatorial libraries. Agents that bind 
specifically to such libraries are candidates to be tested for their 
capacity to block Bak/Bcl-x.sub.L interactions. As discussed above, such 
agents may function as suppressors of apoptosis by either directly 
inhibiting Bak (and/or Bax/Bip1a) function, or by increasing the effective 
activity of endogenous Bcl-2/Bcl-x.sub.L (or other Bcl-2 family member). 
Such agents would be useful for suppressing aberrant apoptosis in 
degenerative disorders or following ischemic injury. 
Antibodies against the GD domain peptides of the invention may be used to 
screen cDNA expression libraries for identifying clones containing cDNA 
inserts encoding structurally related, immunocrossreactive proteins which 
may be members of the GD domain family of proteins. Screening of cDNA and 
mRNA expression libraries is known in the art. Similarly, antibodies 
against GD domain peptides are used to identify or purify 
immunocrossreactive proteins related to this domain, or to detect or 
determine the amount of proteins containing the GD domain in a cell or 
cell population, for example, in tissue or cells, such as lymphocytes, 
obtained from a patient. Known methods for such measurements include 
immunoprecipitation of cell extracts followed by PAGE, in situ detection 
by immunohistochemical methods, and ELISA methods, all of which are well 
known in the art. 
Modulation of apoptosis according to the invention includes methods 
employing specific antisense polynucleotides complimentary to all or part 
of the nucleotide sequences encoding proteins comprising the GD domain 
disclosed herein. Such complimentary antisense polynucleotides may include 
nucleotide additions, deletions, substitutions and transpositions, 
providing that specific hybridization to the target sequence persists. 
Soluble antisense RNA or DNA oligonucleotides which can hybridize 
specifically to mRNA species encoding proteins comprising the GD domain, 
and which prevent transcription of the mRNA species and/or translation of 
the encoded polypeptide are contemplated as complimentary antisense 
polynucleotides according to the invention. Production of proteins 
comprising the GD domain is inhibited by antisense polynucleotides 
according to the invention, and such antisense polynucleotides may inhibit 
apoptosis, senescence and the like, and/or reverse the transformed 
phenotype of cells. A heterologous expression cassette maybe used to 
produce antisense polynucleotides in a transfectant or transgenic cell. 
Antisense polynucleotides also may be administered as soluble 
oligonucleotides to the external environment of the target cell, such as 
the culture medium of cells in vitro or the interstitial fluid (e.g., via 
the circulatory system) in vivo. Antisense polynucleotides and their use 
are known to those of skill, and are described, for example, in Melton, D. 
A., Ed, Antisense RNA and DNA, Cold Spring Harbor Laboratory Press, Cold 
Spring Harbor, N.Y. (1988). 
The predicted biological activity of agents identified according to the 
invention varies depending on the assumptions made regarding the mechanism 
of Bak/Bcl-2 function. For example, an agent which binds tightly to the GD 
domain would be predicted to inhibit Bak (and perhaps Bax/Bip1a) function. 
Assuming Bak (and/or Bax/Bip1a) is the active cell death regulatory 
molecule, an agent that binds tightly to the GD domain may inhibit Bak 
function via a mechanism similar to the action of Bcl-2/Bcl-x.sub.L 
binding. Such agents would comprise "Bcl-2/Bcl-x.sub.L " mimetics and 
might, therefore, exhibit anti-apoptotic activity under conditions in 
which Bcl-2 has a demonstrated protective effect (e.g., protection of 
neurons against injury or cytokine deprivation). Agents in this class 
could have utility in treating diseases characterized by excessive or 
inappropriate cell death, including, for example, neuro-degenerative 
diseases and injury resulting from ischemia. 
If Bcl-2/Bcl-x.sub.L binding actively promotes cell survival, and if Bak 
repression is due simply to its binding and inactivating these survival 
proteins, then an agent that prevented this binding would effectively 
increase the activity of resident Bcl-2/Bcl-x.sub.L in a cell by relieving 
repression by Bak (and/orbyBax/Bip1a). This would also promote cell 
survival, but only in cells that express endogenous Bcl-2/Bcl-x.sub.L. 
Agents that bind to Bcl-x.sub.L and thereby prevent its interaction with 
Bak (and/or with Bax/ Bip1a) might inhibit the cell death suppression 
activity of Bcl-x.sub.L (and/or of Bcl-2). Such agents would comprise "GD 
domain mimetics" and would promote cell death in a fashion mechanistically 
similar to the action of Bak. GD domain mimetic agents would be useful in 
the therapeutic treatment of cancer and viral disease. 
Peptidomimetics of GD domain peptide are also provided by the present 
invention, and can act as drugs for the modulation of apoptosis by, for 
example, blocking the function of proteins comprising the GD domain or 
interfering with GD domain mediated dimerization. Peptidomimetics are 
commonly understood in the pharmaceutical industry to include non-peptide 
drugs having properties analogous to those of those of the mimicked 
peptide. The principles and practices of peptidomimetic design are known 
in the art and are described, for example, in Fauchere J., Adv. Drug Res. 
15: 29 (1986); and Evans et al., J. Med. Chem. 30: 1229 (1987). 
Peptidomimetics which bear structural similarity to therapeutically useful 
peptides may be used to produce an equivalent therapeutic or prophylactic 
effect. Typically, such peptidomimetics have one or more peptide linkages 
optionally replaced bya linkage which may convert desirable properties 
such as resistance to chemical breakdown in vivo. Such linkages may 
include --CH.sub.2 NH--, --CH.sub.2 S--, --CH.sub.2 --CH.sub.2 --, 
--CH.dbd.CH--, --COCH.sub.2 --, --CH(OH)CH.sub.2 --, and --CH.sub.2 SO--. 
Peptidomimetics may exhibit enhanced pharmacological properties 
(biological half life, absorption rates, etc.), different specificity, 
increased stability, production economies, lessened antigenicity and the 
like which makes their use as therapeutics particularly desirable. 
As discussed herein, the GD domain appears to be an area of motifs involved 
in dimerization, and this activity may be related to the regulation of 
apoptosis by proteins comprising the GD domain. Bak possesses a C-terminal 
hydrophobic region which appears to be membrane spanning. Thus, 
sub-cellular localization of proteins containing the GD domain may play a 
role in the regulation of programmed cell death in vivo. It is possible, 
then, to employ the invention for detection or determination of proteins 
comprising the GD domain, for example, in fractions from tissue/organ 
excisions, by means of immunochemical or other techniques in view of the 
antigenic properties thereof. Immunization of animals with peptides 
comprising the GD domain alone or in conjunction with adjuvants by known 
methods can produce antibodies specific for the GD domain peptide. 
Antiserum obtained by conventional procedures may be utilized for this 
purpose. For example, a mammal, such as a rabbit, may be immunized with a 
peptide comprising the GD domain, thereby inducing the formation of 
polyclonal antibodies thereagainst. Monoclonal antibodies also may be 
generated using known procedures. Such antibodies can be used according to 
the invention to detect the presence and amount of peptides comprising the 
GD domain. 
The GD domain peptides of the invention may be used for the detection of 
Bak, Bcl-x.sub.L, Bip1a and other proteins by means of standard assays 
including radioimmunoassays and enzyme immunoassays. 
It will be appreciated by those of skill that the precise chemical 
structure of peptides comprising the GD domain will vary depending upon a 
number of factors. For example, a given protein may be obtained as an 
acidic or basic salt, or in neutral form, since ionizable carboxyl and 
amino groups are found in the molecule. For the purposes of the invention, 
then, any form of the peptides comprising the GD domain which retains the 
therapeutic or diagnostic activity of the naturally occurring peptide is 
intended to be within the scope of the present invention. 
The GD domain peptides and other compositions of the present invention may 
be produced by recombinant DNA techniques known in the art. For example, 
nucleotide sequences encoding the GD domain peptides of the invention may 
be inserted into a suitable DNA vector, such as a plasmid, and the vector 
used to transform a suitable host. The recombinant GD peptide is produced 
in the host by expression. The transformed host may be a prokaryotic or 
eukaryotic cell. Preferred nucleotide sequences for this purpose encoding 
the GD domains of Bak, Bax and Bip1a are set forth in FIG. 8. 
Polynucleotides encoding peptides comprising the GD domain may be genomic 
or cDNA, isolated from clone libraries by conventional methods including 
hybridization screening methods. Alternatively, synthetic polynucleotide 
sequences may be constructed by known chemical synthetic methods for the 
synthesis of oligonucleotides. Such synthetic methods are described, for 
example, in Blackburn, G. M. and Gait, M. J., Ed., Nucleic Acids in 
Chemistry and Biology, IRL Press, Oxford, England (1990), and it will be 
evident that commercially available oligonucleotide synthesizers also may 
be used according to the manufacturer's instructions. One such 
manufacturer is Applied Bio Systems. 
Polymerase chain reaction (PCR) using primers based on the nucleotide 
sequence data disclosed herein may be used to amplify DNA fragments from 
mRNA pools, cDNA clone libraries or genomic DNA. PCR nucleotide 
amplification methods are known in the art and are described, for example, 
in Erlich, H. A., Ed., PCR Technology: Principles and Applications for DNA 
Amplification, Stockton Press, New York, N.Y. (1989); U.S. Pat. No. 
4,683,202; U.S. Pat. No. 4,800,159; and U.S. Pat. No. 4,683,195. Various 
nucleotide deletions, additions and substitutions may be incorporated into 
the polynucleotides of the invention as will be recognized by those of 
skill, who will also recognize that variation in the nucleotide sequence 
encoding GD domain peptides may occur as a result of, for example, allelic 
polymorphisms, minor sequencing errors, and the like. The polynucleotides 
encoding GD domain peptides of the invention may include short 
oligonucleotides which are useful, for example, as hybridization probes 
and PCR primers. The polynucleotide sequences of the invention also may 
comprise a portion of a larger polynucleotide and, through polynucleotide 
linkage, they may be fused, in frame, with one or more polynucleotide 
sequences encoding different proteins. In this event, the expressed 
protein may comprise a fusion protein. Of course, the polynucleotide 
sequences of the invention may be used in the PCR method to detect the 
presence of mRNA encoding GD domain peptides in the diagnosis of disease 
or in forensic analysis. 
cDNAs encoding proteins which interact with the GD domain (or proteins 
containing the GD domain) can be identified by screening cDNA expression 
libraries, employing known methods. Examples of such methods include the 
yeast two-hybrid system (U.S. Pat. No. 5,283,173, inventors Fields and 
Song, issued Feb. 1, 1994; Chien, et al., Proc. Natl. Acad. Sci. 88: 9578 
(1991), and the E. coli/BCCP interactive screening system (Guarente, L., 
Proc. Natl. Acad. Sci. 90: 1639 (1993) and Germino, et al., Proc. Natl. 
Acad. Sci. 90: 933-937 (1993)). Suitable cDNA libraries will include 
mammalian cDNA libraries, such as human, mouse or rat, which may contain 
cDNA produced from RNA and a single cell, tissue or organ type or 
developmental stage, as are know in the art. 
A nucleotide sequence encoding a protein or peptide comprising the GD 
domain may be inserted into a DNA vector in accordance with conventional 
techniques, including blunt-ending or staggered-ending termini for 
ligation, restriction enzyme digestion to provide appropriate termini, 
filling in of cohesive ends as appropriate, alkaline phosphatase treatment 
to avoid undesirable joining, and ligation with appropriate ligases. 
Techniques for such manipulations are disclosed, for example, by 
Sarabrook, J., et al., Molecular Cloning: A Laboratory Manual, 2d Ed., 
Cold Spring Harbor Laboratory Press, Planview, N.Y. (1989), and are well 
known in the art. 
The sequence of amino acid residues in a protein or peptide comprising the 
GD domain is designated herein either through the use of their commonly 
employed three-letter designations or by their single-letter designations. 
A listing of these three-letter and one-letter designations may be found 
in textbooks such as Biochemistry, Second Edition, Lehninger, A., Worth 
Publishers, New York, N.Y. (1975). When the amino acid sequence is listed 
horizontally, the amino terminus is intended to be on the left end whereas 
the carboxy terminus is intended to be at the right end. The residues of 
amino acids in a peptide may be separated by hyphens. Such hyphens are 
intended solely to facilitate the presentation of a sequence. 
The rational design of GD domain mimetics or binding molecules, based on 
modeled (or experimentally determined) peptide structure, may be carried 
out by those of skill, using known methods of rational drug design. 
Therapeutic or prophylactic methods for treating pathological conditions 
such as autoimmune disease, neurodegenerative disease, cancer and the 
like, are accomplished by the administration of an effective amount of a 
therapeutic agent capable of specifically inhibiting GD domain 
homodimerization or heterodimerization, thereby modulating the biological 
activity of GD domain containing proteins and the apoptotic state in a 
patient. 
Truncated Bak molecules comprising the GD domain, such as QVG or PEM, as 
well as other small peptide derivatives that constitute a "minimal" GD 
domain, are demonstrated herein to retain the protein binding and cell 
killing function exhibited by wild-type Bak. These molecules, or 
peptidomimetic derivatives, may induce apoptosis in tumor cells by 
providing the same biological signal produced by high level expression of 
Bak (which has been shown to kill tumor cells in an in vitro assay). Such 
agents comprise a novel class of chemotherapeutic drug that would be 
predicted to operate independently of p53 status. 
If interaction with Bak results in the suppression of the anti-apoptotic 
function of Bcl-x.sub.L and/or other Bcl-2 family members, then GD domain 
peptides, or agents that mimic the GD domain structure, may act as 
inhibitors of the anti-apoptotic function of proteins like Bcl-2. High 
level Bcl-2 expression has been implicated in the resistance of tumor 
cells to a variety chemotherapy drugs (Fisher et al., Cancer Res. 53: 
3321-3326 (1993); Miyashita and Reed, Blood 81: 151-157 (1993); Dole et 
al., Cancer Res. 54: 3253-3259 (1994). Administration of GD domain 
mimetics may suppress Bcl-2 function and restore sensitivity of tumor 
cells to apoptosis induced by traditional chemotherapeutic agents. In 
addition, Bak or GD domain mimetics that inhibit Bcl-2 may themselves be 
selectively toxic to certain tumors, such as follicular lymphoma, that 
depend upon high level Bcl-2 activity for their continued growth and 
survival. 
The GD domain mimetics of the invention may also have utility in combating 
vital infections. Apoptosis of infected cells, with associated DNA 
fragmentation, provides an important defense against viral pathogenesis by 
limiting vital titers and restricting viral propagation (Vaux et al., Cell 
76: 777-779 (1994). For this reason, viruses have evolved diverse 
mechanisms to suppress apoptosis of infected host cells. Certain viral 
proteins, such as Epstein-Barr virus BHRF-1, African Swine Fever Virus 
(ASFV) LHW5-HL, and Adenovirus E1B 19kD, appear to be structural or 
functional homologues of Bcl-2. A second Epstein-Barr virus gene, LMP1, 
transactivates the expression of the cellular bcl-2 gene in latently 
infected cells (Henderson et al., Cell 65: 1107-1115 (1991). In these 
cases, the apoptotic signal triggered by viral infection may be held in 
check by the action of a vital (or cellular) Bcl-2 homolog. A Bak GD 
domain mimetic that opposes the anti-apoptotic function of the 
viral/cellular Bcl-2 homolog would serve to alleviate this block and 
induce apoptosis in infected cells and consequently inhibit vital 
propagation. Anti-apoptotic proteins encoded by at least two unrelated 
viruses (EBV BHRF1 and Adenovirus E1B 19kD) have been demonstrated to 
interact with Bak. Experimental evidence supports the conclusion that 
disrupting the E1B 19kD/Bak interaction (i.e., by competing with a GD 
domain mimetic) would reduce vital titers and productive replication. 
Mutations in E1B 19kD that disrupt the interaction with Bak 
correspondingly abolish the anti-apoptotic function of E1B 19kD. 
Adenovirus strains encoding defective E1B 19kD proteins yield much lower 
progeny virus in vitro, due to apoptosis of infected cells (Pilder et al., 
J. Virol. 52: 664-671 (1984); Subramanian et al., J. Biol. Chem. 259: 
11777-11783 (1984). 
An additional mechanism whereby viruses impose a blockade on the apoptosis 
signal transduction pathway is through the inactivation of the p53 tumor 
suppressor protein. Forced cellular proliferation caused by viral 
infection induces an apoptotic signal that requires p53 function (see 
e.g., Wu and Levine, Proc. Natl. Acad. Sci. USA 91: 3602-3606 (1994). 
Typically, p53 function is abrogated during infection by physical 
interaction with a viral gene product. Examples of viruses that encode p53 
binding proteins include adenoviruses, polyoma viruses, papilloma viruses, 
and cytomegalovirus (Levine et al., Nature 351: 453-456 (1991); Speir et 
al., Science 265: 391-394 (1994). Infected cells are "primed" to undergo 
apoptosis, but cell death is prevented or delayed by viral inhibition of 
p53 function. It is possible that this blockade in the apoptosis signal 
transduction pathway could be relieved, or bypassed, by an agent that 
modulates apoptosis downstream of p53. Bak, or GD domain mimetics, induce 
apoptosis independently of p53, and consequently provide a way to 
implement or restore the cell death signal that is suppressed in infected 
cells. 
Any mode of administration which results in the delivery of the therapeutic 
agent across the cell membrane and into the desired cell is contemplated 
as within the scope of the present invention. The site of administration 
and cells will be selected by one of ordinary skill in the art based upon 
an understanding of the particular disorder being treated. In addition, 
the dosage, dosage frequency, and length of course of treatment, can be 
determined and optimized by one of ordinary skill in the art depending 
upon the particular degenerative disorder being treated. The particular 
mode of administration can also be readily selected by one of ordinary 
skill in the art and can include, for example, oral, intravenous, 
subcutaneous, intramuscular, etc., with the requirement that the 
therapeutic agent cross the cell membrane. Principles of pharmaceutical 
dosage and drug delivery are known and are described, for example, in 
Ansel, H. C. and Popovich, N. G., Pharmaceutical Dosage Forms and Drug 
Delivery Systems, 5th Edition, Lea & Febiger, Publisher, Philadelphia, Pa. 
(1990). It is possible, for example, to utilize liposomes to specifically 
deliver the agents of the invention. Such liposomes can be produced so 
that they contain additional bioactive compounds and the like such as 
drugs, radioisotopes, antibodies, lectins and toxins, which would act at 
the target site. 
Suitable agents for use according to the invention include GD domain 
peptides and mimetics, fragments, functional equivalents and/or hybrids or 
mutants thereof, as well as vectors containing cDNA encoding any of the 
foregoing. Agents can be administered alone or in combination with and/or 
concurrently with other suitable drugs and/or courses of therapy. 
The agents of the present invention are suitable for the treatment of 
degenerative disorders, including disorders characterized by inappropriate 
cell proliferation or inappropriate cell death or in some cases, both. 
Inappropriate cell proliferation will include the statistically 
significant increase in cell number as compared to the proliferation of 
that particular cell type in the normal population. Also included are 
disorders whereby a cell is present and/or persists in an inappropriate 
location, e.g., the presence of fibroblasts in lung tissue after acute 
lung injury. For example, such cells include cancer cells which exhibit 
the properties of invasion and metastasis and are highly anaplastic. Such 
cells include but are not limited to, cancer cells including, for example, 
tumor cells. Inappropriate cell death will include a statistically 
significant decrease in cell number as compared to the presence of that 
particular cell type in the normal population. Such underrepresentation 
may be due to a particular degenerative disorder, includinG, for example, 
AIDS (HIV), which results in the inappropriate death of T-cells, and 
autoimmune diseases which are characterized by inappropriate cell death. 
Autoimmune diseases are disorders caused by an immune response directed 
against self antigens. Such diseases are characterized by the presence of 
circulating autoantibodies or cell-mediated immunity against autoantigens 
in conjunction with inflammatory lesions caused by immunologically 
competent cells or immune complexes in tissues containing the 
autoantigens. Such diseases include systemic lupus erythematosus (SLE), 
rheumatoid arthritis. 
Standard reference works setting forth the heneral principles of immunology 
include Stites, D. P., and Terr, A. I., Basic and Clinical Immunology, 7th 
Ed., Appleton & Lange, Publisher, Norwalk, Conn. (1991); and Abbas, A. K., 
et al., Cellular and Molecular Immunology, W. B. Saunders Co., Publisher, 
Philadelphia, Pa. (1991). 
The GD domain peptides, mimetics, agents and the like disclosed herein, as 
well as vectors comprising nucleotide sequences encoding them or their 
corresponding antisense sequences, and hosts comprising such vectors, may 
be used in the manufacture of medicaments for the treatment of diseases. 
Cells and non-human transgenic animals having one or more functionally 
impaired alleles encoding a protein comprising the GD domain may be 
generated using homologous targeting constructs from genomic clones of 
proteins comprising the GD domain. Methods for the production of 
homologous targeting constructs are known and described, for example, in 
Bradley, et al., Bio/Technology 10: 534 (1992); and Koh, et al., Science 
256: 1210 (1992). For example, "knock-out" mice may be generated which are 
homozygous or heterozygous for an inactivated allele of a protein 
comprising the GD domain byuse of homologous targeting. Such mice are 
useful as research subjects for the investigation of disease and for other 
uses. Methods of producing chimeric targeted mice are known and are 
described, for example, in Robertson, E. J., Ed., Teratocarcinomas and 
Embryonic Stem Cells: A Practical Approach, IRL Press, Washington, D.C. 
(1987), which also describes the manipulation of embryonic stem cells. In 
addition, transgenes for expressing polypeptides comprising the GD domain 
at high levels or under the control of selected transcription control 
sequences may be constructed using the cDNA or genomic gene of a protein 
comprising the GD domain. Transgenes so constructed can be introduced into 
cells and transgenic non-human animals by known methods. Such transgenic 
cells and transgenic non-human animals may be used as screens for agents 
which modulate apoptosis. 
The invention may be appreciated in certain aspects with reference to the 
following examples, offered by way of illustration, not by way of 
limitation. 
EXAMPLES 
A. Methods 
1. Plasmids and DNA Manipulations. 
All recombinant DNA procedures were performed by standard methods. 
Deletions in the bak cDNA were introduced by PCR mutagenesis, and 
truncated Bak species were constructedby PCR (White, B. A., Ed., "PCR 
Protocols: Current Methods and Applications," in, Methods in Molecular 
Biology, Humana Press, Totowa, Conn. (1993). The mutations were confirmed 
by DNA sequence analysis. All Bak derivatives were tagged at the 
amino-terminus with influenza virus hemagglutinin epitope, and expressed 
from the CMV enhancer promoter present in pcDNA-1/Amp, pRcCMV, and pcDNA-3 
(Invitrogen, Inc.). 
2. Transient Transfection Assay. 
The transient transfection assay procedure is similar to that previously 
described for detecting apoptosis induced by IL-1.beta.-converting enzyme 
(Miura et al., Cell 75: 653-660 (1993); Kumar et al., Genes Dev. 8: 
1613-1626 (1994); Wang et al., Cell 78: 739-750 (1994). One day prior to 
transfection, Rat-1 cells were plated in 24 well dishes at 
3.5.times.10.sup.4 cells/well. The following day, the cells were 
transfected with a marker plasmid encoding .beta.-galactosidase (0.16 
.mu.g), in combination with an expression plasmid encoding Bak (0.42 82 
g), by the Lipofectamine procedure (Gibco/BRL). At 24 hours post 
transfection, cells were fixed and stained with X-Gal to detect 
.beta.-galactosidase expression in cells that received plasmid DNA (Miura 
et al., supra). The number of blue cells was counted by microscopic 
examination and scored as either live (flat blue cells) or dead (round 
blue cells). The cell killing activity of Bak in this assay is manifested 
by a large reduction in the number of blue cells obtained relative to 
co-transfection of the .beta.-gal plasmid with a control expression vector 
(i.e., with no bak cDNA insert). 
The interpretation that loss of blue cells reflects the cell killing 
function of Bak is supported by a variety of observations: 
1. Rat-1 cells are rapidly killed by enforced Bak expression in stable 
transfection assays; 
2. Control expression plasmids harboring the bak cDNA in the anti-sense 
orientation, or various unrelated cDNAs, do not eliminate .beta.-gal 
positive cells. In addition, certain Bak mutants (i.e., .DELTA.GD) have 
greatly diminished capacity to eliminate blue cells in this assay; 
3. IL-.beta.-converting enzyme, previously shown to induce apoptosis in 
Rat-1 cells (Miura et al., supra; Kumar et al., supra; Wang et al., 
supra), also eliminates blue cells in this assay when expressed from the 
same vector; 
4. The number of blue cells can be partially restored by co-transfection of 
Bak with Bcl-x.sub.L. Thus, Bak expressing cells can be rescued to some 
degree by the anti-apoptotic function of Bcl-x.sub.L, and Bak expression 
per se does not eliminate .beta.-galactosidase activity. 
3. Detection of Protein/Protein Interactions in Vitro. 
GST and GST-Bcl-x.sub.L were expressed in E. coli and purified by affinity 
chromatography using glutathione-agarose (Smith and Johnson, Gene 67: 
31-40 (1988)). .sup.35 S-Methionine-labeled proteins were expressed in 
vitro using a coupled transcription/translation system in rabbit 
reticulocyte lysates as described by the supplier (Promega). .sup.35 
S-met-labeled proteins were precleared by mixing with 20 ml BSA-washed 
GSH-agarose beads (50% slurry) at 4.degree. C. for 1 hour in 0.1 ml 10 mM 
Hepes buffer, pH 7.2 containing 0 25% NP-40, 142.5 mM NaCl, 5 mM 
MgCl.sub.2, and 1 mM EGTA (NP-40 lysis buffer). The beads were removed by 
centrifugation and the supernatants were incubated with GST or 
GST-Bcl-x.sub.L (final concentration 1 mM) at 4.degree. C. for 1 hour. The 
GST fusion proteins and any interacting proteins were recovered by 
incubation for 1 hour with an additional 20 ml of GSH-agarose beads. The 
beads were washed twice with NP-40 lysis buffer followed by two washes 
with NP-40 lysis buffer without NP-40. Proteins were eluted from the beads 
by incubation in SDS-PAGE sample buffer at 100.degree. C. for 5 min and 
loaded onto 4-20% SDS-polyacrylamide gels. Following electrophoresis, gels 
were fixed and incubated in a fluorography enhancing solution (Amplify; 
Amersham). The gels were dried and subjected to autoradiography at 
-70.degree. C. 
4. Detection of Protein/Protein interactions in Transfected Cells. 
COS cells were grown in Dulbecco's modified Eagle's medium (Life 
Technologies, Inc.) supplemented with 10% bovine calf serum, 2% 
L-glutamine and 1% pen/strep (Life Technologies, Inc.). Cells were seeded 
at 2.0.times.10.sup.5 cells/ 35 mm well and transfected with expression 
plasmids 24 hours later using Lipofectamine as described by the supplier 
(Life Technologies, Inc.). Bak (and Bak mutants) was expressed as a fusion 
protein with the HA epitope tag at its amino terminus. Bcl-x.sub.L was 
also expressed with an amino terminal epitope tag (Flag; Kodak). At 24 
hours post-transfection, cells were washed with phosphate buffered saline 
and lysed in NP-40 Lysis buffer also containing 1 mM PMSF, 1 mM pepstatin, 
and 1 mg/ml leupeptin. The lysates were incubated with anti-HA antibody 
(12CA5, Boehringer Mannheim) for 1 hour and with 20 ml BSA-washed Protein 
A-agarose beads (50% slurry) for an additional hour. The beads were washed 
twice with NP-40 lysis buffer followed by two washes with NP-40 lysis 
buffer without NP-40. Proteins were eluted from the beads by incubation in 
SDS-PAGE sample buffer at 100.degree. C. for 5 min and loaded onto 4-20% 
SDS-polyacrylamide gels. Following electrophoresis, proteins were 
transferred to Immobilon-P membranes (Millipore) and the membranes were 
blocked by incubation for 1 hour with a 1% milk solution in PBS. Primary 
antibody (1 mg/ml 12CA5, Boehringer Mannheim; 1:500 DAKO-bcl-2, 124, DAKO; 
10 mg/ml Anti-FLAG M2, Kodak) was incubated with the membranes for 1 hour, 
followed by secondary antibody (0.8 mg/ml HRP-conjugated goat anti-mouse 
IgG; Jackson Laboratory) for an additional 1 hour. Detection was by 
enhanced chemiluminesence (ECL; Amersham) as described by the supplier 
using X-OMAT AR film (Kodak). 
B. Results 
1. Detection of the Cell Death Function of Bak in Multiple Cell Lines. 
Enforced bak expression induces apoptosis in stable Rat-1 cell lines 
transfected with an inducible bak expression plasmid. In order to more 
rapidly assess the cell killing function of a large number of bak mutants, 
a transient transfection assay was employed. Rat-1 cells were transfected 
with a marker plasmid encoding .beta.-galactosidase, in combination with 
an expression plasmid encoding Bak, or various control plasmids. Cell 
killing activity of Bak in this assay was manifested by a large reduction 
in the number of blue (.beta.-gal expressing) cells obtained relative to 
co-transfection of the .beta.-gal plasmid with a control expression vector 
(FIG. 1). The elimination of blue cells indicated that transfected cells 
were killed by bak prior to expressing detectable levels of 
.beta.-galactosidase. 
Bak cell killing activity was assessed in several additional cell lines. To 
determine whether Bak requires wild-type p53 to induce apoptosis, a 
transient transfection experiment was performed in transformed fibroblasts 
derived from a p53-/- "knockout" mouse. These cells lack functional p53 
and are greatly impaired in their ability to undergo apoptosis in response 
to g-irradiation and DNA-damaging chemotherapeutic drugs (Lowe et al., 
Cell 74: 957-967 (1993); Lowe et al., Nature 362: 847-849 (1993)). 
Co-transfection of Bak with .beta.-gal greatly reduced the number of blue 
cells (FIG. 1) indicating that Bak does not require wild-type p53 to exert 
its cell killing function. Similarly, transient transfection experiments 
performed in the Hela (cervical carcinoma) and BT549 (breast carcinoma) 
cell lines demonstrated that Bak can kill human tumor cells in this 
context (FIG. 1) indicating that its activity is not restricted to rodent 
fibroblasts. 
2. Identification of Bak Domains Required for Cell Killing Function. 
A mutational analysis of Bak was undertaken in order to identify regions of 
the molecule that are necessary and/or sufficient to induce apoptosis. A 
series of deletion mutations spanning the entire Bak protein was 
introduced by PCR mutagenesis and each mutant was tested for cell killing 
activity in a Rat-1 cell transient transfection assay. This analysis 
revealed that much of the Bak molecule is dispensable for its cell death 
function detected by this assay (FIG. 2). Surprisingly, the non-essential 
regions of the Bak protein include the two domains in the carboxyl 
terminal half of the protein that show the highest degree of homology to 
other Bcl-2 family members (Bcl-2 homology domains I and II). 
Deletion of the carboxyl-terminal hydrophobic stretch of amino acids 
(residues 191-211) partially diminished, but did not eliminate, the cell 
killing function of Bak (mutant .DELTA.C). This hydrophobic "tail" likely 
serves as a membrane anchor sequence in Bak. Indeed, immunofluorescence 
studies of .DELTA.C in transiently transfected COS cells showed that the 
intracellular distribution of the AC mutant is altered (diffuse 
cytoplasmic) relative to the wild type Bak, which appears largely 
mitochondrial. The carboxyl terminal hydrophobic tail is not required for 
the cell killing function of Bak, but may contribute indirectly, by 
ensuring proper sub-cellular localization of the protein. 
A segment of the Bak protein encompassed by the .DELTA.GD deletion 
(residues 82-94) is absolutely required for cell death function since this 
mutant is devoid of cell killing activity in the transient transfection 
assay. Specifically, co-transfection of .beta.-gal with Bak AGD yielded as 
many, or more, blue cells relative to co-transfection of .beta.-gal with 
the control vector plasmid. Deletion of adjoining residues (amino acids 
67-81) immediately N-terminal to this domain reduced, but did not 
eliminate, cell death activity (Bak mutant .DELTA.PS). All other deletion 
mutants tested (with the exception of .DELTA.C, discussed above) were 
unaltered in their capacity to kill cells. Taken together, these results 
indicate that a co-linear segment (termed the "GD domain") defined by 
deletion mutants .DELTA.GD and .DELTA.PS (residues 67-94) is uniquely 
required for Bak cell killing function detected in the transient assay. 
To determine if the GD domain is sufficient for cell killing function, two 
truncated Bak protein derivatives, PEM and QVG, corresponding to amino 
acids 58-103 and 73-123, respectively, were tested for activity in the 
transient transfection assay. QVG significantly reduced the number of blue 
cells when co-transfected with .beta.-gal, indicating that it retained 
some capacity to kill Rat-1 cells. While the reduction in blue cell number 
was diminished relative to full length Bak, both PEM and QVG lack the 
carboxyl-terminal membrane anchor and, by analogy to the Bak .DELTA.C 
mutant, would likely not exhibit full cell killing function due to altered 
sub-cellular localization. Indeed, QVG was similar to the Bak .DELTA.C 
mutant with respect to its activity. In an effort to improve the cell 
killing capacity of the truncated Bak species, the hydrophobic tail 
element (amino acids 187-211) was fused to the C-termini of both PEM and 
QVG (PEM+C and QVG+C, respectively). In each case, attachment of the 
putative membrane anchor improved the ability of the truncated Bak mutants 
to eliminate blue cells in the transfection assay, and resulted in 
activity comparable to wild-type Bak (FIG. 2). Thus, these results 
indicate that a protein domain shared by both PEM and QVG (residues 
73-103) is sufficient for the cell killing function of Bak. 
3. Identification of Bak Domains That Mediate the Interaction with 
Bcl-x.sub.L 
Physical interaction with other Bcl-2 family members, such as Bcl-x.sub.L, 
may be essential for Bak to exert its cell death function or may regulate 
Bak activity. Therefore, domains within Bak were examined to determine 
which are necessary and/or sufficient for its Bcl-x.sub.L binding 
activity. The interaction of Bak with Bcl-x.sub.L was measured both by an 
in vitro protein binding assay andby co-immunoprecipitation from 
transfected cells. In vitro translated .sup.35 S labeled Bak binds to a 
purified, bacterially expressed GST-Bcl-x.sub.L fusion protein, and the 
specificity of this in vitro interaction was demonstratedby the failure of 
Bak to bind to purified GST alone (FIG. 3A). A specific Bak/Bcl-x.sub.L 
interaction could also be detected by co-transfecting epitope tagged forms 
of Bak and Bcl-x.sub.L into COS cells. Bak was immunoprecipitated from 
transfected cell lysates and associated Bcl-x.sub.L was detected by 
Western blot analysis of co-precipitated proteins (FIG. 3B). Bcl-x.sub.L 
was not detected in immunoprecipitates in the absence of co-expressed Bak, 
demonstrating that binding is specific. 
The Bak deletion mutants described above were tested for their Bcl-x.sub.L 
binding capacity, both in vitro and in transfected COS cells, and the 
results are summarized in FIG. 4. Deletion of residues 82-94 (.DELTA.GD 
mutant) completely eliminated the ability of Bak to interact with 
Bcl-x.sub.L. Interaction with Bcl-x.sub.L was also diminished by deletion 
of adjoining amino acids 67-81 (.DELTA.PS Bak mutant). All other deletion 
mutants tested, encompassing the entire Bak open reading frame, retained 
the ability to bind Bcl-x.sub.L in these assays. These results identify 
Bak sequences encompassed by the .DELTA.GD and .DELTA.PS mutants 
(maximally, amino acids 67-94) as uniquely important in mediating the 
interaction with Bcl-x.sub.L. The same Bak region, the GD domain, was 
required for the cell killing function of Bak. 
To determine whether the Bak region defined by deletion analysis is 
sufficient for protein binding function, two small truncated Bak species 
(PEM and QVG), encompassing amino acids 58-103 and 73-123 respectively, 
were tested for their ability to interact with Bcl-x.sub.L. Both PEM and 
QVG bound Bcl-x.sub.L, indicating that the region shared by both of these 
truncated Bak species (amino acids 73-103) was sufficient for mediating 
the interaction with Bcl-x.sub.L. Together with the analysis of the 
deletion mutants and truncated species described above, these results 
demonstrate that Bak amino acid sequences spanning residues 73-103 are 
both necessary and sufficient for interaction with Bcl-xL. As described 
above, this region is also implicated in the cell killing function of Bak, 
indicating that protein binding function may linked to cell killing 
function. 
4. Functionally Significant Sequence Elements Resembling the GD Domain Are 
Present in Bax and Bip1la. 
The mutational analysis of Bak described herein demonstrates that the GD 
domain is uniquely involved in both the cell killing and Bcl-x.sub.L 
binding activities of Bak. Two other Bcl-2 interacting proteins, Bax and 
Bip1a, have functional properties that resemble those of Bak. Both Bax and 
Bip1a eliminate blue cells when co-transfected with .beta.-gal in Rat-1 
cells, indicating that they also induce apoptosis in this context. Bax and 
Bip1a also interact specifically with Bcl-x.sub.L, both in vitro and in 
transfected COS cells. These functional similarities prompted the 
examination of whether any structural features are shared by the three 
proteins that contribute to their similar biological functions. 
Specifically, in light of the analysis presented above, Bax and Bip1a were 
examined to determine whether they contain sequences that resemble the Bak 
GD domain and are also important for their biological activities. 
Bax shows extensive homology to Bcl-2 family members (including Bak), with 
the highest degree of sequence homology centered around BH1 and BH2 
(Oltvai et al., Cell 74: 609-619 (1993)). A stretch of amino acids (59-73) 
N-terminal to BH1 in Bax bears homology to sequences (residues 74-88) 
within the GD domain of Bak (FIG. 5). In contrast to Bax, the primary 
sequence of Bip1a does not resemble the known Bcl-2 relatives, and lacks 
sequences homologous to BH1 and BH2 that are characteristic of the Bcl-2 
family. However, Bip1a contains a region (amino acids 57-71) that is 
homologous to the same element within the GD domain in Bak and Bax (FIG. 
5). 
GD domain elements within Bax and Bip1a were evaluated to determine whether 
they are also critical to the cell killing and protein binding functions 
of these proteins. Small deletions that removed the conserved GD domain 
motifs were introduced into Bax and Bip1a, and the mutants were then 
analyzed for their ability to kill Rat-1 cells and bind to Bcl-x.sub.L. 
This analysis revealed that, like Bak .DELTA.GD, the Bax .DELTA.GD and 
Bip1a .DELTA.GD mutants are impaired in their ability to eliminate blue 
cells when co-transfected with .beta.-Gal in Rat-1 cells (FIG. 6). In 
addition, both mutants no longer have the capacity to interact with 
Bcl-x.sub.L (FIG. 6). Thus, function of the GD domain element is conserved 
in Bak, Bax and Bip1a, and is critical to the biological activities of all 
three proteins. 
5. The GD Domain is Sufficient for Homo- and Heterodimer Formation. 
In order to assess whether the GD domain mediates other protein/protein 
interactions which could be relevant to its biological activity, a portion 
of Bak (PEM) encompassing the GD domain (residues 58-103) was fused to 
GST, to create GST-PEM. In vitro translated, .sup.35 S labeled 
Bcl-x.sub.L, Bak, Bax and Bip1a were incubated with either GST alone, or 
GST-PEM bacterially-expressed fusion protein. Interactions of the GD 
domain with Bak and Bax were measured essentially as described herein for 
Bak binding to Bcl-x.sub.L. Complexes were captured with 
glutathione-agarose beads, washed, and bound proteins detected by 
polyacrylamide gel electrophoresis and autoradiography. 
The results of this experiment are shown in FIG. 6. Bcl-x.sub.L, Bak, and 
Bax all interact specifically with GST-PEM, but not with GST alone. These 
results demonstrate that the Bak GD domain is sufficient to bind to Bak 
(homodimerization), Bax (heterodimerization with a different killer 
protein) and Bcl-x.sub.L (heterodimerization with a survival protein). 
Thus, the GD domain is capable of mediating interactions not only with 
Bcl-x.sub.L, but also Bak and Bax. It does not interact with Bip1a. 
All publications mentioned in this specification are herein incorporated by 
reference, to the same extent as if each individual publication was 
specifically and individually indicated to be incorporated by reference. 
It will be understood that the invention is capable of further 
modifications and this application is intended to cover any variations, 
uses, or adoptions of the invention including such departures from the 
present disclosure as come within known or customary practice within the 
art to which the invention pertains, and is intended to be limited only by 
the appended claims. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 34 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acid 
(B) TYPE: amino acid 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GlyAspAspIleAsnArgArgTyrAspSerGluPheGln 
510 
(2) INFORMATION FOR SEQ ID NO:2: 
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(A) LENGTH: 28 amino acid 
(B) TYPE: amino acid 
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(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ProSerSerThrMetGlyGlnValGlyArgGlnLeuAlaIleIleGly 
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AspAspIleAsnArgArgTyrAspSerGluPheGln 
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(2) INFORMATION FOR SEQ ID NO:3: 
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(A) LENGTH: 31 amino acid 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GlnValGlyArgGlnLeuAlaIleIleGlyAspAspIleAsnArgArg 
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TyrAspSerGluPheGlnThrMetLeuGlnHisLeuGlnProThr 
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(2) INFORMATION FOR SEQ ID NO:4: 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
LeuSerGluCysLeuLysArgIleGlyAspGluLeuAspSerAsn 
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(2) INFORMATION FOR SEQ ID NO:5: 
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(A) LENGTH: 9 amino acids 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
LeuLysArgIleGlyAspGluLeuAsp 
(2) INFORMATION FOR SEQ ID NO:6: 
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GlnAspAlaSerThrLysLysLeuSerGluCysLeuLysArgIleGly 
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AspGluLeuAspSerAsnMetGluLeuGln 
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(2) INFORMATION FOR SEQ ID NO:7: 
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(A) LENGTH: 15 amino acids 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
LeuAlaLeuArgLeuAlaCysIleGlyAspGluMetAspValSer 
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(2) INFORMATION FOR SEQ ID NO:8: 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
IleGlyAspGluMet 
5 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
CysMetGluGlySerAspAlaLeuAlaLeuAspLeuAlaCysIleGly 
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AspGluMetAspValSerLeuArgAlaProArgLeu 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ValGlyArgGlnLeuAlaIleIleGlyAspAspIleAsnArgArg 
51015 
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AlaAlaProAlaAspProGluMetValThrLeuProLeuVal 
510 
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IleGlyAspGluMetAspValSerLeuArgAlaProArgLeuAlaGln 
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LeuSerGluVal 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
ValProGlnAspAlaSerThrLysLysLeuSerGluCysLeuLysArg 
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IleGlyAspGluLeuAspSerAsnMetGluLeuGlnArgMetIleAla 
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AlaVal 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
LeuGlnProSerSerThrMetGlyGlnValGlyArgGlnLeuAlaIle 
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IleGlyAspAspIleAsnArgArgTyrAspSerGluPheGlnThrMet 
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LeuGlnHisLeu 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
CAGGTGGGACGGCAGCTCGCCATCATCGGGGACGACATCAACCGACGCTATGACTCAGAG60 
TTCCAGACCATGTTGCAGCACCTGCAGCCCACG93 
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GlnValGlyArgGlnLeuAlaIleIleGlyAspAspIleAsnArgArg 
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TyrAspSerGluPheGlnThrMetLeuGlnHisLeuGlnProThr 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CCTAGCAGCACCATGGGGCAGGTGGGACGGCAGCTCGCCATCATCGGGGACGACATCAAC60 
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ProSerSerThrMetGlyGlnValGlyArgGlnLeuAlaIleIleGly 
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AspAspIleAsnArgArgTyrAspSerGluPheGln 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
ValGlyArgGlnLeuAlaIleIleGlyAspAspIleAsnArgArg 
51015 
(2) INFORMATION FOR SEQ ID NO:21: 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GGGGACGACATCAACCGACGCTATGACTCAGAGTTCCAG39 
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(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
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(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
GlyAspAspIleAsnArgArgTyrAspSerGluPheGln 
510 
(2) INFORMATION FOR SEQ ID NO:23: 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
CAGGATGCGTCCACCAAGAAGCTGAGCGAGTGTCTCAAGCGCATCGGGGACGAACTGGAC60 
AGTAACATGGAGCTGCAG78 
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(A) LENGTH: 26 amino acids 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
GlnAspAlaSerThrLysLysLeuSerGluCysLeuLysArgIleGly 
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AspGluLeuAspSerAsnMetGluLeuGln 
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(A) LENGTH: 45 base pairs 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
CTGAGCGAGTGTCTCAAGCGCATCGGGGACGAACTGGACAGTAAC45 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
LeuSerGluCysLeuLysArgIleGlyAspGluLeuAspSerAsn 
51015 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
CTCAAGCGCATCGGGGACGAACTGGAC27 
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(A) LENGTH: 9 amino acids 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
LeuLysArgIleGlyAspGluLeuAsp 
5 
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(A) LENGTH: 84 base pairs 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
TGCATGGAGGGCAGTGACGCATTGGCCCTGCGGCTGGCCTGCATCGGGGACGAGATGGAC60 
GTGAGCCTGAGGGCCCCGCGCCTG84 
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(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 amino acids 
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(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
CysMetGluGlySerAspAlaLeuAlaLeuArgLeuAlaCysIleGly 
51015 
AspGluMetAspValSerLeuArgAlaProArgLeu 
2025 
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(A) LENGTH: 45 base pairs 
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(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
TTGGCCCTGCGGCTGGCCTGCATCGGGGACGAGATGGACGTGAGC45 
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(A) LENGTH: 15 amino acids 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
LeuAlaLeuArgLeuAlaCysIleGlyAspGluMetAspValSer 
51015 
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(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
ATCGGGGACGAGATG15 
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(A) LENGTH: 5 amino acids 
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(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
IleGlyAspGluMet 
5 
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