DETAILED DESCRIPTION

[0039] The invention provides polypeptides that “modulate apoptosis,” proteins that increase or induce apoptosis or decrease or prevent programmed cell death, including exemplary Bcl-B ( FIG. 1A, e .g., SEQ ID NO:2). The invention also provides nucleic acids encoding polypeptides that “modulate apoptosis,” including exemplary Bcl-B (Example 2, e.g., SEQ ID NO:1). The invention further provides antibodies that bind to Bcl-B including antibodies that increase an activity of Bcl-B (i.e., an agonistic antibody) and antibodies that decrease an activity of Bcl-B (i.e., an antagonistic antibody). Also provided are polynucleotides (e.g., oligonucleotide probes) of various lengths that hybridize to Bcl-B set forth in SEQ ID NO:1. Methods for making these compositions are also provided. Kits containing invention compositions in a container, including instructions for use (e.g., written on a hard copy or a computer readable medium), for example, for modulating apoptosis, for detecting Bcl-B activity, expression or binding, or identifying an agent that modulates Bcl-B activity, expression or binding are provided.

[0040] Invention compositions have various activities including the ability to modulate apoptosis. Thus, invention compositions are useful as therapeutic agents for increasing or decreasing apoptosis. For example, Bcl-B protein, having wild type function or dominant negative activity, can be expressed in a cell, tissue or organ in order to modulate apoptosis. Antisense and antisense like molecules such as ribozymes, DNAzymes or RNAi (RNA interference) molecules (collectively referred to herein as “antisense”) that inhibit expression of Bcl-B can reduce the level of Bcl-B gene expression, thus reducing the level of Bcl-B activity. Accordingly, the compositions can be used to manipulate apoptotic (“cell death”) mechanisms in a variety of cell types, including insect, plant and mammalian, such as human, cells, and organisms. The invention therefore provides methods for increasing or decreasing apoptosis in a cell, tissue or organ in vitro, ex vivo or in vivo (e.g., in a subject in need of increased or decreased apoptosis).

[0041] Invention compositions also are useful as diagnostic markers. In particular, Bcl-B modulates apoptosis and, therefore, may indicate normal or altered apoptosis. For example, greater than normal levels of Bcl-B in a cell, tissue or organ may characterize the cell, tissue or organ as proliferative or hyperproliferative. In contrast, less than normal levels of Bcl-B in a cell, tissue or organ B may characterize the cell, tissue or organ as slowly proliferating, non-proliferating, at risk of apoptosis or undergoing apoptosis. Thus, detecting Bcl-B (nucleic acid or protein) or Bcl-B activity or expression can be used to as a diagnostic marker. Detecting Bcl-B activity or expression may also be useful for assessing a subject's risk of developing altered apoptosis or indicate a prognosis for a condition or disorder associated with apoptosis.

[0042] Bcl-B compositions are also useful for identifying agents, including therapeutic agents for treating disorders associated with apoptosis. In a method of the invention, Bcl-B (protein or nucleic acid) is contacted with a test agent and binding of Bcl-B to the agent is determined. Binding of Bcl-B to binding molecules (e.g., Bax, Bcl-2 or Bcl-X _L ) in the presence of a test agent can be used to identify agents that inhibit or promote Bcl-B binding to a binding molecule in another method of the invention, Bcl-B activity or expression is determined in the presence of a test agent in order to identify agents that modulate Bcl-B activity or expression. Agents that modulate Bcl-B activity, expression or binding to a binding molecule are useful for modulating apoptosis. Agents that bind Bcl-B may also be useful as markers to indicate the presence of Bcl-B.

[0043] Additional invention methods are based on other Bcl-B activities, such as the ability of Bcl-B to bind to cellular molecules associated with apoptosis. Invention compositions are therefore also useful as therapeutic targets. For example, in a method of the invention, contacting Bcl-B with a molecule that binds Bcl-B, for example, contacting Bcl-B with Bax, Bcl-2 or Bcl-X _L can be used to modulate a Bcl-B activity, such as apoptosis (increasing or decreasing). Bcl-B can also be targeted with agents that modulate Bcl-B activity or expression (e.g., antisense or ribozymes or DNAzymes or RNAi), or Bcl-B binding to binding molecules.

[0044] Modulating apoptosis in cells with invention compositions allows treatment of physiological conditions associated with undesirable or insufficient apoptosis, or undesirable cell survival, growth or proliferation (e.g., where apoptosis is less than normal or desired), ex vivo or in vivo in a subject. Conditions associated with undesirable apoptosis/cell death or undesirable cell survival/growth/proliferation include cell degenerative and proliferative disorders. Organ and organ systems affected by degenerative disorders include, for example, the nervous system (brain, spinal cord, neurons associated with tissues and organs) skeletal-muscular system (voluntary muscles) and the cardio-respiratory system (heart and blood vessels). Specific examples of neurodegeneration include cell death caused by stroke and head trauma, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jacob's disease (CJD), Huntington disease (HD), Machado-Joseph disease (MJD), ataxias (Spinocerebellar ataxias 1, 2 and 6; SCA-1, -2 and -6), dentatorubropallidoluysian atrophy (DRPLA) and Kennedy's disease. Specific examples of muscle degeneration include muscular dystrophy. Specific examples of cardiac degeneration include ischemia caused by heart trauma e.g., associated with or resulting from a heart attack, or insufficient blood supply, or oxygen. Such disorders are but a few of the specific examples of disorders treatable with a method of the invention.

[0045] Apoptotic disorders are caused by various molecular mechanisms. For example, particular amino acid sequences are known to confer apoptosis, such as polyglutamine repeat sequences (polyGln) and protein sequences within cell receptors, such as the neurotrophin receptor. Caspases are also known to mediate the apoptotic signaling pathway in cells. Thus, conditions treatable with the compositions of the invention include cells, tissue and organs in which such apoptosis inducing or increasing proteins are expressed or active.

[0046] Bcl-B was initially identified using a genetic screen of a human liver library. A TBLASTN search of the human Expressed Sequence Tag (EST) database using the amino-acid sequence of the mouse Boo/Diva as a query identified partial cDNA having homology with Boo. A human EST clone (Accession no. AA098865) was obtained and sequenced in its entirety, revealing an open reading frame (ORF) encompassing the last 151 residues of a protein with homology to Boo (Bcl-B).

[0047] Structurally, Bcl-B is characterized as having four “BH” domains, denoted BH1, BH2, BH3 and BH4. The sequences of the domains for exemplary Bcl-B are BH1, VLSDSPGPTWGRVVTLVTFAG; BH2, AWLQAQGGWDGFCHF; BH3, EAAVLRSAAARLRQI; and BH4, ERTELLLADYLGYCAREPGTP (SEQ ID NOS: 3-6, respectively). These domains are present in other Bcl-2 family member proteins that inhibit apoptosis. Comparisons of this predicted sequence with all known Bcl-2 family members by BLAST search indicated that it is most similar to the murine Bcl-2-family protein Boo (also known as Diva). Exemplary Bcl-B (SEQ ID NO:2) shares 47% amino-acid sequence identity with Boo.

[0048] The BCL-B gene is comprised of two exons interrupted by a ˜2.3 kbp intron. The location of this intron corresponds precisely to the intronic interruption in the coding region of the anti-apoptotic BCL-2 and BCL-X genes (corresponding to the motif GGW{circumflex over ( )}D in BH2). By comparison to BCL-B, the pro-apoptotic genes BAX and BAK have more complicated exon-intron organizations, in which the coding regions of the gene are spread over 5 (Bak) or 6 (Bax) exons. The similar genomic organization of the BCL-2, BCL-XL, and BCL-B genes thus suggests they evolved from a common ancestor and indirectly implies a similar mechanism of action for their encoded proteins.

[0049] In transfection assays performed in four different human tumor cell lines, anti-apoptotic activity of Bcl-B was observed. Bcl-B binds and suppresses apoptosis-induction by Bax, but does not detectably interact with Bak or modulate apoptosis induced by Bak over-expression. However, because Bcl-B is capable of associating with either anti-apoptotic proteins (e.g., Bcl-2 and Bcl-X _L ) or with the pro-apoptotic proteins (e.g., Bax), it is possible that Bcl-B displays different effects on apoptosis, cell proliferation, survival, growth or differentiation depending on cellular context. A similar phenomenon has been reported for some other Bcl-2 family proteins. For example, Bcl-2 reportedly promotes apoptosis in photoreceptor cells of the retina while Bax can suppress cell death in some types of neurons (Chen et al., Proc. Nat/. Acad. Sci. USA 93, 7042-7047 (1996); Middleton et al., Development 122, 695-701 (1996)). Thus, Bcl-B is likely to inhibit cell apoptosis, proliferation, survival, growth or differentiation, in some cellular contexts and promote cell apoptosis proliferation, survival, growth or differentiation in other cellular contexts.

[0050] The Bcl-B transmembrane (TM) domain is important for apoptosis-inhibiting activity, as well as for intracellular localization to mitochondria. Deletion of the domain reduces Bcl-B ability to inhibit apoptosis as well as reduces association with mitochondria. Replacing the native Bcl-B transmembrane domain with other transmembrane domains, including those targeting mitochondria, will likely restore Bcl-B apoptosis modulating activity. Thus, the invention includes Bcl-B polypeptides and subsequences having non-Bcl-B transmembrane domains, for example, from Bcl-2, Bax, Bak, Bcl-X _L , etc.

[0051] The correlation between membrane-targeting and function is reminiscent of some other Bcl-2 family proteins indicating that the site of action of Bcl-B is close to the intracellular organelles, including mitochondria, with which it associates. Though roughly half the Bcl-BΔTM protein was associated with the HM membrane fraction in cells, this may be due to its dimerization with other resident Bcl-2 family proteins. A membrane site of action for Bcl-B would be consistent with evidence that several Bcl-2 family proteins are capable of forming ion channels or pores in membranes. Indeed, molecular modeling of Bcl-B on the structure of Bcl-X _L suggests that it possesses a similar overall fold and that it contains amphipathic α-helices similar to the putative pore-forming α5 and α6 of Bcl-X _L .

[0052] The differences observed in the activity and protein interaction partners of murine Boo and human Bcl-B proteins indicate that Bcl-B does not represent the human orthologue of mouse Boo/Diva. Also consistent with this interpretation is the difference in the expression patterns of Bcl-B and Boo. For example, although Boo (Diva) is expressed predominantly in ovary, testis, and epididymis in adult mice, Bcl-B mRNA is widely expressed in adult human tissues.

[0053] Weak interactions of Bcl-B with Apaf1 in co-immunoprecipitation assays have been detected but functional analysis has yet to reveal an effect of Bcl-B on Apaf1-induced apoptosis. Since Apaf1 is a soluble cytosolic protein, the inability of Bcl-BΔTM to suppress Bax-induced apoptosis suggests that Bcl-B may not play a significant role in suppressing Apaf1. Moreover, the observation that Bcl-B suppresses apoptosis induced by Bax but not Bak also argues against a role for Bcl-B as an Apaf1 suppresser, given that both Bax and Bak induce mitochondrial release of the Apaf1-activator, cytochrome c. However, though Bcl-B may not regulate Apaf1, it is possible that it binds to and suppresses the activity of some other unidentified Apaf1-like, CED4-family protein, analogous to CED9, with which Bcl-B shares 20% amino-acid sequence identity (53% similarity).

[0054] As used herein, the term “apoptosis” or “programmed cell death” means that the cell (tissue or organ) exhibits one or more characteristics associated with programmed cell death. Characteristics include inhibition of cell survival, growth, death or differentiation, protein/nucleic acid cleavage/fragmentation, chromatin condensation, membrane fragmentation, changes in expression or activity of one or more proteins that promote apoptosis or that inhibit apoptosis, for example, increased caspase (e.g., caspase-3, -7 or -9) or Fas activity or expression, increased expression of proteins containing amplified polyglutamine repeat sequences or amplification of the sequences themselves, protein aggregate formation, etc. “Modulating” apoptosis means increasing, stimulating or inducing, or decreasing, inhibiting, blocking or preventing (e.g., prophylaxis) one or more characteristics of programmed cell death as described herein or known in the art. As described herein, compositions of the invention, including Bcl-B polypeptides, antibodies and subsequences thereof and antisense can have one or more apoptosis modulating activities.

[0055] As used herein, the term “isolated,” when referring to a molecule or composition, such as a nucleic acid, polypeptide or antibody of the invention, means that the molecule or composition is separated from at least one other compound, such as a protein, DNA, RNA, or other contaminants with which it is associated in vivo or in its naturally occurring state. Thus, a nucleic acid sequence is considered isolated when it has been isolated from any other component with which it is naturally associated. Components naturally associated with nucleic acid and protein include cellular components such as membrane, sugars, carbohydrate, lipids, fats, small molecules (e.g., hormones), etc.

[0056] As used herein, “recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in vitro, in cells or in other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide.

[0057] A composition of the invention can also be “substantially pure.” For example, a substantially pure Bcl-B protein will generally be free (30% or less measured by mass) of contaminating cellular components. A substantially pure composition can be free from chemical precursors or other chemicals when chemically synthesized, such as chemical intermediates or byproducts used to synthesize proteins or nucleic acids or that are byproducts of such chemical synthesis procedures. Thus, “substantially pure” can mean Bcl-B protein having less than about 30%, 20%, 10% and more likely 5% (by dry weight), of non-Bcl-B protein (also referred to herein as a “contaminating protein”), of chemical precursors, or of culture medium, i.e., culture medium is less than about 30% of the volume of the protein preparation. An isolated composition can therefore be in a homogeneous state. It can be in a dry or an aqueous solution. The invention includes isolated or purified preparations (protein or nucleic acid) of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight. Purity and homogeneity can be determined, e.g., using analytical chemistry techniques such as, e.g., polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC).

[0058] The terms “nucleic acid,” “nucleic acid sequence” or “polynucleotide” are used interchangeably to refer to a deoxy-ribonucleotide or ribonucleotide oligonucleotide, including single- or double-stranded forms, and coding or non-coding (e.g., “antisense”) forms. Antisense includes single strand non-coding sequence complementary to coding sequence (i.e. sense strand), triplex forming sequences as well as single and double strand ribozymes, DNAzymes and RNAi.

[0059] The term encompasses nucleic acids containing analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described, e.g., by U.S. Pat. Nos. 6,031,092; 6,001,982; 5,684,148; see also, WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompassed by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (see, e.g., U.S. Pat. No. 5,962,674; Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages (see, e.g., U.S. Pat. No. 5,532,226; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156).

[0060] The term nucleic acid is used interchangeably with gene, DNA, RNA, cDNA, mRNA, oligonucleotide primer, probe and amplification product. Nucleic acid molecules encoding Bcl-B polypeptides, proteins or amino acid sequences, hybridize to Bcl-B encoding nucleic acids or have the recited percent identity to Bcl-B set forth in SEQ ID NO:1 are collectively referred to as “nucleic acids or polynucleotides of the invention” or “Bcl-B nucleic acids or polynucleotides.”

[0061] The term “heterologous” when used in reference to a nucleic acid, indicates that the nucleic acid is in a cell or plant where it is not normally found in nature; or, comprises two or more nucleic acid subsequences which are not found in the same relationship to each other as normally found in nature, or is recombinantly engineered so that its level of expression, or physical relationship to other nucleic acids or other molecules in a cell, or structure, is not normally found in nature. For example, a heterologous nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes, such as a recombinant chimeric protein having one or more amino acids from different proteins, arranged in a manner not found in nature; e.g., a promoter sequence operably linked to a nucleic of the invention. As another example, the invention provides recombinant constructs (expression cassettes, vectors, viruses, and the like) comprising various combinations of promoters and sequences of the invention.

[0062] As used herein the terms “polypeptide,” “protein,” and “peptide” are used interchangeably and include compositions of the invention such as Bcl-B protein and antibodies that bind Bcl-B, as well as subsequences thereof. Amino acids comprise the polypeptides, all L- or D-isomers, or mixtures thereof. Amino acids also include circularized forms, such as formed by intra- or intermolecular disulfide bonds, or end-to-end amino-carboxyl linkages.

[0063] Also included are “analogs,” or “conservative variants” and “mimetics” (e.g., “peptidomimetics”) with structures and activity that substantially correspond to the polypeptides of the invention, including the exemplary Bcl-B sequence as set forth in SEQ ID NO:2. Thus, the terms “conservative variant” or “analog” or “mimetic” also refer to a polypeptide or peptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the polypeptide's (the conservative variant's) structure and/or activity (e.g., to modulate apoptosis, bind to a binding molecule, etc.), as defined herein. These include conservatively modified variations of an amino acid sequence, i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity, i.e., they are “substantially the same” in terms of structure or function as an unmodified sequence.

[0064] Conservative substitution tables describing chemically and structurally similar amino acids are well known in the art. For example, an exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): ala/gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. An alternative uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton (1984) Proteins, W. H. Freeman and Company; Schulz and Schimer (1979) Principles of Protein Structure, Springer-Verlag). One of skill in the art will appreciate that the above-described substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative. In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids within an encoded sequence are also considered “conservatively modified variations.”

[0065] A “non-essential” amino acid is a residue that can be altered from the wild-type sequence of Bcl-B (e.g., the sequence of SEQ ID NO:2) without destroying or, without substantially altering a biological activity. An “essential” amino acid is defined as one which when substituted with a non-conservative amino acid or deleted results in significant loss or complete abolition of activity. For example, amino acid residues that are likely to be essential among the polypeptides of the present invention include, for example, amino acids present in one or more of the BH1, BH2, BH3 and BH4 domains. Essential amino acids are unamenable to non-conservative substitution or deletion of more than a few amino acids (e.g., 1-3 amino acids).

[0066] The terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound that has substantially the same structural or functional characteristics of the polypeptides of the invention (e.g., ability to modulate apoptosis, bind to Bax, Bcl-2 or Bcl-X _L , associate with mitochondria, form membrane channels, etc.). The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetics' structure or activity. As with polypeptides of the invention which are conservative variants, routine tests can be used to determine whether a mimetic is within the scope of the invention, i.e., that its function is not substantially altered, e.g., it retains at least part of an activity of native Bcl-B set forth in SEQ ID NO:2. Polypeptide mimetics can contain any combination of natural and non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond (“peptide bond”) linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, etc. A polypeptide can be characterized as a mimetic when one or more of its residues are joined by chemical means other than natural peptide (amide) bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g., —C(═O)-CH ₂ — for —C(═O)—NH—), aminomethylene (CH ₂ —NH), ethylene, olefin (CH═CH), ether (CH ₂ —O), thioether (CH ₂ —S), tetrazole (CN ₄ —), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

[0067] “Expression control elements” are generally cis-acting nucleic acid transcriptional regulatory sequences. Examples include promoters capable of directing expression of a nucleic acid to cells (bacteria, plant, insect, mammalian, yeast, etc.). Examples also include enhancers capable of influencing transcription of a transcribed gene when located at a significant distance from the initiation site, or at the 5′, 3′ ends, or in an intron within the coding sequence. Expression control elements may contain only the minimum elements needed for transcription of the recombinant nucleic acid, such as a minimal promoter sequence.

[0068] An expression control element can be “constitutive,” such that transcription of an operably linked nucleic acid occurs without the presence of a signal or stimuli. Expression control elements can confer expression in a manner that is “regulatable,” that is, a signal or stimuli (external or internal or produced by other cells, such as a hormone produced by one cell that affects gene expression in another cell) that increases or decreases expression of the operably linked nucleic acid. A regulatable element that increases expression of the operably linked nucleic acid in response to a signal or stimuli is also referred to as an “inducible element” (i.e., is induced by a signal, e.g., a hormone). A regulatable element that decreases expression of the operably linked nucleic acid in response to a signal or stimuli is referred to as a “repressible element” (i.e., the signal decreases expression such that when the signal is removed or absent expression increases). Generally, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimuli present. Thus, in the case of an inducible element, the greater an amount of signal or stimuli present the greater the increase in expression. Typically, basal levels of transcription are greater for a repressible element than for an inducible element.

[0069] Expression control elements and promoters include those active in a particular tissue or cell type, referred to herein as a “tissue-specific expression control elements.” Tissue-specific expression control elements are typically active in specific cell or tissue as they are recognized by transcriptional activator proteins, or other regulators of transcription unique to the specific cell or tissue type. Expression control elements also include elements that confer expression at a particular stage of the cell cycle or differentiation. Accordingly, the invention includes expression control elements that confer constitutive, regulatable, tissue-specific, cell cycle specific, and differentiation stage specific expression.

[0070] Expression control elements include other components that influence expression (transcription, translation, RNA or protein stability, etc.), and therefore, include components in addition to promoter and enhancer sequences. Such components include, for example, leader sequences and fusion partner sequences, internal ribosome binding sites (IRES) elements for the creation of multigene, or polycistronic, messages, splice signal for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA, polyadenylation signal for polyadenylation of the transcript of a gene of interest, stop codons, etc. Thus, a combination of expression control elements might include a cis-acting transcriptional control element promoter, an enhancer, transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, and an intron. Expression control elements included herein can be from bacteria, yeast, plant, insect or animal (mammalian or non-mammalian), so long as they function to control expression of an operably linked nucleic acid.

[0071] The term “expression cassette” refers to any recombinant expression system for expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell or organism, including, in addition to insect and plant cells, prokaryotic, yeast, fungal or mammalian (e.g., human) cells. The term includes linear or circular expression systems. The term includes all vectors. The cassettes, when present in a host cell can be episomal, that is maintained apart from the host's genome, or integrate into the genome. Expression cassettes having the ability to self-replicate or not, i.e., drive only transient expression in a cell, are also included. Introduction of expression constructs into target cells can be carried out by conventional methods well known in the art (osmotic shock (e.g., calcium phosphate), electroporation, viral vectors, vesicles or lipid carriers (e.g., lipofection), direct microinjection, etc.

[0072] The term “operable linkage,” “operably linked” and grammatical variations thereof means that the element or elements referred to are in a physical or functional relation that allows them to function in their intended manner. Thus, for an expression control element such as a promoter to be in operable linkage with a gene, the promoter modulates expression of the gene (increasing or decreasing expression, as appropriate). Likewise, enhancers operably linked to a gene influence expression of the gene, which include sequences located 5′ and 3′ of the initiation site and in introns, for example. Thus, nucleic acid relatively close to a gene, within a gene and flanking a gene, e.g., within about 1-10, 10-50, 50-100, 100-500, 500-1000 or more nucleotides from the 5′ or 3′ termini are included. The invention therefore provides nucleic acids of the invention “operably linked” to an expression control element.

[0073] Another example of two elements in operable linkage is where expression of a gene influences expression of another gene. For example, a promoter driving expression of a gene encoding a transcription factor which binds to a promoter that activates expression of a second gene is operably linked to the second gene. Similarly, a Bcl-B polypeptide that binds to Bax is operably linked with a protein (or for that matter signaling pathway) influenced by Bax, through binding, or where, for example, Bax regulates transcription of a gene product. The terms operable linkage, operably linked and the like are therefore functionally defined.

[0074] The term “vector” refers to nucleic acid, such as a plasmid, virus (e.g., viral vector), or other vehicle known in the art that can be manipulated by insertion or incorporation of a polynucleotide, for genetic manipulation (i.e., “cloning vectors”), or can be used to transcribe or translate the inserted polynucleotide (i.e., “expression vectors”). Such vectors are useful for manipulation of nucleic acids of the invention, for producing invention polypeptides (e.g., baculovirus system), for introducing them into cells or whole organisms (ex vivo or in vivo), and expressing the transcribed Bcl-B antisense or encoded Bcl-B protein in cells in vitro or in vivo. A vector generally contains at least an origin of replication for propagation in a cell. Control elements, including expression control elements as set forth herein, present within a vector, are included to facilitate transcription.

[0075] Vectors can include a selection marker. As is known in the art, “selection marker” refers to genes that allow the selection of cells containing the gene. “Positive selection” refers to selecting the cells that survive (contain the positive selection marker) upon exposure to the positive selection agent. For example, drug resistance is a common positive selection marker; cells containing the positive selection marker will survive in culture medium containing the selection drug, and those which do not contain the resistance gene will die. Suitable drug resistance genes are neo, which confers resistance to G418, or hygr, which confers resistance to hygromycin, or puro, which confers resistance to puromycin. Other positive selection marker genes include genes that allow the sorting or screening of cells. These genes include genes for fluorescent proteins, the lacZ gene, the alkaline phosphatase gene, and cell surface markers such CD8 (isolate positive cells by CD8 antibody panning), among others. “Negative selection” refers to a process whereby cells containing a negative selection marker are killed upon exposure to an appropriate negative selection agent which kills cells containing the negative selection marker. For example, cells which contain the herpes simplex virus-thymidine kinase (HSV-tk) gene are sensitive to the drug.

[0076] Vectors applicable in the invention are those based on viral vectors, such as simian virus 40 (SV40) or bovine papilloma virus (BPV), which has the ability to replicate as extra-chromosomal elements (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982; Sarver et al., Mol. Cell. Biol. 1:486 (1981)). Viral vectors include retroviral (lentivirus), adeno-associated virus (see, e.g., Okada (1996) Gene Ther. 3:957-964; Muzyczka (1994) J. Clin. Invst. 94:1351; U.S. Pat. Nos. 6,156,303; 6,143,548 5,952,221, describing AAV vectors; see also U.S. Pat. Nos. 6,004,799; 5,833,993), adenovirus (see, e.g., U.S. Pat. Nos. 6,140,087; 6,136,594; 6,133,028; 6,120,764), reovirus, herpesvirus, rotavirus genomes etc., modified for introducing and directing expression of a polynucleotide or transgene in cells. Retroviral vectors can include those based upon murine leukemia virus (see, e.g., U.S. Pat. No. 6,132,731), gibbon ape leukemia virus (see, e.g., U.S. Pat. No. 6,033,905), simian immuno-deficiency virus, human immuno-deficiency virus (see, e.g., U.S. Pat. No. 5,985,641), and combinations thereof.

[0077] Vectors also include those that efficiently deliver genes to animal cells in vivo (e.g., stem cells) (see, e.g., U.S. Pat. Nos. 5,821,235 and 5,786,340; Croyle, M. A. et al., Gene Ther. 5:645 (1998); Croyle, M. A. et al., Pharm. Res. 15:1348 (1998); Croyle, M. A. et al., Hum. Gene Ther. 9:561 (1998); Foreman, P. K. et al, Hum. Gene Ther. 9:1313 (1998); Wirtz, S. et al., Gut 44:800 (1999)). Adenoviral and adeno-associated viral vectors suitable for in vivo delivery are described, for example, in U.S. Pat. Nos. 5,700,470, 5,731,172 and 5,604,090. Additional vectors suitable for in vivo delivery include herpes simplex virus vectors (see, e.g., U.S. Pat. No. 5,501,979), retroviral vectors (see, e.g., U.S. Pat. Nos. 5,624,820, 5,693,508 and 5,674,703; and WO92/05266 and WO92/14829), bovine papilloma virus (BPV) vectors (see, e.g., U.S. Pat. No. 5,719,054), CMV-based vectors (see, e.g., U.S. Pat. No. 5,561,063) and parvovirus, rotavirus and Norwalk virus vectors. Lentiviral vectors are useful for infecting dividing as well as non-dividing cells (see, e.g., U.S. Pat. No. 6,013,516).

[0078] Vectors for insect cell expression commonly use recombinant variations of baculoviruses and other nucleopolyhedrovirus, e.g., Bombyx mori nucleopolyhedrovirus vectors (see, e.g., Choi (2000) Arch. Virol. 145:171-177). For example, Lepidopteran and Coleopteran cells are used to replicate baculoviruses to promote expression of foreign genes carried by baculoviruses, e.g., Spodoptera frugiperda cells are infected with recombinant Autographa californica nuclear polyhedrosis viruses (AcNPV) carrying a heterologous, e.g., a human, coding sequence (see, e.g., Lee (2000) J. Virol. 74:11873-11880; Wu (2000) J. Biotechnol. 80:75-83). See, e.g., U.S. Pat. No. 6,143,565, describing use of the polydnavirus of the parasitic wasp Glyptapanteles indiensis to stably integrate nucleic acid into the genome of Lepidopteran and Coleopteran insect cell lines. See also, U.S. Pat. Nos. 6,130,074; 5,858,353; 5,004,687.

[0079] Expression vectors capable of expressing proteins in plants are well known in the art, and include, e.g., vectors from Agrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J. 16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene 173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology 169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology 234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993) Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g., Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101), maize Ac/Ds transposable element (see, e.g., Rubin (1997) Mol. Cell. Biol. 17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194), and the maize suppressor-mutator (Spm) transposable element (see, e.g., Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

[0080] Introduction of nucleic acid and polypeptide in vitro, ex vivo and in vivo can also be accomplished using other techniques. For example, a polynucleotide comprising an expression control element in operable linkage with a nucleic acid encoding Bcl-B protein can be incorporated into particles or a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers. A polynucleotide can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules, or poly (methylmethacrolate) microcapsules, respectively, or in a colloid drug delivery system. Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The use of liposomes for introducing various compositions, including polynucleotides, is known to those skilled in the art (see, e.g., U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282). A carrier comprising a natural polymer, or a derivative or a hydrolysate of a natural polymer, described in WO 94/20078 and U.S. Pat. No. 6,096,291, is suitable for mucosal delivery of molecules, such as polypeptides and polynucleotides. Piperazine based amphilic cationic lipids useful for gene therapy also are known (see, e.g., U.S. Pat. No. 5,861,397). Cationic lipid systems also are known (see, e.g., U.S. Pat. No. 5,459,127). Accordingly, vector (viral and non-viral, e.g., naked DNA) and non-vector means of delivery into mucosal cells or tissue, in vitro, in vivo and ex vivo can be achieved and are contemplated.

[0081] As used herein, the term “transgene” means a polynucleotide that has been introduced into a cell or organism by artifice. For example, in a host cell having a transgene, the transgene has been introduced by genetic manipulation or “transformation” of the cell. A cell or progeny thereof into which the transgene has been introduced is referred to as a “transformed cell” or “transformant.” Typically, the transgene is included in progeny of the transformant or becomes a part of the organism (tissue or organs) that develops from the cell. Transgenes may be inserted into the chromosomal DNA or maintained as a self-replicating plasmid, YAC, minichromosome, or the like. Transgenes include any gene that is transcribed into an antisense or encodes a polypeptide.

[0082] A “host cell” includes a cell that expresses a composition of the invention. Host cells include cultured cells (cells that have been adapted for growth in vitro) and primary cells isolated from an organism manipulated to contain a composition of the invention. Host cells also can be present in an animal (subject), either by ex vivo manipulation of cells, transgenic introduction of compositions (non-human animals), or by in vivo introduction of a composition of the invention.

[0083] The term “subject” refers to an animal. Typically, the animal is a mammal, however, any animal in which Bcl-B is present or may modulate apoptosis is encompassed by the term. Specific examples are primates (humans), dogs, cats, horses, cows, pigs, and sheep. Subjects include those having a physiological disorder associated with apoptosis or at risk of developing a disorder associated with apoptosis, although they may not exhibit symptoms of the disorder. Subjects therefore may have a disorder associated with apoptosis or may not. At risk subjects can be identified by screening for genes associated with the disorders; over or under expression of a gene (e.g., caspase, fas, etc.) indicates a subject at risk for a disorder.

[0084] The term “antibody” or “Ab” includes both intact antibodies having at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds and antigen binding fragments thereof, or equivalents thereof, either isolated from natural sources, recombinantly generated or partially or entirely synthetic. Examples of antigen binding fragments include, e.g., Fab fragments, F(ab′)2 fragments, Fd fragments, dAb fragments, isolated complementarity determining regions (CDR), single chain antibodies, chimeric antibodies, partially humanized antibodies, human antibodies made in non-human animals (e.g., transgenic mice) or any form of antigen binding fragment.

[0085] As used herein, the term “bind” or “binding” means that the components referred to specifically interact with each other at a molecular level. Direct binding means physical contact between the components. Indirect binding means binding to one or more components that bind. Thus, the two components need not physically contact each other in order to bind as they may be a part of a oligomeric complex in which an intermediary component binds between the two components. Indirect or direct binding may be relatively stable, such as that which occurs between an antibody and an antigen or be less stable, e.g. a dissociation constant (K _D ) of less than about 10- ₆ . Binding may also be transient, such as the binding that occurs between a transcription factor and DNA for transcription initiation, which does not occur in the absence of transcription. “Specific binding” is where the binding is selective between the components. Specific binding can be detected using methods known in the art, for example, by immunoprecipitation, affinity chromatography, gel shift assays, gene expression assays, etc. “Specific” and “non-specific” binding can be distinguished using appropriate controls.

[0086] The terms “array,” “microarray,” “DNA array” or “nucleic acid array” or “biochip” as used herein mean a plurality of target elements, each target element comprising a defined amount of one or more biological molecules (e.g., nucleic acid, protein, drugs, small organic compounds, natural compounds, libraries thereof, etc.), including the Bcl-B nucleic acids or proteins of the invention, immobilized on a substrate (e.g., solid or semi-solid, permeable or non-permeable surface) for binding with sample nucleic acids, proteins, antibodies, test agents, drugs, organic or natural compounds. The Bcl-B proteins, nucleic acids, antibodies of the invention and subsequences thereof can be incorporated into any form of microarray, such as those described, e.g., in U.S. Pat. Nos. 6,045,996; 6,022,963; 6,013,440; 5,959,098; 5,856,174; 5,770,456; 5,556,752; 5,143,854.

[0087] The term “plant” includes whole plants, plant parts (e.g., leaves, stems, flowers, roots, etc.), plant protoplasts, seeds and plant cells and progeny of same, including progeny obtained through cuttings or through regeneration of a plant or plant tissue from a plant cell. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous states. Plantlets are also included within the meaning of “plant.” Suitable plants for use in the invention include any plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Examples of monocotyledonous plants include, but are not limited to, asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oat, and ornamentals.

[0088] Examples of dicotyledonous plants include, but are not limited to, tomato, potato, arabidopsis, tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops or Brassica (e.g., cabbage, broccoli, cauliflower, brussel sprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers and various ornamentals. The term “plant cell”, as used herein, refers to protoplasts, gamete producing cells, and cells that are capable of regenerating into whole plants. Accordingly, a seed comprising multiple plant cells capable of regenerating into a whole plant is included in the definition of “plant cell”. As used herein, “plant tissue” includes differentiated and undifferentiated tissues of a plant, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture such as single cells, protoplast, embryos, and callus tissue.

[0089] A “biotic insult,” as used herein, refers to plant challenge caused by viable or biologic agents (biotic agents), such as insects, fungi, bacteria, viruses, nematodes, viroids, mycoplasmas, and the like.

[0090] An “abiotic insult,” as used herein, refers to plant challenge by a non-viable or non-living agent (abiotic agent). Abiotic agents that can cause challenge include, for example, environmental factors such as low moisture (drought), high moisture (flooding), nutrient deficiency, radiation levels, air pollution (ozone, acid rain, sulfur dioxide, etc.), temperature (hot and cold extremes), and soil toxicity, as well as herbicide damage, pesticide damage, or other agricultural practices (e.g., over-fertilization, improper use of chemical sprays, etc.).

[0091] The term “pathogen” refers to any biological organism or chemical agent produced by a biological organism that causes a disease or disease state in an animal or plant, including, but not limited to viruses, fungi, bacterium, nematodes, and other related microorganisms.

[0092] “Functional subsequences,” which are portions of the compositions of the invention that retain at least one activity or function, all or in part, of the native full-length composition are included. “Functional subsequences” therefore include portions of Bcl-B protein (e.g., fragments of SEQ ID NO:2) which modulate apoptosis, bind to Bax, Bcl-2 and Bcl-X _L , associate with mitochondria or form a membrane channel alone or in combination with other Bc-2 protein family members. Functional subsequences also include subsequences that are immunogenic.

[0093] Bcl-B protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of SEQ ID NO:2, which include less amino acids than the full length Bcl-B, and exhibit all or part of at least one activity of a Bcl-B protein (e.g., apoptosis modulating activity). Typically, an active portion comprises a domain or motif with at least one activity of the Bcl-B protein, for example, a domain or motif capable of modulating apoptosis; a domain or motif capable of interacting with a binding molecule, such as itself, Bax, Bcl-2 or Bcl-X _L ; a domain or motif capable of associating with mitochondria, such as Bcl-B transmembrane domain; a domain or motif capable of modulating an intracellular molecule (e.g., Bax) that participates in an apoptotic signaling pathway; a domain or motif capable of forming a membrane channel alone, or in combination with other Bc-2 protein family members; a domain or motif capable of modulating a caspase; a domain or motif capable of modulating DNA fragmentation, chromatin condensation or other processes associated with apoptosis; or a domain or motif capable of modulating cell proliferation, survival, growth or differentiation.

[0094] A biologically active portion of a Bcl-B protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Biologically active portions can be used directly as agents that modulate apoptosis or other activities of full length Bcl-B. Bcl-B protein can also be used as targets for developing agents which modulate a Bcl-B mediated activity, e.g., an activity described herein, and are potential therapeutics for treating disorders associated with undesirable or insufficient/deficient apoptosis.

[0095] The terms percent “sequence identity” or “sequence homology,” in the context of two or more nucleic acids or polypeptide sequences, refers to sequences or subsequences thereof that have a specified percentage of nucleotides (or amino acid residues) that are the same, when compared and aligned for maximum correspondence over a comparison window. Percent identity can be measured by manual alignment and visual inspection or using a sequence comparison algorithm, as described herein. This definition also refers to the complement (antisense strand) of a sequence.

[0096] For example, in various embodiments, polypeptides of the invention include those having an amino acid sequence at least about 70%, at least about 80%, at least about 90%, at least about 95% and at least about 99% identical to an exemplary sequence set forth in SEQ ID NO:2. Polypeptide subsequences are also included. In additional embodiments, a subsequence is at least about 15, 20, 25, 30, 40, 50, 75, 125, 150 or 200, or greater amino acids (e.g., 300, 350, 400, 450, 500, 550, etc.) in length. Thus, a polypeptide sequence having the requisite sequence identity to SEQ ID NO:2, or a subsequence thereof, also is a polypeptide of the invention. In various additional embodiments, the polypeptides, including subsequences have one or more activities of a sequence set forth in SEQ ID NO:2. Thus, in various aspects a polypeptide has apoptosis modulating activity, oligomerization (heteromerizes or forms homodimers, trimers, etc.) activity, binds to Bcl-2, B

[0097] Nucleic acids of the invention include those having at least about 70%, at least about 80%, at least about 90%, at least about 95%, and at least about 99% identity to the exemplary sequence set forth in SEQ ID NO:1, wherein the sequence is distinct from Accession no. AA098865. Thus, if a nucleic acid sequence has the requisite sequence identity to SEQ ID NO:1, or a subsequence thereof, it also is a polynucleotide sequence of the invention. In various aspects, the sequence is less than about 50 Kb, 25 Kb, 10 Kb, 5 Kb or 2.5 Kb. In additional aspects, the sequence is between about 2.5-1.0 Kb, 1.0-0.5 Kb, 0.25-0.1 Kb and 100-15 base pairs. In yet other aspects, the sequence is selected from SEQ ID NO:1; SEQ ID NO:1 where one or more T's are U's; nucleic acids complementary to these sequences and subsequences of the aforementioned sequences at least 15 base pairs long.

[0098] In one aspect, the percent identity exists over a region of the sequence that is at least about 25 nucleotides or amino acid residues in length, or, over a region that is at least about 50 to 100 nucleotides or amino acids in length. Parameters (including, e.g., window sizes, gap penalties and the like) to be used in calculating