MXPA00007645A - Novel molecules of the card-related protein family and uses thereof - Google Patents

Abstract

Novel CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, and murine CARD-4L polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, and murine CARD-4L proteins, and the invention further provides CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, and murine CARD-4L fusion proteins, antigenic peptides and anti-CARD-3, anti-CARD-4L and anti-CARD-4S, anti-CARD-4Y, anti-CARD-4Z, and anti-murine CARD-4L antibodies. The invention also provides CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, and murine CARD-4L nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, and murine CARD-4L gene has been introduced or disrupted. The invention further provides CARD-3 and CARD-4 target proteins that bind to CARD-3 or CARD-4 and allelic variants of human CARD-4. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

Description

NOVELTY MOLECULES OF THE CARD RELATED PROTEIN FAMILY AND ITS USES CROSS REFERENCE TO RELATED REQUESTS This application is a continuation in part of the North American Application Serial No. 09 / 207,359 filed on December 8, 1998, which is a continuation in Part of the North American Application Serial No. 09 / 099,041, filed on June 17, 1998, which is a continuation in part of the North American Application Serial No. 09 / 019,942, filed on February 6, 1998. The content of each of these applications is incorporated herein by reference. BACKGROUND OF THE INVENTION In multicellular organisms, homeostasis is maintained by balancing the rate of cell proliferation and the rate of cell death. Cell proliferation is influenced by numerous growth factors and the expression of proto-oncogenes, which typically promote progression through the cell cycle. In contrast, numerous events, including the expression of tumor suppressor genes, can cause a suspension of cell proliferation.

In differentiated cells, a particular type of cell death occurs which is known as apoptosis when an internal suicide program is activated. This program can be initiated by several external signals as well as signals generated within the cell in response, for example, to genetic damage. For many years, the magnitude of apoptotic cell death was not recognized since dead cells are rapidly eliminated by phagocytes, without an inflammatory response. The mechanisms that mediate apoptosis have been studied intensively. These mechanisms include the activation of endogenous proteases, the loss of mitochondrial function, and structural changes such as cytoskeletal disruption, cellular shrinkage, membrane bladder formation, and nuclear condensation due to DNA degradation. It is believed that the various signals that cause apoptosis cause these events by their convergence in a common cell death pathway regulated by the expression of highly conserved genes from worms such as C. elegans, to humans. In fact, invertebrate model systems have been very valuable tools to identify and characterize the genes that control apoptosis. Through the study of invertebrates and more evolved animals, numerous genes associated with cell death have been identified, but the way in which their products interact to execute the apoptotic program is known to a limited extent. Caspases, a class of central proteins in the apoptotic program, are cysteine proteases that have a specificity for aspartate at the substrate dissociation site.

These proteases are primarily responsible for the degradation of cellular proteins that cause morphological changes observed in cells subjected to apoptosis. For example, one of the caspases identified in humans was previously known as the interleukin-la (IL-la) conversion enzyme (ICE), a cysteine protease responsible for the processing of pro-LA in active cytosine. Overexpression of ICE in rat fibroblasts-1 induces apoptosis (Miura et al., Cell 75: 653, 1993). Many caspases and proteins that interact with caspases have domains of approximately 60 amino acids known as the caspase recruitment domain (CARD). Hof ann et al. (TIBS 22: 155, 1997) and others have postulated that certain apoptotic proteins bind to each other through their CARDs and that different subtypes of CARDs can provide binding specificity, regulation of the activity of several caspases, for example. The functional importance of CARDs has been demonstrated in recent publications. Duan et al. (Nature 385: 86, 1997) showed that the removal of CARD at the N-terminus of RAIDD, a newly identified protein involved in apoptosis, it canceled RAIDD's ability to join caspases. In addition, Li et al. (Cell 91: 479, 1997) showed that the N-terminal 97 amino acids of apoptotic protease activation factor-1 (Apaf-1) were sufficient to confer the binding capacity of caspase-9. Inohara et al. (J. Biol. Chem. 273: 12296-12300, 1998) showed that Apaf-1 can bind with several other caspases such as caspase-4 and caspase-8. Apaf-1 can interact with caspases through the CARD-CARD interaction (Li et al., Supra, Hu et al., PNAS, 95: 4386-4391, 1998). The nuclear factor -? B (NF-? B) is a transcription factor expressed in many cell types and that activates homologous or heterologous genes that have KB sites in their promoters. Quiescent NF-? B receives in the cytoplasm as a heterodimer between proteins known as p50 and p65 and forms complexes with the regulatory protein I? B. The binding of NF-? B with I? B causes NF-? B to remain in the cytoplasm. At least two dozen stimuli that activate NF-? B are known (New England Journal of Medicine 336: 1066, 1997) and include cytokines, protein kinase C activators, oxidants, viruses, as well as immune system stimuli. The stimuli that activate NF-? B activate specific I? B kinases that phosphorylate I? B that lead to their degradation. Once released from I? B, they are translocated to the nucleus and activate genes with the KB sites in their promoters. How all these stimuli that activate NF-? B act are currently unknown and it is considered that components of novel NF-? B pathways are involved. NF-KB and the NF-? B pathway have been indicated in the mediation of chronic inflammation in inflammatory diseases such as asthma, ulcerative colitis, rheumatoid arthritis (New England Journal of Medicine 336: 1066, 1997) and inhibition of NF- ? BO well of the routes of NF-? B can be an effective way to treat these diseases. NF-? B and the NF-KB pathway have also been implicated in atherosclerosis (American Journal of Cardiology 76: 18C, 1995), especially in the mediation of the formation of fatty parts, and the inhibition of NF-? BO from the NF-? B pathways may represent an effective therapy for atherosclerosis. COMPENDIUM OF THE INVENTION The present invention is based, at least partially, on the discovery of genes encoding CARD-3 and CARD-4. The CARD-4 gene can express a long transcript encoding CARD-4L, a short transcript encoding partial CARD-4S, or two splice variants of CARD-4. A murine total length cDNA sequence for the murine ortholog of CARD-4L is also presented. CARD-3 and CARD-4 are intracellular proteins that are predicted to be involved in the regulation of caspase activation. CARD-4 activates the NF-KB pathway and enhances cell death mediated by caspase 9. In addition, proteins that bind with CARD-4 are present, including CARD-3 and hNUDC. The CARD-3 cDNA described below (SEQ ID NO: 1) has an open reading frame of 1620 (nucleotides 214 to 1833 of SEQ ID NO: 1).; SEQ ID NO: 3) that encodes a protein of 540 amino acids (SEQ ID NO: 2). CARD-3 contains a kinase domain extending from amino acid 1 to amino acid 300 of SEQ ID NO: 2; SEQ ID NO: 4, followed by a linker domain at amino acid 301 to amino acid 431 of SEQ ID NO: 2; SEQ ID NO: 5 and a CARD at amino acid 432 to amino acid 540 of SEQ ID NO: 2; SEQ ID NO: 6. There are at least four forms of CARD-4 in the cell, a long form, CARD-4L, a short form, CARD-4S, and two variants that are spliced, CARD-4Y and CARD-4Z. The CARD-4L cDNA described below (SEQ ID NO: 7) has an open reading frame of 2859 nucleotides (nucleotides 245-3103 of SEQ ID NO: 7; SEQ ID NO: 9) which encodes a 953 amino acid protein (SEQ ID NO: 8). The CARD-4L protein possesses a CARD domain (amino acids 15-114; SEQ ID NO: 10). The nucleotide sequence of the full-length cDNA corresponding to the murine ortholog of human CARD-4L is presented (SEQ ID NO: 42) as well as the predicted amino acid sequence of murine CARD-4L (SEQ ID NO: 43). A comparison between the predicted amino acid sequences of human CARD-4L and murine CARD-4L is also shown in figure 17. It is also predicted that human CARD-4L has a nucleotide binding domain that extends from about the amino acid 198 to about amino acid 397 of SEQ ID NO: 8; SEQ ID NO: 11, a Walker Table "A", which extends approximately from amino acid 202 to amino acid 209, approximately, of SEQ ID NO: 8, SEQ ID NO: 12, a Walker Table "B", which extends approximately from amino acid 280 to approximately amino acid 284, of SEQ ID NO: 8; SEQ ID NO: 13, a kinase sub-domain (loop P) extending approximately from amino acid 127 to about amino acid 212 of SEQ ID NO: 8; SEQ ID NO: 46, a kinase 2 sub-domain extending approximately from amino acid 273 to approximately amino acid 288 of SEQ ID NO: 8; SEQ ID NO: 47, a sub-domain of kinase 3a, extending approximately from amino acid 327 to approximately amino acid 338 of SEQ ID NO: 8; SEQ ID NO: 14, and ten leucine-rich repeats ranging from about amino acid 674 to about amino acid 950 of SEQ ID NO: 8. The first leucine-rich repeat extends from about amino acid 674 to about amino acid 701 of SEQ ID NO: 8; SEQ ID NO: 15. The second leucine-rich repeat extends from about amino acid 702 to about amino acid 727 of SEQ ID NO: 8; SEQ ID NO: 16. The third leucine-rich repeat extends from about amino acid 728 to about amino acid 754 of SEQ ID NO: 8; SEQ ID? O: 17. The fourth leucine-rich repeat extends from about amino acid 755 to about amino acid 782 of SEQ ID? O: 8; SEQ ID NO: 18. The fifth leucine-rich repeat extends from about amino acid 783 to about amino acid 810 of SEQ ID NO: 8; SEQ ID NO: 19. The sixth leucine-rich repeat extends from about amino acid 811 to about amino acid 838 of SEQ ID NO: 8; SEQ ID NO: 20 The seventh leucine-rich repeat extends from about amino acid 839 to about amino acid 866 of SEQ ID NO: 8; SEQ ID NO: 21. The eighth leucine-rich repeat extends from about amino acid 867 to about amino acid 894 of SEQ ID NO: 8; SEQ ID NO: 22. The ninth leucine-rich repeat extends from about amino acid 895 to about amino acid 922 of SEQ ID NO: 8; SEQ ID NO: 23, and the leucine-rich repeat repeat extends from about amino acid 923 to about amino acid 950 of SEQ ID NO: 8; SEQ ID NO: 24 The partial CARD-4S cDNA described below (SEQ ID NO: 25) has an open reading frame of 1470 nucleotides (nucleotides 1-1470 of SEQ ID NO: 25).; SEQ ID NO: 27) that encodes a protein of 490 amino acids (SEQ ID NO: 26). The CARD-4S protein possesses a CARD domain (amino acid 1-74 of SEQ ID NO: 26, SEQ ID NO: 28). It is predicted that CARD-4S has a P loop extending from about amino acid 163 to about amino acid 170 of SEQ ID NO: 26; SEQ ID NO: 29, and a Walker Table "B" extending from about amino acid 241 to about amino acid 245 of SEQ ID NO: 26; SEQ ID NO: 30. A nucleotide cDNA sequence CARD-4Y is presented (SEQ ID NO: 38) as the amino acid sequence of the predicted CARD-4Y product (SEQ ID NO: 39). A human CARD-4Z nucleotide cDNA sequence is presented (SEQ ID NO: 40) as well as the amino acid sequence of the predicted CARD-4Z product (SEQ ID NO: 41). A comparison of the amino acid sequences of CARD-4Y, CARD-4Z and human CARD-4L is also shown in Figure 14. Like other proteins that contain a CARD domain, both CARD-3 and CARD-4 are expected to participate in the network of interactions that leads to caspasa activity. Each human CARD-4L is expected to play a role in caspase activation similar to that of Apaf-1 (Zou et al., Cell, 90: 405-413, 1997). For example, when activated, CARD-4L can bind a nucleotide, thus allowing CARD-4L to bind and activate a caspase containing CARD through a CARD-CARD interaction, leading to the apoptotic dead cell. Accordingly, molecules CARD-3 and CARD-4 are useful as modulating agents to regulate various cellular processes including cell growth and cell death. In one aspect, this invention offers isolated nucleic acid molecules encoding CARD-4 or CARD-4 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of nucleic acids. encoding CARD-3 or CARD-4, The invention encompasses methods for diagnosis and treatment of patients suffering from a disorder associated with an abnormal level or an abnormal rate (undesirable can or undesirably low) of apoptotic cell death, an abnormal activity of the complex of Fas / APO-1 receptor, abnormal activity of the TNF receptor complex, or abnormal activity of a caspase by administration of a compound that modulates the expression of CARD-3 or CARD-4 (in DNA, ANRm or at the protein level, for example, by altering the splicing mRNA) or by altering CARD-3 or CARD-4 activity. Examples of compounds of this type include small molecules, antisense nucleic acid molecules, ribozymes, and polypeptides. Some disorders are related to an increased number of surviving cells that are produced and continue to survive or proliferate when apoptosis is inhibited. These disorders include cancer (particularly follicular lymphomas, carcinomas associated with mutations in p53, and hormone-dependent tumors such as breast cancer, prostate cancer, and ovarian cancer), autoimmune disorders (such as systemic lupus erythematosis, glomerulonephritis) mediated by the immune system), as well as viral infections (such as those caused by herpes viruses, poxviruses, and adenoviruses). The non-removal of autoimmune cells that arise during development or that develop as a result of somatic mutation during an immune response may cause an autoimmune disease. One of the molecules that play the critical role in the regulation of cell death in lymphocytes is the cell surface receptor for Fas. Cell populations are frequently depleted in the case of a viral infection, with perhaps the most dramatic example, being the cellular depletion caused by the human immunodeficiency virus (HIV). Surprisingly, most T cells that die during HIV infections do not appear to be infected with HIV. Even when numerous explanations were proposed, recent evidence suggests that stimulation of the CD4 receptor results in increased susceptibility of uninfected T cells to apoptosis. A wide variety of neurological diseases is characterized by the gradual loss of specific sets of neurons. Such disorders include Alzhemeir's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, spinal muscular atrophy, and several cerebellar degeneration. The loss of cells in these diseases does not cause an inflammatory response, and apoptosis seems to be the mechanism of cell death. In addition, numerous hematological diseases are related to a decreased production of blood cells. These disorders include anemia associated with chronic disease, aplastic anemia, chronic neutropenia, and ipsilateral syndromes. Disorders of blood cell production such as myelodysplastic syndrome and other forms of aplastic anemia are associated with increased apoptotic cell death in the bone marrow. These disorders could result from the activation of genes that promote apoptosis, deficiencies acquired in stromal cells or hematopoietic survival factors, or the direct effects of toxins and mediators of immune responses. Two common disorders associated with cell death are myocardial infarction and stroke. In both disorders the cells within the central area of ischemia, which occurs in case of acute loss of blood flow, seem to die rapidly as a result of necrosis. However, outside the central ischemic area, cells die over a long period of time and morphologically seem to die of apoptosis. The invention features a nucleic acid molecule that is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42, the nucleotide sequence of the cDNA insert of the plasmid deposited with ATCC with the Access Number (the "ATCC cDNA"), the nucleotide sequence of the plasmid cDNA insert deposited with ATCC as the Accession Number (the "ATCC cDNA"), the nucleotide sequence of the plasmid cDNA insert deposited with ATCC as Accession Number (the "ATCC cDNA") or a complement thereof The invention features a nucleic acid molecule that includes a fragment of at least 150 (300, 325, 350, 375, 400, 425, 450, 500, 550, 600 , 650, 700, 800, 900, 1000, 1300, 1600, or 1931) nucleotide of the nucleotide sequence illustrated in SEQ ID NO: 1, or SEQ ID NO: 3, or in the nucleotide sequence of the cDNA of ATCC, or a complement thereof The invention also features a nucleic acid molecule that includes a fragment of at least 150 (350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000 , 1300, 1600, 1900, 2100, 2400, 2700, 3000, or 3382) nucleotides of the nucleotide sequence illustrated in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 43 either in the nucleotide sequence of the ATCC cDNA or a complement thereof, likewise within the present is a nucleic acid molecule that includes a fragment of at least 150 (350, 400, 450, 500, 550 , 600, 650, 700, 800, 900, 1000, 1300, 1600, 1900, 2100, 2400, 2700, or 3080) nucleotides of the nucleotide sequence illustrated in SEQ ID NO: 25, SEQ ID NO: 38, SEQ ID NO: 40, or the nucleotide sequence of the ATCC cDNA, or a complement thereof. The invention features a nucleic acid molecule that includes a sequence of nucleotides that encode the protein having an amino acid sequence that is at least 45%, (or 55%, 65%, 75%, 85% 95%, or 98%) identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 41, and SEQ ID NO: 43, or the amino acid sequence encoded by the AD? c of ATCC, the amino acid sequence encoded by the AD? c of ATCC, or the amino acid sequence encoded by the AD? c of ATCC. In one embodiment, a CARD-3 nucleic acid molecule has the nucleotide sequence shown in SEQ ID? O: 1, or SEQ ID? O: 3, or the nucleotide sequence of the AD? C of ATCC. In another embodiment, a nucleic acid molecule of CARD-4L has the nucleotide sequence illustrated in SEQ ID? O: 7, or SEQ ID? O: 9, or the nucleotide sequences of the AD? C of ATCC. In another embodiment a CARD-4S nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 25 or SEQ ID NO: 27, or the nucleotide sequence of the cDNA of ATCC. In another embodiment, a murine CARD-4L nucleic acid molecule has the nucleotide sequence illustrated in SEQ ID NO: 42. In another embodiment, a CARD-4Y nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. : 38 or the nucleotide sequence of the ATCC cDNA. In another embodiment, a CARD 4-Z nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 40 or the cDNA nucleotide sequence of ATCC. Also, within the invention, there is a nucleic acid molecule encoding a fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 8 or SEQ ID NO: 26 or SEQ ID NO. : 39 or SEQ ID NO: 41 or SEQ ID NO: 43, the fragment including at least 15 (25, 30, 50, 100, 150, 300, 400, or 540, 600, 700, 800, 953) contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 8 or SEQ ID NO: 26 or SEQ ID NO: 39 or SEQ ID NO: 31 or SEQ ID NO: 43 or the polypeptide encoded by the Access Number cDNA ATCC, or the polypeptide encoded by the cDNA of the ATCC Accession Number, or the polypeptide encoded by the cDNA of ATCC Accession Number The invention includes a nucleic acid molecule encoding a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 39 or SEQ ID NO: 41 or SEQ ID NO: 43 or one amino acid encoded by the cDNA of ATCC Accession Number, wherein the nucleic acid molecule hybridizes with a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 38 or SEQ ID NO: 40 or SEQ ID NO: 42 under stringent conditions. The invention also includes a nucleic acid molecule encoding an allelic variant that naturally occurs from a polypeptide comprising the amino acid sequence SEQ ID NO: 8 or an amino acid sequence encoded by the access code cDNA ATCC, wherein the nucleic acid molecule hybridizes with a nucleic acid molecule comprising SEQ ID NO: 7 or SEQ ID NO: 9, under stringent conditions. The invention also includes a nucleic acid molecule encoding an allelic variant that naturally occurs from a polypeptide comprising the amino acid sequence SEQ ID NO: 26 or an amino acid sequence encoded by the ATCC accession cDNA, wherein the nucleic acid is hybridized with a nucleic acid molecule comprising SEQ ID NO: 25, or SEQ ID NO: 27 under stringent conditions. In general, an allelic variant of a gene will be easily identifiable since it is traced in the same chromosomal location as said gene. For example, in example 6, the chromosomal location of the human CARD-4 gene is chromosome 7 near the genetic marker SHGC-31928. Allelic variants of human CARD-4 will be easily identifiable since they are found in the human CARD-4 locus on chromosome 7 near the genetic marker SHGC-31928. Within the invention is the following: an isolated CARD-3 protein having an amino acid sequence which has an identity level of at least about 65%, preferably 75%, 85%, 95%, or 98% with the amino acid sequence SEQ ID NO: 2; an isolated CARD-3 protein having an amino acid sequence having an identity level of at least about 85%, 95%, or 98% relative to the kinase domain SEQ ID NO: 2 (eg, about the residues of amino acids 1 to 300 of SEQ ID NO: 2; SEQ ID NO: 4); and an isolated CARD-3 protein having an amino acid sequence that exhibits an identity level of at least about 85%, 95% or 98% with the linker domain SEQ ID NO: 2 (e.g. amino acids 301 to 431 of SEQ ID NO: 2; SEQ ID NO: 5); an isolated CARD-3 protein having an amino acid sequence that is at least about 85%, 95% or 98% identical with the CARD domain of SEQ ID NO: 2 (e.g., about amino acid residues 432 to 540 of SEQ ID NO: 2; SEQ ID NO: 6); an isolated CARD-4L protein having an amino acid sequence that is at least about 65%, preferably 75%, 85%, 95% or 98% identical with the amino acid sequence of SEQ ID NO: 8; an isolated CARD-4L protein having an amino acid sequence that is at least about 85%, 95% 98% identical with the CARD domain SEQ ID NO: 8 (e.g., about amino acid residues 15 to 114 of SEQ ID NO: 8) NO: 8; SEQ ID NO: 10); a CARD-4L protein having an amino acid sequence that is at least about 85%, 95% or 98% identical to the nucleotide linkage domain of SEQ ID NO: 8 (e.g., about amino acid residue 198 a) 397 of SEQ ID NO: 8, SEQ ID NO: 11: an isolated CARD-4L protein having an amino acid sequence that is at least about 85% 95% or 98% identical with the kinase subdomain the (P loop) SEQ ID NO: 8 (for example, about amino acid 127 to about amino acid 212 of SEQ ID NO: 8, SEQ ID NO: 46), an isolated CARD-4L protein having an amino acid sequence that is at least about 85% 95% or 98% identical with the kinase 2 subdomain of SEQ ID NO: 8 (for example, approximately from amino acid 273 to approximately amino acid 288 of SEQ ID NO: 8; SEQ ID NO: 47); Isolated 4L having an amino acid sequence that is at least about 85%, 95% or 98 % identical to a kinase domain 3a of SEQ ID NO: 8 (for example about residual amino acids 327 to 338 of SEQ ID NO: 8; SEQ ID NO: 14); an isolated CARD-4L protein having an amino acid sequence that is at least about 85%, 95% or 98% identical to the leucine-rich repeats of SEQ ID NO: 8 (e.g., about 674 amino acid residues) to 701 of SEQ ID NO: 8; SEQ ID NO: 15; of amino acids 702 to amino acid 727 of SEQ ID NO: 8; SEQ ID NO: 16; which extends from amino acid 728 to amino acid 754 SEQ ID NO: 8; SEQ ID NO: 17; from amino acid 755 to amino acid 782 of SEQ ID NO: 8; SEQ ID NO: 18; from amino acid 783 to amino acid 810 of SEQ ID NO: 8; SEQ ID NO: 19; from amino acid 811 to amino acid 838 of SEQ ID NO: 8; SEQ ID NO: 20; from amino acid 839 to amino acid 866 of SEQ ID NO: 8; SEQ ID NO: 21; from amino acid 867 to amino acid 894 of SEQ ID NO: 8; SEQ ID NO: 22; from amino acid 895 to amino acid 922 of SEQ ID NO: 8; SEQ ID NO: 23; and from amino acid 923 to amino acid 923 to amino acid 950 of SEQ ID NO: 8; SEQ ID NO: 24); an isolated CARD-4S protein having an amino acid sequence that is at least about 65%, preferably 75%, 85%, 95% or 98% identical with the amino acid sequence of SEQ ID NO: 26; an isolated CARD-4S protein having an amino acid sequence that is at least about 85%, 95% or 98% identical with the CARD domain of SEQ ID NO: 26 (for example, from amino acid residues 1 to 74 of SEQ ID NO: 26; SEQ ID NO: 28). Also, within the present invention are contemplated the following: an isolated murine CARD-4L protein having an amino acid sequence that is at least about 65%, preferably 75%, 85%, 95% or 98% identical with the amino acid sequence of SEQ ID NO: 43. Likewise, the following are contemplated within the invention: an isolated CARD-4Y protein having an amino acid sequence that is at least about 65%, preferably 75%, 85% , 95%, or 98% identical to the amino acid sequence SEQ ID NO: 39. Also, within the present invention, the following are contemplated: an isolated CARD-4Z protein having an amino acid sequence that is at least about 65%, preferably 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 41. Also, within the present invention the following is contemplated: a CARD-3 protein isolated which is encoded by a nucleic acid molecule that has a nucleotide sequence that is at least 65%, preferably 75%, 85% or 95% identical to SEQ ID NO: 3 or to the ATCC cDNA; an isolated CARD-3 protein that is encoded by a nucleic acid molecule that is encoded by a nucleic acid molecule having a nucleotide sequence of at least about 65%, preferably 75%, 85% or 95% identical with the of kinase domain coding of SEQ ID NO: 1 (for example, about nucleotides 213 to 1113 of SEQ ID NO: 1); an isolated CARD-3 protein encoded by a nucleic acid molecule having a nucleotide sequence at least about 65%, preferably 75%, 85%, or 95% identical with the linker domain coding portion SEQ ID NO: 1 (for example, about nucleotides 114 to 1506 of SEQ ID NO: 1); and an isolated CARD-3 protein encoded by a nucleic acid molecule having a nucleotide sequence at least about 65%, preferably 75%, 85% or 95% identical with the CARD domain coding portion of SEQ ID NO: 1 (for example, about nucleotides 1507 to 1833 of SEQ ID NO: 1); and an isolated CARD-3 protein encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes under stringent conditions of hybridization with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 3 or the non-coding coding of ATCC cDNA.

Also, within the invention, the following is contemplated: an isolated CARD-4Y protein encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65%, preferably 75%, 85% or 95% identical to SEQ ID NO: 38 or to the ATCC cDNA. Also, within the invention, there are nucleic acid molecules that include from about nucleotides 2759 to 2842 of SEQ ID NO: 7; from about nucleotides 2843 to 2926 of SEQ ID NO: 7; from about nucleotides 2927 to 3010 of SEQ ID NO: 7; from about nucleotides 3011 to 3094 of SEQ ID NO: 7; and an isolated CARD-4L protein encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes, under stringent hybridization conditions, with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 9 or the non-coding ATCC cDNA chain. Also, within the present invention, the following is contemplated: an isolated CARD-4S protein encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65%, preferably 75%, 85%, or % identical with SEQ ID NO: 27 or the ATCC cDNA; an isolated CARD-3 protein that is encoded by a nucleic acid molecule having a nucleotide sequence of at least about 65%, preferably 75%, 85% or 95% identical to the CARD domain coding portion of SEQ ID NO: 25 (for example, approximately from nucleotides 1 to 222 of SEQ ID NO: 25); an isolated CARD-3 protein encoded by a nucleic acid molecule having a nucleotide sequence at least about 65%, preferably 75%, 85% or 95% identical to the coding portion of P loop of SEQ ID NO: 25 ( for example of about nucleotides 485 to 510 of SEQ ID NO: 25). Also within the present invention is a polypeptide that is an allelic variant that occurs naturally from a polypeptide that includes the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence encoded by the insert cDNA of the plasmid deposited with ATCC with number of Access, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes with a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions. Also, within the present invention is a polypeptide that is an allelic variant that naturally occurs from a polypeptide that includes the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC with Accession Number, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes with a nucleic acid molecule comprising SEQ ID NO: 7 or SEQ ID NO: 9 under stringent conditions. Also, within the present invention is a polypeptide that is an allelic variant that naturally occurs from a polypeptide that includes the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence encoded by the cDNA insert of the plasmid deposited before ATCC with Accession Number, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes with a nucleic acid molecule comprising SEQ ID NO: 25 or SEQ ID NO: 27 under stringent conditions. Another embodiment of the present invention features CARD-3 or CARD-4 nucleic acid molecules that specifically detect CARD-3 or CARD-4 nucleic acid molecules, relative to nucleic acid molecules encoding other members of the CARD superfamily. For example, in one embodiment, a CARD-4L nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO: 9, or the cDNA of ATCC, or a complement of it. In another embodiment, the nucleic acid molecule of CARD-4L has a length of at least 300 (350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1300, 1600, 1900, 2100, 2400, 2700, 3000, or 3382) nucleotides and hybrid under strict conditions to a molecule of nucleic acid comprising the nucleotide sequence illustrated in SEQ ID NO: 7, SEQ ID NO: 9, the ATCC cDNA, or a complement thereof. In another embodiment, an isolated CARD-4L nucleic acid molecule comprises nucleotides 287 to 586 of SEQ ID NO: 7, which encode CARD-4L CARD domain, or a complement thereof. In another embodiment, the invention provides an isolated nucleic acid molecule that is antisense to the coding strand of a CARD-4L nucleic acid. Another aspect of the invention offers a vector, for example a recombinant expression vector, comprising a CARD-3 or CARD-4L nucleic acid molecule of the invention. In another embodiment, the invention offers a host cell that contains such a vector. The invention also provides a method for producing by culture a CARD-3 or CARD-4 protein in a suitable medium, producing a host cell of the invention containing a recombinant expression vector such as a CARD-3 or CARD-4 protein. . Another aspect of this invention features isolated or recombinant CARD-3 or CARD-4 proteins and polypeptides. Preferred CARD-3 or CARD-4 proteins and polypeptides possess at least one biological activity possessed by CARD-3 or human CARD-4 occurring in nature, for example, (1) the ability to form protein: protein interactions with proteins in the apoptotic signaling path; (2) the ability to form CARD-CARD interactions with proteins in the apoptotic signaling pathway; (3) the ability to bind the ligand of CARD-3 or CARD-4, (4) the ability to bind to an intracellular target. Other activities include: (1) modulation of cell proliferation, (2) modulation of cell differentiation and (3) modulation of cell death (4) modulation of the NF-? B pathway. The CARD-3 or CARD-4 proteins of the present invention or biologically active portions thereof can be operably linked to a non-CARD-3 or non-CARD-4 polypeptide (e.g., heterologous amino acid sequences) to form proteins of fusion CARD-3 or CARD-4, respectively. The invention also presents antibodies that specifically bind CARD-3 or CARD-4 protein, such as, for example, monoclonal or polyclonal antibodies. In addition, the CARD-3 or CARD-4 proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers. In another aspect, the present invention provides a method for detecting the presence of CARD-3 or CARD-4 activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of CARD activity. -3 or CARD-4 in such a way that the presence of CARD-3 or CARD-4 activity is detected in the biological sample. In another aspect, the invention provides a method for modulating CARD-3 or CARD-4 activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) CARD-3 or CARD-4 activity or expression. in such a way that the activity or expression of CARD-3 or CARD-4 in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds with CARD-3 or CARD-4 protein. in another embodiment, the agent modulates the expression CARD-3 or CARD-4 by modulating the transcription of a CARD-3 or CARD-4 gene by splicing a CARD-3 or CARD-4 mRNA, or by transferring a mRNA from CARD-3 or CARD-4. in another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of CARd-3 or CARD-4 mRNA, or the CARD-3 gene or the CARD gene. -4. In one embodiment, the methods of the present invention are used to treat a patient having a disorder characterized by a CARD-3 or CARD-4 protein or a nucleic acid expression or an aberrant activity by administering an agent that is a CARD-3 or CARD-4 modulator to the patient. In one embodiment, the CARD-3 or CARD-4 modulator is a CARD-3 or CARD-4 protein. In another embodiment the modulator CARD-3 or CARD-4 is a nucleic acid molecule CARD-3 or CARD-4. In other embodiments, the CARD-3 or CARD-4 modulator is a peptide, peptidomimetic, or another small molecule. The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of the following: (i) aberrant modification or mutation of a gene encoding a CARD-3 or CARD-protein 4 (ii) erroneous regulation of a gene encoding a CARD-3 or CARD-4 protein, (iii) aberrant RNA splicing; and (iv) aberrant post-translational modification of CARD-3 or CARD-4 protein, where a wild-type form of the gene encodes a protein with a CARD-3 or CARD-4 activity. In another aspect, the invention provides a method for identifying a compound that binds or modulates the activity of a CARD-3 or CARD-4 protein. In general, such methods incorporate the measurement of a biological activity of a CARD-3 or CARD-4 protein in the presence and absence of a test compound and the identification of compounds that alter the activity of the CARD-3 or CARD-3 protein. Four. The invention also presents methods for identifying a compound that modulates CARD-3 expression or CARD-4 by measuring CARD-3 or CARD-4 expression in the presence and absence of a compound. Other features and advantages of the invention will be apparent from the following detailed description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents the cDNA sequence (SEQ ID NO: 1) of human CARD-3. The open reading frame of CARD-3 (SEQ ID NO: 1) extends from nucleotide 213 to nucleotide 1833 (SEQ ID NO: 3). Figure 2 illustrates the predicted amino acid sequence (SEQ ID NO: 2) of human CARD-3. Figure 3 illustrates the cDNA sequence (SEQ ID NO: 7) of CARD-4L. The open reading frame of SEQ ID NO: 7 extends from nucleotide 245 to nucleotide 3103 (SEQ ID NO: 9). Figure 4 illustrates the predicted amino acid sequence (SEQ ID NO: 8) of human CARD-4L. Figure 5 illustrates the partial cDNA sequence (SEQ ID NO: 5) of CARD-4S and the predicted amino acid sequence (SEQ ID NO: ) of human CARD-4S. The open reading frame of CARD-4 (SEQ ID NO: 25) extends from nucleotide 1 to nucleotide 1470 (SEQ ID NO: 27). Figure 6 represents the predicted amino acid sequence (SEQ ID NO: 26) of human CARD-4S. Figure 7 illustrates an alignment of the CARD domains of CARD-4 (SEQ ID NO: 10), CARD-3 (SEQ ID NO: 6), ARC-CARD (SEQ ID NO: 31), cIAPl-CARD (SEQ ID NO: 32) and CIAP2-CARD (SEQ ID NO: 33). Figure 8 is a graph showing the predicted structural characteristics of human CARD-4L. Figure 9 is a graph showing the predicted structural characteristics of human CARD-4S. Figure 10 illustrates the cDNA sequence (SEQ ID NO: 38) of the human CARD-4Y splicing variant clone. The predicted open reading frame of the human CARD-4Y splice variant clone extends from nucleotide 438 to nucleotide 1184. Figure 11 shows the amino acid sequence (SEQ ID NO: 39) of the protein of which predicts that it is encoded by the human CARD-4Y open cDNA reading frame. Figure 12 shows the cDNA sequence (SEQ ID NO: 40) of the human CARD-4Z splice variant clone. The predicted open reading frame of the human CARD-4Z splice variant clone extends from nucleotide 489 to nucleotide 980. Figure 13 shows the amino acid sequence (SEQ ID NO: 41) of the protein of which it is predicted which is encoded by the open reading frame of human CARD-4Z cDNA. Figure 14 shows an alignment of human CARD-4L (SEQ ID NO: 8), the predicted amino acid sequence of human CARD-4Y (SEQ ID NO: 39), and the predicted amino acid sequence of human CARD-4Z (SEQ. ID NO: 41). Figure 15 shows the nucleotide sequence of the murine CARD-4L cDNA (SEQ ID NO: 42). Figure 16 shows the predicted amino acid sequence of murine CARD-4L (SEQ ID NO: 43). Figure 17 shows an alignment of human CARD-4L (SEQ ID NO: 8) and the predicted amino acid sequence of murine CARD-4L (SEQ ID NO: 43). Figure 18 shows a genomic sequence of 32042 nucleotides of CARD-4. DETAILED DESCRIPTION OF THE INVENTION The present invention is based, in part, on the discovery that the cDNA molecules encoding human CARD-3, human CARD-4 and partial murine CARD-4L proteins. A nucleotide sequence encoding a human CARD-3 protein is shown in Figure 1 (SEQ ID NO: 1; SEQ ID NO: 3 includes only the open reading frame). A predicted amino acid sequence of CARD-3 protein is also shown in Figure 2 (SEQ ID NO: 2). CARD-4 has at least two forms, a long form, CARD-4L and a short form CARD-4S, as well as two or more splice variants. A nucleotide sequence encoding a human CARD-4L protein is shown in Figure 3 (SEQ ID NO: 7; SEQ ID NO: 9 includes only the open reading frame). A predicted amino acid sequence of CARD-4L protein is also shown in Figure 4 (SEQ ID NO: 8). A nucleotide sequence encoding a human CARD-4S protein is shown in Figure 5 (SEQ ID NO: 25; SEQ ID NO: 27 includes only the open reading frame). A predicted amino acid sequence of CARD-4S protein is also shown in Figure 6 (SEQ ID NO: 26). Two additional splice variants of human CARD-4 are provided in Figures 10 and 11 (human CARD-4Y) and Figures 12 and 13 (human CARD-4Z) (predicted amino acid sequences: SEQ ID NO: 39 and SEQ ID NO : 41 and the nucleic acid sequences: SEQ ID NO: 38 and SEQ ID NO: 40). It is predicted that these two splice variants contain 249 and 164 amino acids, respectively. Figure 14 shows an alignment of human CARD-4Y, human CARD-4Z and human CARD-4L. In addition to the human CARD-4 proteins, a murine orthologous total-length nucleotide sequence of human CARD-4L _ is provided in Figure 15 (SEQ ID NO: 42). An alignment of murine CARD-4L with human CARD-4L is shown in Figure 17. The human CARD-3 cDNA of Figure 1 (SEQ ID NO: 1), which has a length of about 1931 nucleotides including the regions untranslated, it encodes a protein amino acid that has a molecular weight of approximately 61 KDa (excluding post-translational modifications). A plasmid containing a cDNA encoding a human CARD-3 (named cDNA insert of ...) was deposited with the American Type Culture Collection (ATCC), Manasass, VA, on the day ... and received the Access Number ... This deposit will be maintained in accordance with the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. This deposit was made only for the convenience of those skilled in the art and is not an admission requiring a deposit in accordance with 35 U.S.C. §112. The human CARD-4L cDNA of Figure 3 (SEQ ID NO: 7), which has a length of about 3382 nucleotides including the non-translated regions, encodes an amino acid of protein having a molecular weight of approximately 108 KDa (excluding modifications). post-transnational). A plasmid containing a cDNA encoding human CARD-4L (with the name of cDNA insert of ...) was deposited with the American Type Culture Collection (ATCC), Manasass, VA, the day ... and received the Number of Access ... That deposit will be maintained in accordance with the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. This deposit was made only as a convenience to those skilled in the art and does not constitute an admission that such a deposit is required in accordance with 35 U.S.C. §112. The human CARD-4S cDNA of Figure 5 (SEQ ID NO: 25), which has a length of about 3082 nucleotides including the non-translated regions. A plasmid containing a cDNA encoding human CARD-4S (with the cDNA insert name of ....) was deposited with the American Type Culture Collection (ATCC), Manasass, VA, on the day ... and received the Access Number ... This deposit will be maintained in accordance with the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. This deposit was made simply as a convenience for those skilled in the art and does not constitute an admission in the sense that a deposit is required according to 35 U.S.C. §112. A region of human CARD-4L protein (SEQ ID NO: 8) bears some similarity to a CARD domain of CARD-3 (SEQ ID NO: 6), ARC-CARD (SEQ ID NO: 31), cIAPl-CARD (SEQ ID NO: 32), and CIAP2-CARD (SEQ ID NO: 33). This comparison is illustrated in Figure 7. CARD-3 or human CARD-4 are members of a family of molecules (the "CARD family") that have retained certain structural and functional characteristics. The term "family" when referring to the protein and nucleic acid molecules of the present invention means two or more protein or nucleic acid molecules having a common structural domain and having an identity of amino acid or nucleotide sequences sufficient in accordance with what is defined here. Such family members can occur naturally and can come from either the same species or from different species. For example, a family may contain a first protein of human origin and a homolog of this protein of murine origin, as well as a second distinct protein of human origin and a murine homologue of this protein. Members of a family can also common functional characteristics. In one embodiment, a CARD-3 or CARD-4 protein includes a CARD domain that is at least about 65%, preferably at least about 75%, and more preferably about 85%, 95%, or 98% sequence identity of amino acids with the CARD domain of SEQ ID NO: 6 or the CARD domain of SEQ ID NO: 10 or the CARD domain of SEQ ID NO: 28. The preferred CARD-3 or CARD-4 polypeptides of the present invention have a sequence of amino acid sufficiently identical with the CARD domain consensus amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 28, respectively. The CARD-3 polypeptide also has an amino acid sequence sufficiently identical with the consensus domain kinase sequence SEQ ID NO: 4 and an amino acid sequence sufficiently identical with the linker domain SEQ ID NO: 5. The CARD-4L polypeptide has a sequence of amino acids sufficiently identical with the nucleotide binding domain SEQ ID NO: 11, an amino acid sequence sufficiently identical with the Walker Chart "A" of SEQ ID NO: 12 or with the Walker Chart "B" of SEQ ID NO: 13, a sequence of amino acids sufficiently identical with the kinase subdomain of SEQ ID NO: 46, a sequence of amino acids sufficiently with the kinase 2 subdomain of SEQ ID NO: 47, or a sufficiently identical amino acid sequence with the kinase 3a subdomain of SEQ ID NO: 14, or an amino acid sequence sufficiently identical with the leucine-rich repeats of SEQ ID NO: 14, SEQ ID NO 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO 23. As used herein, the term "sufficiently identical" refers to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of nucleotides or identical or equivalent amino acid residues (e.g., an amino acid residue having a similar side chain) with a second amino acid or nucleotide sequence such that the first amino acid or nucleotide sequence and the second amino acid or nucleotide sequence have a common structural domain and / or a common functional activity. For example, amino acid or nucleotide sequences containing a common structural domain having approximately 65% identity, preferably 75% identity, more preferably 85%, 95% or 98% identity are defined herein as sufficiently identical. As used interchangeably herein, a "CARD-3 or CARD-4 activity", "CARD-3 or CARD-4 biological activity" or "CARD-3 or CARD-4 functional activity" refers to an activity performed by a protein, polypeptide or nucleic acid molecule of CARD-3 or CARD-4 on a cell that responds to CARD-3 or CARD-4 in accordance with that determined in vivo, or in vitro, according to standard techniques. A CARD-3 or CARD-4 activity can be a direct activity, such as, for example, association with a second protein or enzymatic activity in a second protein, or an indirect activity, such as, for example, a cell signaling activity mediated by the interaction of the CARD-3 or CARD-4 protein. In one embodiment, a CARD-3 or CARD-4 activity includes at least one or several of the following activities: (i) interaction with proteins in the apoptotic signaling pathway (ii) interaction with a CARD-3 or CARD- ligand 4; or (iii) interaction with an intracellular target protein; (iv) indirect interaction with caspazas. For example, in Example 4, proteins containing CARD-3 are shown to be associated with proteins containing CARD-4. In example 9, CARD-4 proteins that induce NF-éB mediated transcription are shown. In example 10, CARD-3 and CARD-4 are shown enhancing the caspase activity 9. In another embodiment of the present invention there are isolated characteristics of CARD-3 or CARD-4 proteins and polypeptides having a CARD-3 activity or CARD-4.

In the following subsections various aspects of the invention are described in detail. I. Isolated Nucleic Acid Molecules One aspect of the present invention relates to isolated nucleic acid molecules encoding CARD-3 or CARD-4 proteins or biologically active portions thereof, as well as sufficient nucleic acid molecules to be used as hybridization probes for identifying nucleic acids encoding CARD-3 or CARD-4 (e.g., CARD-3 or CARD-4 mRNA) and fragments for use as polymerase chain reaction primers for the amplification or mutation of molecules of CARD-3 or CARD-4 nucleic acid. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., AR? M) and AD analogues? or AR? generated using nucleotide analogues. The nucleic acid molecule can be single chain or double chain, but preferably it is AD? double chain. An "isolated" nucleic acid molecule is a molecule separated from other nucleic acid molecules present in the natural source of the nucleic acid. Preferably an "isolated" nucleic acid is free of sequences (preferably protein coding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA in the organism from which the nucleic acid is derived. For example, in various embodiments, the CARD-3 or CARD-4L / S ISOLATED nucleic acid molecule may contain less than about 5kb, 4kb, 3kb, 2kb, lkb, 0.5kb or O.lkb of naturally flanking nucleotide sequences. the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. In addition, an "isolated" nucleic acid molecule, for example the cDNA molecule may be substantially free of other cellular material or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemical substances when synthesized chemically. A nucleic acid molecule of the present invention, for example, a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, the ATCC cDNA or ATCC cDNA, as a hybridization probe, acid molecules can be isolated CARD-3 or CARD-4 nucleic acid using standard hybridization and cloning techniques (eg, as described in Sanbrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd, ed., Cold Spring Harbor Laboratory , Cold Sprin Harbor Laboratory Press, Cold Sprin Harbor, NY, 1989).

A nucleic acid of the present invention can be amplified using cDNA, mRNA or genomic DNA as a template and employing suitable oligonucleotide primers according to standard polymerase chain reaction amplification techniques. The nucleic acid amplified in this way can be cloned into an appropriate vector and characterized by analysis of DNA sequences. In addition, oligonucleotides corresponding to oligonucleotide sequences of CARD-3 or CARD-4 can be prepared by standard synthetic techniques, for example, using an automated DNA synthesizer. In another embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleic acid molecule that is a complement to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, the ATCC cDNA or ATCC cDNA, or a portion thereof. A nucleic acid molecule complementary to a given nucleotide sequence is a molecule sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex. further. , the nucleic acid molecule of the present invention can encompass only a portion of a nucleic acid sequence encoding CARD-3 or CARD-4, for example, a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion of CARD-3 or CARD-4. The nucleotide sequence determined from the cloning of CARD-3 or human CARD-4, and the partial murine CARD-4 gene allows the generation of probes and primers designed to be used to identify and / or clone CARD-3 homologs or CARD-4 in other cell types, for example, from other tissues as well as CARD-3 or CARD-4 homologs and CARD-3 or CARD-4 orthologs from other mammals. The probes / primer specifically comprise substantially purified oligonucleotide. The oligonucleotide specifically comprises a region of nucleotide sequences that hybridizes under stringent conditions with at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 300, 350, or 400 consecutive nucleotides of the sense or anti-sense sequence SEQ ID NO: l, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, the ATCC cDNA, the ATCC cDNA, or of a mutant that occurs in nature SEQ ID NO: l, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, the cDNA of ATCC or the ATCC cDNA. Probes based on the nucleotide sequences in human CARD-3, human CARD-4 or murine CARD-4 can be used to detect transcripts or genomic sequences that encode the same proteins or identical proteins. The probe comprises a label group bound therein, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit to identify allelic and orthologous variants of the CARD-3 or CARD-4 proteins of the present invention, to identify cells or tissue that erroneously express a CARD-3 protein. or ACRD-4, as for example by measuring a level of nucleic acid encoding CARD-3 or CARD-4 in a sample of cells from a subject, for example, by detecting CARD-mRNA levels. 3 or CARD-4 or by determining whether an ACRD-3 or genomic CARD-4 gene has been mutated or removed. A nucleic acid fragment encoding a "biologically active portion of CARD-3 or CARD-4L" can be prepared by isolating a portion of SEQ ID? 0: 1, SEQ ID? 0: 3, SEQ ID? 0: 7 , SEQ ID? 0: 9, either the nucleotide sequence of the AD? C of ATCC, or the nucleotide sequence of AD? C of ATCC encoding a polypeptide having a biological activity CARD-3 or CARD-4, expressing the purified portion of CARD-3 or CARD-4 protein (for example, by recombinant expression in vitro) and evaluating the activity of the encoded portion of CARD-3 or CARD-4. For example, a nucleic acid fragment encoding a biologically active portion of CARD-3 or CARD-4 includes a CARD domain, for example, SEQ ID NO: 6 and SEQ ID NO: 10 or SEQ ID NO: 26. The invention encompasses in addition nucleic acid molecules that differ from the nucleotide sequence SEQ ID NO: l, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, the ATCC cDNA or the ATCC cDNA due to the degeneracy of the genetic code and therefore encode the same CARD-3 or CARD-4 protein as the encoded by the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, the ATCC cDNA or the ATCC cDNA. In addition to the nucleotide sequence of CARD-3 or human CARD-4 shown in SEQ ID NO: l, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO : 26, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, the ATCC cDNA, the ATCC cDNA, or the ATCC cDNA, and the murine CARD-4L cDNA sequence shown in SEQ ID NO: 42 will be observed by those skilled in the art that cDNA sequence polymorphisms that cause changes in the amino acid sequences of CARD-3 or CARD-4 may exist within the population (e.g., the human population). Such a genetic polymorphism in the CARD-3 or CARD-4 gene may exist between individuals within a population due to natural allelic variation. As used herein the term "gene" and the term "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a CARD-3 or CARD-4 protein, preferably a CARD-3 protein or CARD-4 of mammal. Such natural allelic variants can typically result in a variation of 1-5% in the nucleotide sequence of the CARD-3 or CARD-4 gene. Each and every one of the nucleotide variations of this equipment and the resulting amino acid polymorphisms in CARD-3 or CARD-4 that result from a natural allelic variation and that do not alter the functional activity of CARD-3 or CARD-4 are found within the scope of this invention. In addition, nucleic acid molecules that encode CARD-3 or CARD-4 proteins from other species (orthologs / CARD-3 or CARD-4 homologs) that have a nucleotide sequence that differs from the sequence of a CARD-3 or human CARD-4, are within the scope of the present invention. For example, Example 5 describes an ortholog of murine CARD-4. Nucleic acid molecules corresponding to natural allelic variants and CARD-3 or CARD-4 cDNA homologs of the present invention can be isolated on the basis of their identity with the nucleic acids of human CARD-3 or human or murine CARD-4 disclosed here by employing murine or human cDNA or a portion thereof as a hybridization probe in accordance with standard hybridization techniques under stringent hybridization conditions. In general, an allelic variant of a gene will be easily identifiable as being traced in the same chromosomal location as said gene. For example, in Figure 6 the chromosomal location of the human CARD-4 gene is chromosome 7 near the genetic marker SHGC-31928. Allelic variants of human CARD-4 will be easily identifiable by tracing at the CARD-4 locus on chromosome 7 near the genetic marker SHGC-31928. Accordingly, in another embodiment, an isolated nucleic acid molecule of the present invention has a length of at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1300, 1600 or 1931) nucleotides and hybrid under strict conditions with the nucleic acid molecule comprising the nucleotide sequence preferably the coding sequence of SEQ ID NO: 1, SEQ.

ID NO: 3, or ATCC cDNA. In another embodiment, an isolated nucleic acid molecule of the invention has a length of at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1300, 1640, 1900, 2200, 25010, 2800, 3100, or 3382) nucleotides and hybrid under strict conditions with the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 38, SEQ ID NO: 40, or the cDNA of ATCC. Accordingly, in another embodiment, an isolated nucleic acid molecule of the present invention has at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1300, 1640, 1900, 2200, 2500, 2800, 3100, 3300, 3600, 3900, 4200, or 4209) nucleotides in length and hybrid under stringent conditions with the nucleic acid molecule comprising the nucleotide sequences, preferably the coding sequences of SEQ ID NO: 42. As used in the term "hybrid under stringent conditions" is intended to describe hybridization and washing conditions in which at least 60% nucleotide sequences (65%, 70% , preferably 75%) identical among them typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A very limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride / sodium citrate (SSC) at a temperature of about 45 ° C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at a temperature 50-65 ° C. Preferably an isolated nucleic acid molecule of the present invention that hybridizes under stringent conditions to the SEQ ID sequence NO: l, SEQ ID NO: 3, the ATCC cDNA corresponds to a naturally occurring nucleic acid molecule. As used herein, a nucleic acid molecule (which occurs naturally) refers to a RNA molecule 0 AD? which has a sequence of nucleotides that occur in nature (for example that encodes a natural protein). In addition to naturally occurring allelic variants of the CARD-3 or CARD-4 sequence that may exist in the population, the person skilled in the art will additionally observe which changes can be introduced by mutation in the nucleotide sequence SEQ ID? 0: 1, SEQ ID NO: 3, SEQ ID? 0: 7, SEQ ID? 0: 9, SEQ ID? O: 25, SEQ ID? O: 26, SEQ ID? O: 38, SEQ ID? O: 40, SEQ ID? O: 42, the AD? C of ATCC the AD? C of ATCC, or the AD? C of ATCC, thus causing changes in the amino acid sequence of the CARD-3 protein CARD-4L / S encoded , the splice variant of CARD-4 or murine CARD-4 without altering the functional capacity of the splice variant of CARD-3, CARD-4L / S, CARD-4, or murine CARD-4 protein. For example, nucleotide substitutions can be made that cause amino acid substitutions in "non-essential" amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild type sequence of CARD-3, CARD-4L / S, CARD-4 splice variant, or murine CARD-4 protein (for example, the sequence of SEQ ID? O: 2, SEQ ID? O: 8, SEQ ID? O: 26, SEQ ID? O: 39, SEQ ID? O: 41, and SEQ ID? O: 43) without altering biological activity, while, that an "essential" amino acid residue is required for biological activity. For example, amino acid residues conserved between CARD-3, CARD-4L / S, CARD-4 splice variant, or murine CARD-4 proteins of several species are predicted to be especially difficult to alter. For example, preferred CARD-3 or CARD-4 proteins of the present invention contain at least one CARD domain. In addition, a CARD-3 protein also contains a kinase domain or at least one linker domain. A CARD domain contains at least one nucleotide binding domain or leucine-rich repeats. Such conserved domains are less likely to be able to present mutations. Other amino acid residues, however (for example, those that are not conserved or only semi-preserved between CARD-3 or CARD-4 of several species) may not be special for the activity and therefore are more likely to be altered. Accordingly, another aspect of the present invention relates to nucleic acid molecules that encode CARD-3 or CARD-4 proteins that contain changes in amino acid residues that are not essential for activity. Such CARD-3 or CARD-4 proteins differ in terms of the amino acid sequence SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 25, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43, and yet retain their biological activity. In the embodiment, the isolated nucleic acid molecule includes a nucleotide sequence that encodes a protein that includes an amino acid sequence that is at least about 45% identical, 65%, 75%, 85%, 95% or 98% identical with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43. An isolated nucleic acid molecule encoding a CARD-3 protein or a CARD-4 protein having a sequence that differs from the sequence SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 41 or Well SEQ ID NO: 43, can be created by introducing one or more nucleotide substitutions, additions or deletions in the nucleotide sequence of CARD-3 (SEQ ID NO: 1, SEQ ID NO: 3, the ATCC cDNA ) Either CARD-4L (SEQ ID NO: 7, SEQ ID NO: 9, the ATCC cDNA), or CARD-4S (SEQ ID NO: 25, SEQ ID) NO: 27, the ATCC cDNA), or splice variants of Human CARD-4 (SEQ ID NO: 38, SEQ ID NO: 40, the ATCC cDNA or the ATCC cDNA), or murine CARD-4 (SEQ ID NO: 42) in such a way that one or more substitutions, additions or removals of amino acids are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and mutagenesis mediated by polymerase chain reaction. Preferably, conservative amino acid substitutions are made in one or more predicted nonessential amino acid residues. A "conservative amino acid substitution" is a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, licina, arginine, histidine), acid side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine). , tyrosine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains in beta (eg, threonine, valine, isoleucine), as well as chains aromatic sides (for example, tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue predicted in CARD-3 or CARD-4 is preferably replaceable with another amino acid residue of the same side chain family. Alternatively, mutations can be randomly introduced together with all or a part of a coding sequence of CARD-3 or CARD-4, such as by saturation mutagenesis, and the resulting mutants can be screened for the biological activity of CARD-3 or CARD-4 to identify mutants that retain their activity. After mutagenesis the encoded protein can be expressed recombinantly and the activity of the protein can be determined. In the modality a CARD-3 or a mutant CARD-4 protein can be tested for the following: (1) Determine the ability to form protein: protein interactions with proteins in the apoptotic signaling pathway; (2) the ability to bind a CARD-3 or CARD-4 ligand; or (3) the ability to bind with an intracellular white protein. For example (1) in Example 7, a 2-hybrid screening assay for the physical interaction of CARD-3 or CARD-4 is shown, (2) in Example 8 a 2-hybrid system assay for interaction is described between CARD-4 and its hNUDC ligand, (39 in Example 12, a co-immunoprecipitation assay is shown for the interaction of CARD-3 with its CARD-4 ligand. The mutant can be tested for the ability to modulate cell proliferation, cell differentiation, or cell death, for example, in Example 10, assays for the regulation of cell death (apoptosis) by CARD-3 are described. or CARD-4 In another embodiment, the CARD-3 or mutant CARD-4 protein can be tested for the regulation of a cellular signal transduction path, for example, in example 9, an assay for regulation by CARD-4 of the NF-KB pathway The present invention encompasses molecules of antisense nucleic acid, ie as molecules that are complementary to a sense nucleic acid encoding a protein, for example, complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, the antisense nucleic acid can bind hydrogen on a sense nucleic acid. The antisense nucleic acid may be complementary to a total CARD-3 or CARD-4 coding strand, or only a part thereof, for example, all of part of the protein coding region (or open reading frame) an antisense nucleic acid molecule can be antisense to a non-coding region of the coding strand of a nucleotide sequence is encoded CARD-3 or CARD-4. The non-coding regions ("5 'and 3' untranslated regions") are the 5 'and 3' sequences that flank the coding region and are not translated into the amino acids. Given the coding strand sequences encoding CARD-3 or CARD-4 disclosed herein (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42), antisense nucleic acids of the present invention can be designed in accordance with the rules of Watson &; Crick. The antisense nucleic acid molecule may be complementary to the entire CARD-3 or CARD-4L / S mRNA coding region, but is preferably an oligonucleotide that is antisense only to a portion of the coding region or not. mRNA coding CARD-3 or CARD-4. For example, the antisense oligonucleotide may be complementary to the region surrounding the CARD-3 mRNA translation start site, for example, an oligonucleotide having the sequence CCCTGGTACTTGCCCCTCCGGTAG (SEQ ID NO: 34) or CCTGGTACTTGCCCCTCC (SEQ ID NO. : 35), or of the CARD-4L mRNA, for example, TCGTTAAGCCCTTGAAGACAGTG (SEQ ID NO: 36) and TCGTTAGCCCTTGAAGACCAGTGAGTGTAG (SEQ ID NO: 37). An antisense oligonucleotide can be, for example, an oligonucleotide having a length of about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides. An antisense nucleic acid of the present invention can be constructed using enzymatic ligation and chemical synthesis reactions using methods known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using nucleotides that occur in nature or modified nucleotides in various ways designed to increase the biological stability of the molecules or to increase stability The physics of the duplexes formed between the antisense and sense nucleic acids, for example, phosphorothioate derivatives and nucleotides substituted by acridine can be used. Examples of modified nucleotides that can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl -2-thiouridine, 5-carboxylmethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil , 5-methoxyminomethyl-2-thiouracil, beta-D-mannosylkeosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wibutoxosine, pseudouracil, kerosine, 2-thiocytosine , 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically by employing an expression vector in which a nucleic acid has been subcloned in the antisense orientation (i.e., RNA transcribed from the inserted nucleic acid which will be of an antisense orientation with relationship to a target nucleic acid of interest, which is described more fully in the following subsection). The antisense nucleic acid molecules of the present invention are typically administered to a subject or generated in situ such that they hybridize or bind to cellular mRNA and / or genomic DNA encoding a CARD-3 or CARD-protein. 4 to thereby inhibit the expression of the protein as, for example, by inhibiting transcription and / or translation. Hybridization can be carried out or complementary to a conventional nucleotide to form a stable duplex or, for example, in the case of an antisense nucleic acid molecule that binds with DNA duplexes through specific interaction in the main cleft of the double helix. An example of a route of administration of antisense nucleic acid molecules of the present invention and includes direct injection into a tissue site. Alternatively, nucleic acid and antisense molecules can be modified to push selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified in such a way that they bind specifically to receptors or antigens expressed on the selected cell surface, for example, by binding the antisense nucleic acid molecules to peptides or antibodies that are bound on cell surface receptors or antigens. The antisense nucleic acid molecules can also be administered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs are preferred in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter. An antisense nucleic acid molecule of the present invention can be an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms double-stranded hybrids with complementary RNA in which, unlike the usual beta units, the chains are parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15: 6625- 6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al (1987) Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analog (Inoue et al., 1987). ) FEBS Lett 215: 327-330). The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that can dissociate a single-stranded nucleic acid, such as mRNA to which they have a complementary region. Thus, as ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591)) can be used to catalytically dissociate mRNA transcripts from CARD-3 or CARD-4 to inhibit this forms the translation of CARD-3 or CARD-4 mRNA. A ribozyme having specificity for nucleic acid encoding CARD-3 or CARD-4 can be designed based on the nucleotide sequence of a CARD-3 or CARD-4 cDNA disclosed herein (eg, SEQ ID N0: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42). For example, a derivative of an IVS RNA tetrahimena L-19 can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be dissociated in an mRNA encoding CARD-3 O card-4. See, for example, Cech et al American patent? 4,987,071; and Cech et al. North American patent? 5,116,742. Alternatively, CARD-3 or CARD-4 mRNA can be used to select an AR? Catalytic that has a ribonuclear activity is specified from a set of AR molecules ?. See, for example Bartel and Szostack (1993) Science 261: 1411-1418. The invention also encompasses nucleic acid molecules that form triple helical structures. For example, the expression of the CARD-3 or CARD-4 gene can be inhibited by targeting complementary nucleotide sequences to the regulatory region of CARD-3 or CARD-4 (eg, the promoter and / or CARD-enhancers). 3 or CARD-4) to form triple helical structures that prevent transcription of the CARD-3 or CARD-4 gene in target cells. See generally, Helene (1991) Anticancer Drug Des.6 (6): 569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660: 27-36; and Maher (1992) Bioassays 14 (12): 807-15. In embodiments, the nucleic acid molecules of the present invention can be modified in the base portion, in the sugar portion, or in the phosphate structure to improve, for example, the stability, hybridization or solubility of the molecule . For example, the deoxyribose phosphate structure of nucleic acids can be modified to generate peptide nucleic acids (See Hyrup et al (1996) Bioorganic &Medicinal Chemistry 4 (1): 5-23 As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, for example , DNA mimics, where the deoxyribose phosphate structure is replaced by a pseudopeptide structure and only the four natural nucleobases are conserved.The neutral structure of PNAs allows, as has been shown, the specific hybridization of DNA and RNA under conditions of low force Ionic The synthesis of PNA oligomers can be carried out using standard solid phase peptide synthesis protocols in accordance with that described in Hyrup et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Nati Acad. Sci. USA 93: 14670-675. CARD-3 or CARD-4 PNAs can be used for therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigenic agents for the sequence-specific modulation of gene expression by, for example, the induction of transcription or translational suspension or replication inhibition. CARD-3 or CARD-4 PNAs can also be employed, for example, in the analysis of single base-pair mutations in a gene by, for example, agitation by PNA-directed polymerase chain reaction; as "artificial restriction enzymes when used in combination with other enzymes such as, for example, Sl nucleases (Hyrup (1996) supra), either as probes or primers for DNA sequences and hybridization (Hyrup (1996) Proc. Nati. Acad. Sci. USA 93: 14670-675) In another embodiment, PNAs of CARD-3 or CARD-4 can be modified, for example, to increase their stability or cellular absorption, by fixing lipophilic groups or other auxiliary groups. PNA, wherein the formation of PNA-DNA chimeras, either by the use of liposomes or other pharmacological administration techniques known in the art, such as, for example, CARD-3 or CARD-PNA-DNA chimeras can be generated. 4 that can combine the very own of PNA and DNA.Such chimeras allow DNA recognition enzymes, for example, RNase H and ADβ polymerases to interact with the portion of ADβ while the portion provides a high affinity of binding. e and specificity. Chimeras P? A-AD? they can be joined by using linkers of appropriate lengths and selected in terms of base stacking, number of links between the nucleobases, and orientation (Hyrup (1996) supra). The synthesis of P? A-AD chimeras? it can be carried out in accordance with that described in Hyrup (1996) supra Finn et al. (1996)? Ucleic Acids Research 24 (17): 3357-63. For example, a chain of AD? can be synthesized on a solid support using standard phosphoramidite coupling chemistry as well as modified nucleoside analogs, for example, 5 '- (4-methoxytrityl) amino-5'-deoxy-thymidinephosphoramidite, can be used between P? A and extreme 5 'AD? (Mag et al., 1989) The P? A monomer is then progressively coupled to produce a chimeric molecule with a P? A segment of 5? And a segment of AD? Of 3? Finn et al. 1996)? Ucleic Acids Research 24 (17): 3357-63). Alternatively, the chimeric molecules can be synthesized with a segment of AD? of 5 'and a segment of P? A of 3' (Petersen et al. (1975) Bioorganic Med. Chem lett.5: 1119-11124). In other embodiments, the oligonucleotide can include other adjoining groups such as peptides (for example, for cell receptors or white host in vivo) or agents that facilitate transport across the cell membrane (see, for example, Letsinger et al. (1989) Proc. Nati, Acad. Sci. USA 84: 648-652, PCT publication No. WO 88/09810) or the blood-brain barrier (see, for example, PCT publication No. WO 89/10134). In addition, the oligonucleotides can be modified with cleavage agents activated by hybridization (see, for example Krol et al. (1988) Bio / Techniques 6: 958-976) or intercalating agents (see, for example, Zon (1988) Pharm.Res.5: 539-549). For this purpose, the oligonucleotide can be conjugated to another molecule, for example, a peptide, a crosslinking agent activated by hybridization, transport agent, dissociation agent activated by hybridization, etc. II. CARD-3 or CARD-4 proteins isolated and anti-CARD-3 or CARD-4 antibodies. One aspect of the present invention relates to isolated CARD-3 or CARD-4 proteins, and to biologically active portions thereof, as well as to fragments of polypeptides suitable for use as immunogens for raising anti-CARD-3 antibodies and in CARD-4. In one embodiment, native CARD-3 or CARD-4 proteins can be isolated from cells or tissue sources through an appropriate purification scheme employing standard protein purification techniques. In another embodiment, CARD-3 or CARD-4 proteins are produced by recombinant DNA techniques. Alternatively to recombinant expression, a CARD-3 or CARD-4 protein or polypeptide can be chemically synthesized using standard peptide synthesis techniques. An "isolated" or "purified" protein or a biologically active portion thereof is substantially free of cellular material or other contaminating proteins from a cell or woven source from which the CARD-3 or CARD protein is derived. -4, or substantially free of chemical precursors or other chemical substances when chemically synthesized. The expression "substantially free of cellular material" includes preparations of CARD-3 or CARD-4 protein wherein the protein is separated from cellular components of the cells from which it is isolated or produced recombinantly. Thus, the CARD-3 or CARD-4 protein substantially free of cellular material includes protein preparations of CARD-3 or CARD-4 having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-CARD-3 or CARD-4 protein (also referred to herein as a "contaminating protein"). When the CARD-3 or CARD-4 protein or a biologically thereof is produced recombinantly, it is also preferably substantially free of culture medium, ie the culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the CARD-3 or CARD-4 protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, for example, it is separated from chemical precursors or other chemicals involved in the synthesis of the chemical. protein. Accordingly, such CARD-3 or CARD-4 protein preparations generate less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or non-CARD-3 O CARD-4 chemicals. Biologically active portions of CARD-3 or CARD-4 protein include peptides comprising amino acid sequences sufficiently identical or derived from the amino acid sequence of the CARD-3 or CARD-4 protein (for example, the amino acid sequence illustrated in SEQ ID. NO: 2, SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 41, and SEQ ID NO: 43), which include fewer amino acids than CARD-3 or CARD-4 proteins of full length, and present at least a CARD-3 or CARD-4 protein activity. Typically the biologically active portions comprise a domain or motif with at least one activity of the CARD-3 or CARD-4 protein. A biologically active portion of CARD-3 or CARD-4 may be a polypeptide having, for example, a length of 10, 25, 50, 100 or more amino acids. Preferred biologically active polypeptides include one or more structural domains of CARD-3 or CARD-4 identified, for example, the CARD domain (SEQ ID NO: 6 or SEQ ID NO: 10 or SEQ ID NO: 7). In addition, other biologically active portions in which other regions of the protein are removed can be prepared by recombinant techniques and evaluated through one or more of the functional activities of a native CARD-3 or CARD-4 protein. A CARD-3 or CARD-4 protein has the amino acid sequence illustrated in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43. Other useful CARD-3 or CARD-4 proteins are substantially identical to SEQ ID NO: 2 , SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43 and retain the functional activity of the protein SEQ ID NO: 2 or SEQ ID NO: 8 or SEQ ID NO: 26 or SEQ ID NO: 39 or SEQ ID NO: 41 or SEQ ID NO: 43, and yet differ in terms of amino acid sequence due to natural allelic variation or mutagenesis. CARD-3 and CARD-4 are involved in the activation of caspases in the apoptotic pathway. For example, CARD-4 is shown in Example 10 which enhances caspase activity 9. Accordingly, a useful CARD-3 or CARD-4 protein is a protein that includes an amino acid sequence of at least about 45%, preferably 55%, 65%, 75%, 85%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 2 OR SEQ ID NO: 8 OR SEQ ID NO: 26 OR SEQ ID NO: 39 OR SEQ ID NO: 41 OR SEQ ID NO: 43 and retains the functional activity of the CARD-3 or CARD-4 proteins of SEQ ID NO: 2 OR SEQ ID NO: 8 OR SEQ ID NO: 26 OR SEQ ID NO: 39 OR SEQ ID NO: 41 OR SEQ ID NO: 43. In other cases, the CARD-3 or CARD-4 protein is a protein that has an amino acid sequence of 55%, 65%, 75%, 85%, 95% or 98%. % identical to the CARD-3 or CARD-4L CARD domains (SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 27). In the embodiment, the CARD-3 or CARD-4 protein retains a functional activity of the CARD-3 O CARD-4 protein of SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 26, SEQ ID NO: 39 O SEQ ID NO: 41 OR SEQ ID NO: 43. To determine the percentage identity of 2 amino acid sequences or 2 amino acids, the sequences are aligned for optimal comparison purposes (for example, spaces can be inserted in the sequence of A first amino acid or nucleic acid sequence for optimal alignment with a second nucleic acid or amino acid sequence The amino acid or nucleotide residues at corresponding amino acid positions or corresponding nucleotide positions are then compared When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical in this position. uencias depends on the number of identical positions shared by the sequence (ie, percentage of identity equal to the number of identical positions / total number of positions x 100). The determination of the percentage homology between two sequences can be achieved using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm employing for the comparison of 2 sequences is the algorithm of Karlin and Altschul (1990) Proc. Nat'l Acad. Sci. USA 87: 2264-2268, modified by Karlin Altschuln (1993) Proc. Nat'l Acad. Sci. USA 90: 5873-5877. Said algorithm is incorporated in the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215: 403-410. Search for nucleotides according to BLAST can be carried out with the NBLAST program, result = 100, word length = 12 to obtain similar nucleotide sequences or homologous the CARD-3 or CARD-4 nucleic acid molecules of the invention. For example, Example 5 describes the use of the TBLASTN program to investigate a database of full length and partial length cDNA sequence sequences with the human CARD-4 polypeptide sequence leading to the discovery of murine CARD-4 and the Example 4 describes the use of BLASTN to investigate a proprietary ETS database or with the untranslated sequence of CARD-4 leading to the discovery of 2 splice variants of human CARD-4. Searches of protein with BLAST can be carried out with the XBLAST program, result = 50, length = 3 to obtain amino acid sequences homologous to the CARD-3 or CARD-4 protein molecules of the invention. To obtain spaced alignments for comparison purposes, spaced BLAST can be employed as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When using the spaced BLAST and BLAST programs, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used. See http: // www. ncbi. nlm.nih. gov. Another preferred non-limiting example of a mathematical algorithm used for the comparison of sequences is the algorithm Myers and Miller, CABIOS (1989). This algorithm is incorporated into the ALIGN program (version 2.0) that is part of the GCG sequence alignment software package. When the ALIGN program is used to compare amino acid sequences, a PAM 120 weighted residual table can be used. A space length penalty of 12, and a space penalty of 4. The percentage identity between two sequences can be determined using techniques similar to the techniques described above, with or without spaces. To calculate the percentage identity, only exact correspondences are counted. The invention also offers CARD-3 or CARD-4 fusion or chimeric proteins. As used herein, a "chimeric protein" or "fusion protein" of CARD-3 or CARD-4 comprises a CARD-3 or CARD-4 polypeptide operably linked to a non-CARD-3 or CARD-4 polypeptide. A "CARD-3 or CARD-4 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to CARD-3 or CARD-4L / S, splice variants of murine CARD-4 or human CARD-4 , while a "non-CARD-3 or CARD-4 polypeptide" refers to a polypeptide having an amino acid sequence that corresponds to a protein not substantially identical to a CARD-3 or CARD-4L / S, CARD-4 protein of murine, or variant of splicing of human CARD-4, etc., a protein different from CARD-3 or CARD-4 proteins and that is derived from the same organism or from a different organism. Within a fusion protein of CARD-3 or CARD-4L, the CARD-3 or CARD-4 polypeptide may correspond to all or a portion of a CARD-3 or CARD-4 protein, preferably at least a portion biologically active of a CARD-3 or CARD-4 protein. Within the fusion protein, the term "operably linked" is intended to indicate that the CARD-3 or CARD-4 polypeptide and the non-CARD-3 or non-CARD-4 polypeptide are fused in frame with each other. The non-CARD-3 or non-CARD-4 polypeptide can be fused to the N-terminus or the C-terminus of the CARD-3 or CARD-4 polypeptide. A useful fusion protein is a GST-CARD-3 or GST-CARD-4 fusion protein wherein the CARD-3 or CARD-4 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of CARD-3 or recombinant CARD-4. In another embodiment, the fusion protein contains a signal sequence for another protein. In certain host cells (e.g., mammalian host cells), the expression and / or secretion of CARD-3 or CARD-4 can be increased through the use of a heterologous signal sequence. For example, the secretory gp67 sequence of a baculovirus envelope protein can be cooled as the heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., Eds., John Wiley & amp;; Sons, 1992). Other examples of heterologous eukaryotic signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene, La Jolla, California). In another example, useful eukaryotic heterologous signal sequences include a phoA secretory signal (Molecular cloning, Sambrook et al, second edition, Cold Spring Harbor Laboratory Press, 1989) and the protein A secretory signal (Pharmacia Biotech, Piscataway, New Jersey). In another embodiment, the fusion protein is a fusion protein of CARD-3 or CARD-4-immunoglobulin wherein all or part of CARD-3 or CARD-4 is fused onto sequences derived from a member of the protein family of immunoglobulin. The CARD-3 or CARD-4-immunoglobulin fusion proteins of the present invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit the interaction between a CARD-3 or CARD-4 ligand and a CARD-3 or CARD- 4 on the surface of a cell, to thereby suppress in vivo transduction of signal mediated by CARD-3 or CARD-4. The CARD-3 or CARD-4-immunoglobulin fusion proteins can be used to affect the bioavailability of cognate ligand of CARD-3 or CARD-4. The inhibition of the ligand interaction of CARD-3 / CARD-3 or CARD-4 / CARD-4 ligand may be useful therapeutically both for the treatment of proliferative and differentiation disorders and for modulating cell survival (for example, promoting or inhibit). In addition, the CARD-3 or CARD-4-immunoglobulin fusion proteins of the present invention can be used as immunogens to produce anti-CARD-3 or CARD-4 antibodies in a subject, to purify CARD-3 or CARD-4 ligands. in screening tests to identify molecules that inhibit the interaction of CARD-3 or CARD-4 with a CARD-3 or CARD-4 ligand. Preferably, a chimeric or fusion protein of CARD-3 or CARD-4 of the present invention is produced by standard recombinant DNA techniques. For example, DNA fragments encoding the different polypeptide sequences are joined together in a frame in accordance with conventional techniques, for example, by using flat ends or stepped ends for ligation, digestion with restriction enzyme to provide appropriate ends, filling of cohesive ends as appropriate, treatment with alkaline phosphatase to avoid undesirable binding, as well as enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, polymerase chain reaction amplification of gene fragments can be carried out by the use of anchor primers that provide complementary projections between two consecutive gene fragments that can be subsequently quenched and reamplified to generate a chimeric gene sequence (see , for example, Current Protocols in Molecular Biology, Ausubel et al., Eds., John Wiley &Sons: 1992). In addition, many expression vectors are commercially available which already encode a fusion portion (eg, GST polypeptide). A nucleic acid encoding CARD-3 or CARD-4 can be cloned into an expression vector of this type such that the fusion portion is bound in frame to the CARD-3 or CARD-4 protein. The present invention also relates to variants of the CARD-3 or CARD-4 proteins that function either as CARD-3 or CARD-4 (mimetics) agonists or as CARD-3 or CARD-4 antagonists. Variants of the CARD-3 or CARD-4 protein can be generated by mutagenesis, for example, discrete point or truncated mutation of the CARD-3 or CARD-4 protein. An agonist of the CARD-3 or CARD-4 protein can retain substantially the same biological activities or a subset of the biological activities in the naturally occurring form of CARD-3 or CARD-4 protein. A CARD-3 or CARD-4 protein antagonist can inhibit one or more of the activities of the naturally occurring form of the CARD-3 or CARD-4 protein by, for example, competitive binding on a downstream member or upstream of a cellular signaling cascade that includes the CARD-3 or CARD-4 protein. A) Yes, specific biological effects can be caused by treatment with a variant of limited function. The treatment of a patient with a variant that has a subset of the biological activities of the protein form occurring in nature may have fewer side effects in a patient relative to the treatment with the naturally occurring form of the CARD proteins. 3 or CARD-4. Variants of the CARD-3 or CARD-4 protein that function either as CARD-3 or CARD-4 (mimetics) agonists or as CARD-3 or CARD-4 antagonists should be identified by screening combination libraries of mutants, for example, truncated mutants of the CARD-3 or CARD-4 protein for the CARD-3 or CARD-4 protein agonist or antagonist activity. In one embodiment, a varied library of variants of CARD-3 or CARD-4 is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a varied gene library. A varied library of variants of CARD-3 or CARD-4 can be produced, for example, by the enzymatic ligand of a mixture of synthetic oligonucleotides in gene sequences in such a way that a degenerate set of potential sequences of CARD-3 or CARD -4 can be expressed as individual polypeptides, or alternatively, as a larger fusion protein set (e.g., for phage display) that it contains in the set of CARD-3 or CARD-4 sequences therein. There are several methods that can be employed to produce libraries of potential CARD-3 or CARD-4 variants from a degenerate sequence of oligonucleotides. The chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic gene can be ligated into an appropriate expression vector. The use of a degenerate set of genes allows the delivery, in admixture of all the sequences encoding the desired set of potential sequences of CARD-3 or CARD-4. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11: 477). Useful fragments of CARD-3 or CARD-4 INCLUDE fragments comprising or consisting of a domain or subdomain described herein, for example, a kinase domain or a CARD domain. In addition, libraries of fragments of the CARD-3 or CARD-4 protein coding sequence can be used to generate a varied population of CARD-3 or CARD-4 fragments for screening and subsequent selection of variants of a CARD-3 protein or CARD-4. In one embodiment, a library of coding sequence fragments can be generated by treating a double-stranded polymerase chain reaction fragment of a coding sequence of CARD-3 or CARD-4 with a nuclease at conditions in the which cut occurs only about once per molecule, denaturing the double helix DNA, renaturing the DNA to form double helix DNA that can include sense / antisense pairs from different cut products, remove the individual cut portions of the reformed duplexes by treatment with nuclease Sl, and ligand the resulting fragment library in an expression vector. Through this method, an expression library encoding N-terminal and internal fragments of various sizes of the CARD-3 or CARD-4 protein can be derived. Several techniques are known for screening genetic products from combinatorial libraries made by dot or truncation mutations, and for screening cDNA libraries for genetic products having a selected property. Such techniques are adaptable for rapid screening of gene libraries generated by the combinatorial mutagenesis of CARD-3 or CARD-4 proteins. The most widely used techniques that can be employed with high throughput analysis for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting vector library, and expression of the combinatorial genes under conditions in which the detection of a desired activity facilitates the isolation of the vector encoding the gene whose product was detected. Recursive assembly mutagenesis (REM), a technique that covers the frequency of functional mutants in libraries, or can be used in combination with the screening assay to identify variants of CARD-3 or CARD-4 (Arkin and Yourvan (1992) Proc). Nati, Acad Sci USA 89: 7811-7815, Delgrave et al. (1993) Protein Engineering 6 (3): 327-331). An isolated CARD-3 or CARD-4 protein, or a portion thereof, can be used as an immunogen to generate CARD-3 or CARD-4 binding antibodies using standard techniques for the preparation of polyclonal and monoclonal antibodies. The full length CARD-3 or CARD-4 protein can be used or, alternatively, the invention offers antigenic peptide fragments of CARD-3 or CARD-4 for use as immunogens. The antigenic peptide of CARD-3 or CARD-4 comprises at least 8 (preferably 10, 15, 20 or 30) amino acid residues of the amino acid sequence illustrated in SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 26, or SEQ ID NO: 39 or SEQ ID NO: 41 or SEQ ID NO: 43 or polypeptides including amino acids 128-139 or 287-298 of CARD-4L and encompasses an epitope of CARD-3 or CARD-4 in such a way that an antibody against the peptide forms a specific immune complex with CARD-3 or CARD-4. Useful antibodies include antibodies that bind over a domain or subdomain of CARD-3 or CARD-4 described herein, for example, a kinase domain or a CARD domain. Preferred epitopes encompassed by the antigenic peptide are CARD-3 or CARD-4 regions located on the surface of the protein, e.g., hydrophilic regions. Other important criteria include a preference for a terminal sequence, high antigenic index (e.g., as predicted by the Jameson-Wolf algorithm), ease of peptide synthesis (e.g., avoiding prolines); and high surface probability (for example, in accordance with that predicted by the Emini algorithm, figure 8 and figure 9). A CARD-3 or CARD-4 immunogen is typically used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation may contain, for example, a CARD-3 or CARD-4 protein expressed recombinantly or a chemically synthesized CARD-3 or CARD-4 polypeptide. The preparation may further include an adjuvant, such as a complete or incomplete Freund's adjuvant, or a similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic preparation of CARD-3 or CARD-4 induces a polyclonal anti-CARD-3 or CARD-4 antibody response. For example, polypeptides including amino acids 128-139 or 287-298 of human CARD-4L were conjugated to KLH and the resulting conjugates were used to immunize rabbits and polyclonal antibodies were generated that specifically recognize the two immunogenic peptides. Accordingly, another aspect of the invention relates to anti-CARD-3 or CARD-4 antibodies. The term "antibody" as used herein refers to immunoglobulin molecules and to immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that specifically binds an antigen, such as, for example, CARD-3 or CARD-4. A molecule that binds specifically to CARD-3 or CARD-4 is a molecule that binds to CARD-3 or CARD-4, but does not bind substantially to other molecules in a sample, for example, a biological sample, which contains naturally CARD-3 or CARD-4. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as, for example, pepsin. The invention offers polyclonal and monoclonal antibodies that are linked with CARD-3 or CARD-4. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of inreaction with a particular epitope of CARD-3 or CARD-4. A monoclonal antibody composition therefore typically exhibits a unique binding affinity for a particular CARD-3 or CARD-4 protein with which it immunoreacts. Polyclonal anti-CARD-3 or CARD-4 antibodies can be prepared in accordance with that described above by immunizing a suitable subject with a CARD-3 or CARD-4 immunogen. The anti-CARD-3 or CARD-4 antibody titer in the immunized subject can be monitored over time by standard techniques, such as for example with an enzyme-linked immunosorbent assay (ELISA) using CARD-3 or CARD- 4 immobilized. If desired, antibody molecules directed against CARD-3 or CARD-4 can be isolated from the mammal (eg, from the blood) and further purified by well-known techniques, such as, for example, protein A chromatography to obtain the fraction of IgG. At an appropriate time after immunization, for example, when the anti-CARD-3 or CARD-4 antibody titers are the highest, cells that produce antibodies from the subject can be obtained and used to separate monoclonal antibodies by techniques standards, such as for example the hybridoma technique generally described by Koller and Milstein (1975) Nature 256: 495-497, the human B-cell hybridoma technique (Kosbor et al. (1983) Immunol Today 4:72), the technique of EBV-hybridoma (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or the trioma techniques. The technology for the production of several anti-mydromalomas of monoclonal antibodies is well known (see in general Current Protocols in Immunology (1994) Coligan et al. (Eds.) JWiley & amp;; Sons, Inc., New York, NY). Briefly, a line of immortal cells (typically a myeloma) is fused with lymphocytes (typically splenocytes) from a mammal immunized with a CARD-3 or CARD-4 immunogen in accordance with that described above, and culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma that produces a monoclonal antibody that binds with CARD-3 or CARD-4. Any of the many known protocols used to fuse lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-CARD-3 or CARD-4 monoclonal antibody (see, for example, Current Protocols in Immunology, supra; Galfre et al. (1977) Nature 266: 55052; RH Kenneth, in Monoclonal Antibodies: A New Dimension in Biological Analyzes, Plenum Publishing Corp., New York, New York (1980); and Lerner (1981) Yale J. Biol. Med., 54 : 387-402 However, the person with certain knowledge in the art will note that there are numerous variations of such methods that could also be useful.Typically, the immortal cell line (e.g., myeloma cell line) is derived from the same mammalian species and in lymphocytes, for example, murine hybridomas can be made by melting lymphocytes from an immunized mouse with an immunogenic preparation of the present invention with a line of c mouse cells 1 immortalized, such as, for example, a myeloma cell line that is sensitive to a culture medium containing hypoxanthine, aminopterin and thymidine ("medium HAT"). Any of numerous myeloma cell lines can be used as a fusion partner in accordance with standard techniques, for example, the myeloma lines P3-NSl / l-Ag4-1, P3-x63-Ag8.653 or Sp2 / 0- Agl4. These lines of myeloma are available from ATCC. Typically, HAT sensitive mouse myeloma cells are fused onto mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using a HAT medium, which kills the unfused myeloma cells and the myeloma cells fused unproductively (the unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the present invention are detected by screening supernatants from the culture of hybridomas for antibodies that bind with CARD-3 or CARD-4, for example, using a standard ELISA assay. As an alternative to the preparation of hybridomas secreting monoclonal antibodies, an anti-CARD-3 or CARD-4 monoclonal antibody can be identified and can be isolated by screening a recombinant combinatorial immunoglobulin library (e.g. antibody phages) with CARD-3 or CARD-4 to thereby isolate immunoglobulin library members that bind with CARD-3 or CARD-4. Kits for generating and screening phage display libraries are commercially available (for example, the Pharmacia Recombinant Phage Antibody System, catalog No. 27-9400-01, and the Stratagene SurfZAP Phage Display Kit, catalog No. 240612) . In addition, examples of methods and reagents particularly suitable for use in generating and screening antibody visualization libraries can be found, for example, in U.S. Pat. 5,223,409; in the PCT publication no. WO 92/18619; in the PCT publication no. WO 91/17271; in the PCT publication no. WO 92/20791; in the PCT publication no. WO 92/15679; in the PCT publication no. WO 93/01288; in the PCT publication no. WO 92/01047; in the PCT publication no. WO 92/09690; in the PCT publication no. WO 90/02809; Fuchs et al. (1991) Bio Technology 9: 1370-1372; Hay et al. (1992) Hum.

Antibod. Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al. (1993) EMBO J 12: 725-734. In addition, recombinant anti-CARD-3 or CARD-4 antibodies, such as chimeric and humanized monoclonal antibodies, which comprise both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. present invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using methods described in PCT publication no. WO 87/02971; in European patent application 184,187; in European patent application 171,496; in European patent application 173,494; in the PCT publication no. WO 86/01533; in the North American patent no. 4,816,567; in European patent application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Nati Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526; Sun et al. (1987) Proc. Nati Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Nati. Cancer Inst. 80: 1553-1559); Morrison, (1985) Science 229: 1202-1207; Oi et al. (1986) Bio / Techniques 4: 214; in the North American Patent no. 5,225,539; Jones et al. (1986) Nature 321: 552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beilder et al. (1988) J. Immunol. 141: 4053-4060. An anti-CARD-3 or CARD-4 antibody (for example, a monoclonal antibody) can be used to isolate CARD-3 or CARD-4 by standard techniques, eg, affinity chromatography or immunoprecipitation. An anti-CARD-3 or CARD-4 antibody can facilitate the purification of native CARD-3 or CARD-4 from cells and CARD-3 or CARD-4 produced recombinantly expressed in host cells. In addition, an anti-CARD-3 or CARD-4 antibody can be used to detect a CARD-3 or CARD-4 protein (e.g., in a cell lysate or on either cell supernatant) in order to evacuate abundance and expression pattern of the CARD-3 or CARD-4 protein. Anti-CARD-3 or CARD-4 antibodies can be used for diagnostic purposes to monitor protein levels in tissue as part of a clinical test procedure, for example, to determine the efficacy of given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes influence sour radish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase, examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, aequorin, and examples of a suitable radioactive material include 125 I, 131 I, 35 S or 3 H.

III. Recombinant Expression Vectors and Host Cells Another aspect of the present invention relates to vectors, preferably expression vectors containing a nucleic acid encoding CARD-3 or CARD-4 (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid" that refers to a circular double-stranded DNA loop in which additional DNA segments can join. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and vectors of episomal mammals). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and are therefore replicated together with the host genome. In addition, some vectors, expression vectors, are capable of directing the expression of genes to which they are operatively linked.

In general, expression vectors of utility in recombinant DNA techniques are frequently in the form of plasmids (vectors) However, the invention includes other forms of expression vectors such as viral vectors (e.g., retroviruses, adenoviruses, and adeno-associated viruses with defective replication), which serve equivalent functions. The recombinant expression vectors of the present invention comprise a nucleic acid of the present invention in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected with base in the host cells to be employed for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, the term "operably linked" is intended to indicate that the nucleotide sequence of interest is linked to the sequence (s) in a manner that allows for the expression of the nucleotide sequence ( for example, in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described as, for example, in Goedel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those sequences that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct the expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will note that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of protein expression desired, etc. The expression vectors of the present invention can be produced in host cells to thereby produce protein or peptides, including proteins or fusion peptides, encoded by nucleic acid as described herein (for example CARD-3 or CARD-4 proteins). , mutant forms of CARD-3 or CARD-4, fusion proteins, etc.). The recombinant expression vectors of the invention can be designed for the expression of CARD-3 or CARD-4 in prokaryotic or eukaryotic cells, for example, bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) , yeast cells or mammalian cells. Suitable host cells are further discussed in Goedel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using regulatory sequences of T7 promoter and T7 polymerase. Expression of proteins in prokaryotes is most frequently carried out in E. Coli with vectors containing constitutive or inducible promoters that direct the expression of fusion proteins or non-fusion proteins. The fusion vectors add a number of amino acids to an encoded protein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) increase the expression of recombinant protein; 2) increase the solubility of the recombinant protein; and 3) helping to purify the recombinant protein by acting as a ligand in affinity purification. Frequently, in fusion expression vectors a proteolytic cleavage site is introduced at the junction of the fusion portion and the recombinant protein to allow separation of the recombinant protein from the fusion portion after purification of the fusion protein. Such enzymes, and their recognition sequences, include factor Xa, thrombin and enterokinase. Typical expression and fusion vectors include pGEX (Pharmacia Biotech Ine; Smith and Johnson (1998) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) that fuse glutathione S-transferase (GST), maltose binding protein E, or protein A, respectively, on the white recombinant protein. Examples of suitable inducible non-fusion E. Coli expression vectors include pTrc (Mann et al., (1998) Gene 69: 301-315) and pET lid (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). The expression of the target gene from the pTrc vector is based on the transcription of host RNA polymerase from a hybrid trp-lac fusion promoter. The expression of the target gene from the pET lid vector is based on transcription from a g7-lac T7 fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by BL21 (DE3) or HMS174 (DE3) host strains from a resistant prototype harboring a T7 gnl gene under the transcriptional control of the lacUV5 promoter. One strategy to optimize the expression of recombinant protein in E. coli is to express the protein in a host bacterium with an affected ability to proteolytically dissociate the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector such that individual strands for each amino acid are those preferentially employed in E. Coli (Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118). Said alteration of nucleic acid sequences of the present invention can be carried out by standard techniques of DNA synthesis. In another embodiment, the expression vector of CARD-3 or CARD-4 is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRYdd ( Schultz et al. (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA), pGBT9 (Clontech, Palo Alto, CA), pGADlO (Clontech, Palo Alto, CA), pYADE4 and pYGAE2 and pYPGE (Brunelli and Pall, (1993) Yeast 9: 1299-1308), pYPGE15 (Brunelli and Pall, (1993) Yeast 9: 1309-1318), pACTII (Dr. S.E. Elledge, Baylor College of Medicine), and picZ (Invitrogen Corp, San Diego, CA). For example, Example 7 describes the expression of a fusion protein comprising amino acids 1-145 of human CARD-4L fused to the DNA binding domain of the transcription factor of S. cerevisiae GAL4 from the vector of yeast expression pGBT9. In another example, Example 8 describes the expression e a fusion protein comprising amino acids 406-953 of human CARD-4L fused to the DNA binding domain of transcription factor of S. cerevisiae GAL-4 from the vector of yeast expression pGBT9. In another example, Example 7 describes the expression of a fusion protein comprising CARD-3 fused over the transcriptional activation domain of the transcription factor of S. cerevisiae GAL-4 from the expression vector of yeast pACTII. Alternatively, CARD-3 or CARD-4 can be expressed in insect cells using baculovirus expression vectors. Baculovirus expression vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39). In another embodiment, a nucleic acid of the invention is expressed in mammalian cells that employ a mammalian expression vector. Examples of mammalian expression vectors include pCDMd (Seed (1987) Nature 329: 840), pCI (Promega), and pMT2PC (Kaufman et al (1987) EMBO J. 6: 187-195). When used in mammalian cells, expression vector control functions are frequently provided by viral regulatory elements. For example, frequently used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook et al. (supra). For example, Example 9, Example 10 and Example 12 describe the expression of human CARD-4 or fragments thereof, CARD-3, or both from the mammalian expression vector pCI. In another embodiment, the recombinant mammalian expression vector can direct expression of the nucleic acid preferably in a particular cell type (for example, tissue-specific regulatory elements are used to express the nucleic acid). Regulatory elements specific for tissue are known in the art. Non-limiting examples of specific promoters for suitable tissue include the albumin promoter (specific for liver; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Caiame and Eaton (1988) Adv. Immunol. 43: 235-275), particularly T-cell receptor promoters (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g. neurofilament; Byrne and Ruddle (1989) Proc. Nati, Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230: 912-916), and specific mammary gland promoters (e.g., whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also encompassed, for example murine hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilgman (1989) Genes Dev. 3: 537-546 ). The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner that allows the expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to CARD-4 or CARD-4 mRNA. Regulatory sequences operably linked with a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in various cell types, for example viral promoters and / or enhancers, or regulatory sequences can be chosen. which direct direct constitutive expression, specific for tissue or specific cell-type antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, whose activity can be determined by the type of cell in question. where the vector is entered. For a comment on the regulation of gene expression using antisense genes, see Weintraub et al. (Reviews - Trends in Genetics, Vol. 1 (1) 1986). Another aspect of the present invention pertains to host cells in which a recombinant expression vector of the invention or isolated nucleic acid molecules of the invention were introduced. The terms "host cells" and "recombinant host cells" are used interchangeably herein. It is understood that these terms refer not only to the particular cell but to the progeny or potential progeny of said cell. Since certain modifications may occur in successive generations due either to mutation or to environmental influences, said progeny may in fact not be identical to the stem cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, CARD-3 or CARD-4 proteins can be expressed in bacterial cells such as for example E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary (CHO) cells or COS cells). . Other suitable host cells are known to those skilled in the art. For example, in Example 7 a Saccharomyces cerevisiae host cell is described for the expression of CARD-4 and recombinant CARD-3 and in Examples 9, 10, and 12 CARD-4 293T host cells or fragments thereof are described. or CARD-3.The vector DNA or an isolated nucleic acid molecule of the invention can be introduced into prokaryotic or eukaryotic cells through conventional transformation or transfection techniques. As used herein, the term "transformation" and the term "transfection" are intended to refer to various techniques known to introduce foreign nucleic acid (eg, DNA) into a host cell, including coprecipitation of calcium phosphate or calcium, transfection mediated by DEAE-dextran, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (supra), and other laboratory manuals. For a stable transfection of mammalian cells, it is known that, depending on the expression vector and the transfection technique employed, only a small fraction of the cells can integrate the foreign DNA into their genome. In some cases the vector DNA is retained by the host cell. In other cases, the host cell does not retain the vector DNA and retains only an isolated nucleic acid molecule of the invention carried by the vector. In some cases, the isolated nucleic acid molecule of the invention is employed to transform a cell without the use of a vector. In order to identify and select these integrants, a gene encoding a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells together with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. The nucleic acid encoding a selectable marker can be introduced into a host cell in the same vector as the nucleic acid encoding CARD-3 or CARD-4 or it can be introduced into a separate vector. Stably transfected cells with the introduced nucleic acid can be identified by drug selection (for example, cells that have the selectable marker gene incorporated will survive, while the other cells will perish). A host cells of the present invention, such as for example a prokaryotic or eukaryotic host cell in culture, can be used to produce a CARD-3 or CARD-4 protein (ie, express). Accordingly, the invention further provides methods for producing CARD-3 or CARD-4 protein using the host cells of the invention. In one embodiment, the method comprises culturing a host cell of the invention (in which a recombinant expression vector or an isolated nucleic acid molecule encoding CARD-3 or CARD-4 has been introduced) in a suitable medium of such that the CARD-3 or CARD-4 protein is produced. In another embodiment, the method further comprises the isolation of CARD-3 or CARD-4 from the medium or host cell. The host cells of the present invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the present invention is a fertilized oocyte or an embryonic stem cell into which CARD-3 or CARD-4 coding sequences were introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous CARD-3 or CARD-4 sequences were introduced into their genome or homologous recombinant animals in which the exogenous CARD-3 or CARD-4 sequences were disrupted. Such animals are useful for studying the function and / or activity of CARD-3 or CARD-4 and for identifying and / or evaluating modulators of CARD-3 or CARD-4 activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or a mouse, in which one or more of the cells of the animal includes (n) a transgen Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thus directing the expression of a genetic product encoded in one or several types of cells or tissues. of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous CARD-3 or CARD-4 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into an animal cell, for example, an embryonic cell of the animal, prior to the development of the animal. A transgenic animal of the present invention can be created by introducing nucleic acid encoding CARD-3 or CARD-4 into the male pronuclei of a fertilized oocyte, for example, by microinjection, retroviral infection and allowing. that the oocyte develops in a pseudo-pregnant female receptor animal. The cDNA sequence of CARD-3 or CARD-4, for example the sequence of (SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 42, or the ATCC cDNA, either the ATCC cDNA, or the ATCC cDNA) can be introduced as a transgene in the genome of a non-human animal. Alternatively, a homologue or non-human ortholog of the human CARD-3 or CARD-4 gene, such as the CARD-3 or mouse CARD-4 gene, can be isolated based on hybridization in the CARD-3 cDNA or Human CARD-4 and used as transgene. For example, the mouse ortholog of CARD-4, Figure 15 and SEQ ID No. 42 can be used to make a transgenic animal using standard methods. Intronic sequences and polyadenylation signals can also be included in the frangen to increase the efficiency of transgene expression. A specific regulatory sequence (s) for tissue can be operatively linked to the CARD-3 or CARD-4 transgene to direct the expression of CARD-3 or CARD-4 protein in particular cells. Methods for the generation of transgenic animals through embryo manipulation and microinjection, especially animals such mice, are conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, in the U.S. Patent.

Number 4, 873, 191 and in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1986). Similar methods are used for the production of other transgenic animals. A transgenic founder animal can be identified based on the presence of the CARD-3 or CARD-4 transgene in its genome and / or by the expression of CARD-3 or CARD-4 mRNA in animal tissues or cells. A transgenic founder animal can then be used to create additional animals that carry the transgene. In addition, transgenic animals carrying a transgene encoding CARD-3 or CARD-4 can be further crossed with other transgenic animals carrying other transgenes. To create a homologous recombinant animal, a vector is prepared that contains at least a portion of a CARD-3 or CARD-4 gene (eg, a human or non-human homologue of the CARD-3 or CARD-4 gene, as per example, a CARD-3 or murine CARD-4 gene) in which a removal, addition or substitution has been introduced to alter in this way, for example, functionally interrupting the CARD-3 or CARD-4 gene. In one embodiment, the vector is designed in such a way that, upon homologous recombination, the endogenous CARD-3 or CARD-4 gene is functionally disrupted (ie, it no longer encodes a functional protein, it is also known as a vector "knocked out") . Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous CARD-3 or CARD-4 gene is mutated or otherwise altered but continues to encode a functional protein. For example, the upstream regulatory region can be altered and therefore alter the expression of endogenous CARD-3 or CARD-4 protein). In the homologous recombination vector, the altered portion of the CARD-3 or CARD-4 gene is flanked at its 5 'and 3' ends by additional nucleic acid from the CARD-3 or CARD-4 gene to allow homologous recombination between the exogenous CARD-3 or CARD-4 gene carried by the vector and an endogenous CARD-3 or CARD-4 gene in an embryonic stem cell. The additional flank CARD-3 or CARD-4 nucleic acid is of sufficient length to allow successful homologous recombination with the endogenous gene. Typically, several kilobases of flank DNA are included (both at the 5 'end and at the 3' end in the vector) in the vector (see, for example, Thomas and Capecc i (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and the cells in which the introduced CARD-3 or CARD-4 gene has been recombined in a manner homologous to the endogenous CARD-3 gene or CARD-4 are selected (see, for example, Li et al (1992) Cell 69: 915). The selected cells are then injected into an animal's blastócisto (for example, a mouse) to form aggregation chimeras (see, for example, Bradley in Teratocarcinomas and Embryonic Stem Cells: A practical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo may then be implanted into a suitable pseudopregnant female recipient animal and the embryo brought to term. The progeny containing the homologously recombined DNA in their germ cells can be used to breed animals in which all the cells of the animal contain the homologously recombined DNA by germ line transcription of the Transgen. Methods for the construction of homologous recombination vectors and homologous recombinant animals are further described in Bradley (1991) Current Opinion in Bio / Technology 2: 823-829 and in PCT Publication Nos. WO 90/11354, WO 01140, WO 92/0968 , and WO 93/04169. In another embodiment, non-human transgenic animals can be produced which contain selected systems that allow regulated expression of the transgene. An example of such a system is the cre / loxP recombinase system of bacteriophage Pl. For a description of the cre / loxP recombined system, see, for example, Lakso et al. (1992) Proc. Nati Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O 'Gorman et al (1991) Science 251: 1351-1355). If a cre / loxP recombinase system is used to regulate transgene expression, animals that contain transgenes encoding both Cre recombinase and a selected protein are required. Such animals can be provided through the "double" transgenic construct, for example, by crossing two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Clones of the non-human transgenic animals described herein can also be produced in accordance with the methods described in Wilmut et al. (1997) Nature 385: 810-813 and in PCT Publication Nos. WO 97/07668 and WO 97/07669. In summary, a cell, for example, a somatic cell, from a transgenic animal can be isolated and introduced to exit the growth cycle and enter the Go phase. The quiescent cell can then be fused, for example, through the use of electrical impulses, over an enucleated oocyte of an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured in such a way that it develops into a morula or blastocyst and is then transferred to a pseudopregnant female recipient animal. The offspring born of this female receptor animal will be a clone of the animal from which the cell is isolated, as for example, the somatic cell. IV. Pharmaceutical Compositions CARD-3 or CARD-4 nucleic acid molecules, CARD-3 or CARD-4 proteins, and anti-CARD-3 or CARD-4 antibodies (also known herein as "active compounds") of the present invention can be incorporated in pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid, protein or antibody molecule of a pharmaceutically vehicle. As always here, the term "pharmaceutically acceptable carrier" has the purpose of including each and every one of the solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption and isotonic delay agents, and the like, compatible with pharmaceutical administration. . The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as a conventional medium or agent is incompatible with the active compound, its use in the compositions is contemplated. Complementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Exemplary routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; regulators such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be found in ampules, disposable syringes or multi-dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (soluble in water) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include a physiological saline solution, bacteriostatic water, Cremophor EL? (BASF; Parsippany, NJ) or a phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to allow easy syringability. From presenting stability in the conditions of manufacture and storage and must be preserved against the action of contamination of microorganisms such as bacteria and fungi. The vehicle can be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be achieved through various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride in the composition. The prolonged absorption of the injectable compositions can be realized by the inclusion in the composition of an agent that retards absorption, such as for example aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (eg, a CARD-3 or CARD-4 protein, or anti-CARD-3 or CARD-4 antibody in the required amount in a suitable solvent with an ingredient or a combination of the ingredients mentioned above, as required, followed by filtered sterilization Generally, the dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the other ingredients required among those mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization which provides a powder of the active ingredient plus any additional desired ingredient of a sterile, pre-filtered solution of the Oral compositions they generally include an inert diluent or an edible vehicle. It can be found in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, dragees or capsules. Oral compositions can also be prepared by employing a fluid vehicle for use as a mouthwash, where the compound in the fluid vehicle is applied orally and is spit on or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, dragees and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as for example microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as, for example, starch or lactose, a disintegrating agent such as, for example, alginic acid, Primogel, or corn starch; a lubricant such as, for example, magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, or orange flavor. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a container under pressure or a dispenser containing a suitable driving agent, for example, a gas such as carbon dioxide or an atomizer. Systemic administration can also be effected through transmucosal or transdermal means. For transmucosal or transdermal administration, appropriate penetration agents for the permeate barrier are employed in the formulation. Such penetration agents are generally known in the art and include, for example, in the case of transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. In the case of transdermal administration, the active compounds are formulated in ointments, gels or creams as is generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enema for rectal administration. In one embodiment, the active compounds are prepared with carriers that offer protection to the compound against rapid elimination from the body, as in the case of a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biodegradable polymers can be used, such as ethylene, vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for the preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes focused on cells infected with monoclonal antibodies to viral antigens) can also be employed as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art as, for example, in accordance with that described in US Pat. No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in the form of a dosage unit to facilitate administration and uniformity of dosage. The dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined amount of active compound which is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention directly depend on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and on the limitations inherent in the art of forming compounds with such an active compound for the treatment of individuals. The nucleic acid molecules of the present invention can be inserted into vectors and used as vectors for gene therapy. Genetic therapy vectors can be administered to a subject, for example by intravenous injection, local administration (US Pat. No. 5,328,470) or by stereotactic injection (see, for example, Chen et al (1994) Proc Nati. Acad. Sci USA 91 : 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or it can comprise a slow release matrix in which the gene delivery vehicle is integrated. Alternatively, when the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

The pharmaceutical compositions may be included in a container, package or dispenser together with instructions for administration. V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologs, and antibodies described herein may be employed in one or more of the following methods: a) screening assays; b) detection assays (eg, chromosomal mapping, tissue typology, forensic biology), c) predictive medicine (eg, diagnostic tests, prognostic trials, clinical monitoring trials, and far-reach hospitality, and d) methods of treatment (for example, therapeutic and prophylactic). A CARD-3 or CARD-4 protein interacts with other cellular proteins and can therefore be used to (i) regulate cell proliferation; (ii) regulate cell differentiation; (iii) regulate cell survival. The isolated nucleic acid molecules of the present invention can be used to express CARD-3 or CARD-4 protein (e.g., through a recombinant expression vector in a host cell in gene therapy applications), to detect CARD mRNA -3 or CARD-4 (for example in a biological sample), or a genetic lesion in a CARD-3 or CARD-4 gene and to modulate the activity of CARD-3 or CARD-4 and to modulate CARD activity -3 or CARD-4. in addition, the CARD-3 or CARD-4 proteins can be used to screen drugs or compounds that modulate CARD-3 or CARD-4 activity or its expression as well as to treat disorders characterized by insufficient or excessive production of CARD-3 protein or CARD-4 or the production of CARD-3 or CARD-4 protein forms that have a decreased or aberrant activity compared to wild-type CARD-3 or CARD-4 protein. In addition, anti-CARD-3 or CARD-4 antibodies of the present invention can be used to detect and isolate CARD-3 or CARD-4 proteins and modulate CARD-3 or CARD-4 activity. This invention also relates to novel agents identified by the screening assays described above and their use for treatment in accordance with what is described herein. A. Screening Assays The invention provides a method (also known as a "screening assay") for identifying modulators, ie candidate or test compounds or agents (eg, peptides, peptomimetics, small molecules or other drugs) that they bind with the CARD-3 or CARD-4 proteins or biologically active portions thereof or have a stimulation or inhibition effect, for example of the expression CARD-3 or CARD-4 or of the CARD-3 activity or CARD-4. an example of a biologically active portion of human CARD-4 is amino acids 1-145 encoding the CARD domain which is sufficient to show a CARD-3 binding activity in accordance with that described in example 7. Amino Acids 406-953 of human CARD-4L comprising the LRR domain represent a biologically active portion of CARD-4L since they possess a binding activity with hNUDC in accordance with that described in the example. In one embodiment, the invention provides assays for screening test compounds or candidates that bind CARD-3 or CARD-4 proteins or polypeptides or biologically active portions thereof or that modulate the activity of said CARD-3 proteins or CARD-4 or polypeptides or biologically active portions thereof. The test compounds of the present invention can be obtained by employing any of numerous approaches in combination library methods known in the art, including: biological libraries, spatially steerable parallel phase solution or solid phase libraries; methods of synthetic libraries that require deconvolution; the library method of "one pearl one compound"; and methods of synthetic libraries that employ selection by affinity chromatography. The biological library approach is limited to the peptide libraries, while the other four approaches apply to libraries of peptide compounds, non-peptide oligomers, or small molecules (Lam (1997) Anticancer Drug Des. 12: 145) . Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Nati Acad. Sci. U.S.A. 90: 6909; Erb et al. (1994) Proc. Nati Acad. Sci. USA 91: 11422; Zuckermann et al. (1994). J. Med. Chem. 37: 2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) J. Med. Chem. 37: 1233. Compound libraries can be presented in solution (eg, Hougten (1992) Bio / Techniques 13: 412-421), or in beads (Lam (1991) Nature 354: 82-84), in bacteria (US Patent number 5,223,409), in spores (Patents Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Culi et al. (1992) Proc. Nati. Acad. Sci. USA 89: 1865-1869) or on phage) (Scott and Smith (1990) Science 249 : 386-390; Devlin (1990) Science 249: 404-406; Cwirla et al. (1990) Proc. Nati. Acad. Sci. 87: 6378-6382; and Felici (1991) J. Mol. Biol. 222: 301-310). The determination of the ability of the test compound to modulate the activity of CARD-3 or CARD-4 of a biologically active portion can be achieved, for example, by determining the ability of the CARD-3 or CARD-4 protein to bind or interact with a white molecule CARD-3 or CARD-4. As used herein, a "white molecule" is a molecule with which a CARD-3 or CARD-4 protein binds or interacts in nature, for example, a molecule associated with the inner surface of a cell membrane or a molecule cytoplasmic A white CARD-3 or CARD-4 molecule can be a non-CARD-3 or CARD-4 molecule or a CARD-3 or CARD-4 protein or polypeptide of the present invention. In one embodiment, a white CARD-3 or CARD-4 molecule is a component of an apoptotic signal transduction pathway, for example, CARD-3 and CARD-4. The blank, for example, may be a second intracellular protein having a catalytic activity or a protein that facilitates the association of downstream signaling molecules with CARD-3 or CARD-4. In another embodiment, target molecules of CARD-3 or CARD-4 include CARD-3 since it was found that CARD-3 bound to CARD-4 (example 7 and 12) and hNUDC because it was found that hNUDC binds to CARD- 4 (example 8). The determination of the ability of the test compound to modulate the activity of CARD-3 or CARD-4 or a biologically active portion thereof can be achieved, for example, by determining the capacity of the CARD-3 or CARD-3 protein. 4 to bind or interact with any of the specific proteins listed in the previous paragraph as white molecules of CARD-3 or CARD-4. In another embodiment, target molecules of CARD-3 or CARD-4 include all proteins that bind with a CARD-3 or CARD-4 protein or fragment thereof in a two-hybrid system binding assay that can be employed without Exaggerated experimentation to isolate said proteins from genomic two-hybrid system libraries or cDNA. For example, Example 7 describes the use of the CARD-4 CARD domain region to identify CARD-3 in a two-hybrid screening and Example 8 describes the use of the LRR region of CARD-4 to identify hNUDC in a sieving of two hybrids. The binding assays described in this section could be cell-based or cell-free (described below). The determination of the ability of the CARD-3 or CARD-4 protein to bind or interact with a target molecule of CARD-3 or CARD-4 can be achieved by one of the methods described above to determine a direct link. In one embodiment, the determination of the ability of the CARD-3 or CARD-4 protein to bind or interact with a target molecule of CARD-3 or CARD-4 can be achieved by determining the capacity of the target molecule. For example, the activity of the target molecule can be determined by detection of induction of a second target cell messenger (eg, intracellular Ca2 +, diacylglycerol, IP3, etc.), detection of target catalytic / enzymatic activity on an appropriate substrate detecting the induction of a reporter gene (eg, a regulatory element that responds to CARD-3 or CARD-4 operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase) or by detecting a cellular response, for example, cell survival, cell differentiation or cell proliferation. For example, in example 12, CARD-4 binds with CARD-3 and in example 10, by monitoring a cellular response, it is shown that CARD-4 enhances caspase 9 activity, cell death or apoptosis. Since CARD-3 and CARD-4 enhance caspase 9 activity, the activity of CARD-3 and CARD-4 can be monitored by caspase-9-mediated apoptosis cell response assay or caspase-9 enzymatic activity. , and in another modality, genes induced by the expression of CARD-3 and CARD-4 could be identified through the expression of CARD-3 or CARD-4 in a cell line and carrying out a transcriptional profile experiment where the patterns of mRNA expression of the cell line transformed with an empty expression vector and the cell line transformed with an expression vector of CARD-3 or CARD-4 are compared. Promoters of genes induced by the expression of CARD-3 or CARD-4 would be operatively linked on reporter genes suitable for screening such as for example luciferase, secreted alkaline phosphatase, or beta-galactosidase and the resulting constructs could be introduced into vectors of appropriate expression. A line of recombinant cells containing CARD-3 or CARD-4. and transfected with an expression vector containing a promoter responsive to CARD-3 or CARD-4 operatively linked to a reporter gene could be used to identify test compounds that modulate the activity of CARD-3 or CARD-4 by the assay of expression of the reporter gene in response to contacting the recombinant cell line with the test compounds. CARD-3 or CARD-4 agonists can be identified by increasing the expression of the reporter gene and antagonists of CARD-3 or CARD-4 can be identified as decreasing the expression of the reporter gene. In another embodiment of the invention, the ability of a test compound to modulate the activity of CARD-3, CARD-4, or biologically active portions thereof can be determined by assaying the ability of the test compound to modulate routes dependent on CARD-3 or CARD-4 or processes in which the CARD-3 or CARD-4 target proteins that mediate the effect of CARD-3 or CARD-4 are known or unknown. Pathways or processes dependent on potential CARD-3 or CARD-4 include, but are not limited to, the modulation of cellular signal transduction pathways and their second related messenger molecules (eg, intracellular Ca2 +, diacylglycerol, IP3, cAMP etc.) , cellular enzymatic activities, cellular responses (for example, cell survival, cell differentiation, or cell proliferation), or the induction or repression of cellular or heterologous mRNAs or proteins. Pathways dependent on CARD-3 or CARD-4 or CARD-3 or CARD-4 dependent processes could be assayed by standard cell-based or cell-free assays appropriate for the specific pathway or specific process under study. For example, Example 9 describes how the expression of CARD-4S or CARD-4L in 293T cells induces the? F-? B pathway in accordance with that determined by the measurement of a? F-path luciferase reporter gene. ? B co-transfected. In another embodiment, cells co-transfected with CARD-4 and the? F-? B luciferase reporter gene could come into contact with a test compound and test compounds that block CARD-4 activity could be identified by their reduction of luciferase reporter gene expression from the? F-? B pathway dependent on CARD-4. One would expect that test compounds that agonize CARD-4 increase reporter gene expression. In another embodiment, CARD-4 could be expressed in a cell line and the cell line expressing recombinant CARD-4 could come into contact with a test compound. Test compounds that inhibit the activity of CARD-4 could be identified by their reduction of the stimulation of the CARD-4-dependent? F-? B pathway of 12 * 0 conformance with that measured by the assay of a NF-? B route reporter gene, NF-? B nuclear localization, I? B phosphorylation or proteolysis, or other standard assays for activation of the NF- pathway? B known to those skilled in the art. In another embodiment, an assay of the present invention is a cell-free assay comprising contacting a CARD-3 or CARD-4 protein or a biologically active portion thereof with a test compound and determining the the ability of the test compound to bind with the CARD-3 or CARD-4 protein or a biologically active portion thereof. The binding of the test compound to the CARD-3 or CARD-4 protein can be determined either directly or indirectly in accordance with that described above. In one embodiment, a competitive binding assay includes contacting the CARD-3 or CARD-4 protein or a biologically active portion thereof with a compound known to bind CARD-3 or CARD-4 to form a mixture. of test, contacting the test mixture with a test compound, and determining the ability of the test compound to interact with a CARD-3 or CARD-4 protein, where the determination of the ability of the test compound to interact with CARD-3 or CARD-4 comprises the determination of the ability of the test compound to preferentially bind with CARD-3 i or CARD-4 or a biologically active portion thereof in comparison with the known binding compound. In another embodiment, an assay is a cell-free assay comprising contacting a CARD-3 or CARD-4 protein or a biologically active portion thereof with a test compound and determining the capacity of the compound of test to modulate (eg, stimulate or inhibit) the activity of the CARD-3 or CARD-4 protein or a biologically active portion thereof. The determination of the ability of the test compound to modulate the activity of CARD-3 or CARD-4 can be achieved, for example, by determining the ability of the CARD-3 or CARD-4 protein to bind to a white molecule of CARD-3 or CARD-4 by one of the methods described above to determine a direct bond. In an alternative embodiment, the determination of the ability of the test compound to modulate the activity of CARD-3 or CARD-4 can be carried out by determining the ability of the CARD-3 or CARD-4 protein to further modulate a target molecule of CARD-3 or CARD-4. For example, the catalytic / enzymatic activity of the target molecule in an appropriate substrate can be determined in accordance with what has been previously described. In another embodiment, the cell-free assay comprises contacting the CARD-3 or CARD-4 protein or a protein. biologically active portion thereof with a known compound that binds CARD-3 or CARD-4 to form an assay mixture, contact the test mixture with a test compound, and determine the ability of the test compound to interact with a CARD-3 or CARD-4 protein, where the determination of the ability of the test compound to interact with a CARD-3 or CARD-4 protein comprises the determination of the capacity of the CARD-3 or CARD-4 protein to preferentially bind or modulate the activity of a target molecule of CARD-3 or CARD-4. The cell-free assays of the present invention can be employed with both the soluble form and the membrane-associated form of CARD-3 or CARD-4. A membrane-associated form of CARD-3 or CARD-4 refers to CARD-3 or CARD-4 that interacts with a membrane bound white molecule. In the case of cell-free assays comprising the membrane associated form of CARD-3 or CARD-4, it may be desirable to use a solubilizing agent in such a way that the membrane-associated form of CARD-3 or CARD-4 is keep in solution. Examples of such solubilizing agents include nonionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecyl maltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit® , isotridecipoli (ethylene glycol ether) n, 3- [(3-colamidopropyl) dimethylaminium] -1-propane 1,3-sulphonate (CHAPS), sulfonate 3- [(3-cholamidopropyl) dimethylamminio] -2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl sulfonate = N, N-dimethyl-3-ammonio-l-propane. In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either CARD-3 or CARD-4 or its target molecule to facilitate the separation of complex forms from non-complex forms of one or both of proteins, as well as to allow the automation of the assay. The binding of a test compound with CARD-3 or CARD-4, or the interaction of CARD-3 or CARD-4 with a target molecule in the presence and absence of a compound can be achieved in any suitable vessel to contain the reagents. Examples of such containers include microtiter plates, test tubes as well as microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both proteins to be (are) bound to a matrix. For example, fusion proteins can be adsorbed glutathione S-transferase / CARD CARD-3 or-4 fusion proteins or glutathione S-transferase / white sepharose glutathione beads (Sigma Chemical; St. Louis, MO) or well microtiter plates derived from glutathione, which are then combined with the test compound or the test compound and either the target protein unadsorbed or CARD-3 or CARD-4 protein, and the mixture is incubated under conditions that promote the formation of complexes, for example, under physiological conditions for salt and pH). After incubation, the beads or wells of microtitre plates are washed to remove unbound components, the matrix is immobilized in the case of beads, complexes are determined either directly or indirectly, for example, in accordance with what is described above. . Alternatively, the complexes can be dissociated from the matrix, and the level of linkage or activity of CARD-3 or CARD-4 can be determined using standard techniques. In an alternative embodiment, fusion proteins CARD-3 or CARD-4 labeled MYC epitope or HA or proteins white tagged fusion MYC epitope or HA can be adsorbed microbeads coated with anti-MYC antibody or anti-HA or in microtitre plates coated with anti-MYC or anti-HA antibody, which are then combined with the test compound or the test compound and either the non-adsorbed white protein or the CARD-3 or CARD-4 protein, and the mixture is incubated under conditions that lead to the formation of complexes (for example, under physiological conditions for salt and pH). After incubation, the beads or wells of microtitre plates are washed to remove unbound components, the matrix is immobilized in the case of beads, complexes are determined either directly or indirectly, for example, in accordance with what is described above. . Alternatively, the complexes can be dissociated from the matrix, and the level of linkage or activity of CARD-3 or CARD-4 can be determined using standard techniques. Example 12 describes a CARD-4 protein labeled with HA epitope, which physically interacts in a co-immunoprecipitation assay with a CARD-3 protein labeled with MYC epitope. In one embodiment of the invention, the HA-epitope tagged CARD-4 protein can be used in combination with a MYC epitope-tagged CARD-3 protein in the form of a protein-protein interaction assay described above in this paragraph. Other techniques for immobilizing the proteins in the matrices can also be employed in the screening assays of the present invention. For example, either CARD-3 or CARD-4 or its white molecule can be immobilized using conjugation of biotin and streptavidin. CARD-3 or biotinylated CARD-4 or white molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using well-known techniques (for example, biotinylation kit, Pierce, Chemical; Rockford, IL), and immobilized in wells of 96-well plates coated with streptavidin (Pierce Chemical). Alternatively, antibodies that react with CARD-3 or CARD-4 or target molecules but do not interfere with the binding of the CARD-3 or CARD-4 protein with its target molecule can be derived to the wells of the plate, and white does not bound or CARD-3 or CARD-4 trapped in the wells by conjugation of antibodies. Methods to detect such complexes, in addition to those described above for immobilized complexes with GST and immobilized complexes tagged with epitopes, include the immunodetection of complexes using antibodies that react with the target molecule or CARD-3 or CARD-4, as well as bound assays with enzyme that are based on the detection of an activated enzyme associated with CARD-3 or CARD-4 or white molecule. In another embodiment, modulators of CARD-3 or CARD-4 expression are identified in a method in which a cell is contacted with a candidate compound and expression of the CARD-3 or CARD-4 promoter is determined, MRNA or protein in the cell. The level of expression of CARD-3 or CARD-4 mRNA or protein in the presence of the candidate compound is compared to the level of expression of CARD-3 or CARD-4 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as modulating the expression of CARD-3 or CARD-4 based on this comparison. For example, when the expression of CARD-3 or CARD-4 mRNA or protein is higher (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as an AR-stimulator? of CARD-3 or CARD-4 or protein expression. Alternatively, when the expression of AR? M of CARD-3 or CARD-4 or protein is lower (statistically significantly lower) in the presence of the candidate compound than in the absence of said compound, the candidate compound is identified as an inhibitor of CARD mRNA. -3 or CARD-4 or protein expression. The level of AR? M of CARD-3 or CARD-4 or protein expression in the cells can be determined by methods described herein to detect CARD-3 or CARD-4 mRNA or proteins. The activity of the CARD-3 or CARD-4 promoter can be tested through the binding of the CARD-3 or CARD-4 promoter with a reporter gene such as luciferase, secreted alkaline phosphatase, or beta-galactosidase and by introduction of the resulting construct into an appropriate vector, transfecting a line of host cells, and measuring the activity of the reporter gene in response to the test compounds. For example, two AR? M specific for CARD-4 were detected in a Northern blot experiment, one of 4.6 kilobases and the other of 6.5-7.0 kilobases (example 11). In example 11, mRNA species specific for CARD-4 were found widely distributed in the tissues and cell lines studied. In another aspect of the invention, the CARD-3 or CARD-4 proteins can be used as "bait proteins" in a two-hybrid assay or in a three-hybrid assay (see, for example, US Patent No. 5,283,317).; Zervos et al. (1993) Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054; Bartel et al. (1993) Bio / Techniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and PCT Publication No. WO 94/10300), to identify other proteins that bind to or interact with CARD-3 or CARD-4 ("CARD-3 or CARD-4 binding proteins" or "CARD-3 or CARD -4-bp ") and modulate the activity of CARD-3 or CARD-4. Such binding proteins with CARD-3 or CARD-4 are probably involved in signal propagation by the CARD-3 or CARD-4 proteins for example, as elements upstream or downstream of the CARD-3 or CARD-3 pathway. Four. For example, Example 7 describes the construction of a two-hybrid screening bait construct that includes amino acids 1-145 of human CARD-4L comprising the CARD domain and the use of this bait construct to screen two-hybrid libraries of human mammary gland and prostate gland, which results in the identification that human CARD-3 as a protein that interacts with CARD-4. In another example, Example 8 describes the construction of a two-hybrid screening bait construct that includes amino acids 406-953 of CARD-4 comprising the LRR domain and the use of this bait construct to screen a two-hybrid library of human mammary gland which results in the identification of hNUDC as an interacting protein with CARD-4.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA binding and activation domains. In summary, the assay employs two different DNA constructs. In a construct, the gene encoding CARD-3 or CARD-4 is fused to a gene encoding the DNA binding domain of a known transcription factor (eg, GAL-4). In another construct, a DNA sequence from a library of DNA sequences encoding an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the proteins of "bait" and "prey" can interact, in vivo, forming a complex that depends on CARD-3 or CARD-4, the domains of DNA binding and activation of the transcription factor are close. This closeness allows the transcription of a reporter gene (for example, LacZ) operably linked over a transcription regulation site that responds to the transcription factor. The expression of the reporter gene can be detected and colonies of cells that contain the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein that interacts with CARD-3 or CARD-4. In one embodiment of the invention, the ability of a test compound to modulate the activity of CARD-3 or CARD-4, or a biologically active portion thereof can be determined by assaying the ability of the test compound to block the link of CARD-3 and CARD-4 on white proteins in a two-hybrid system assay. Example 7 describes a two-hybrid system assay for the interaction between CARD-3 and CARD-4 and example 8 describes a two-hybrid system assay for the interaction between CARD-4 and its blank protein hNUDC. To screen test compounds that block the interaction between CARD-3 and CARD-4 and its target proteins, including, but not limited to, CARD-3, CARD-4 and hNUDC, a yeast two-hybrid screening strain co-expresses the interacting bait and prey constructs, eg, a CARD-4 bait construct and a CARD-3 prey construct in accordance with that described in example 7, comes into contact with the test compound and the activity of the reporter gene of two hybrids is tested, usually HIS3, lacZ, or URA3. If the strain remains viable but exhibits a significant decrease in reporter gene activity, this could indicate that the test compound has inhibited the interaction between the bait and prey proteins. This assay could be automated for high performance screening purposes. In another embodiment of the invention, CARD-3 or CARD-4 and its target proteins could be configured in the reverse two-hybrid system (Vidal et al. (1996) Proc. Nati. Acad. Sci.

USA 93: 10321-6 and Vidal et al. (1996) Proc. Nati Acad. Sci. USA 93: 10315-20 designed specifically for efficient drug screening. In the two-hybrid reverse system, inhibition of a physical interaction of CARD-3 or CARD-4 with a target protein would result in the induction of a reporter gene in contrast to the normal two-hybrid system where inhibition of the interaction Physics of CARD-3 or CARD-4 with a white protein would cause a repression of the reporter gene. The reverse two-hybrid system is preferred to screen drugs since the induction of the reporter gene is more easily assayed than the repression of the reporter gene. Alternative embodiments of the invention are proteins that physically interact with proteins that bind with CARD-3 or CARD-4. Interactors of CARD-3 or CARD-4, including but not limited to hNUDC and CARD-3, could be configured in two-hybrid system baits and used in two-hybrid screens to identify additional members of the CARD-3 and CARD routes -4. The interactors of CARD-3 or CARD-4 identified in this way could be useful targets for therapeutic intervention in diseases related to CARD-4 and pathologies related to CARD-4 and an assay of their enzymatic or binding activity could be useful for identification of test compounds that modulate the activity of CARD-3 or CARD-4.

This invention also relates to novel agents identified through the sieving tests described above and uses thereof for treatments in accordance with what is described herein. B. Detection assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in various ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, consequently, locate gene regions associated with genetic disease; (ii) identify an individual from a small biological sample (tissue typing); and (iii) assist forensic identification of a biological sample. These applications are described in the following subsections. 1. Mapping of chromosomes Once the sequence (or a part of the sequence) of a gene is isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, the CARD-3 or CARD-4 nucleic acid molecules described herein or fragments thereof can be used to map the location of CARD-3 O CARD-4 genes on a chromosome. The mapping of the CARD-3 or CARD-4 sequences on chromosomes is an important first step to correlate these sequences with genes associated with the disease. In summary, the CARD-3 or CARD-4 genes can be mapped onto chromosomes by preparing polymerase chain reaction primers (preferably 15-25 base pairs in length) from CARD-3 sequences. CARD-4. A computer analysis of sequences of CARD-3 or CARD-4 can be used to quickly select primers that do not cover more than one exon in genomic DNA, thus complicating the amplification process. These primers can then be used for sieving by polymerase chain reaction of somatic cell hybrids containing individual human chromosomes. Only these hybrids containing the human gene corresponding to the CARD-3 or CARD-4 sequences will provide an amplified fragment. For example, in Example 6, polymerase chain reaction primers specific for human CARD-4 were used to screen DNA from a hybrid panel of somatic cells showing that human CARD-4 is located on chromosome 7 near the genetic marker SHGC-31928. Hybrids of somatic cells were prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose their human chromosomes in random order, but retaining mouse chromosomes. By using media in which mouse cells can not grow since they do not have a particular enzyme but in which human cells can grow, the human chromosome containing the gene encoding the required enzyme will be retained. Through the use of several media panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a complete set of mouse chromosomes, allowing easy mapping of individual genes into specific human chromosomes (D 'Eustachio et al. 1983) Science 220: 919-924). Hybrids of somatic cells are only contained in fragments of human chromosomes can also be produced by human chromosomes with translocations and removals. The mapping by polymerase chain reaction of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a unique thermal cycle device. Using CARD-3 or CARD-4 sequences to design oligonucleotide primers, a sub-location with panels of fragments can be achieved from specific chromosomes. Other mapping strategies that can be similarly employed to map a sequence of CARD-3 or CARD-4 to its chromosome include in situ hybridization (described in Fan et al. (1990) Proc. Nati. Acad. Sci. USA 87 : 6223-27), pre-stamped with labeled flux-labeled chromosomes and pre-selection by hybridization in chromosome-specific cDNA libraries. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal extension can be used to provide a precise chromosomal location in one step. Chromosomal extensions can be made using cells whose division has been blocked in metaphase by a chemical substance such as, for example, colzamide that disrupts the mitotic spindle. Chromosomes can probably be treated with trypsin and then labeled with Giemsa. A pattern of light and dark bands develops on each chromosome, such that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence from 500 or 600 bases. However clones greater than 1000 bases are more likely to bind to a single chromosomal location with sufficient signal strength for simple detection. Preferably 1000 bases and more preferably 2000 bases will be sufficient to provide good results in a reasonable time. For a review of this technique see See et al., (Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York, 1988)). Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on this chromosome, or panels of reagents can be used to mark multiple sites and / or multiple chromosomes. Reagents corresponding to the non-coding regions of the genes are preferred for mapping purposes. It is more likely that the coding sequences are conserved within the gene families, thus increasing the possibility of cross-hybridizations during chromosomal mapping. Once a sequence at a precise chromosomal location is mapped, the physical position of the sequence on the chromosome can be correlated with genetic map data, (such data are found, for example, in V. Muckusick, Mendelian Inheritance in Man, available at line through the Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped in the same chromosomal region, can then be identified through linkage analysis (coinherence of physically adjacent genes), as described, for example, Egeland et al. (1987) Nature, 325: 783-787. In addition Differences in DNA sequences between affected and unaffected individuals with a disease associated with the CARD-3 or CARD-4 gene can be determined. If a mutation is observed in some or all of the affected individuals but not in the unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. The comparison between the affected individuals and the unaffected individuals usually involves first seeing the structural alterations in the chromosomes as for example removals or translocations visible from the chromosome extensions or detectable using a polymerase chain reaction based on this DNA sequence . Finally, complete sequencing of genes from several individuals can be carried out to confirm the presence of a mutation and to distinguish between mutations and polymorphisms. 2. Tissue typing The CARD-3 or CARD-4 sequences of the present invention can also be used to identify individuals from minute biological samples. The US military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for the identification of its personnel. In this technique, a person's genomic DNA is digested with one or more restriction enzymes, and tested with Southern blot to provide unique bands for identification. This method does not suffer from the current limitations of "dog tags" that can be lost, changed or stolen, complicating positive identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057). In addition, the sequences of the present invention can be used to provide an alternative technique which determines the base DNA sequence per current basis of selected portions of an individual's genome. Thus, the CARD-3 or CARD-4 sequences described herein can be used to prepare two polymerase chain reaction primers from the 5 'and 3' ends of the sequences. These primers can then be used to amplify the DNA of an individual and to sequence it subsequently. Panels of corresponding DNA sequences from individuals prepared in this way may offer unique identifications of an individual, since each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain said identification sequences from individuals and from tissue. The CARD-3 or CARD-4 sequences of the present invention uniquely represent portions of the human genome. Allelic variations occur to some extent in the coding regions of these sequences, and to a greater degree in the non-coding regions. It is estimated that an allelic variation between humans occurs with a frequency of approximately one time per 500 bases. Each of the sequences described herein can to some extent be used as a standard against which a person's DNA can be compared for identification purposes. Due to the large number of polymorphisms that occur in the non-coding regions, a smaller number of sequences are required to differentiate the individuals. The non-coding sequences SEQ ID NO: l, SEQ ID NO: 7, SEQ ID NO: 25 and SEQ ID NO: 42, can comfortably provide positive identification of an individual with a panel of perhaps 10 to 1000 initiators who each provide an amplified non-coding sequence of 100 bases. If predicted coding sequences are used as for example SEQ ID NO: 3, SEQ ID NO: 9 and SEQ ID NO: 27, their most appropriate number of primers for positive identification of an individual would be 500-2,000. If a panel of reagents from the CARD-3 or CARD-4 sequences described herein is used to generate a unique identification database for an individual, these same reagents can then be used to identify tissue from this individual. Using the unique identification database, a positive identification of the person, living or dead, can be made from extremely small tissue samples. 3. Use of Partial Sequences CARD-3 or CARD-4 in Forensic Biology DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field that employs the genetic typing of biological evidence found in a crime scene as a means to positively identify, for example, the person who performed a crime. To make such an identification, the polymerase chain reaction technology can be used to amplify the DNA sequences taken from very small biological samples such as tissues, hair or skin, for example, blood, saliva or semen found in the scenario of a crime. The amplified sequence can then be compared with a standard allowing thus the identification of the origin of the biological sample. The sequences of the present invention can be used to provide polynucleotide reagents, for example, polymerase chain reaction primers, focused on specific loci in the human genome, which can increase the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (ie, another DNA sequence that is unique to a specific person) ). As mentioned above, the actual base sequence information can be used for identification as an accurate alternative to the patterns formed by fragments generated by restriction enzymes. The sequences focused towards non-coding regions SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 25 are especially appropriate for this use since a greater number of polymorphisms occur in the non-coding regions, making it easier to differentiate the individuals using this technique. Example of polynucleotide reagents include the sequences of CARD-3 or CARD-4 or portions thereof, for example, fragments derived from the non-coding regions of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 25, which have a length of at least 20 or 30 bases. The CARD-3 or CARD-4 sequences described herein can be further employed to provide nucleotide reagents, for example, labeled or labeled probes which can be employed, for example, in the in situ hybridization technique, to identify a tissue specific, for example, brain tissue. This can be very useful in cases in which a forensic pathologist obtains a tissue of unknown origin. Panels of such ARD-3 or CARD-4 probes can be used to identify a tissue by species and / or by type of organ. Similarly, these reagents, e.g., CARD-3 or CARD-4 primers or probes can be used to screen tissue culture for contamination (ie, screen for the presence of a mixture of different cell types in a culture) . C. Predictive medicine The present invention also relates to the field of predictive medicine where diagnostic tests, prognostic tests, pharmacogenomas, and monitoring of chemical tests are used for predictive (predictive) purposes in order to treat an individual of prophylactic way. As follows, one aspect of the present invention relates to diagnostic assays for determining the expression of CARD-3 or CARD-4 and / or nucleic acid as well as CARD-3 or CARD-4 activity, in the context of a biological sample (eg, blood, serum, cells, tissues) to determine in this way if an individual is suffering from a disease or disorder or if he is at risk of developing a disorder, associated with the expression or aberrant activity of CARD -3 or CARD-4. The invention also provides prognostic (or predictive) assays to determine whether an individual is at risk of developing a disease associated with a CARD-3 or CARD-4 protein, nucleic acid expression or nucleic acid activity. For example, mutations in the CARD-3 or CARD-4 gene can be tested in the biological sample. Such assays can be used for prognostic or predictive purposes to prophylactically treat an individual before the onset of a disorder characterized by CARD-3 or CARD-4 protein, nucleic acid expression or activity or associated with said CARD protein. -3 or CARD-4, nucleic acid expression or activity. Another aspect of the invention offers methods for determining a CARD-3 or CARD-4 protein, nucleic acid expression or CARD-3 or CARD-4 expression in an individual to thereby select appropriate therapeutic or prophylactic agents for this person ( known here as "pharmacogenomics"). Pharmacogenomics allows the selection of agents (e.g., drugs) for the therapeutic or prophylactic treatment of a person based on the genotype of the individual (e.g., the genotype of the individual examined to determine the individual's ability to respond to a particular agent ). Another aspect of the invention relates to monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of CARD-3 or CARD-4 in clinical trials. These and other agents are described in more detail in the following sections. 1.- Diagnostic assays An exemplary method for detecting the presence or absence of CARD-3 or CARD-4 in a biological sample includes obtaining a biological sample from a test subject and contacting the biological sample. with a compound or agent capable of detecting CARD-3 or CARD-4 protein or nucleic acid (e.g., mRNA, genomic DNA) encoding the CARD-3 or CARD-4 protein such that the presence of CARD-3 or CARD-4 is detected in the biological sample. An agent for detecting CARD-3 or CARD-4 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing with CARD-3 or CARD-4 mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length CARD-3 or CARD-4 nucleic acid, such as the nucleic acid of SEQ ID NO: 3, SEQ ID NO: 7 or 9, SEQ ID NO: 25 or 27, or a portion thereof such as for example an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and in sufficient to hybridize specifically under stringent conditions with CARD-3 or CARD mRNA -4 either genomic DNA, or a splicing variant of human CARD-4 such as, for example, the nucleic acid of SEQ ID NO: 38, or SEQ ID NO: 40. 0 after probes suitable for use in the assays of diagnosis of the invention are described here. For example, Example 11 describes the use of a nucleic acid probe to detect CARD-4 mRNA in human tissues and cell lines, and the probe used in this experiment could be used for a diagnostic assay.

An agent for detecting CARD-3 or CARD-4 protein can be an antibody capable of binding to CARD-3 or CARD-4 protein, preferably an antibody with a detectable label. Antibodies may be polyclonal, or preferably monoclonal. For example, polypeptides corresponding to amino acids 128-139 and 287-298 of human CARD-4L were used to immunize rabbits and produce polyclonal antibodies that specifically recognize human CARD-4L. An intact antibody, or a fragment thereof (for example, Fab or F (ab ') 2) can be used. The term "labeled", with reference to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physical binding), a detectable substance on the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within the subject. That is, the detection method of the present invention can be used to detect CARD-3 OR CARD-4 mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for the detection of CARD-3 or CARD-4 mRNAs include Northern hybridizations as well as in situ hybridization. For example, Example 11 contains the use of a human CAR-4L nucleic acid probe for a Northern Blot analysis after mRNA encoded by human CARD-4L detected in RNA samples from human tissues and cell lines. In vitro techniques for the detection of CARD-3 or CARD-4 proteins include enzyme-linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for the detection of genomic DNA of CARD-3 or CARD-4 include Southern hybridizations. In addition, in vivo techniques for the detection of CARD-3 or CARD-4 protein include the introduction into a subject of a labeled anti-CARD-3 or CARD-4 antibody. For example, the antibody can be labeled with a radioactive label whose presence and location in a subject can be detected by standard imaging techniques. In the embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample may contain mRNA molecule from the test subject or genomic DNA molecules from the test subject. A biological sample is a sample of peripheral blood leukocytes isolated by conventional means of a subject. In another embodiment, the methods further include obtaining a biological control sample from a control subject, contacting the control sample with a compound or agent capable of detecting a CARD-3 or CARD-4 protein. , MRNA, or genomic DNA, in such a way that the presence of a CARD-3 or CARD-4 protein, mRNA or genomic DNA can be detected in the biological sample, and the presence of the protein, mRNA or genomic DNA can be compared of CARD-3 or CARD-4 in the control sample with the presence of CARD-3 or CARD-4 protein, mRNA or genomic DNA in the test sample. This invention also encompasses kits for detecting the presence of CARD-3 or CARD-4 in a biological sample (a test sample). Such kits can be used to determine whether a subject is at or is at risk of developing a disorder associated with the aberrant expression of CARD-3 or CARD-4 (e.g., an immunological disorder). For example, the kit can comprise a compound labeled an agent capable of detecting a CARD-3 or CARD-4 protein or mRNA in a biological sample and means for determining the amount of CARD-3 or CARD-4 in the sample (e.g. anti-CARD-3 or CARD-4 antibody or an oligonucleotide probe that binds to DNA encoding CARD-3 or CARD-4, eg, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25 or SEQ ID NO: 27). The kits may also include instructions to observe that the test subject is suffering from or is at risk of developing a disorder associated with the aberrant expression of CARD-3 or CARD-4 if the amount of CARD-3 or CARD-3 protein or mRNA 4 is above or below a normal level. For antibody-based kits, the kit may comprise, for example: (1) a first antibody (e.g., fixed on a solid support) that binds a CARD-3 or CARD-4 protein; and, optionally, (2) a second, different antibody that binds to a CARD-3 or CARD-4 protein or the first antibody and is conjugated to a detectable agent. In the case of oligonucleotide-based kits, the kit may comprise, for example: (1) an oligonucleotide, eg, a detectably-labeled oligonucleotide, which hybridizes to a nucleic acid sequence of CARD-3 or CARD-4 or (2) a pair of primers useful for amplifying a nucleic acid molecule of CARD-3 or CARD-4. The kit may also comprise, for example, a regulating agent, a preservative, or a protein stabilizing agent. The kit can also comprise the components necessary to detect the detectable agent (e.g., an enzyme or a substrate). The kit may also contain a control sample or a series of control samples that can be tested and compared to the contained test sample. Each component of the kit is usually contained within a single container and all containers are contained in a single package along with instructions to observe whether the test subject is at or is at risk of developing a disease associated with the aberrant expression of CARD. 3 or CARD-4. 2. Prognostic assays The methods described herein may also be used as diagnostic or prognostic assays to identify subjects who have or are at risk of developing a disease or disorder associated with the expression or aberrant activity of CARD-3 or CARD-4. . For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be used to identify a subject that has or is at risk of developing a disorder associated with protein, nucleic acid expression or CARD activity. -3 or CARD-4. Alternatively, prognostic assays can be used to identify a subject who has or is at risk of developing said disease or disorder. Thus, the present invention offers a method in which a test sample is obtained from a subject and a protein or nucleic acid of CARD-3 or CARD-4 (for example, mRNA, genomic DNA) is detected, where the The presence of protein or nucleic acid of CARD-3 or CARD-4 is a diagnosis for a subject who has or is at risk of developing a disease or disorder associated with the expression or aberrant activity of CARD-3 or CARD-4. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample may be a biological fluid (e.g., serum), a cell sample, or a tissue. In addition, the prognostic assays described herein can be used to determine whether a subject can receive an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or another candidate drug to treat a disease or condition). disorder associated with the expression or aberrant activity of CARD-3 or CARD-4 For example, such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (eg, agents of a type which decreases the activity of CARD-3 or CARD-4.) Thus, the present invention offers methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or aberrant activity of CARD-3 or CARD-4 where a test sample is obtained and protein or nucleic acid of CARD-3 or CARD-4 is detected (for example, where the presence of protein or nucleic acid of CARD-3 or CARD-4 is a diagnosis for a subject who can receive the agent to treat a disorder associated with the expression or aberrant activity of CARD-3 or CARD-4). The methods of the invention can also be used to detect lesions or genetic mutations in a CARD-3 or CARD-4 gene, thus determining whether a subject with the injured gene is at risk of a disorder characterized by proliferation and / or differentiation. aberrant of the cells. In preferred modalities, methods include the detection, in a sample of cells from the subject, of the presence or absence of a genetic lesion characterized by at least one of the following: alteration that affects the integrity of a gene encoding a CARD-3 protein or CARD-4, or erroneous expression of the CARD-3 or CARD-4 gene. For example, such genetic lesions can be detected by determining the existence of at least one of the following: 1) a removal of one or several nucleotides from the CARD-3 or CARD-4 gene; 2) the addition of one or several nucleotides to a CARD-3 or CARD-4 gene; 3) replacement of one or more nucleotides of a CARD-3 or CARD-4 gene, 4) a chromosomal rearrangement of a CARD-3 or CARD-4 gene; 5) the alteration of the level of a messenger RNA transcript of a CARD-3 or CARD-4 gene, 6) the aberrant modification of a CARD-3 or CARD-4 gene, such as the DNA methylation pattern genomic, 7) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of a CARD-3 or CARD-4 gene (eg, caused by mutation at a splice donor site or splice acceptor ), 8) a non-wild type level of CARD-3 or CARD-4 protein, 9) allelic loss of a CARD-3 or CARD-4 gene, and 10) inappropriate post-translational modification of a CARD-3 protein or CARD-4. As described herein, there are numerous known assay techniques that can be employed to detect lesions in a CARD-3 or CARD-4 gene. A biological sample is a peripheral blood leukocyte sample isolated by conventional means from a sample. In certain embodiments, the detection of the lesion includes the use of a probe / primer in a polymerase chain reaction (PCR) (see, for example, U.S. Patent Nos. 4,683,195 and 4,683,202), such as, for example, polymerase chain reaction. anchor either RACE polymerase chain reaction, or, alternatively, in a ligation chain reaction (LCR) (see, for example, Landegran et al (1988) Science 241: 1077-1080; and Nakazawa et al. (1994) Proc. Nati, Acad. Sci. USA 91: 360-364), the latter is exclusively useful for detecting point mutations in the CARD-3 or CARD-4 gene (see, for example, Abravaya et al. (1995) Nucleic Acids Res. 23: 675-682). This method may include the steps of collecting a sample of cells from a patient, isolating the nucleic acid (e.g., genomic, mRNA, or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that hybridize specifically with a CARD-3 or CARD-4 gene under conditions such that hybridization and amplification of the CARD-3 or CARD-4 gene (if present) occurs, and detect the presence or absence of an amplification product, or detect the size of the amplification product and compare the length with the control sample. It is anticipated that the polymerase chain reaction and / or LCR can be used in a desirable manner as a preliminary amplification step in combination with any of the techniques employed to detect the mutations described herein. Alternative amplification methods include: self-sustained sequence replication (Guatelli et al (1990) Proc. Nati, Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh, et al. (1989) Proc. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio / Technology 6: 1197), or any other method of nucleic acid amplification, followed by detection of the Amplified molecules employing techniques well known to those skilled in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In an alternative embodiment, mutations in a CARD-3 or CARD-4 gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, the sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and the fragment length sizes are determined by gel electrophoresis and compared. The differences in fragment lengths between the sample and control DNA indicate mutations in the sample DNA. In addition, the use of sequence-specific ribozymes (see, eg, U.S. Patent No. 5,498,531) can be used to qualify the presence of specific mutations by developing or losing a ribozyme cleavage site. In other embodiments, genetic mutations in CARD-3 or CARD-4 can be identified by hybridizing sample and control nucleic acids, eg, DNA or RNA, to high-density pools containing hundreds or thousands of oligonucleotide probes ( Cronin et al. (1996) Human Mutation 7: 244-255; Kozal et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in CARD-3 or CARD-4 can be identified in two-dimensional arrays containing DNA probes generated by light as described in Cronin et al. Supra. In summary, a first set of hybridization probes can be used to scan long segments of DNA in a sample and control to identify base changes between sequences or splicing linear sets of sequential probes. This step allows the identification of point mutations. This step is followed by a second set of hybridizations that allows the characterization of specific mutations through the use of specialized, smaller, complementary probe sets for all variants or mutations detected. Each set of mutations is composed of parallel sets of probes, or one complementary to the wild-type gene, and the other complementary to the mutant gene. In another embodiment, any of several sequencing reactions known in the art can be employed to directly sequence the CARD-3 or CARD-4 gene and detect mutations by comparing the CARD-3 or CARD-4 sequence of sample with the corresponding sequence of wild type (control). Examples of sequencing reactions include reactions based on techniques developed by Maxam and Gilbert ((1977) Proc. Nati, Acad. Sci. USA 74: 560) or Sanger ((1977) Proc. Nati. Acad. Sci. USA 74: 5463). It is also contemplated that any of several automated sequencing methods may be employed when carrying out diagnostic assays ((1995) Bio / Techniques 19: 448), including sequencing by mass spectrometry (see, for example, PCT publication no. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36: 127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38: 147-159). Other methods for detecting mutations in the CARD-3 or CARD-4 gene include methods in which protection against dissociation agents is used to detect non-corresponding bases in RNA / RNA heteroduplexes or RNA / AD? (Myers Oet al. (1985) Science 230: 1242). In general, the "non-corresponding dissociation" technique begins by supplying heteroduplexes formed by hybridization (marked) of AR? or AD? which contains the sequence of CARD-3 or CARD-4 wild type with AR? or AD? potentially mutant of a tissue sample. Double-stranded duplexes are treated with an agent that dissociates single-stranded regions of the duplex as they exist due to lack of base-pair matching between the control and sample chains. For example, duplexes AR? / AD? Can they be treated with R? asa and hybrids of AD? / AD? treated with nuclease Sl to enzymatically digest the regions that do not correspond. In other modalities, either duplexes of AD? / AD? or RNA / AD duplexes? they can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest the regions that do not correspond. After digestion of the regions that do not correspond, the resulting material is then separated by site in denaturing polyacrylamide gels to determine the site of the mutation. See, for example, Cotton et al. (1998) Proc. Nati Acad Sci USA 85: 4397; Saleeba et al. (1992) Methods Enzimol. 217: 286-295. In one embodiment, the control DNA or RNA can be labeled for detection. In another embodiment, the mismatch dissociation reaction employs one or more proteins that recognize base pairs that do not correspond to double-stranded DNA (what are known as "DNA mismatch repair" enzymes) in defined systems to detect and map point mutations in CARD-3 or CARD-4 cDNA from cell samples. For example, the mutY enzyme of E. coli dissociates A in the mismatches G / A and the thymidine DNA glycosidase of HeLa cells dissociates T in the mismatches G / T (Hsu et al. (1994) Carcinogenesis 15: 1657-1662). According to an exemplary embodiment, a probe based on a sequence of CARD-3 or CARD-4, for example, a wild-type CARD-3 or CARD-4 sequence, is hybridized to a cDNA or other DNA product from of one test cell (s). The duplex is treated with a DNA mismatch repair enzyme, and the dissociation products, if any, can be detected from electrophoresis protocols or the like. See, for example, US Patent no. ,459,039. In other modalities, alterations in electrophoretic mobility will be used to identify mutations in CARD-3 or CARD-4 genes. For example, a single chain conformation polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc Nati. Acad. Sci USA: 86: 2766, see also Cotton (1993) Mutat, Res. 285: 125-144; and Hayashi (1992) Genet Anal Tech Appl 9: 73-79). Single-stranded DNA fragments of CARD-3 or CARD-4 nucleic acids from sample and control will be denatured and their renaturation will be allowed. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility allows the choice of changing up to a single base. DNA fragments can be labeled or detected with labeled probes. The sensitivity of the assay can be increased by the use of RNA (instead of DNA), where the secondary structure is more sensitive even in sequence change. In one embodiment, the method of the present invention employs heteroduplex analysis to separate double-stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al (1991) Trends Genet 7: 5). In another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a denaturant gradient is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985) Nature 313: 495). When DGGE is used as a method of analysis, the DNA will be modified to ensure that it is not completely denatured, for example, by the addition of a GC staple of approximately 40 base pairs of high-melting GC-rich DNA by polymerase chain reaction. In a further embodiment, a temperature gradient is used instead of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaun and Reissner (1987) Biophys Chem 265: 12753). Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective extension of primers. For example, oligonucleotide primers can be prepared in which the known mutation is centrally placed and then hybridized to a white DNA under conditions that allow hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324: 163 ); Siki et al. (1989) Proc. Nati Acad. Sci USA 86: 6230). Such allele-specific oligonucleotides are hybridized to white DNA amplified by polymerase chain reaction or several different mutations when the oligonucleotides are fixed on the hybridization membrane and hybridized with labeled white DNA. Alternatively, an allele-specific amplification technology that depends on selective amplification in polymerase chain reaction can be used in combination with the present invention. The oligonucleotides used as primers for specific amplification can carry the mutation of interest in the center of the molecule (such that the amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17: 2437-2448) or at the 3 'end of an initiator where, under appropriate conditions, a mismatch can be avoided, or the polymerase extension can be reduced (Prossner (1993) Tibtech 11: 238). In addition, it may be desirable to introduce a novel restriction site in the region of the mutation to create a detection based on dissociation (Gasparini et al. (1992) Mol. Cell Probes 6: 1). It is anticipated that in certain embodiments, the amplification can also be carried out using Taq ligase for amplification (Barany (1991) Proc. Nati, Acad. Sci USA 88: 189). In such cases, ligation can only occur if there is a perfect match at the 3 'end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

The methods described herein can be carried out, for example, by the use of pre-packaged diagnostic kits comprising at least one probe nucleic acid or one antibody reagent described herein, which can be conveniently employed, for example, in clinical settings to diagnose symptoms that present patients or family history a disease that involves a CARD-3 or CARD-4 gene. In addition, any cell or tissue type, preferably peripheral blood leukocytes, wherein CARD-3 or CARD-4 is expressed may be employed in the prognostic assays described herein. 3. Pharmacogenomics Agents, or modulators that have an effect of stimulation or inhibition on the activity of CARD-3 or CARD-4 (for example, expression of the CARD-3 or CARD-4 gene) in accordance with that identified by an assay screening methods described here can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., an immunological disorder) associated with the aberrant activity of CARD-3 or CARD-4). In combination with such treatment, pharmacogenomics (ie, the study of the relationship between the genotype of an individual and the response of said individual to a foreign compound or drug) of the individual can be considered. Differences in the metabolism of therapeutic agents can cause severe toxicity or therapeutic failures by altering the relationship between the dose and the blood concentration of the pharmacologically active substance. Thus, the pharmacogenomics of the individual allows the selection of effective agents (for example, drugs) for prophylactic or therapeutic treatments based on the consideration of the genotype of the individual. Said pharmacogenomics can be further employed to determine appropriate dosages and appropriate therapeutic regimens. Accordingly, the activity of CARD-3 or CARD-4 protein, the expression of CARD-3 O CARD-4 nucleic acid, or the mutation content of CARD-3 or CARD-4 genes in an individual can be determined to select in this way the appropriate agent (s) for the therapeutic or prophylactic treatment of the individual. Pharmacogenomics focuses on the clinically significant hereditary variations in response to drugs due to an altered disposition to the drug and due to an abnormal action in the affected persons. See, for example, Linder (1997) Clin. Chem. (2): 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor that alters the way drugs act in the body (altered pharmacological action) or genetic conditions transmitted as individual factors that alter the way the body acts on drugs (altered metabolism of drugs ). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is an inherited common enzyme disease in which the main clinical complication is haemolysis after the ingestion of oxidizing drugs (anti-malaria)., sulfonamides, analgesics, nitrofuran) and the consumption of beans. As an illustrative modality, the activity of the enzymes that metabolize drugs is a major factor in both the intensity and the duration of the pharmacological action. The discovery of genetic polymorphisms of enzymes that metabolize drugs (for example, N-acetyltransferase 2 (NAT 2) and cytochrome P450 in enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected pharmacological effects or show an exaggerated pharmacological response and severe toxicity after taking the standard and safe dose of the drug. These polymorphisms are expressed in two genotypes in the population, the extensive metabolizer (EM) and the limited metabolizer (PM). The prevalence of PM is different between different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations in PM have been identified, all leading to the absence of CYP2D6. The limited metabolisers of CYP2D6 and CYP2C19 very often experience an exaggerated response to drugs and side effects when receiving standard doses. If a metabolite is the active therapeutic portion, PM shows no therapeutic response as demonstrated for the analgesic effect of codeine mediated by its metabolite formed of CYP2D6, morphine. The other extreme is what is known as ultra-rapid metabolizers that do not respond to standard doses. Recently, the molecular basis of ultrafast metabolism has been identified as the amplification of the CYP2D6 gene. Thus, the activity of CARD-3 0 CARD-4 protein, the nucleic expression of CARD-3 or CARD-4, or the mutation content of CARD-3 or CARD-4 genes in an individual can be determined in order to select in this way the appropriate agent (s) for the therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply the genotyping of polymorphic alleles that encode enzymes that metabolize drugs for the identification of an individual's drug response phenotype. This knowledge when applied to the dosage or selection of drug, can avoid adverse reactions or therapeutic failures and therefore can increase the therapeutic or prophylactic efficiency when treating a patient with a modulator of CARD-3 or CARD-4, as per example a modulator identified by one of the exemplary screening tests described herein. 4. Monitoring effects during clinical trials Monitoring the influence of agents (eg, drugs, compounds) on the expression or activity of CARD-3 or CARD-4 (eg, the ability to modulate proliferation or differentiation of aberrant cells) can be applied not only in the basic screening of drugs, but also clinical trials. For example, the effectiveness of an agent determined by a screening assay according to that described herein in the sense of increasing the expression of the CARD-3 O CARD-4 gene, protein levels or ascending regulation of ACRD-3 activity. or CARD-4 can be monitored in clinical trials of patients showing a diminished expression of the CARD-3 or CARD-4 gene, protein levels or a down-regulation of CARD-3 or CARD-4 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease the expression of the CARD-3 or CARD-4 gene, protein levels or down-regulation of CARD-3 or CARD-4 activity, can be monitored in clinical trials. of patients showing an increased expression of the CARD-3 or CARD-4 gene, protein levels or an up-regulated activity of CARD-3 O CARD-4. In such clinical trials, the expression or activity of CARD-3 or CARD-4 and, preferably, other genes that have been implicated for example in a cell proliferation disorder can be employed as a "reading" or response markers. immune from a particular cell. For example, and not to limit the invention, genes including CARD-3 or CARD-4, which are modulated in cells through treatment with an agent (e.g., compound, drug or small molecule) that modulates the activity of CARD-3 or CARD-4 (for example, identified in a screening test in accordance with what is described here) can be identified. Thus, to study the effect of agents on cell proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA can be prepared and analyzed for the expression levels of CARD-3 or CARD-4 and other genes involved in the disorder. The levels of gene expression (ie, a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described here, or alternatively by measuring the amount of the protein produced by one of the methods described herein or by measuring the activity levels of CARD-3 or CARD-4 or other genes. In this way, the gene expression pattern can serve as a marker, an indicator of the physiological response of the cells to the agent. Accordingly, the state of the response can be determined in advance, and at various points during the treatment of the individual with the agent. In one embodiment, the present invention provides a method for monitoring the effectiveness of a patient's treatment with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or another candidate drug identified to through screening assays described herein) comprising the steps of (i) obtaining a sample prior to administration of a subject prior to administration of the agent; (ii) detecting the level of expression of a CARD-3 or CARD-4 protein of mRNA or genomic DNA in the sample prior to administration; (iii) obtaining one or more samples after administration of the subject; (iv) detecting the level of expression or activity of the mRNA protein or ACRD-3 or CARD-4 genomic DNA in the samples after administration; (V) comparing the level of expression or activity of the mRNA protein either CARD-3 or CARD-4 genomic DNA in the sample prior to administration with the mRNA protein, or CARD-3 or CARD-4 genomic DNA in the sample or samples after administration; (vi) alter the administration of the agent to the subject in a corresponding manner. For example, increased administration of the agent may be desirable to increase the expression or activity of CARD-3 or CARD-4 at levels higher than those detected, ie, to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease the expression or activity of CARD-3 or CARD-4 at levels higher than those detected, ie, to decrease the effectiveness of the agent. C. Methods of Treatment The present invention offers both prophylactic and therapeutic methods for the treatment of a patient at risk (or susceptible) to suffer from a disorder because he has a disorder associated with the expression or aberrant activity of CAR-3 or CARD-4. . 1. Prophylactic methods In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with the expression or aberrant activity of CARD-3 or CARD-4, by administering to the subject an agent that modulates the expression of CARD-3 or CARD-4 or at least one activity of CARD-3 or CARD-4. Subjects at risk for a disease caused or contributed by the aberrant expression or activity of CARD-3 or CARD-4 can be identified by, for example, any or a combination of diagnostic or prognostic tests described herein. The administration of a prophylactic agent can occur before the manifestation of the symptoms characteristic of the expression or aberrant activity of CARD-3 or CARD-4, in such a way that a disease or a disorder can be prevented or alternatively, retard its progression . Depending on the type of aberration of CARD-3 or CARD-4, for example, a CARD-3 or CARD-4 agonist or a CARD-3 or CARD-4 antagonist can be used for the treatment of the subject. The appropriate Agent can be determined based on screening tests described herein. Activities of CARD-3 or CARD-4 that can be modulated for prophylactic purposes include but are not limited to 1) CARD-3 or CARD-4 protein expression or gene, for example, see example 11 for a description of the expression pattern of human CARD-4 mRNA; 2) Binding of CARD-3 or CARD-4 on a target protein, for example, see example 7.8 and 12 for a description of proteins known to bind with CARD-3 or CARD-4; 3) Regulation of CARD-4 of? F-? B in accordance with that described in example 9; and 4) Increase by CARD-3 or CARD-4 of caspase activity 9 in accordance with that described in example 10. 2. Therapeutic methods Another aspect of the invention relates to methods for modulating the expression or activity of CARD-3 or CARD-4 for therapeutic purposes. The modulatory method of the present invention includes contacting a cell with an agent that modulates one or more of the CARD-3 or CARD-4 protein activities associated with the cell. An agent that modulates the activity of CARD-3 or CARD-4 protein can be an agent according to that described herein, such as for example a nucleic acid or a protein a cognate ligand that naturally occurs from a CARD-3 O CARD protein. 4, a peptide, a peptodomimetic of CARD-3 or CARD-4, or another small molecule. In one embodiment, the agent stimulates one or several of the biological activities of CARD-3 O CARD-4 protein. Example of such stimulatory agents include active CARD-3 or CARD-4 protein and a nucleic acid molecule encoding CARD-3 or CARD-4 that has been introduced into the cell. In another modality, the agent inhibits one or more of the biological activities of a CARD-3 or CARD-4 protein. Examples of such inhibitory agents include antisense nucleic acid molecules of CARD-3 or CARD-4 as well as antibodies, anti-CARD-3 or CARD-4. These modulatory methods can be carried out in vitro (for example, by culturing the cell with the agent), or alternatively in vivo (for example, by administering the agent to some subject). As such, the present invention offers methods for treating an individual suffering from a disease or disorder characterized by the aberrant expression or activity of a protein or nucleic acid molecule of CARD-3 O CARD-4. In one embodiment the method includes the administration of an agent (eg, an agent identified through a screening assay described herein) or a combination of agents that it modulates (e.g., up-regulates or down-regulates) ) expression or activity CARD-3 0 CARD-4. In another embodiment the method includes the administration of a protein or nucleic acid molecule of CARD-3 or CARD-4 as therapy to compensate for the expression or reduced or aberrant activity of CARD-3 O CARD-4. CARD-3 0 CARD-4 activities that can be modulated for therapeutic purposes include, but are not limited to, 1) CARD-3 O CARD-4 protein or gene expression, for example, see example 11 for a description of the pattern of mRNA expression of human CARD-4; 2) Binding of CARD-3 or CARD-4 on a target protein, for example, see example 7.8 and 12 for a description of proteins known to bind on CARD-3 or CARD-4; 3) Regulation by CARD-4 of NF-KB in accordance with that described in example 9; and 4) Increase by CARD-3 or CARD-4 of caspase activity 9 in accordance with that described in example 10. The stimulation of CARD-3 or CARD-4 activity is desirable in situations in which CARD-3 or CARD-4 is regulated downward in a normal manner and / or in which an increased activity of CARD-3 or CARD-4 will probably have a beneficial effect. Conversely, inhibition of CARD-3 or CARD-4 activity is desirable in situations in which CARD-3 or CARD-4 is abnormally upregulated, for example, in myocardial infarction and / or where the activity of CARD-3 or decreased CARD-4 will probably have a beneficial effect. Since CARD-4 may be involved in the processing of cotosine, the inhibition of the activity or expression of CARD-4 may be beneficial in patients who have aberrant inflammation. This invention is further illustrated through the following examples that should not be considered as limiting. The contents of the published references, patents and patent publications mentioned in this application are incorporated herein by reference. EXAMPLES Example 1: ISOLATION AND CHARACTERIZATION OF COMPLETE LENGTH CARD-3 AND CARD-4L / S HUMAN cDNAs. A known CARD domain profile was used to search databases of cDNA sequences and partial cDNA sequences using TBLAST? (Washington University, version 2.0, search nuance BLOSUM62). This search led to the identification of CARD-3. Using CARD-3 to search databases of sequences of AD? C as well as partial sequences of AD? C, AD? C potential of CARD was found. This sequence of AD? it was used to screen a human umbilical vein endothelial library (HUVE) and a clone containing the partial CARD-4S sequence was identified. The human umbilical vein endothelial library was then screened again using a probe designed against the partial CARD-4S sequence and a clone containing the upper CARD-4L sequence was identified. Example 2: CHARACTERIZATION OF PROTEINS CARD-3 AND CARD-4L / S In this example, the predicted amino acid sequences of human CARD-3 and CARD-4L / S proteins were compared with the amino acid sequences of known proteins and several were identified motives For example, the CARD domains of CARD-3 and CARD-4 were aligned (Figure 7) with the CARD domains of ARC-CARD (SEQ ID NO: 31), cIAPl-CARD (SEQ ID NO: 32) and CIAP2- CARD (SEQ ID NO: 33). In addition, the molecular weight of the human CARD-3 and CARD-4L / S proteins was predicted. The human CARD-3 cDNA was isolated in accordance with that described above (Figure 1; SEQ ID NO: 1) and encodes a protein of 540 amino acids (Figure 2; SEQ ID NO: 2). CARD-3 also includes a predicted kinase domain (amino acid 1 to amino acid 300 of SEQID NO: 2, SEQ ID NO: 4), followed by a predicted linker domain (from amino acid 301 to amino acid 431 of SEQ ID NO: 2; SEQ ID NO: 5) and a predicted CARD domain (from amino acid 432 to amino acid 540 of SEQ ID NO: 2; SEQ ID NO: 6). The human CARD-4L cDNA was isolated in accordance with that described above (Figure 3; SEQ ID NO: 7) and have an open reading frame of 2859 nucleotides (nucleotides 245-3103 of SEQ ID NO: 7; SEQ ID; NO: 9) which encodes a protein of 953 amino acids (Figure 4; SEQ ID NO: 8). The CARD-4L protein has a CARD domain (amino acids 15-114; SEQ ID NO: 10). It was also predicted that CARD-4L have a nucleotide binding domain extending from about amino acid 198 to about amino acid 397 of SEQ ID NO: 8; SEQ ID NO: 11, a Walker chart predicted "A", which extends from about amino acid 202 to about amino acid 209 of SEQ ID NO: 8; SEQ ID NO: 12, a Walker "B" chart predicted, ranging from about amino acid 280 to about amino acid 284 of SEQ ID NO: 8; SEQ ID NO: 13, a predicted kinase domain (loop-P) extending from about amino acid 197 to about amino acid 212 of SEQ ID NO: 8; SEQ ID NO: 46, a predicted kinase 2 domain extending from about amino acid 273 to about amino acid 288 of SEQ ID NO: 8; SEQ ID NO: 47, a predicted kinase 3a subdomain extending from about amino acid 327 to about amino acid 338 of SEQ ID NO: 8; SEQ ID NO: 14, 10 predicted leucine-rich repeats extend from about amino acid 674 to about amino acid 950 of SEQ ID NO: 8. The first leucine-rich repeat is predicted to extend from about amino acid 674 to about amino acid 701 of SEQ ID NO: 8; SEQ ID NO: 15, the second leucine-rich repeat is predicted to extend from about amino acid 702 to about amino acid 727 of SEQ ID NO: 8; SEQ ID NO: 16. The third leucine-rich repeat is predicted to extend from about amino acid 728 to about amino acid 754 of SEQ ID NO: 8; SEQ ID NO: 17. The fourth leucine-rich repeat is predicted to extend from about amino acid 755 to about amino acid 782 of SEQ ID NO: 8; SEQ ID NO: 18. The fifth leucine-rich repeat is predicted to extend from about amino acid 783 to about amino acid 810 of SEQ ID NO: 8; SEQ ID NO: 19. The sixth leucine-rich repeat is predicted to extend from about amino acid 811 to about amino acid 838 of SEQ ID NO: 8; SEQ ID NO: 20. The seventh leucine-rich repeat is predicted to extend from about amino acid 839 to about amino acid 866 of SEQ ID NO: 8; SEQ ID NO: 21. The eighth leucine-rich repeat is predicted to extend from about amino acid 867 to about amino acid 894 of SEQ ID NO: 8; SEQ ID NO: 22. The ninth leucine-rich repeat is predicted to extend from about amino acid 895 to about amino acid 922 of SEQ ID NO: 8; SEQ ID NO: 23, and the tenth leucine-rich repeat is predicted to extend from about amino acid 923 to about amino acid 950 of SEQ ID NO: 8; SEQ ID NO: 24. The human partial CARD-4S cDNA isolated as described above (Figure 5, SEQ ID NO: 25) encodes a protein of 490 amino acids (Figure 6, SEQ ID NO: 26). CARD-4S includes a predicted partial CARD domain (amino acids 1-74 of SEQ ID NO: 26). It is also predicted that CARD-4s has a P loop extending from about amino acid 163 to about amino acid 170 of SEQ ID NO: 26; SEQ ID NO: 29, and a Walker Table "B" predicted to extend from about amino acid 241 to about amino acid 245 of SEQ ID NO: 26; SEQ ID NO: 30. A graph like this the predicted features of CARD-4L is presented in Figure 8. This figure shows the predicted alpha regions (Gaernier-Robinson and Chou-Fasman), the predicted beta regions (Gaernier-Robinson and Chou-Fasman), the predicted return regions (Gaernier-Robinson and Chou-Fasman) and the predicted helical regions (Gaernier-Robinson and Chou-Fasman). Also included in the figure is a graph of hydrophilicity (Kyte-Doolittle), the predicted alpha and beta amphase regions (Eisenberg), the predicted flexible regions (Karplus-Schulz), the predicted antagonistic index (Ja eson-Wolf) and the graph predicted surface probability (Emini) A graph like this the predicted structural features of CARD-4S also appear in figure 9. This figure shows the predicted alpha regions (Gaernier-Robinson and Chou-Fasman), the predicted beta regions (Gaernier- Robinson and Chou-Fasman), the predicted return regions (Gaernier-Robinson and Chou-Fasman) and the predicted helical regions (Gaernier-Robinson and Chou-Fasman). Also included in the figure is a hydrophilicity graph (Kyte-Doolittle), the predicted alpha and beta amphatic regions (Eisenberg), the predicted flexible regions (Karplus-Schulz), the predicted antigenic index (Jameson-Wolf) and the predicted surface probability graph (Emini). The predicted molecular weight of CARD-3 is approximately 61 kDa. The predicted molecular weight of CARD-4L is approximately 108 kDa. Example 3: Preparation of CARD-3 and CARD-4 proteins. Recombinant CARD-3 and CARD-4 can be produced in various expression systems. For example, the CARD-3 and CARD-4 peptides can be expressed as a fusion protein. Recombinant glutathione-S-transferase (GST) from E. coli and the fusion protein can be isolated and characterized. Specifically in accordance with what is described above, CARD-3 or CARD-4 can be fused to GST and the fusion protein can be expressed in PEB1999 strain of E. coli. Expression of the fusion protein GST-CARD-3 or GST-CARD-4 in PEB199 can be induced with IPTG. The recombinant fusion protein can be purified from crude bacterial lysates of strain PEB199 induced by affinity chromatography on glutathione beads. Example 4: Identification of splice variants of CARD4. The untranslated 5 'sequence of CARD-4L was used to search databases of cDNA sequences and partial cDNA sequences using BLASTN (Washington University, version 2.0, search BLOSUM62) for additional CARD-4 cDNA clones. This search led to the identification of two cDNA clones, the Z clone from a human lymph node library and the Y clone from a human brain cDNA library. Both clones were sequenced and found to represent probable splice variants of CARD-4 encoding truncated CARD-4 proteins, Y encoding a protein of 249 amino acids and Z encoding a protein of 164 amino acids. Figure 10 shows the nucleotide (SEQ ID NO: 38) and Figure 11 shows the predicted amino acid sequences (SEQ ID NO: 39) of CARD-4Y; Figure 12 shows the nucleotide (SEQ ID NO: 40) and Figure 13 shows the amino acid sequences (SEQ ID NO: 41) of human CARD-4Z; and Figure 14 shows an alignment of the amino acid sequences of CARD-4L, when CARD-4Y, and CARD-4Z generated by the Crustal program using a table of PAM250 residue weights. Example 5: Identification of murine CARD-4. The CARD-4 polypeptide sequence was used to search databases of cDNA sequences and partial cDNA sequences using the TBLASTN program (version 1.4, search matrix BLOSUM62, and a word length of 3) for cDNA clones from CARD-4 of murine. This search led to the identification of a murine CARD-4 partial murine CARD-4 clone. Rapid identification of the cDNA terminus procedure (RACE) was applied to the 5 'end of the murine CARD-4L clone to elucidate the 5' end of the murine CARD-4L cDNA. Figure 15 shows the nucleotide sequence of murine CARD-4L (SEQ ID NO: 42), Figure 16 shows the amino acid sequence of murine CARD-4 (SEQ ID NO: 43), and Figure 17 shows a alignment of the amino acid sequences of murine CARD-4L and human CARD-4L generated by the Crustal program using a PAM250 waste weight table. Example 6. Identification of the chromosomal location of human CARD-4. To determine the chromosomal location of the human CARD-4 gene, a polymerase chain reaction was carried out with the primers specific for CARD-4 human CARD-4T, with the 5 'to 3' sequence agaaggtctggtcggcaaa (SEQ ID NO: 44), and card4k, with the sequence of 5 'a 3' aagccctgagtggaagca (SEQ ID NO: 45), was used to screen DNA from a commercially available hybrid panel of somatic cells. This analysis showed that human CARD4 is traced on chromosome 7 near the genetic marker SHGC-31928. Example 7: Identification of CARD-3 in a sieving of two yeast hybrids for proteins that physically interact with the CARD domain of human CARD-4 The DNA encoding amino acids 1-145 of human CARD-4 comprising the CARD domain was cloned into a 2-hybrid yeast screening vector to create a DNA-binding fusion CARD-4, 1-145-GAL4 for two-hybrid screening. The fusion fusion DNA domain CARD-4, 1-145-GAL4 was used to screen two hybrid libraries of human mammary gland and human prostate for genetic products that could be physically related to CARD-4, 1-145. Twelve plasmids from libraries expressing interaction proteins with CARD-4, 1-145 contained the CARD domain containing the CARD-3 protein, thus establishing a direct or indirect physical interaction between CARD-4 and CARD-3. In addition, the DNA encoding CARD-3 amino acids 434-540 comprising the CARD domain of CARD-3 (SEQ ID NO: 6) was cloned into a GAL4 transcription activation domain fusion vector of two-hybrid yeast to create a transcriptional activation domain fusion CARD-3, 435-540-GAL4. To test whether the CARD domain of CARD-3 binds with CARD-4, 1-145, the fusion expression vector of the transcriptional activation domain of CARD-3, 435-540-GAL4 and the domain fusion vector of DNA linkage CARD-4, 1-145-GAL4 were counter-formed in a Saccharomyces cerevisiae separator (yeast) of two hybrid screening. The resulting cotransformated yeast strain expressed the two reporter genes that indicate a physical interaction between the two hybrid proteins in the experiment, in this case, the transcriptional activation domain fusion protein CARD-3, 435-540-GAL4 and the protein DNA linkage fusion domain CARD-4, 1-145-GAL4. This experiment established a physical interaction between the CARD domain of CARD-3 and the CARD domain of CARD-4. Example 8: Identification of hNUDC in a yeast two hybrid screening for proteins that physically interact with the LRR domain of human CARD-4 The AD? encoding amino acids 406-953 of human CARD-4L comprising the LRR domain was cloned into a yeast two-hybrid screening vector to create an AD? CARD-4, 406-953-GAL4 for two hybrid screening. The link domain fusion of AD? CARD-4, 406-953-GAL4 was used to screen a two-hybrid library of human mammary gland for gene products that could be physically related to CARD-4, 406-953. A library plasmid expressing a protein that interacts CARD-4, 406-953 contained the hNUDC protein, the human ortholog of the rat? UDC protein that had been implicated in the nuclear movement (Morris et al., Curr. Biol. 8: 603 [1998], Morris et al., Exp. Cell Res. 238: 23 [1998]), thus establishing a physical interaction between CARD-4 and h? UDC. Example 9: Discovery of regulation by CARD-4 of? F-? B. The first group of experiments described in this example was carried out to determine if CARD-4 can activate the? F-? B pathway. The regulation of CARD-4 from the route of? F-? B is interesting because the route of? F-? B is involved in many diseases described in (? Ew England Journal of Medicine 336: 1066 [1997]) and (American Journal of Cardiology 76: 18C [1995]), and other preferences known to those skilled in the art. The participation of CARD-4 in the route of? F-? B would make CARD-4 an attractive target for drugs that modulate the route of? F-? B for the treatment of diseases, conditions and biological processes dependent on the route of? F-? B. The first group of experiments showed that the induction of the? F-? B pathway mediated by CARD-4 specific. The second group of experiments described in this example were carried out to determine whether CARD-3, the serine / threonine protein kinase of? IK (Su et al., EMBO J. 16: 1279 [1997]) or the transduction protein of TRAF6 signal (Cao et al., Nature 383: 443 [1996]), proteins known to participate in the induction of NF-? B (McCarthy et al., J. Biol. Chem. 273: 16968 [1998]), are involved in the transduction of the NF-? B path induction signal dependent on CARD-4. It was found that CARD-3, NIK, and TRAF6 are all involved in the transduction of the NF-? B path induction signal mediated by CARD-4. In nine transfection experiments, 293T cells coexpressing a reporter plasmid NF-? B and either pCI, pCI-CARD-4L (expressing CARD-4L), pCI-CARD-4S (expressing CARD.4S), pCI- APAFL (expressing Apaf-1), pCI-APAFS (expressing an Apaf-1 variant that does not have WD repeats), pCI-CARD-4LnoCARD (expressing CARD-4L without a CARD domain), pCI-CARD- 4LnoLRR (expressing CARD-4L without an LRR), pCI-CARD4LCARD only (expressing only the CARD-4L CARD domain) or pCI-CARD4NBS only (expressing only the nucleotide binding sequence of CARD-4L) were created. 293T cells were placed in 6 well plates (35 mm wells) and transfected two days later (90% confluence) with 1 μg reporter plasmid of NF-KB luciferase (pNF-? B-Luc, Stratagene), 200 ng of pCMV ß-gal, 600 ng of pCI vector and 200 ng of indicated expression plasmids using a Superfect transfection reagent (Qiagen). For dominant negative experiments, 2 ng of CARD4 express the plasmid and 800 ng of dominant negative plasmid were used. The cells were harvested at 48 hours after transfection and the luciferase activity in extracts of cells diluted 1000 times was determined by using the Luciferase Assay System (Promega). In addition, ß-galactosidase activities were determined and used to normalize the efficiency of transfection. The relative luciferase activity was determined at the end of the experiment to evaluate the activation of the NF-? B pathway by the gene expressed by the pCI-based plasmid in each line of transfected cells. Cell lines containing pCI, pCI-APAFS, pCI-APAFL, non-CARD pCI-CARD-4L, and pCI-CARD4NBS only had similar baseline levels of luciferase expression but cell lines containing pCI-CARD-4L , pCI-CARD4LnoLRR, and pCI-CARD-4L CARD only had an expression of luciferase about 9 months higher in relation to the baseline and the cell line containing pCI-CARD4S had a luciferase expression 16 times higher with relation to the baseline. This result demonstrates the induction by CARD-4S and CARD-4L of the NF-? B pathway. This induction of the NF-? B pathway mediated by CARD-4 is independent of the CARD domain of CARD-4 since the non-CARD pCI-CARD-4 construct expressing CARD-4 that does not have its CARD domain did not induce Luciferase reporter gene and pCI-CARD4LCARD alone expressing the CARD domain of CARD-4 induced the reporter gene of luciferase. Likewise, the LRR domains of CARD-4 are not required for the activation of the NF-? B pathway since pCI-CARD4 LnoLRR expressing a mutant CARD-4 protein that does not have LRR domains can induce the reporter gene of luciferase. In addition, the NBS domain of CARD-4 is not sufficient for the activation of the NF-? B pathway since only pCI-CARD-4NBS expressing the NBS domain of CARD-4 can not induce the luciferase reporter gene. In addition, the induction of the NF-? B pathway by CARD-4 is specific, since no construct expressing Apaf in this experiment induced the activation of luciferase. In five transfection experiments, 293T cells that coexpress a reporter plasmid of NF-KB (NF-? B-luciferase, Stratagene), and pCI-CARD-4L and no vector, pCI-TRAF6-DN (expressing a dominant negative version of TRAF- 6), pCI-NIK-DN (expressing a dominant negative version of NIK kinase), pCI-CARD-3 CARD only (expressing the CARD-3 domain, which acts as the dominant negative reaction of CARD-3), or pCI-Bcl-XL (expressing the anti-apoptotic protein Bcl-XL) were created. TRAF6-DN, NIK-DN, and CARD-3-CARD are only dominant negative alleles of the TRAF6, NIK, and CARD3 genes, respectively. After 48 hours, the cells were used and the relative luciferase activity (Promega kit) was determined to evaluate the activation of the NF-EB pathway by the genes expressed by the plasmid or the two pCI-based plasmids in each cell line transfected. Cell lines containing pCI-CARD-4 Only or pCI-CARD and pCI-Bcl-XL had a relative expression of luciferase reporter gene of approximately 18 units. The cell lines containing pCI-CARD-4L and pCI-TRAF6-DN, pCI-CARD-4L and pCI-NIK-DN, or pCI-CARD-4L and pCI-CARD-3-CARD only showed gene expression reporter of relative luciferase of approximately 4 units. The inhibition of the induction of the NF-? B pathway mediated by CARD-4L by TRF6-DN, NIK-DN and CARD-3-CARD is only specific since Bcl-XL did not inhibit the induction of the NF- pathway. ? B mediated by CARD-4L. These results demonstrate that dominant negative alleles of TRAF6, NIK and CARD-3 expressed respectively from pCI-TRAF6-DN, pCI-NIK-DN, and pCI-CARD-3-CARD only block the induction of the reporter gene of NF- ? B by CARD-41 expression (pCI-CARD-4L) and suggest that TRAF6, NIK, and CARD-3 act downstream of CARD-4L to transduce the induction stimulus of the CARD NF-? B pathway. 4L. In a further experiment, the coexpression of CARD-4 and the CARD domain of RICK revealed that the RICK CARD domain functions as a dominant negative mutant which suggests that RICK is a current mediator below the CARD-4 function. Example 10. Discovery of CARD-4 increase in caspase activity 9 In 10 transfection experiments, 293T cells co-expressing a plasmid expressing beta galactosidase (pCMV β-gal from Stratagene) as a marker for viable cells and either pCI, pCI -CARD-3, pCI-APAF, pCI-CARD-4L, pCI-CARD-4S, pCI-CARD4LnoLRR, pCI-CARD4NBS only, pCI-CARD4LCARD, pCI-CARD-4LnoCARD or pCI-casp9 (expressing caspase-9) They were created. Transfections included 400 ng pCMV ß-gal, 800 ng expression plasmid and Superect transfection reagent from Qiagen, and were carried out in accordance with the manufacturer's instructions. After 40-48 hours, the cells were fixed and had for beta galactosidase expression and cell viability was determined by counting the number of beta galactosidase-positive cells. The expression of pCI, pCI-CARD-3, pCI-APAF, pCI-CARD-4L, pCI-CARD-4S, pCI ~ CARD4LnoLRR, pCI-CARD4NBS only, pCI-CARD4LCARD, and pCI-CARD-4LnoCARD did not result in loss of cellular viability. As expected, the expression of pCI-casp9 in 293T cells resulted in a loss of viability of approximately 75% of the cells in the experiment. It was then tested whether pCI, pCI-CARD-3, pCI-APAF, pCI-CARD-4L, pCI-CARD-4S, pCI-CARD4LnoLRR, pCI-CARD4NBS only, pCI-CARD4LCARDs alone, or pCI-CARD-4LnoCARD could regulate the caspase-mediated apoptosis 9. In nine transfection experiments, 293T cells co-expressing a plasmid expressing beta galactosidase as a marker for viable cells, pCI-casp9, and either pCI, pCI-CARD-3, pCI-APAF, pCI-CARD -4L, pCI-CARD-4S, pCI-CARD4LnoLRR, pCI-CARD4NBS, and pCI-CARD-4LnoCARD were created. After 40-48 hours, cells were fixed and stained for beta-galactosidase expression and cell viability was determined by counting the number of beta galactosidase-positive cells. The expression of pCI, pCI-CARD-4LnoCARD, and pCI-CARD4NBS only in 293T cells expressing caspase 9 had no effect on apoptosis induced by caspase 9. However, pCI-CARD-3, pCI-CARD-4L, pCI-CARD-4S , pCI-CARD4LnoLRR, pCI-CARD4LCARD and, as expected, pCI-APAF increased the caspase-induced apoptosis level 9 to 20 or less beta-galactosidase-positive cell per experiment from approximately 100 beta-galactosidase-positive cells per experiment. This experiment demonstrated that CARD-4 can increase caspase-mediated apoptosis 9 because the co-expression of CARD-4L or CARD-4S with caspase 9 dramatically increases apoptosis mediated by caspase-9. In addition, the CARD domain of CARD-4 (SEQ ID NO: 10) is necessary and sufficient for the CARD-4-mediated increase in apoptosis enhanced by caspase-9 because CARD-4L does not have its CARD domain (pCI-CARD -4LnoCARD) does not increase apoptosis mediated by caspase-9 while the CARD domain of CARD-4 expressed alone (pCI-CARD4LCARD) only if it induces apoptosis mediated by caspase-9. In addition, the LRR present in CARD-4 is not required to increase by CARD-4 caspase-9 mediated apoptosis since the expression of a CARD-4 protein that does not have the LRR (pCI-CARD4LnoLRR) continues to increase apoptosis mediated by caspasa-9. The NBS of CARD-4 is not enough to increase the caspase-9 mediated apoptosis through CARD-4 because the expression of NBS only of CARD-4 (pCI-CARD4NBS) does not increase the apoptosis mediated by caspase-9. This experiment also shows that CARD-3 can increase apoptosis mediated by caspase-9. As presented in details below in example 12, CARD-4 does not appear to interact directly with caspase-9, suggesting that potentiation of caspase-9 activity by CARD-4 is mediated by the activation of downstream days . Example 11: Identification and tissue distribution of mRNA species expressed by the human CARD-4 gene. Northern analysis of mRNAs extracted from adult human tissues revealed a band of 4.6 kilobase mRNA that was expressed in most of the tissues examined. Higher expression was observed in heart, spleen, placenta and lung.

CARD-4 was also expressed in kidney, liver, lung and fetal brain. Lines of cancer cells expressing the 4.6 kilobase CARD-4 mRNA include HeLa, K562, Molt4, SW480, A549 and melanoma. A larger CARD4 mRNA of 6.5 to 7.0 kilobases was expressed in heart, spleen, lung, fetal lung, fetal liver, and in the Molt4 and SW480 cell lines. Example 12: Physical association of CARD-4 with CARD-3 Polymerase chain reaction primers specific for CARD-4 with the 3 'primer encoding the HA epitope tag were used to amplify the labeled CARD-4L gene epitope with HA and this polymerase chain reaction product was cloned into the mammalian expression vector pCI. The CARD-3 specific polymerase chain reaction primers with the 5 'primer encoding the MYC epitope tag were used to amplify the MYC-tagged CARD-3 gene epitope and this polymerase chain reaction product was cloned in the mammalian expression vector pCI. The polymerase chain reaction primers specific for CARD-3 with the 5 'primer encoding the MYC epitope tag were used to amplify the CARD-3 gene that does not have the CARD domain (SEQ ID NO: 6) epitope tagged with MYC and this polymerase chain reaction product was cloned into the mammalian expression vector pCI. Polymerase chain reaction primers specific for caspase 9 with the 3 'primer encoding the MYC epitope tag were used to amplify the caspase 9 gene epitope labeled with MYC and this polymerase chain reaction product was cloned in the mammalian expression vector pCI. In three transfection experiments, 293T cells co-expressing pCI-CARD-4LcHA and either pCI-CARD-3nMYC, pCI-CARD-3 non-CARD-nMYC, or pCI-casp9cMYC were created. Cell from each transfected line was used and an immunoprecipitation procedure was carried out on each lysate with an antibody labeled with anti-MYC epitope to precipitate in CARD-4LCHA expressed by each cell line and any physically associated protein. The immunoprecipitated proteins were separated by electrophoresis on denaturing polyethylamide gels, transferred to nylon filters, probed with an antibody labeled with anti-HA epitope in a Western blotting experiment to determine whether the MYC-labeled protein co-expressed with the CARD protein. 4LcHA had co-immunoprecipitated with the CARD-4LcHA protein. In this experiment, it was found that CARD-3 is co-immunoprecipitated with CARD-4 while CARD-3 does not have * its CARD domain and caspase-9 was not co-immunoprecipitated with CARD-4. This experiment demonstrates that CARD-4 and CARD-3 are physically associated and that CARD-3 requires its CARD domain pair to associate with CARD-4. In addition, CARD-4 does not seem to associate with caspase-9. Example 13: Genomic sequence of CARD-4 Figure 18 shows the genomic sequence of 32042 nucleotides of CARD-4. This sequence is based on the cDNA sequence of CARD-4 described above and a BAC sequence (DBEST accession number AC006027). The CARD-4 cDNA sequence described above was used to correct 3 errors in the BAC sequence including an error that results in a change of frame. The genomic sequence of CARD-4 of Figure 18 includes the following introns and exons: exon 1: nucleotides 364-685, which encode amino acids 1-67 (initial codon at nucleotides 485-487); intron 1: nucleotides 686-2094; exon 2: nucleotides 2095-2269, which encode amino acids 67-126; intron 2: nucleotides 2270-4365; exon 3: nucleotides 366-6190, which encodes amino acids 126-734; intron 3: nucleotides 6191-9024; exon 4: nucleotides 9025-9108, which encode amino acids 734-762; intron 4: nucleotides 9109-10355; exon 5: nucleotides 10356-10439, which encodes amino acids 762-790; intron 5: nucleotides 10440-11181; exon 6: nucleotides 1182-11265, which codes for amino acids 790-818; intron 6: nucleotides 11266-19749; exon 7: nucleotides 19750-19833, which encodes amino acids 818-846; intron 7: nucleotides 19834-21324; exon 8: nucleotides 21325-21408, which encodes amino acids 846-874; intron 8: nucleotides 21409-24226; exon 9: nucleotides 24227-24310, amino acids 874-903; intron 9: nucleotides 24311-27948; exon 10: nucleotides 27949-28032, amino acids 903-930; intron 10: nucleotides 28033-31695; exon 11: nucleotides 31696-32024, which encode amino acids 930-953 (retention codon in nucleotides 31766-31768). Introns in the genomic sequence of CARD-4 contain donor splice acceptor and consensus sites (Molecular Cell Biology, Darnell et al., Eds., 1996). The genomic sequence of CARD-4 is useful for genetic identification and mapping and identifies mutations, for example, mutations in splice donor sites or splice acceptors. Equivalents Those skilled in the art will recognize or may determine using no more than routine experiments many equivalents to the specific embodiments of the invention described herein. Such equivalents are encompassed in the following claims.

Claims (22)

1. CLAIMS An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 38 or a complement thereof; b) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 40 or a complement thereof; c) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 42, or a complement thereof; d) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 38; e) a nucleic acid molecule encoding a polypeptide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 40; f) a nucleic acid molecule encoding a polypeptide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 42; g) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 38 or a complement thereof; h) a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 40 or a complement thereof; i) a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 42 or a complement thereof; j) a nucleic acid molecule encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 38; k) a nucleic acid molecule encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 40; and 1) a nucleic acid molecule encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 42. A host cell containing one of the nucleic acid molecules of claim 1. An isolated polypeptide selected from the group which consists of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 39; b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 41; c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 43; d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 39; e) a polypeptide comprising the amino acid sequence of SEQ ID NO: 41; f) a polypeptide comprising the amino acid sequence of SEQ ID NO: 43; g) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 39, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 39; h) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 41, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 41; i) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 43, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 43; ) an allelic variant that naturally occurs from a polypeptide consisting of the amino acid sequence SEQ ID NO: 39, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes with a nucleic acid molecule consisting of SEQ ID NO: 38, under stringent conditions; k) an allelic variant occurring naturally of a polypeptide consisting of the amino acid sequence SEQ ID NO: 41, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes with a nucleic acid molecule consisting of SEQ ID NO : 40, under strict conditions; and 1) a naturally occurring allelic variant of a polypeptide consisting of the amino acid sequence SEQ ID NO: 43, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes with a nucleic acid molecule consisting of SEQ ID NO: 43. NO: 42, under strict conditions. 4. An antibody that selectively binds to any of the polypeptides of claim 3. 5. A method for the production of a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO : 39; b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 41; c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 43; d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 39; e) a polypeptide comprising the amino acid sequence of SEQ ID NO: 41; f) a polypeptide comprising the amino acid sequence of SEQ ID NO: 43; g) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 39, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 39; h) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 41, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 41; i) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 43, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 43; j) an allelic variant occurring naturally of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 39, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes with a nucleic acid molecule consisting of SEQ ID NO: 39. NO: 38 under strict conditions; k) an allelic variant occurring naturally of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 41, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule consisting of SEQ ID NO: 41. NO: 40 under strict conditions; 1) an allelic variant that naturally occurs from a polypeptide consisting of the amino acid sequence of SEQ ID NO: 43, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule consisting of SEQ ID NO: 43. NO: 42 under strict conditions; comprising the step of culturing the host cell of claim 2 under conditions in which the nucleic acid molecule is expressed. A method for detecting the presence of a polypeptide of claim 2 in a sample, comprising: a) contacting the sample with a compound that selectively binds with a polypeptide of claim 2; and b) determining whether the compound binds to the polypeptide of claim 2 in the sample. A method for detecting the presence of a nucleic acid molecule of claim 2 in a sample, comprising: a) contacting the sample with a nucleic acid probe or an initiator that hybridizes selectively to the nucleic acid molecule; and b) determining whether the nucleic acid probe or the primer binds to a nucleic acid molecule in the sample. A method for identifying a compound that binds with a polypeptide of claim 2, comprising the steps of: a) contacting a polypeptide, or a cell expressing a polypeptide of claim 2 with a test compound; and b) determining whether the polypeptide binds to the test compound. The method according to claim 8, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detecting the link by direct detection of the test compound / polypeptide linkage; b) detecting the link through the use of a competition assay assay; and c) detecting the link using an assay for signal transduction mediated by CARD-4L or CARD-4S. A method for modulating the activity of a polypeptide according to claim 2 comprising contacting a polypeptide or a cell expressing a polypeptide according to claim 2 with a compound that binds on the polypeptide in a sufficient concentration to modulate the activity of the polypeptide. A method for identifying the compound that modulates the activity of a polypeptide of claim 2 comprising: a) contacting a polypeptide of claim 2 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound that modulates the activity of the polypeptide. 12. A method for identifying a compound that blocks the interaction between a CARD-4 protein comprising a CARD-4 domain and a protein that interacts with CARD-4, comprising the steps of: a) incubating said CARD-4 protein and said agent that interacts in the presence and absence of a test agent; b) determining whether said test agent reduces the binding of said CARD-4 protein and said interacting agent; and c) identifying a compound that desires the interaction of said CARD-4 protein with said agent that interacts when said compound reduces the binding of said CARD-4 protein with said interacting agent; wherein said interacting agent is selected from the group consisting of CARD-3 and hNUDC and wherein said CARD-4 domain comprises amino acids 1-145 of an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 43. 13. The method according to claim 12, wherein the CARD-4 protein comprising a CARD-4 domain is selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 8; b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 39; c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 41; and d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 43. The method according to claim 12, wherein the CARD-4 protein and the interacting agent are expressed in a prokaryotic or recombinant eukaryotic cell line or where the CARD-4 protein and the interacting agent are proteins isolated or present in cell-free protein extracts. A method for identifying a compound that inhibits the induction of the NF-? B pathway by a CARD-4 protein, comprising the steps of: a) incubating a recombinant cell line containing a vector expressing CARD-4 in the presence and absence of a test agent; b) determining whether said test agent inhibits the induction of the NF-? B pathway by CARD-4; and c) identifying a compound that inhibits the induction of the NF-KB pathway by CARD-4. The method according to claim 15, wherein the CARD-4 protein comprising a CARD-4 domain is selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 8; b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 39; c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 41; and d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 43. The method according to claim 15, further comprising: a) incubating a line of recombinant cells expressing CARD-4 and also expressing a reporter gene of the NF-? B path in the presence and absence of a test agent; b) determining whether said test agent inhibits the induction of the reporter gene of the NF-éB pathway by CARD-4; and c) identifying a compound that inhibits the reporter gene induction of NF-? B pathway by CARD-4. A method to identify a compound that inhibits the increase of caspase 9 activity by a protein CARD-4 comprising a CARD-4 domain, comprising the steps of: a) incubating a line of recombinant cells expressing caspase 9 and CARD-4 in the presence and absence of a test agent; b) determining whether said test agent inhibits caspase 9 activity; and c) identifying a compound that inhibits the increase of caspase 9 activity by a CARD-4 protein. The method according to claim 18, further comprising: a) incubating a line of recombinant cells expressing caspase 9 and CARD-4 and a beta-galactosidase expression vector in the presence and absence of a test agent; b) determining whether the presence of said test agent increases the proportion of cells staining positively for beta-galactosidase; and c) identifying a compound that inhibits the increase of caspase-9 activity by a CARD-4 protein by identifying a compound that increases the proportion of cells that stain positively for beta-galactosidase. The method according to claim 18, wherein the CARD-4 protein comprising a CARD-4 domain is selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 8; b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 39; c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 41; and d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 43. 21. A method for identifying a compound that inhibits the enhancement of caspase-9 activity by a CARD-3 protein comprising a CARD-3 domain, which comprises the steps of: a) incubating a line of recombinant cells expressing caspase 9 and CARD-3 in the presence and absence of a test agent; b) determining whether said test agent inhibits caspase 9 activity; and c) identifying a compound that inhibits the increase of caspase 9 activity by a CARD-3 protein. 22. The method according to claim 21 further comprising: a) incubating a line of recombinant cells expressing caspase 9 and CARD-3 and a beta-galactosidase expression vector in the presence and absence of a test agent; b) determining whether the presence of said test agent increases the proportion of cells staining positively for beta-galactosidase; and c) identifying a compound that inhibits the increase of caspase-9 activity by a CARD-3 protein by identifying a compound that increases the proportion of cells that stain positively for beta-galactosidase.