WO1999000499A1 - Human fadd-interacting protein (fip) - Google Patents

Abstract

A novel gene encoding a human FADD-interacting protein (FIP) is disclosed. FIP physically interacts with FADD and is involved in the human apoptotic pathway. Transfection of FIP into human cells induces apoptosis. FIP protein and coding sequences can be used to treat diseases characterized by defects in the regulation of cell proliferation and to screen for drugs which affect the apoptotic pathway.

Description

HUMAN FADD-INTERACTING PROTEIN (FTP)

This application claims the benefit of applications Serial Nos. 60/050,792, filed June 26, 1997, and 60/087,886, filed June 3, 1998, which are incorporated herein by reference.

TECHNICAL AREA OF THE INVENTION

The invention relates to the area of apoptosis (programmed cell death). More particularly, the invention relates to the regulation of apoptosis.

BACKGROUND OF THE INVENTION

Programmed cell death, or apoptosis, is involved in the normal development of multicellular organisms. The cellular pathways involved in apoptosis are not well understood, but diseases which involve defects in the control of cellular proliferation and death may involve defects in proteins involved in apoptosis. The ability to manipulate the apoptotic pathway would be useful in the control of these diseases. Thus, there is a need in the art to identify proteins involved in apoptotic pathways so that these pathways can be exploited to control disease.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide protein and nucleotide sequences which can be used to affect apoptosis. This and other objects of the invention are provided by one or more of the embodiments described below. One embodiment of the invention provides an isolated and purified human

FADD-Interacting Protein (FIP protein) having an amino acid sequence which is at least 85% identical to the amino acid sequence shown in SEQ LD NO:2. Another embodiment of the invention provides an isolated and purified human FIP polypeptide comprising at least 8 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2.

Even another embodiment of the invention provides a fusion protein comprising a first protein segment and a second protein segment fused to each other by means of a peptide bond. The first protein segment comprises at least 8 contiguous amino acids of a human FLP protein selected from the amino acid sequence shown in SEQ ID NO:2.

Still another embodiment of the invention provides a preparation of antibodies which specifically bind to a human FIP protein.

Yet another embodiment of the invention provides an isolated and purified subgenomic polynucleotide encoding an amino acid sequence of a human FIP protein or a human FIP protein variant. The nucleotide sequence of the subgenomic polynucleotide is at least 85% identical to the nucleotide sequence shown in SEQ ED NO:l.

Another embodiment of the invention provides an expression construct. The expression construct comprises a subgenomic polynucleotide and a promoter. The subgenomic polynucleotide comprises at least 11 contiguous nucleotides selected from the nucleotide sequence shown in SEQ ID NO: 1. The subgenomic polynucleotide is located downstream from the promoter. Transcription of the subgenomic polynucleotide initiates at the promoter.

Even another embodiment of the invention provides a homologously recombinant cell having incorporated therein a new transcription initiation unit. The new transcription initiation unit comprises an exogenous regulatory sequence, an exogenous exon, and a splice donor site. The transcription initiation unit is located upstream of a coding sequence of a FIP gene. The exogenous regulatory sequence directs transcription of the coding sequence of the FTP gene.

Yet another embodiment of the invention provides a host cell which comprises an expression construct. The expression construct comprises (1) a subgenomic polynucleotide comprising at least 11 contiguous nucleotides selected from the nucleotide sequence shown in SEQ ID NO: 1 and (2) a promoter. The subgenomic polynucleotide is located downstream from the promoter. Transcription of the subgenomic polynucleotide initiates at the promoter.

Even yet another embodiment of the invention provides a method of inducing apoptosis in a cell. The cell is contacted with a polypeptide comprising amino acids 348-727 of SEQ ID NO:2. Apoptosis of the cell is induced.

Still another embodiment of the invention provides a method of preventing apoptosis of a cell. The cell is contacted with a composition comprising a polynucleotide encoding a reagent which specifically binds to a wild-type human FIP expression product and a pharmaceutically acceptable carrier. Apoptosis of the cell is prevented.

A further embodiment of the invention provides a composition. The composition comprises a polynucleotide encoding a reagent which specifically binds to a wild-type human FIP expression product and a pharmaceutically acceptable carrier.

Another embodiment of the invention provides a composition. The composition comprises all or a portion of a protein having the amino acid sequence shown in SEQ ID NO:2 or all or a portion of a gene having the nucleotide sequence shown in SEQ ID NO: 1 and a pharmaceutically acceptable carrier. Even another embodiment of the invention provides a method of screening test compounds for the ability to induce or prevent apoptosis. A cell is contacted with a test compound. The cell comprises (i) a first expression construct comprising a subgenomic polynucleotide encoding at least a portion of FADD comprising amino acids 1-110 of SEQ ID NO:4, wherein the portion of FADD binds to a portion of FIP and (ii) a second expression construct comprising a subgenomic polynucleotide encoding at least a portion of FIP, wherein the portion of FIP comprises amino acids 348-727 of SEQ ID NO:2 and wherein the portion of FIP binds to the portion of FADD. The ability of the test compound to decrease or increase binding of the portions of FIP to the portion of FADD is measured. A test compound which increases the binding being a potential drug for inducing apoptosis. A test compound which decreases the binding being a potential drug for preventing apoptosis.

Still another embodiment ofthe invention provides a method of screening test compounds for the ability to induce or prevent apoptosis. A cell is contacted with a test compound. The cell comprises a first fusion protein and a second fusion protein. The first fusion protein comprises (1) at least a portion of a FIP protein comprising amino acids 348-727 of SEQ ID NO: 2 and (2) either a DNA binding domain or a transcriptional activating domain. The second fusion protein comprises at least a portion of a FADD protein comprising amino acids 1-110 of SEQ ID NO:4. The portion ofthe FIP protein binds to the portion ofthe FADD protein. If the first fusion protein comprises a DNA binding domain, then the second fusion protein comprises a transcriptional activating domain. If the first fusion protein comprises a transcriptional activating domain, then the second fusion protein comprises a DNA binding domain. The interaction ofthe first and second fusion proteins reconstitutes a sequence-specific transcription activating factor. The cell also comprises a reporter gene comprising a DNA sequence to which the DNA binding domain specifically binds. The expression ofthe reporter gene is measured. A test compound which decreases the expression ofthe reporter gene is identified as a potential agent for preventing apoptosis in the cell. A test compound which increases the expression ofthe reporter gene is identified as a potential agent for inducing apoptosis in the cell.

Another embodiment ofthe invention provides a method of screening test compounds for the ability to induce or prevent apoptosis. A test compound is contacted with a first polypeptide comprising a FADD-binding site and a second polypeptide comprising a FTP-binding site. The FADD-binding site comprises at least a portion of a FIP protein comprising amino acids 348-727 of SEQ ID NO:2. The FlP-binding site comprises amino acids 1-110 of SEQ ID NO:4. The amount of binding between the first and second polypeptides in the presence ofthe test compound is measured. A test compound which increases the amount of binding between the first and second polypeptides is identified as a potential drug for inducing apoptosis. A test compound which decreases the amount of binding between the first and second polypeptides is identified as a potential drug for preventing apoptosis.

Yet another embodiment ofthe invention provides a method of determining the identity of a body sample of a human. The body sample is assayed for the presence of a polypeptide consisting of amino acids 348-727 of SEQ ID NO:2 or a 2.5 kb FIP mRNA encoding the FIP polypeptide. The presence ofthe FIP polypeptide or the 2.5 kb FIP mRNA indicates that the body sample originates from a tissue selected from the group consisting of brain, pancreas, spleen, and peripheral blood lymphocytes.

Still another embodiment ofthe invention provides a method of screening for test compounds which are useful for enhancing transfer of FIP subgenomic polynucleotides to a cell. A FIP subgenomic polynucleotide is delivered to a cell. The cell is contacted with a test compound. Transfer ofthe FIP subgenomic polynucleotide to the cell is measured or a biological effect ofthe FIP subgenomic polynucleotide within the cell is monitored. A test compound which enhances the transfer to the cell or the biological effect within the cell ofthe FIP subgenomic polynucleotide identifies a compound which is useful for enhancing transfer ofthe FIP subgenomic polynucleotide to the cell. Another embodiment ofthe invention provides a method of expressing a

FIP subgenomic polynucleotide in a cell. The FIP subgenomic polynucleotide is delivered to the cell. The FIP subgenomic polynucleotide is expressed.

The present invention thus provides the art with the information that FIP, a heretofore unknown protein, binds to FADD and is involved in the regulation of programmed cell death, or apoptosis. Alteration of FIP protein levels in a cell can be used to induce or prevent apoptosis in the cell. FIP protein can also be used, inter alia, in assays to screen for substances which have the ability to induce or prevent apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Figure 1 shows the results of transfection experiments in which the C-terminal portion of FLP (amino acids 348-727 of SEQ ID NO:2) induced cell death, but full-length FIP did not.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is a discovery ofthe present invention that the novel human protein FIP (FADD-Interacting Protein) binds to FADD (Fas-associated protein with a novel death domain), a protein involved in the Fas-mediated cell death pathway (Chinnaiyan et al, Cell 81, 505-512 1995). FLP is located in the cytoplasm of human tissues in two forms. Full-length FLP is found in all tissues investigated.

Additionally, a shorter form of full-length FLP, consisting ofthe C-terminal 348-727 amino acids of full-length FLP (FLP_34g.₇₂₇), is found in brain, pancreas, peripheral blood lymphocytes, and spleen. Both forms of FLP protein bind to FADD. In addition, FD?₃₄₈.₇₂₇ induces apoptosis. As used herein, "FLP protein" refers to both full-length FLP and to

Sequences ofthe human FLP gene (SEQ LD NO: 1) and protein (SEQ LD NO:2), as well as the human FADD gene (SEQ LD NO:3) and protein (SEQ LD NO:4), are disclosed herein. Reference to each of these nucleotide or amino acid sequences includes variants which have one or more ofthe same biological activities, as described below. SEQ LD NOS: 5 and 6 are oligonucleotide probes

(see Example 1).

Full-length human FLP has the sequence disclosed in SEQ LD NO:2. Any naturally occurring biologically active variants of this sequence which occur in human tissues are within the scope of this invention. Naturally occurring biologically active variants of full-length FLP bind to FADD, are expressed in at least one human tissue, and do not induce apoptosis. Naturally occurring biologically active variants of FLP^.^ bind to FADD, are found in brain, pancreas, peripheral blood lymphocytes, and spleen, and induce apoptosis. FLP polypeptides differ in length from full-length FLP or FIP₃₄g_₇₂₇ and contain 6, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 75, 80, 90, or 100 or more contiguous amino acids of a FLP protein. Variants of FLP protein and FLP polypeptides can also occur. FLP variants can be naturally or non-naturally occurring. Naturally occurring FLP variants are found in humans or other species and comprise amino acid sequences which are substantially identical to the amino acid sequence shown in SEQ LD NO:2. Non-naturally occurring FLP variants which retain substantially the same biological activities as naturally occurring FLP variants are also included here. Preferably, naturally or non-naturally occurring FLP variants have amino acid sequences which are at least 85%, 90%, or 95% identical to amino acid sequences shown in SEQ ID NO:2 and have similar biological properties, including the ability to bind to FADD and, for FLP_34g.₇₂₇, to induce apoptosis. More preferably, the molecules are at least 98% or 99% identical. Percent sequence identity between a wild-type protein or polypeptide and a variant is calculated by counting the number of amino acid matches between the wild-type and the variant and dividing the total number of matches by the total number of amino acid residues ofthe wild-type sequence.

Preferably, amino acid changes in FLP variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. It is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological properties ofthe resulting FLP variant. Properties and functions of FLP variants are ofthe same type as a F P protein or polypeptide comprising amino acid sequences of SEQ ID NO:2, although the properties and functions of variants can differ in degree. Whether an amino acid change results in a functional FLP variant can readily be determined. For example, binding of a full-length FIP or FLP₃₄₈.₇₂₇ variant to FADD can be detected using specific antibodies, which are disclosed herein. The ability of a

or polypeptide variant to induce apoptosis can be assayed by transfecting cultures of cells, such as HeLa cells, with subgenomic polynucleotides encoding FLP variants and examining the cultures for apoptotic cells, as described below in Example 4. Apoptotic cells can be recognized by well known morphological features, such as cell shrinkage, membrane blebbing, and chromatin condensation. Cohen, 1993, Immunol Today 14, 126-30.

FLP variants include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties. FLP variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions which do not affect the binding of FLP to FADD or the ability of FLP_34g_₇₂₇ to induce apoptosis are also FLP variants. Covalent variants can be prepared by linking functionalities to groups which are found in the amino acid chain or at the N- or C-terminal residue, as is known in the art. Full-length FLP can be extracted, using standard biochemical methods, from

FLP-producing human cells, such as spleen, thymus, prostate, testis, small intestine, colon, peripheral blood lymphocytes, heart, brain, placenta, lung, liver, skeletal muscle, kidney, or pancreas.

can be extracted from spleen, peripheral blood lymphocytes, brain, or pancreas cells. An isolated and purified FLP protein or polypeptide is separated from other compounds which normally associate with a

FLP protein or polypeptide in a cell, such as certain proteins, carbohydrates, lipids, or subcellular organelles. A preparation of isolated and purified F P proteins or polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. FLP proteins and polypeptides can also be produced by recombinant DNA methods or by synthetic chemical methods. For production of recombinant FLP proteins or polypeptides, coding sequences selected from the FIP nucleotide sequence shown in SEQ LD NO:l, or variants of that sequence which encode FLP protein, can be expressed in known prokaryotic or eukaryotic expression systems

(see below). Bacterial, yeast, insect, or mammalian expression systems can be used, as is known in the art.

Alternatively, synthetic chemical methods, such as solid phase peptide synthesis, can be used to synthesize a FLP protein or polypeptide. General means for the production of peptides, analogs or derivatives are outlined in CHEMISTRY

AND BIOCHEMISTRY OF AMΓNO ACIDS, PEFΠDES, AND PROTEINS ~ A SURVEY OF RECENT DEVELOPMENTS, Weinstein, B. ed., Marcell Dekker, Inc., publ, New York (1983). Moreover, substitution of D-amino acids for the normal L-stereoisomer can be carried out to increase the half-life ofthe molecule. FLP variants can be similarly produced.

Non-naturally occurring fusion proteins comprising at least 6, 8, 10, 12, 15, 18, 20, 25, 50, 60, 75, 80, 90, or 100 or more contiguous FLP amino acids can also be constructed. Human FLP fusion proteins are useful for generating antibodies against FLP amino acid sequences and for use in various assay systems. For example, FLP fusion proteins can be used to identify proteins which interact with

FLP protein and influence its function or which interfere with the binding of FADD to FLP or Fff ₃₄₈.₇₂₇. Physical methods, such as protein affinity chromatography, or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can also be used for this purpose. Such methods are well known in the art and can also be used as drug screens.

A FLP fusion protein comprises two protein segments fused together by means of a peptide bond. The first protein segment comprises at least 6, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 75, 80, 90, or 100 or more contiguous amino acids of a FLP protein. The amino acids can be selected from the amino acid sequence shown in SEQ LD NO:2 or from a biologically active variant of that sequence, such as those described above. The first protein segment can also comprise full-length FLP or FLP_34g_₇₂₇.

The second protein segment can be a full-length protein or a protein fragment or polypeptide. The fusion protein can be labeled with a detectable marker, as is known in the art, such as a radioactive, fluorescent, chemiluminescent, or biotinylated marker. The second protein segment can be an enzyme which will generate a detectable product, such as β-galactosidase. The first protein segment can be N-terminal or C-terminal, as is convenient.

Techniques for making fusion proteins, either recombinantly or by covalently linking two protein segments, are also well known. Recombinant DNA methods can be used to prepare FIP fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO: 1 in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as described below. Isolated and purified FLP proteins, polypeptides, variants, or fusion proteins can be used as immunogens, to obtain preparations of antibodies which specifically bind to FLP protein. The antibodies can be used, inter alia, to detect wild-type FLP protein in human tissue and fractions thereof. The antibodies can also be used to detect the presence of mutations in the FIP gene which result in under- or over- expression of a FLP protein or in expression of a FLP protein with altered size or electrophoretic mobility.

Preparations of polyclonal or monoclonal antibodies can be made using standard methods. Single-chain antibodies can also be prepared. Single-chain antibodies which specifically bind to FLP proteins, polypeptides, variants, or fusion proteins can be isolated, for example, from single-chain immunoglobulin display libraries, as is known in the art. The library is "panned" against FLP protein amino acid sequences, and a number of single chain antibodies which bind with high-affinity to different epitopes of FLP protein can be isolated. Hayashi et al, 1995, Gene 160:129-30. Single-chain antibodies can also be constructed using a DNA amplification method, such as the polymerase chain reaction (PCR), using hybridoma cDNA as a template. Thirion et al, 1996, Eur. J. Cancer Prev. 5:507-

11.

Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught in Coloma and Morrison, 1997, Nat. Biotechnol. 75:159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender and Voss, 1994, J.

Biol. Chem. 269:199-206.

A nucleotide sequence encoding the single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into DNA expression constructs using standard recombinant DNA methods, and introduced into cells which express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology. Verhaar et al, 1995, Int. J. Cancer 67:497-501; Nicholls et al,

1993, J. Immunol. Meth. 765:81-91. FLP-specific antibodies specifically bind to epitopes present in a full-length

FLP protein having the amino acid sequence shown in SEQ LD NO: 2, to FLP^.^, to FLP polypeptides, or to FLP variants, either alone or as part of a fusion protein.

Preferably, FLP epitopes are not present in other human proteins. Typically, at least

6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least

15, 25, or 50 amino acids.

Antibodies which specifically bind to FLP proteins, polypeptides, fusion proteins, or variants provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in Western blots or other immunochemical assays. Preferably, antibodies which specifically bind to FLP epitopes do not detect other proteins in immunochemical assays and can immunoprecipitate a FTP protein, polypeptide, fusion protein, or variant from solution.

Antibodies can be purified by methods well known in the art. Preferably, the antibodies are affinity purified, by passing the antibodies over a column to which a FLP protein, polypeptide, variant, or fusion protein is bound. The bound antibodies can then be eluted from the column, for example, using a buffer with a high salt concentration.

Subgenomic polynucleotides contain less than a whole chromosome. Preferably, the polynucleotides are intron-free. Purified and isolated FIP subgenomic polynucleotides can comprise at least 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more contiguous nucleotides selected from the nucleotide sequence shown in SEQ LD NO: 1 or its complement. SEQ LD NO: 1 is the coding sequence of a human FIP gene. The complement ofthe nucleotide sequence shown in SEQ LD NO: 1 is a contiguous nucleotide sequence which forms Watson-Crick base pairs with the contiguous nucleotide sequence shown in SEQ LD NO: 1. The complement ofthe nucleotide sequence shown in SEQ LD NO:l (the antisense strand) is also a subgenomic polynucleotide, and can be used provide FIP antisense ohgonucleotides. FIP subgenomic polynucleotides can comprise at least 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more contiguous nucleotides selected from the nucleotide sequence contained within plasmid pSPORT-FLP, deposited with the ATCC under Accession No. 209099 (see below). FIP subgenomic polynucleotides also include polynucleotides which encode FLP-specific single-chain antibodies and ribozymes, or fusion proteins comprising FLP amino acid sequences. Degenerate nucleotide sequences encoding amino acid sequences of FIP protein and or variants, as well as homologous nucleotide sequences which are at least 85%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence shown in SEQ LD NO: 1, are also FIP subgenomic polynucleotides. Percent sequence identity between the sequence of a wild-type subgenomic polynucleotide and a homologous nucleotide sequence is calculated by counting the number of nucleotide matches between the wild-type and the homolog and dividing the total number of matches by the total number of nucleotides ofthe wild-type sequence. Typically, homologous FIP sequences can be confirmed by hybridization under stringent conditions, as is known in the art. FIP subgenomic polynucleotides can be isolated and purified free from other nucleotide sequences using standard nucleic acid purification techniques. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise nucleotide sequences encoding a FLP protein. Isolated and purified subgenomic polynucleotides are in preparations which are free or at least 90% free of other molecules.

Complementary DNA molecules which encode FLP proteins can be made using reverse transcriptase, with FIP mRNA as a template. The polymerase chain reaction (PCR) or other amplification techniques can be used to obtain FIP subgenomic polynucleotides, using either human genomic DNA or cDNA as a template, as is known in the art. Alternatively, synthetic chemistry techniques can be used to synthesize FIP subgenomic polynucleotides which comprise coding sequences for regions of FLP proteins, single-chain antibodies, or ribozymes, or which comprise antisense ohgonucleotides. The degeneracy ofthe genetic code allows alternate nucleotide sequences to be synthesized which will encode a FLP protein comprising amino acid sequences of SEQ LD NO:2.

Purified and isolated FIP subgenomic polynucleotides can be used as primers to obtain additional copies ofthe polynucleotides or as probes for identifying wild-type and mutant FIP coding sequences. FIP subgenomic polynucleotides can be used to express FIP mRNA, protein, polypeptides, or fusion proteins and to generate FIP antisense ohgonucleotides and ribozymes.

A. FIP subgenomic polynucleotide comprising FIP coding sequences can be used in an expression construct. Preferably, the FIP subgenomic polynucleotide is inserted into an expression plasmid (for example, the Ecdyson system, pLND, In Vitro Gene). FIP subgenomic polynucleotides can be propagated in vectors and cell lines using techniques well known in the art. FIP subgenomic polynucleotides can be on linear or circular molecules. They can be on autonomously replicating molecules or on molecules without replication sequences. They can be regulated by their own or by other regulatory sequences, as are known in the art. A host cell comprising a FIP expression construct can then be used to express all or a portion of a FLP protein. Host cells comprising FIP expression constructs can be prokaryotic or eukaryotic. A variety of host cells are available for use in bacterial, yeast, insect, and human expression systems and can be used to express or to propagate FIP expression constructs (see below). Expression constructs can be introduced into host cells using any technique known in the art. These techniques include transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, and calcium phosphate-mediated transfection.

A FIP expression construct comprises a promoter which is functional in a chosen host cell. The skilled artisan can readily select an appropriate promoter from the large number of cell type-specific promoters known and used in the art. The expression construct can also contain a transcription terminator which is functional in the host cell. The expression construct comprises a polynucleotide segment which encodes all or a portion ofthe FLP protein, variant, fusion protein, antibody, or ribozyme. The polynucleotide segment is located downstream from the promoter. Transcription ofthe polynucleotide segment initiates at the promoter. The expression construct can be linear or circular and can contain sequences, if desired, for autonomous replication.

Bacterial systems for expressing FIP expression constructs include those described in Chang et al, Nature (1978) 275: 615, Goeddel et al, Nature (1979) 281: 544, Goeddel etal, Nucleic Acids Res. (1980) 8: 4057, EP 36,776, U.S. 4,551,433, deBoer etal, Proc. Natl Acad Sci. USA (1983) 80: 21-25, and Siebenlist et al, Cell (1980) 20: 269.

Expression systems in yeast include those described in Hinnen et al, Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Lto etal, J. Bacteriol (1983) 755: 163; Kurtz et al, Mol. Cell Biol (1986) 6: 142; Kunze et al, J. Basic Microbiol (1985) 25: 141; Gleeson etal, J. Gen. Microbiol. (1986) 132: 3459, Roggenkamp et al, Mol Gen. Genet. (1986) 202 :302) Das et al, J. Bacteriol. (1984) 755: 1165; De Louvencourt et al, J. Bacteriol. (1983) 754: 737, Van den Berg et al, Bio/Technology (1990) 8: 135; Kunze et al, J. Basic Microbiol. (1985) 25: 141; Cregg et al, Mol. Cell. Biol (1985) 5: 3376, U.S. 4,837,148, US 4,929,555; Beach and Nurse, Nature (1981) 300: 706; Davidow et al, Curr. Genet. (1985) 10: 380, Gaillardin et al, Curr. Genet. (1985) 10: 49, Ballance et al, Biochem. Biophys.

Res. Commun. (1983) 772: 284-289; Tilburn et al, Gene (1983) 26: 205-221, Yelton et al, Proc. Natl. Acad Sci. USA (1984) 81: 1470-1474, Kelly and Hynes, ΕMBO J. (1985) 4: 475479; EP 244,234, and WO 91/00357.

Expression of FIP expression constructs in insects can be carried out as described in U.S. 4,745,051, Friesen et al. (1986) "The Regulation of Baculovirus

Gene Expression" in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.), EP 127,839, EP 155,476, and Ylak etal, J. Gen. Virol (1988) 69: 765-776, Miller etal, Ann. Rev. Microbiol. (1988) 42: 177, Carbonell etal, Gene (1988) 73: 409, Maeda et al, Nature (1985) 375: 592-594, Lebacq-Verheyden et al, Mol. Cell. Biol (1988) 8: 3129; Smith et al, Proc. Natl Acad Sci. USA (1985) 82:

8404, Miyajima et al, Gene (1987) 58: 273; and Martin et al, DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts are described in Luckow et al, Bio/Technology (1988) 6: 47-55, Miller et l, in GENETIC ENGINEERING (Setlow, J.K. etal eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al, Nature, (1985) 575: 592-594.

Mammalian expression of FIP expression constructs can be achieved as described in Dijkema et al, EMBO J. (1985) 4: 761, Gorman et al, Proc. Natl Acad Sci. USA (1982b) 79: 6777, Boshart etal, Ce//(1985) 41: 521 and U.S. 4,399,216. Other features of mammalian expression of FIP expression constructs can be facilitated as described in Ham and Wallace, Meth. Era. (1979) 58: 44,

Barnes and Sato, Anal Biochem. (1980) 702: 255, U.S. 4,767,704, US 4,657,866, US 4,927,762, US 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.

Subgenomic polynucleotides ofthe invention can also be used in gene delivery vehicles, for the purpose of delivering &FIP mRNA or oligonucleotide (either with the sequence of native FIP mRNA or its complement), full-length FLP protein, FLP₃₄g_₇₂₇, FLP fusion protein, FLP polypeptide, or FLP-specific ribozyme or single-chain antibody, into a cell preferably a eukaryotic cell. According to the present invention, a gene delivery vehicle can be, for example, naked plasmid DNA, a viral expression vector comprising a FIP subgenomic polynucleotide, or a FIP subgenomic polynucleotide in conjunction with a liposome or a condensing agent.

In one embodiment ofthe invention, the gene delivery vehicle comprises a promoter and a FIP subgenomic polynucleotide. Preferred promoters are tissue- specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters which are activatable by infection with a virus, such as the α- and β-interferon promoters, and promoters which are activatable by a hormone, such as estrogen. Other promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter.

A FTP gene delivery vehicle can comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus. In a preferred embodiment, the FIP gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al,

Cell 35:153, 1983, Cane and Mulligan, Proc. Natl Acad Sci. USA 81:6349, 1984, Miller etal, Human Gene Therapy 1:5-14, 1990, U.S. Patent Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos. WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral gene delivery vehicles can be utilized in the present invention, including for example those described in EP

0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 55:3860-3864, 1993; Vile and Hart, Cancer Res. 55:962-967, 1993; Ram etal, Cancer Res. 55:83-88, 1993; Takamiya et al, J. Neurosci. Res. 55:493-503, 1992; Baba et al, J. Neurosurg. 79:729-735, 1993 (U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91/02805).

Particularly preferred retroviruses are derived from retroviruses which include avian leukosis virus (ATCC Nos. VR-535 and VR-247), bovine leukemia virus (VR-1315), murine leukemia virus (MLV), mink-cell focus-inducing virus

(Koch etal, J. Vir. 9:828, 1984; and Oliffe? α/., J. Vir. 48:542, 1983), murine sarcoma virus (ATCC Nos. VR-844, 45010 and 45016), reticuloendothehosis virus (ATCC Nos VR-994, VR-770 and 45011), Rous sarcoma virus, Mason-Pfizer monkey virus, baboon endogenous virus, endogenous feline retrovirus (e.g., RDl 14), and mouse or rat gL30 sequences used as a retroviral vector. Particularly preferred strains of MLV from which recombinant retroviruses can be generated include 4070A and 1504A (Hartley and Rowe, J. Vir. 79:19, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi (Ru et al, J. Vir. 67:4722, 1993; and Yantchev Neoplasma 26:391, 1979), Gross (ATCC No. VR- 590), Kirsten (Albino et al, J. Exp. Med 164:1110, 1986), Harvey sarcoma virus

(Manly et al, J. Vir. 62:3540, 1988; and Albino etal, J. Exp. Med 164:1710, 1986) and Rauscher (ATCC No. VR-998), and Moloney MLV (ATCC No. VR- 190). A particularly preferred non-mouse retrovirus is Rous sarcoma virus. Preferred Rous sarcoma viruses include Bratislava (Manly et al, J. Vir. 62:3540, 1988; and Albino et al, J. Exp. Med 164:1710, 1986), Bryan high titer (e.g.,

ATCC Nos. VR-334, VR-657, VR-726, VR-659, and VR-728), Bryan standard (ATCC No. VR-140), Carr-Zilber (Adgighitov et al, Neoplasma 27:159, 1980), Engelbreth-Holm (Laurent et al, Biochem BiophysActa 908:241, 1987), Harris, Prague (e.g., ATCC Nos. VR-772, and 45033), and Schmidt-Ruppin (e.g. ATCC Nos. VR-724, VR-725, VR-354) viruses.

Any ofthe above retroviruses can be readily utilized in order to assemble or construct retroviral FIP gene delivery vehicles given the disclosure provided herein and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989, and Kunkle, PNAS S2:488, 1985) known in the art. Portions of retroviral FIP expression vectors can be derived from different retroviruses. For example, retrovector LTRs can be derived from a murine sarcoma virus, a tRNA binding site from a Rous sarcoma virus, a packaging signal from a murine leukemia virus, and an origin of second strand synthesis from an avian leukosis virus. These recombinant retroviral vectors can be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see Serial No. 07/800,921, filed November 29, 1991). Recombinant retroviruses can be produced which direct the site-specific integration ofthe recombinant retroviral genome into specific regions ofthe host cell DNA. Such site-specific integration can be mediated by a chimeric integrase incorporated into the retroviral particle (see Serial No. 08/445,466 filed May 22, 1995). It is preferable that the recombinant viral gene delivery vehicle is a replication-defective recombinant virus. Packaging cell lines suitable for use with the above-described retroviral gene delivery vehicles can be readily prepared (see Serial No. 08/240,030, filed May 9, 1994; see also WO 92/05266) and used to create producer cell lines (also termed vector cell lines or "VCLs") for production of recombinant viral particles. In particularly preferred embodiments ofthe present invention, packaging cell lines are made from human (e.g., HT1080 cells) or mink parent cell lines, thereby allowing production of recombinant retroviral gene delivery vehicles which are capable of surviving inactivation in human serum. The construction of recombinant retroviral gene delivery vehicles is described in detail in WO 91/02805. These recombinant retroviral gene delivery vehicles can be used to generate transduction competent retroviral particles by introducing them into appropriate packaging cell lines (see Serial No. 07/800,921). Similarly, adenovirus gene delivery vehicles can also be readily prepared and utilized given the disclosure provided herein (see also Berkner,

Biotechniques 6:616-627, 1988, and Rosenfeld et al, Science 252:431-434, 1991, WO 93/07283, WO 93/06223, and WO 93/07282).

A FIP gene delivery vehicle can also be a recombinant adenoviral gene delivery vehicle. Such vehicles can be readily prepared and utilized given the disclosure provided herein (see Berkner, Biotechniques 6:616, 1988, and Rosenfeld et al, Science 252:431, 1991, WO 93/07283, WO 93/06223, and WO 93/07282). Adeno-associated viral FIP gene delivery vehicles can also be constructed and used to deliver FLP amino acids or nucleotides. The use of adeno-associated viral gene delivery vehicles in vitro is described in Chatterjee etal, Science 258: 1485-1488 (1992), Walsh et al, Proc. Nat 7. Acad Sci. 89: 7257-7261 (1992), Walsh et al, J.

Clin. Invest. 94: 1440-1448 (1994), Flotte et al, J. Biol. Chem. 268: 3781-3790 (1993), Ponnazhagan et al, J. Exp. Med 179: 733-738 (1994), Miller et al, Proc. Nat'lAcad Sci. 91: 10183-10187 (1994), Einerhand et al, Gene Ther. 2: 336-343 (1995), Luo et al, Exp. Hematol. 23: 1261-1267 (1995), and Zhou et al, Gene Therapy 3: 223-229 (1996). In vivo use of these vehicles is described in Flotte et al, Proc. Nat'lAcad Sci. 90: 10613-10617 (1993), and Kaplitt etal, Nature Genet. 8: 148-153 (1994).

In another embodiment ofthe invention, a FIP gene delivery vehicle is derived from a togavirus. Preferred togaviruses include alphaviruses, in particular those described in U.S. Serial No. 08/405,627, filed March 15, 1995, WO

95/07994. Alpha viruses, including Sindbis and ELVS viruses can be gene delivery vehicles for FIP polynucleotides. Alpha viruses are described in WO 94/21792, WO 92/10578 and WO 95/07994. Several different alphavirus gene delivery vehicle systems can be constructed and used to deliver FIP subgenomic polynucleotides to a cell according to the present invention. Representative examples of such systems include those described in U.S. Patents 5,091,309 and 5,217,879. Particularly preferred alphavirus gene delivery vehicles for use in the present invention include those which are described in WO 95/07994, and U.S. Serial No. 08/405,627. Preferably, the recombinant viral vehicle is a recombinant alphavirus viral vehicle based on a Sindbis virus. Sindbis constructs, as well as numerous similar constructs, can be readily prepared essentially as described in U.S. Serial No. 08/198,450. Sindbis viral gene delivery vehicles typically comprise a 5' sequence capable of initiating Sindbis virus transcription, a nucleotide sequence encoding Sindbis non-structural proteins, a viral junction region inactivated so as to prevent subgenomic fragment transcription, and a Sindbis RNA polymerase recognition sequence. Optionally, the viral junction region can be modified so that subgenomic polynucleotide transcription is reduced, increased, or maintained. As will be appreciated by those in the art, corresponding regions from other alphavimses can be used in place of those described above.

The viral junction region of an alphavirus-derived gene delivery vehicle can comprise a first viral junction region which has been inactivated in order to prevent transcription ofthe subgenomic polynucleotide and a second viral junction region which has been modified such that subgenomic polynucleotide transcription is reduced. An alphavirus-derived vehicle can also include a 5' promoter capable of initiating synthesis of viral RNA from cDNA and a 3' sequence which controls transcription termination.

Other recombinant togaviral gene delivery vehicles which can be utilized in the present invention include those derived from Semliki Forest vims (ATCC VR- 67; ATCC VR-1247), Middleberg vims (ATCC VR-370), Ross River vims (ATCC

VR-373; ATCC VR-1246), Venezuelan equine encephalitis vims (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in US. Patents 5,091,309 and 5,217,879 and in WO 92/10578. The Sindbis vehicles described above, as well as numerous similar constructs, can be readily prepared essentially as described in U. S. Serial No. 08/198,450.

Other viral gene delivery vehicles suitable for use in the present invention include, for example, those derived from poliovirus (Evans et al, Nature 559:385, 1989, and Sabin etal, J. Biol Standardization 7:115, 1973) (ATCC VR-58); rhinovi s (Arnold et al, J. Cell Biochem. L401, 1990) (ATCC VR-1110); pox viruses, such as canary pox vims or vaccinia vims (Fisher-Hoch et al, PNAS

86:317, 1989; Flexner et al, Ann. NY. Acad Sci. 569:86, 1989; Flexner et al, Vaccine 8:11, 1990; U.S. 4,603,112 and U.S. 4,769,330; WO 89/01973) (ATCC VR-111; ATCC VR-2010); SV40 (Mulligan et al, Nature 277:108, 1979) (ATCC VR-305), (Madzak etal, J. Gen. Vir. 75:1533, 1992); influenza vims (Luytjes et al, Cell 59: 1107, 1989; McMicheal et al, The New England Journal of Medicine 509:13, 1983; and Yap et al, Nature 275:238, 1978) (ATCC VR-797); parvovirus such as adeno-associated vims (Samulski et al, J. Vir. 65:3822, 1989, and Mendelson etal, Virology 766:154, 1988) (ATCC VR-645); herpes simplex vims (Kit etal, Adv. Exp. Med Biol. 275:219, 1989) (ATCC VR-977; ATCC VR-260); Nature 277: 108, 1979); human immunodeficiency vims (EPO 386,882,

Buchschacher ef α/., J. Vir. 66:2731, 1992); measles vims (EPO 440,219) (ATCC VR-24); A (ATCC VR-67; ATCC VR-1247), Aura (ATCC VR-368), Bebam vims (ATCC VR-600; ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya vims (ATCC VR-64; ATCC VR-1241), Fort Morgan (ATCC VR-924), Getah vims (ATCC VR-369; ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC

VR-66), Mucambo vims (ATCC VR-580; ATCC VR-1244), Ndumu (ATCC VR- 371), Pixuna vims (ATCC VR-372; ATCC VR-1245), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Whataroa (ATCC VR-926), Y-62- 33 (ATCC VR-375), O"Nyong vims, Eastern encephalitis vims (ATCC VR-65; ATCC VR-1242), Western encephalitis vims (ATCC VR-70; ATCC VR-1251;

ATCC VR-622; ATCC VR-1252), and coronavirus (Hamre etal, Proc. Soc. Exp. Biol. Med 727:190, 1966) (ATCC VR-740).

A subgenomic FIP polynucleotide ofthe invention can also be combined with a condensing agent to form a gene delivery vehicle. In a preferred embodiment, the condensing agent is a polycation, such as polylysine, polyarginine, polyornithine, protamine, spermine, spermidine, and putrescine. Many suitable methods for making such linkages are known in the art (see, for example, Serial No. 08/366,787, filed December 30, 1994).

Ln an alternative embodiment, &FIP subgenomic polynucleotide is associated with a liposome to form a gene delivery vehicle. Liposomes are small, lipid vesicles comprised of an aqueous compartment enclosed by a lipid bilayer, typically spherical or slightly elongated structures several hundred Angstroms in diameter. Under appropriate conditions, a liposome can fuse with the plasma membrane of a cell or with the membrane of an endocytic vesicle within a cell which has internalized the liposome, thereby releasing its contents into the cytoplasm. Prior to interaction with the surface of a cell, however, the liposome membrane acts as a relatively impermeable barrier which sequesters and protects its contents, for example, from degradative enzymes. Additionally, because a liposome is a synthetic stmcture, specially designed liposomes can be produced which incorporate desirable features. See Stryer, Biochemistry, pp. 236-240, 1975 (W.H. Freeman, San

Francisco, CA); Szoka et al, Biochim. Biophys. Acta 600:1, 1980; Bayer etal, Biochim. Biophys. Acta. 550:464, 1979; Rivnay et al., Meth. Enzymol 149:119, 1987; Wang etal, PNAS 84: 7851, 1987, Plant et al, Anal Biochem. 776:420, 1989, and U.S. Patent 4,762,915. Liposomes can encapsulate a variety of nucleic acid molecules including DNA, RNA, plasmids, and expression constructs comprising FIP subgenomic polynucleotides such those disclosed in the present invention.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid

DNA (Feigner etal, Proc. Natl Acad Sci. USA 84:1413-1416, 1987), mRNA (Malone etal, Proc. Natl Acad Sci. USA 56:6077-6081, 1989), and purified transcription factors (Debs et al, J. Biol. Chem. 265:10189-10192, 1990), in functional form. Cationic liposomes are readily available. For example, N[l-2,3- dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. See also Feigner etal, Proc. Natl. Acad Sci. USA 91: 5148-5152.87, 1994. Other commercially available liposomes include Transfectace (DDAB/DOPE) and DOTAP DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka etal, Proc. Natl. Acad Sci. USA 75:4194-4198, 1978; and WO 90/11092 for descriptions ofthe synthesis of DOTAP (l,2-bis(oleoyloxy)-3- (trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various Hposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger etal, METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527;

Szoka etal, Proc. Natl Acad Sci. USA 57:3410-3414, 1990; Papahadjopoulos et al, Biochim. Biophys. Acta 594:483, 1975; Wilson et al, Cell 17:11, 1979; Deamer and Bangham, Biochim. Biophys. Acta 443:629, 1976; Ostro et al, Biochem. Biophys. Res. Commun. 76:836 , 1977; Fraley et al, Proc. Natl Acad Sci. USA 76:3348, 1979; Enoch and Strittmatter, Proc. Natl Acad Sci. USA

76:145, 1979; Fraley etal, J. Biol Chem. 255:10431, 1980; Szoka and Papahadjopoulos, Proc. Natl. Acad Sci. USA 75:145, 1979; and Schaefer-Ridder et al, Science 215:166, 1982.

In addition, lipoproteins can be included with a FIP subgenomic polynucleotide for delivery to a cell. Examples of such lipoproteins include chylomicrons, HDL, LDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Modifications of naturally occurring lipoproteins can also be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are included with a polynucleotide, no other targeting hgand is included in the composition.

Ln another embodiment, naked FIP subgenomic polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Patent 5,580,859. Such gene delivery vehicles can be either FIP DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et al, Hum. Gene. Ther. 3: 147-154, 1992. Other suitable vehicles include DNA-ligand (Wu et al, J. Biol Chem. 264:16985-16987, 1989), lipid-DNA combinations (Feigner et al, Proc. Natl. Acad Sci. USA 84:7413 7417, 1989), liposomes (Wang etal, Proc. Natl. Acad Sci. 54:7851-7855, 1987) and microprojectiles (Williams et al, Proc. Natl. Acad Sci. 55:2726-2730, 1991).

One can increase the efficiency of naked FIP subgenomic polynucleotide uptake into cells by coating the polynucleotides onto biodegradable latex beads. This approach takes advantage ofthe observation that latex beads, when incubated with cells in culture, are efficiently transported and concentrated in the perinuclear region ofthe cells. The beads will then be transported into cells when injected into muscle. FIP subgenomic polynucleotide-coated latex beads will be efficiently transported into cells after endocytosis is initiated by the latex beads and thus increase gene transfer and expression efficiency. This method can be improved further by treating the beads to increase their hydrophobicity, thereby facilitating the dismption ofthe endosome and release of FIP subgenomic polynucleotides into the cytoplasm.

According to the present invention, apoptosis of a cell can be prevented by contacting the cell with a composition which can decrease the

Apoptosis of cells which are dying prematurely in a disease state such as Alzheimer's Disease, ALDS, muscular dystrophy, amyotrophic lateral sclerosis, or other muscle wasting diseases, autoimmune diseases, or a disease in which the cell is infected with a pathogen, such as a vims, bacterium, fungus, mycoplasm, or protozoan, can be prevented using such a composition. The composition comprises a reagent which specifically binds to a wild-type human FLP expression product so as to decrease the

in the cell.

Ln one embodiment ofthe invention, the reagent is a ribozyme, an RNA molecule with catalytic activity. See, e.g., Cech, Science 236: 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59:543-568; 1990, Cech, Curr. Opin. Struct. Biol 2: 605-609; 1992, Couture and Stinchcomb, Trends Genet. 12: 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673).

The coding sequence of a FIP gene can be used to generate ribozymes which will specifically bind to mRNA transcribed from the FIP gene. Methods of designing and constmcting ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff, J. et l Nature 554:585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific FIP RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target FIP RNA and thus specifically hybridizes with the target (see, for example, Gerlach, et al, EP 321,201). The nucleotide sequence shown in SEQ LD NO: 1 provides a source of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the FIP ribozyme can be integrally related; thus, upon hybridizing to the target FIP

RNA through the complementary regions, the catalytic region ofthe ribozyme can cleave the target.

FIP ribozymes can be introduced into cells as part of a DNA construct, as is known in the art and described above. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce the ribozyme-containing DNA construct into cells in which it is desired to decrease 7P expression, as described above. Alternatively, if it is desired that the cells stably retain the DNA construct, it can be supplied on a plasmid and maintained as a separate element or integrated into the genome ofthe cells, as is known in the art. The DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of FIP ribozymes in the cells.

As taught in Haseloff et al, U.S. Patent 5,641,673, FIP ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression ofthe FIP gene. Ribozymes can also be engineered to provide an additional level of regulation, so that destmction of FIP mRNA occurs only when both a FIP ribozyme and a FIP gene are induced in the cells.

In another embodiment ofthe invention, the level of FLP_34g.₇₂₇ is decreased using an antisense oligonucleotide sequence. The antisense sequence is complementary to at least a portion ofthe sequence encoding

selected from the nucleotide sequence shown in SEQ LD NO: 1. Preferably, the antisense oligonucleotide sequence is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences can also be used. FIP antisense oligonucleotide molecules can be provided in a DNA construct and introduced into cells as described above to decrease the level of FLP_34g-727 in the cells.

FIP antisense ohgonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Ohgonucleotides can be synthesized manually or by an automated synthesizer, by covalently Unking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol Biol 20:1-8,

1994; Sonveaux, Meth. Mol Biol. 26:1-72, 1994; Uhlmann etal, Chem. Rev. 90:543-583, 1990.

Although precise complementarity is not required for successful duplex formation between &FIP antisense molecule and the complementary coding sequence of a FIP gene, antisense molecules with no more than one mismatch are preferred. One skilled in the art can easily use the calculated melting point of a FIP antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular FIP coding sequence. FIP antisense ohgonucleotides can be modified without affecting their ability to hybridize to a FIP coding sequence. These modifications can be internal or at one or both ends ofthe antisense molecule. For example, intemucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.

Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, can also be employed in a modified antisense oligonucleotide. These modified ohgonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol. 70:152-158, 1992; Uhlmann ef /.,

Chem. Rev. 90:543-584, 1990; Uhlmann et /., Tetrahedron. Lett. 275:3539-3542, 1987.

Antibodies ofthe invention which specifically bind to FLP₃₄g.₇₂₇, particularly single-chain antibodies, can also be used to alter

The antibodies bind to

and prevent the protein from inducing apoptosis. Polynucleotides encoding single-chain antibodies ofthe invention can be introduced into cells as described above.

Preferably, the mechanism used to decrease the

whether ribozyme, antisense oligonucleotide sequence, or antibody, decreases the level of FI.P₃₄g.₇₂₇ by at least 50%, 60%, 70%, or 80%. Most preferably, the level of FLP₃₄₈.

₇₂₇ is decreased by at least 90%, 95%, 99%, or 100%. The effectiveness ofthe mechanism chosen to decrease the

can be assessed using methods well known in the art, such as hybridization of nucleotide probes to FIP mRNA, quantitative RT-PCR, or detection of FIP protein using FLP-specific antibodies of the invention.

Compositions comprising FLP antibodies, ribozymes, or antisense ohgonucleotides can optionally comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those in the art. Such carriers include, but are not limited to, large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive vims particles. Pharmaceutically acceptable salts can also be used in FLP compositions, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates. FLP compositions can also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes, such as those described in U.S. Patent 5,422,120, WO 95/13796, WO 91/14445, or EP 524,968 Bl, can also be used as a carrier for a FLP composition. Typically, a FLP composition is prepared as an injectable, either as a liquid solution or suspension; however, solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. A FLP composition can also be formulated into an enteric coated tablet or gel capsule according to known methods in the art, such as those described in U.S. Patent 4,853,230, EP 225,189, AU 9,224,296, and AU 9,230,801.

Alternatively, a composition comprising all or a

or a nucleotide sequence encoding

can be introduced into a cell in order to induce apoptosis in cells which are proliferating abnormally in a disease state such as neoplasia. Such compositions can also comprise a pharmaceutically acceptable carrier, as described above. Prohferative disorders, such as neoplasias, dysplasias, and hyperplasias, and their symptoms can be treated by administration of a FLP composition comprising coding sequences for

or comprising

protein or polypeptide fragments. Neoplasias which can be treated with such FLP compositions include, but are not limited to, melanomas, squamous cell carcinomas, adenocarcinomas, hepatocellular carcinomas, renal cell carcinomas, sarcomas, myosarcomas, non-small cell lung carcinomas, leukemias, lymphomas, osteosarcomas, central nervous system tumors such as gliomas, astrocytomas, oligodendrogliomas, and neuroblastomas, tumors of mixed origin, such as Wilms' tumor and teratocarcinomas, and metastatic tumors. Prohferative disorders, such as anhydric hereditary ectodermal dysplasia, congenital alveolar dysplasia, epithelial dysplasia ofthe cervix, fibrous dysplasia of bone, and mammary dysplasia, can also be treated according to the invention. Hyperplasias, for example, endometrial, adrenal, breast, prostate, or thyroid hyperplasias, or pseudoepitheliomatous hyperplasia ofthe skin can be treated with such FLP compositions. An entire FLP₃₄g_₇₂₇ coding sequence or protein can be introduced, as described above. Alternatively, a portion of a FJLP protein which induces apoptosis can be identified, and that portion or a nucleotide sequence encoding it can be introduced into the cell. Portions of a

which induce apoptosis can be identified by introducing expression constmcts which express different portions of the protein into cells and observing increased apoptosis, as described in Example 4, below.

Administration of FLP compositions ofthe invention can include local or systemic administration, including injection, oral administration, particle gun, or catheterized administration, and topical administration. Various methods can be used to administer a FLP composition directly to a specific site in the body. For inducing apoptosis in a tumor, for example, an appropriate FLP composition injected several times in several different locations within the body ofthe tumor. Alternatively, arteries which serve the tumor can be identified, and a FLP composition can be injected into such an artery in order to deliver the composition to the tumor.

A tumor which has a necrotic center can be aspirated, and a FLP composition can be injected directly into the now empty center ofthe tumor. A FLP composition can also be administered directly to the surface of a tumor, for example, by topical application ofthe composition. X-ray imaging can be used to assist in certain of these delivery methods. Combination therapeutic agents, including a FTP^^ or polypeptide or a subgenomic FLP polynucleotide, can be administered simultaneously or sequentially together with other therapeutic agents.

FLP compositions can be delivered to specific tissues using receptor- mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al Trends in Biotechnol. 11, 202-05, (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICAΉONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24, 1988; Wu et al, J. Biol. Chem. 269, 542-46, 1994; Zenke etal, Proc. Natl Acad Sci. U.S.A. 87, 3655-59, 1990; Wu etal, J. Biol Chem. 266, 338-42, 1991. Both the dose of a particular FLP composition and the means of administering the composition can be determined based on specific qualities ofthe FLP composition, the condition, age, and weight ofthe patient, the progression of the particular disease being treated, and other relevant factors. If the composition contains FLP proteins, polypeptides, or antibodies, effective dosages ofthe composition are in the range of about 5 μg to about 50 μg/kg of patient body weight, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg.

Compositions containing FIP subgenomic polynucleotides, including antisense ohgonucleotides and ribozyme-or antibody-encoding sequences, can be administered in a range of about 100 ng to about 200 mg of DNA for local administration. Suitable concentrations range from about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA. Factors such as method of action and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy ofthe FLP composition. If greater expression is desired over a larger area of tissue, larger amounts of a FLP composition or the same amount administered successively, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.

Expression of an endogenous FIP gene in a cell can be altered by introducing in frame with the endogenous P7P gene a DNA construct comprising a FIP targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site by homologous recombination, such that a homologously recombinant cell comprising a new FIP transcription unit is formed. The new transcription unit can be used to turn the T^T gene on or off as desired. This method of affecting endogenous gene expression is taught in U.S. Patent 5,641,670, which is incorporated herein by reference.

The targeting sequence is a segment of at least 10, 12, 15, 20, or 50 contiguous nucleotides selected from the nucleotide sequence shown in SEQ LD

NO: 1. The transcription unit is located upstream of a coding sequence ofthe endogenous FIP gene. The exogenous regulatory sequence directs transcription of the coding sequence ofthe FIP gene.

The present invention provides assays which can be used to screen test compounds for the ability to induce or prevent apoptosis. The basis for all of these assays is the discovery ofthe binding interaction between FLP and FADD. Both full-length FLP, FLP₃₄g_₇₂₇ portions of full-length FLP which comprise

and

which bind to FADD can be used in the following methods. The test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants. Test compounds can be produced recombinantly or synthesized by chemical methods known in the art.

In one embodiment ofthe invention, a cell is contacted with a test compound. The cell can be any cell capable of being maintained in vitro. It can be freshly isolated from a human tissue or can be obtained from a cell line such as HeLa cells. Methods of culturing cells, for example as monolayers, explants, or cellular reaggregates, are well-known in the art. The test compound can be a component ofthe culture medium or can be added separately. At the time of contacting, the cell comprises two expression constructs.

The first expression construct comprises a subgenomic polynucleotide encoding at least a portion of FADD selected from the sequence shown in SEQ LD NO:4. The portion of FADD binds to a portion of FLP. The second expression constmct comprises a subgenomic polynucleotide encoding at least a portion of FLP selected from the sequence shown in SEQ LD NO:2. The portion of FLP comprises at least amino acids 348-727 of SEQ LD NO:2 and binds to FADD. The expression constmcts can be assembled using standard recombinant DNA techniques. The expression constmcts are introduced into the cell by methods well known in the art, as disclosed above. The ability ofthe test compound to decrease the binding ofthe portion of

FLP to the portion of FADD is then measured. A number of methods can be used to measure the binding. For example, the relative concentration of FLP-FADD complexes can be detected by examining the apparent molecular masses ofthe molecules by size exclusion chromatography or by polyacrylamide gel electrophoresis under non-reducing conditions. The complexes can be visualized using antibodies that specifically bind to FIP or to FADD epitopes. Antibodies which specifically bind to FADD epitopes can be prepared, for example, using standard polyclonal or monoclonal antibody techniques. Lf expression constmcts encoding FLP or FADD fusion proteins are used, binding can be monitored by means of radioactive, fluorescent, or enzymatic tags on at least one ofthe fusion proteins. Other methods of measuring the amount binding will readily occur to those of ordinary skill in the art and can be used.

A test compound which increases the amount of binding is a potential dmg for inducing apoptosis. Such a dmg can be used, for example, to treat biological conditions or disease states which are characterized by an abnormal proliferation of cells, such as neoplasias. A test compound which decreases the amount of binding is a potential dmg for preventing apoptosis. Such a dmg can be used to treat biological conditions or disease states which are characterized by abnormal levels of cell death, such as Alzheimer's Disease, AIDS, muscle wasting diseases, autoimmune diseases, or diseases in which a cell is infected with a pathogen, such as a bacterium, vims, mycoplasm, fungus, or protozoan. Preferably, the test compound increases or decreases binding by at least 30-40%. More preferably, the test compound increases or decreases binding by at least 40-60%, 50-70%, 60- 80%, 70-90%, 75-95%, or 80-98%. According to one particular embodiment ofthe present invention, the yeast two-hybrid technique can be used to screen for test compounds which induce or prevent apoptosis. The yeast two-hybrid technique is generically taught in Fields, S. and Song, O., Nature 340, 245-46, 1989. In a preferred embodiment, a cell is contacted with a test compound. The test compound can be part ofthe cell culture medium or it can be added separately. The cell comprises two expression constmcts. Each expression constmct encodes a fusion protein. The first expression constmct encodes a fusion protein comprising a DNA binding domain and either (1) all or at least a portion of a human FIP protein comprising a contiguous sequence of amino acids selected from the amino acid sequence shown in SEQ ID NO:2 and capable of binding to FLP or (2) all or at least a portion of a human FADD protein with the sequence shown in SEQ LD NO:4.

The second expression constmct encodes a fusion protein comprising a transcriptional activating domain and either (1) all or at least a portion of a human FADD protein with the sequence shown in SEQ LD NO:4 or (2) all or a portion of a human FLP protein with the sequence shown in SEQ LD NO:2. When the first expression constmct encodes FLP amino acids, the second expression constmct encodes FADD amino acids. When the first expression constmct encodes FADD amino acids, the second expression constmct encodes FIP amino acids. The cell also comprises a reporter gene comprising a DNA sequence to which the DNA binding domain specifically binds.

When the portions of FIP and FADD bind, the DNA binding domain and the transcriptional activating domain will be in close enough proximity to reconstitute a transcriptional activator capable of initiating transcription ofthe detectable reporter gene in the cell. The expression ofthe reporter gene in the presence ofthe test compound is then measured. A test compound which increases the expression of the reporter gene is a potential dmg for preventing apoptosis. A test compound which decreases the expression ofthe reporter gene is a potential dmg for inducing apoptosis. Preferably, the test compound increases or decreases reporter gene expression by at least 30-40%. More preferably, the test compound increases or decreases reporter gene expression by at least 40-60%, 50-70%, 60-80%, 70-90%, 75-95%, or 80-98%.

Many DNA binding domains and transcriptional activating domains can be used in this system, including the DNA binding domains of GAL4, LexA, and the human estrogen receptor paired with the acidic transcriptional activating domains of

GAL4 or the herpes vims simplex protein VP16 (See, e.g., G.J. Hannon et al, Gene Dev. 7, 2378, 1993; A.S. Zervos etal, Cell 72, 223, 1993; A.B.Votjet et al, Cell 74, 205, 1993; J.W. Harper et al, Cell 75, 805, 1993; B. Le Douarin et al, Nucl Acids Res. 23, 876, 1995). A number of plasmids known in the art can be constructed to contain the coding sequences for the fusion proteins using standard laboratory techniques for manipulating DNA (see Example 1, infra). Suitable detectable reporter genes include the E. coli lacZ gene, whose expression can be measured colorimetrically (e.g., Fields and Song, supra), and yeast selectable genes such as HIS3 (Harper et al, supra; Votjet et al, supra; Hannon et al, supra) or URA3 (Le Douarin et al, supra). Methods for transforming cells are also well known in the art. See, e.g., Hinnen et al, Proc. Natl. Acad Sci. U.S.A. 75, 1929- 1933, 1978.

Ln another embodiment ofthe invention, a test compound is contacted with a first polypeptide comprising a FLP-binding site and a second polypeptide comprising a FADD-binding site. Contacting can occur in vitro. The FADD- binding site of FLP is contained within amino acids 348-727 of SEQ LD NO:2. The FLP-binding site of FADD is contained within the N-terminus of FADD (the "death effector domain," amino acids 1-110 of SEQ LD NO:4). Polypeptides comprising the binding sites can be produced recombinantly, isolated from human cells, or synthesized by standard chemical methods. The binding sites can be located on full- length proteins, fusion proteins, polypeptides, or protein fragments.

Binding or dissociation ofthe first and second polypeptides in the presence ofthe test compound can be measured as described above. Proteins or polypeptides comprising the FLP and/or FADD binding sites can be radiolabeled or labeled with fluorescent or enzymatic tags and can be detected, for example, by scintillation counting, fluorometric assay, monitoring the generation of a detectable product, or by measuring the apparent molecular mass ofthe bound or unbound proteins by gel filtration or electrophoretic mobility. Proteins or polypeptides comprising either a FLP- or a FADD-binding site can be bound to a solid support, such as a column matrix or a nylon membrane.

A test compound which increases the amount of binding between the first and second polypeptides is a potential dmg for inducing apoptosis. A test compound which decreases the amount of binding between the first and second polypeptides is a potential dmg for preventing apoptosis. Preferably, the test compound increases or decreases binding by at least 30-40%. More preferably, the test compound increases or decreases binding by at least 40-60%, 50-70%, 60- 80%, 70-90%, 75-95%, or 80-98%.

The invention also provides a method for determining the tissue source of a body sample of a human. Ln one embodiment, the body sample is assayed for the presence of a polypeptide consisting of amino acids 348-727 of SEQ LD NO:2

(FLP₃₄g.₇₂₇). FLP amino acids can be detected, for example, using FLP-specific antibodies in Western blots. In another embodiment, the body sample is assayed for the presence of a. FIP mRNA encoding FLP_34g_₇₂₇. Such mRNA has a size of about 2.5 kb. Subgenomic polynucleotides ofthe invention can be radiolabeled or labeled with fluorescent or enzymatic tags and used to detect FIP mRNA in Northern blots.

kb FIP mRNA indicates that the body sample originates from either brain, pancreas, spleen, or peripheral blood lymphocytes, all of which express

The body sample can be normal tissue or can be a tumor. The tumor can be a metastatic lesion. Determination of the origin of a tumor or metastatic lesion is useful in selecting appropriate therapeutic interventions.

A FIP subgenomic polynucleotide can also be delivered to subjects for the purpose of screening test compounds for those which are useful for enhancing transfer of FIP subgenomic polynucleotides to the cell or for enhancing subsequent biological effects of FIP subgenomic polynucleotides within the cell. Such biological effects include hybridization to complementary FIP mRNA and inhibition of its translation, expression of a FIP subgenomic polynucleotide to form FIP mRNA and/or FLP protein, and replication and integration of a FIP subgenomic polynucleotide. The subject can be a cell culture or an animal, preferably a mammal, more preferably a human.

Test compounds which can be screened include any substances, whether natural products or synthetic, which can be administered to the subject. Libraries or mixtures of compounds can be tested. The compounds or substances can be those for which a pharmaceutical effect is previously known or unknown. The compounds or substances can be delivered before, after, or concomitantly with a

FIP subgenomic polynucleotide. They can be administered separately or in admixture with P7P subgenomic polynucleotide.

Integration of a delivered FIP subgenomic polynucleotide can be monitored by any means known in the art. For example, Southern blotting ofthe delivered FIP subgenomic polynucleotide can be performed. A change in the size of the fragments of a delivered polynucleotide indicates integration. Replication of a delivered polynucleotide can be monitored inter alia by detecting incorporation of labeled nucleotides combined with hybridization to a FIP probe. Expression of a FIP subgenomic polynucleotide can be monitored by detecting production of FIP mRNA which hybridizes to the delivered polynucleotide or by detecting FLP protein. FLP protein can be detected immunologically. Thus, the delivery of FIP subgenomic polynucleotides according to the present invention provides an excellent system for screening test compounds for their ability to enhance transfer of FIP subgenomic polynucleotides to a cell, by enhancing delivery, integration, hybridization, expression, replication or integration in a cell in vitro or in an animal, preferably a mammal, more preferably a human.

The following plasmid, containing an isolated polynucleotide which encodes FLP, was deposited with the American Type Culture Collection: Name Deposit Date Accession No. CMCC Accession No. plasmid pSPORT-FLP June 6, 1997 209099 4742

The above material has been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, U.S.A., under the accession number indicated. This deposit will be maintained under the terms ofthe

Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for purposes of Patent Procedure. The deposit will be maintained for a period of 30 years following issuance of this patent, or for the enforceable life ofthe patent, whichever is greater. Upon issuance ofthe patent, the deposit will be available to the public from the ATCC without restriction.

The deposit is provided merely as a convenience to those of skill in the art, and is not an admission that a deposit is required under 35 U.S.C. § 112. The sequence ofthe polynucleotides contained within the deposited material, as well as the amino acid sequence ofthe polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the written description of sequences herein. A license may be required to make, use, or sell the deposited material, and no such license is granted hereby.

The following are provided for exemplification purposes only and are not intended to limit the scope ofthe invention which has been described in broad terms above.

EXAMPLE 1

A yeast two-hybrid system was used to isolate a protein which interacts with FADD.

A fusion gene encoding the GAL4 DNA-binding domain (amino-acids 1- 147), HA epitope tag, and the full-length human Fas-associated Death Domain

(FADD) cDNA was cloned into the yeast bait pASl-CYH vector. This constmct was co-transformed with a human placenta cDNA library (Clonetech) fused to the activation domain ofthe GAL4 in the pACT2 vector (prey). Interaction between bait and prey encoded proteins in the Y190 yeast strain reconstitutes an active GAL4 transcriptional complex. The complex activates both a histidine biosynthetic gene and a β-galactosidase gene. Thus, if the GAL4 transcriptional complex is reconstituted, the Y190 cells will grow in the absence of histidine and express β- galactosidase.

Two million clones were screened and three independent, but identical, partial clones of human FADD Interacting Protein (FLP) were found to encode proteins which interact specifically with full-length FADD and not with negative controls (Lamin, RIP, TRADD, Fas, and TNF-R1). These 3 clones encoded the C- terminal 380 amino acids (amino-acids 348-727) of full-length FIP (FTP_Mi.m). Gibco-BRL's GeneTrap kits were used to clone full-length human FLP. Oligos S'-GCAAGTCCATGTACACGC-S' (SEQ LD NO:5) and S'-GAAGCTTGGGTTCATTAA-S' (SEQ LD NO:6) were used to trap and repair the full-length cDNA, respectively, from a pCMV-SPORT human liver cDNA library (Gibco-BRL). Full-length FLP is an 85 kD protein with no strong homology with currently characterized proteins.

Full-length FLP was subcloned back into pASl-CYH and was used to retest its binding specificity. The full-length (amino acids 1-208), N-terminus (amino acids 1-110, death effector domain), and C-terminus (amino acids 110-208, death domain) of human FADD were subcloned into the pACT2 vector and individually co-transformed with full-length FLP into the Y190 yeast strain. FLP interacted strongly with both full-length FADD and the N-terminus of FADD and mildly with the C-terminus, as measured by β-galactosidase reporter gene assays. Thus, FLP is a novel FADD-binding protein which binds most strongly to the

N-terminus (the death effector domain) of FADD.

EXAMPLE 2

This example demonstrates the detection of FIP mRNA splice variants in human tissue. Radioactive probes were synthesized using cDNA encoding the C-terminal of FLP (amino-acids 348-727) and Stratagene's Prime-It kits. Clonetech's Human Multi-Tissue Northern Blots I and II were used as instmcted by manufacturer. MTN blot I includes RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas while MTN blot II has RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocyte.

A 4 kb FIP mRNA encoding the 85 kD protein, was detected in all tissues tested, while a 2.5 kb mRNA encoding the 55 kD C-terminal portion of FLP was detected only in brain, pancreas, spleen, and peripheral blood lymphocytes. Thus, there are two splice variants of FIP mRNA which are differentially expressed in human tissues.

EXAMPLE 3

This example demonstrates the co-immunoprecipitation of FIP and FADD proteins. Partial cDNA of FLP was subcloned into Novagen's pET-23b vector and recombinant protein was induced using 1 mM LPTG in BL21 E. coli cells. Recombinant protein was purified from the insoluble fraction. Three mg ofthe fusion protein was n on SDS-PAGE gels and stained with Coomassie blue. The 45 kD fusion protein was cut out and used for polyclonal antibody production. FADD monoclonal antibodies were used to co-immunoprecipitate FTP-

FADD complexes from Jurkat cells. 10⁷ Jurkat cells were lysed with 150 mM NaCl, 1% Triton X-100, 20 mM Tris-Cl pH 7.5, 1 mM EDTA 1 mM PMSF, and protease inhibitors. Pre-cleared lysates were immunoprecipitated using anti-human FADD monoclonal antibodies (Transduction Laboratories). The immuno- precipitated products were n on SDS-PAGE gels, Western blotted, and probed with 1/1000 dilution of anti-FLP polyclonal antibodies. Binding was detected using Amersham's goat-anti-rabbit HRP secondary antibodies and ECL chemiluminescent kits.

The results of these experiments demonstrated that FLP and FADD form complexes in vivo which can be coimmunoprecipitated with FADD antibodies and detected with FLP antibodies.

EXAMPLE 4

This example demonstrates that the C-terminal portion of FLP induces apoptosis in cultured HeLa cells, while full-length FLP does not.

HeLa cells were transiently transfected with a total of 3 μg of DNA using PanVera's TRANSIT-LT1 lipid mediated transfection kits. The pCGN8.1 vector expresses the subcloned cDNA as an HA fusion protein under a CMV promoter. When co-transfecting two polynucleotides at once, 1.5 μg of each plasmid was used without the blank vector. Test plasmids were pCGN8.1 vectors encoding DN-

FADD (a dominant-negative FADD mutant, amino acids 110-208, which inhibits apoptosis), full-length FADD, the C-terminal portion of FIP (amino acids 348-727), or full-length FIP . Some cultures were transfected with both FADD and DN- FADD or with both partial FLP and DN-FADD. All transfections included 0.5 μg of Clontech's pGFP, which encodes green fluorescent protein. Control cultures were transfected with pGFP alone.

Twelve to eighteen hours post-transfection, the cells were observed by fluorescence microscopy. The total number of green fluorescent cells were counted in a field and live or apoptotic green fluorescent cells were determined based upon morphology. Apoptotic cells look rounded, are barely attached to the surface, and contain refractory nuclei, while live cells are firmly attached to the plate with normal, clear nuclei. Results are shown in Figure 1.

Cultures of HeLa cells transfected with either the clone encoding the shortened form of FLP or the clone encoding FADD contained at least 60% apoptosis cells, whereas less than 20% ofthe cells in cultures transfected with clones encoding DN-FADD, full-length FLP, FADD and DN-FADD, partial FLP and DN-FADD, or pGFP alone were apoptotic. Thus, the C-terminal portion of FLP, encoded by the smaller 2.5 kb FIP mRNA induces apoptosis. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Chen, Tseng-hui Timothy Williams, Lewis T.

(ii) TITLE OF INVENTION: Human FADD-Interacting Protein

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Chiron Corporation

(B) STREET: 4560 Horton Street

(C) CITY: Emeryville (D) STATE: California

(E) COUNTRY: U.S.A.

(F) ZIP: 94608

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Jane Potter

(B) REGISTRATION NUMBER: 33,332

(C) REFERENCE/DOCKET NUMBER: 1380.001

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202-508-9100

(B) TELEFAX: 202-508-9299

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2184 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

ATGCCGCCAC CCGCGGACAT CGTCAAGGTG GCCATAGAAT GGCCGGGCGC CTACCCCAAA 60

CTCATGGAAA TTGATCAGAA AAAACCACTG TCTGCAATAA TAAAGGAAGT CTGTGATGGG 120

TGGTCTCTTG CCAACCATGA ATATTTTGCA CTCCAGCATG CCGATAGTTC AAACTTCTAT 180

ATCACAGAAA AGAACCGCAA TGAGATAAAA AATGGCACTA TCCTTCGATT AACCACATCT 240

CCAGCTCAGA ACGCCCAGCA GCTCCATGAA CGAATCCAGT CCTCGAGTAT GGATGCCAAG 300

CTGGAAGCCC TGAAGGACTT GGCCAGCCTC TCCCGGGATG TCACGTTTGC CCAGGAGTTT 360

ATAAACCTGG ACGGTATCTC TCTCCTCACG CAGATGGTGG AGAGCGGCAC TGAGCGATAC 420

CAGAAATTGC AGAAGATCAT GAAGCCTTGC TTTGGAGACA TGCTGTCCTT CACCCTGACG 480

GCCTTCGTTG AGCTGATGGA CCATGGCATA GTGTCCTGGG ATACATTTTC GGTGGCGTTC 540

ATTAAGAAGA TAGCAAGTTT TGTGAACAAG TCAGCCATAG ACATCTCGAT CCTGCAGCGG 600

TCCTTGGCCA TTTTGGAGTC GATGGTGCTC AATAGCCATG ACCTCTACCA GAAAGTGGCG 660

CAGGAGATCA CCATCGGCCA GCTCATTCCA CACCTGCAAG GGTCAGATCA AGAAATCCAA 720

ACCTATACTA TTGCAGTGAT TAATGCGCTT TTCCTGAAGG CTCCTGATGA GAGGAGGCAG 780

GAGATGGCGA ATATTTTGGC TCAGAAGCAA CTGCGTTCCA TCATTTTAAC ACATGTCATC 8 0

CGAGCCCAGC GGGCCATCAA CAATGAGATG GCGCACCAGC TGTATGTTCT ACAAGTGCTC 900

ACCTTTAACC TCCTGGAAGA CAGGATGATG ACCAAAATGG ACCCCCAGGA CCAGGCTCAG 960

AGGGACATCA TATTTGAACT TCGAAGAATT GCTTTTGATG CTGAGTCTGA ACCTAACAAC 1020

AGCAGTGGCA GCATGGAGAA ACGCAAGTCC ATGTACACGC GAGATTATAA GAAGCTTGGG 1080

TTCATTAATC ATGTCAACCC TGCCATGGAC TTCACGCAGA CTCCACCTGG GATGTTGGCT 1140

CTGGACAACA TGCTGTACTT TGCCAAGCAC CACCAAGATG CCTACATCCG GATTGTGCTT 1200

GAGAACAGTA GTCGAGAAGA CAAGCATGAA TGTCCCTTTG GCCGCAGTAG TATAGAGCTG 1260

ACCAAGATGC TATGTGAGAT CTTGAAAGTG GGCGAGTTGC CTAGTGAGAC CTGCAACGAC 1320

TTCCACCCGA TGTTCTTCAC CCACGACAGA TCCTTTGAGG AGTTTTTCTG CATCTGTATC 1380

CAGCTCCTGA ACAAGACATG GAAGGAAATG AGGGCAACTT CTGAAGACTT CAACAAGGTA 1440

ATGCAGGTGG TGAAGGAGCA GGTTATGAGA GCACTTACAA CCAAGCCTAG CTCCCTGGAC 1500

CAGTTCAAGA GCAAACTGCA GAACCTGAGC TACACTGAGA TCCTGAAAAT CCGCCAGTCC 1560

GAGAGGATGA ACCAGGAAGA TTTCCAGTCC CGCCCGATTT TGGAACTAAA GGAGAAGATT 1620

CAGCCAGAAA TCTTAGAGCT GATCAAACAG CAACGCCTGA ACCGCCTTGT GGAAGGGACC 1680

TGCTTTAGGA AACTCAATGC CCGGCGGAGG CAAGACAAGT TTTGGTATTG TCGGCTTTCG 1740

CCAAATCACA AAGTCCTGCA TTACGGAGAC TTAGAAGAGA GTCCTCAGGG AGAAGTGCCC 1800

CACGATTCCT TGCAGGACAA ACTGCCGGTG GCAGATATCA AAGCCGTGGT GACGGGAAAG 1860

GACTGCCCTC ATATGAAAGA GAAAGGTGCC CTTAAACAAA ACAAGGAGGT GCTTGAACTC 1920

GCTTTCTCCA TCTTGTATGA CTCAAACTGC CAACTGAACT TCATCGCTCC TGACAAGCAT 1980

GAGTACTGTA TCTGGACGGA TGGACTGAAT GCGCTACTCG GGAAGGACAT GATGAGCGAC 2040

CTGACGCGGA ATGACCTGGA CACCCTGCTC AGCATGGAAA TCAAGCTCCG CCTCCTGGAC 2100

CTGGAAAACA TCCAGATCCC TGACGCACCT CCGCCGATTC CCAAGGAGCC CAGCAACTAT 2160 GACTTCGTCT ATGACTGTAA CTGA 2184

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 727 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Pro Pro Pro Ala Asp lie Val Lys Val Ala lie Glu Trp Pro Gly 1 5 10 15

Ala Tyr Pro Lys Leu Met Glu lie Asp Gin Lys Lys Pro Leu Ser Ala 20 25 30 lie lie Lys Glu Val Cys Asp Gly Trp Ser Leu Ala Asn His Glu Tyr 35 40 45

Phe Ala Leu Gin His Ala Asp Ser Ser Asn Phe Tyr lie Thr Glu Lys 50 55 60

Asn Arg Asn Glu lie Lys Asn Gly Thr lie Leu Arg Leu Thr Thr Ser 65 70 75 80

Pro Ala Gin Asn Ala Gin Gin Leu His Glu Arg lie Gin Ser Ser Ser 85 90 95

Met Asp Ala Lys Leu Glu Ala Leu Lys Asp Leu Ala Ser Leu Ser Arg 100 105 110

Asp Val Thr Phe Ala Gin Glu Phe lie Asn Leu Asp Gly lie Ser Leu 115 120 125

Leu Thr Gin Met Val Glu Ser Gly Thr Glu Arg Tyr Gin Lys Leu Gin 130 135 140

Lys lie Met Lys Pro Cys Phe Gly Asp Met Leu Ser Phe Thr Leu Thr 145 150 155 160

Ala Phe Val Glu Leu Met Asp His Gly lie Val Ser Trp Asp Thr Phe 165 170 175

Ser Val Ala Phe lie Lys Lys lie Ala Ser Phe Val Asn Lys Ser Ala 180 185 190 lie Asp lie Ser lie Leu Gin Arg Ser Leu Ala lie Leu Glu Ser Met 195 200 205 Val Leu Asn Ser His Asp Leu Tyr Gin Lys Val Ala Gin Glu lie Thr 210 215 220 lie Gly Gin Leu lie Pro His Leu Gin Gly Ser Asp Gin Glu lie Gin 225 230 235 240

Thr Tyr Thr lie Ala Val lie Asn Ala Leu Phe Leu Lys Ala Pro Asp 245 250 255

Glu Arg Arg Gin Glu Met Ala Asn lie Leu Ala Gin Lys Gin Leu Arg 260 265 270

Ser lie lie Leu Thr His Val lie Arg Ala Gin Arg Ala lie Asn Asn 275 280 285

Glu Met Ala His Gin Leu Tyr Val Leu Gin Val Leu Thr Phe Asn Leu 290 295 300

Leu Glu Asp Arg Met Met Thr Lys Met Asp Pro Gin Asp Gin Ala Gin 305 310 315 320

Arg Asp lie lie Phe Glu Leu Arg Arg lie Ala Phe Asp Ala Glu Ser 325 330 335

Glu Pro Asn Asn Ser Ser Gly Ser Met Glu Lys Arg Lys Ser Met Tyr 340 345 350

Thr Arg Asp Tyr Lys Lys Leu Gly Phe He Asn His Val Asn Pro Ala 355 360 365

Met Asp Phe Thr Gin Thr Pro Pro Gly Met Leu Ala Leu Asp Asn Met 370 375 380

Leu Tyr Phe Ala Lys His His Gin Asp Ala Tyr He Arg He Val Leu 385 390 395 400

Glu Asn Ser Ser Arg Glu Asp Lys His Glu Cys Pro Phe Gly Arg Ser 405 410 415

Ser He Glu Leu Thr Lys Met Leu Cys Glu He Leu Lys Val Gly Glu 420 425 430

Leu Pro Ser Glu Thr Cys Asn Asp Phe His Pro Met Phe Phe Thr His 435 440 445

Asp Arg Ser Phe Glu Glu Phe Phe Cys He Cys He Gin Leu Leu Asn 450 455 460

Lys Thr Trp Lys Glu Met Arg Ala Thr Ser Glu Asp Phe Asn Lys Val 465 470 475 480

Met Gin Val Val Lys Glu Gin Val Met Arg Ala Leu Thr Thr Lys Pro 485 490 495

Ser Ser Leu Asp Gin Phe Lys Ser Lys Leu Gin Asn Leu Ser Tyr Thr 500 505 510

Glu He Leu Lys He Arg Gin Ser Glu Arg Met Asn Gin Glu Asp Phe 515 520 525 Gin Ser Arg Pro He Leu Glu Leu Lys Glu Lys He Gin Pro Glu He 530 535 540

Leu Glu Leu He Lys Gin Gin Arg Leu Asn Arg Leu Val Glu Gly Thr 545 550 555 560

Cys Phe Arg Lys Leu Asn Ala Arg Arg Arg Gin Asp Lys Phe Trp Tyr 565 570 575

Cys Arg Leu Ser Pro Asn His Lys Val Leu His Tyr Gly Asp Leu Glu 580 585 590

Glu Ser Pro Gin Gly Glu Val Pro His Asp Ser Leu Gin Asp Lys Leu 595 600 605

Pro Val Ala Asp He Lys Ala Val Val Thr Gly Lys Asp Cys Pro His 610 615 620

Met Lys Glu Lys Gly Ala Leu Lys Gin Asn Lys Glu Val Leu Glu Leu 625 630 635 640

Ala Phe Ser He Leu Tyr Asp Ser Asn Cys Gin Leu Asn Phe He Ala 645 650 655

Pro Asp Lys His Glu Tyr Cys He Trp Thr Asp Gly Leu Asn Ala Leu 660 665 670

Leu Gly Lys Asp Met Met Ser Asp Leu Thr Arg Asn Asp Leu Asp Thr 675 680 685

Leu Leu Ser Met Glu He Lys Leu Arg Leu Leu Asp Leu Glu Asn He 690 695 700

Gin He Pro Asp Ala Pro Pro Pro He Pro Lys Glu Pro Ser Asn Tyr 705 710 715 720

Asp Phe Val Tyr Asp Cys Asn 725

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1582 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GCCAGCGAGC CGAGGACAGA GGGCGCACGG AGGGCCGGGC CGCAGCCCCG GCCGCTTGCA 60 GACCCCGCCA TGGACCCGTT CCTGGTGCTG CTGCACTCGG TGTCGTCCAG CCTGTCGAGC 120 AGCGAGCTGA CCGAGCTCAA GTTCCTATGC CTCGGGCGCG TGGGCAAGCG CAAGCTGGAG 180 CGCGTGCAGA GCGGCCTAGA CCTCTTCTCC ATGCTGCTGG AGCAGAACGA CCTGGAGCCC 240 GGGCACACCG AGCTCCTGCG CGAGCTGCTC GCCTCCCTGC GGCGCCACGA CCTGCTGCGG 300 CGCGTCGACG ACTTCGAGGC GGGGGCGGCG GCCGGGGCCG CGCCTGGGGA AGAAGACCTG 360 TGTGCAGCAT TTAACGTCAT ATGTGATAAT GTGGGGAAAG ATTGGAGAAG GCTGGCTCGT 420 CAGCTCAAAG TCTCAGACAC CAAGATCGAC AGCATCGAGG ACAGATACCC CCGCAACCTG 480 ACAGAGCGTG TGCGGGAGTC ACTGAGAATC TGGAAGAACA CAGAGAAGGA GAACGCAACA 540 GTGGCCCACC TGGTGGGGGC TCTCAGGTCC TGCCAGATGA ACCTGGTGGC TGACCTGGTA 600 CAAGAGGTTC AGCAGGCCCG TGACCTCCAG AACAGGAGTG GGGCCATGTC CCCGATGTCA 660 TGGAACTCAG ACGCATCTAC CTCCGAAGCG TCCTGATGGG CCGCTGCTTT GCGCTGGTGG 720 ACCACAGGCA TCTACACAGC CTGGACTTTG GTTCTCTCCA GGAAGGTAGC CCAGCACTGT 780 GAAGACCCAG CAGGAAGCCA GGCTGAGTGA GCCACAGACC ACCTGCTTCT GAACTCAAGC 840 TGCGTTTATT AATGCCTCTC CCGCACCAGG CCGGGCTTGG GCCCTGCACA GATATTTCCA 900 TTTCTTCCTC ACTATGACAC TGAGCAAGAT CTTGTCTCCA CTAAATGAGC TCCTGCGGGA 960 GTAGTTGGAA AGTTGGAACC GTGTCCAGCA CAGAAGGAAT CTGTGCAGAT GAGCAGTCAC 1020 ACTGTTACTC CACAGCGGAG GAGACCAGCT CAGAGGCCCA GGAATCGGAG CGAAGCAGAG 1080 AGGTGGAGAA CTGGGATTTG AACCCCCGCC ATCCTTCACC AGAGCCCATG CTCAACCACT 1140 GTGGCGTTCT GCTGCCCCTG CAGTTGGCAG AAAGGATGTT TTGTCCCATT TCCTTGGAGG 1200 CCACCGGGAC AGACCTGGAC ACTAGGGTCA GGCGGGGTGC TGTGGTGGGG AGAGGCATGG 1260 CTGGGGTGGG GGTGGGGAGA CCTGGTTGGC CGTGGTCCAG CTCTTGGCCC CTGTGTGAGT 1320 TGAGTCTCCT CTCTGAGACT GCTAAGTAGG GGCAGTGATG GTTGCCAGGA CGAATTGAGA 1380 TAATATCTGT GAGGTGCTGA TGAGTGATTG ACACACAGCA CTCTCTAAAT CTTCCTTGTG 1440 AGGATTATGG GTCCTGCAAT TCTACAGTTT CTTACTGTTT TGTATCAAAA TCACTATCTT 1500 TCTGATAACA GAATTGCCAA GGCAGCGGGA TCTCGTATCT TTAAAAAGCA GTCCTCTTAT 1560 TCCTAAGGTA ATCCTATTAA AA 1582

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 208 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens ( i ) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser

1 5 10 15

Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly 20 25 30

Lys Arg Lys Leu Glu Arg Val Gin Ser Gly Leu Asp Leu Phe Ser Met 35 40 45

Leu Leu Glu Gin Asn Asp Leu Glu Pro Gly His Thr Glu Leu Leu Arg 50 55 60

Glu Leu Leu Ala Ser Leu Arg Arg His Asp Leu Leu Arg Arg Val Asp 65 70 75 80

Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala Ala Pro Gly Glu Glu Asp 85 90 95

Leu Cys Ala Ala Phe Asn Val He Cys Asp Asn Val Gly Lys Asp Trp 100 105 110

Arg Arg Leu Ala Arg Gin Leu Lys Val Ser Asp Thr Lys He Asp Ser 115 120 125

He Glu Asp Arg Tyr Pro Arg Asn Leu Thr Glu Arg Val Arg Glu Ser 130 135 140

Leu Arg He Trp Lys Asn Thr Glu Lys Glu Asn Ala Thr Val Ala His 145 150 155 160

Leu Val Gly Ala Leu Arg Ser Cys Gin Met Asn Leu Val Ala Asp Leu 165 170 175

Val Gin Glu Val Gin Gin Ala Arg Asp Leu Gin Asn Arg Ser Gly Ala 180 185 190

Met Ser Pro Met Ser Trp Asn Ser Asp Ala Ser Thr Ser Glu Ala Ser 195 200 205

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCAAGTCCAT GTACACGC 18 (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GAAGCTTGGG TTCATTAA 18

Claims

1. An isolated and purified human F ADD-Interacting Protein (FLP protein) having an amino acid sequence which is at least 85% identical to the amino acid sequence shown in SEQ LD NO:2.

2. An isolated and purified human FLP polypeptide comprising at least 8 contiguous amino acids selected from the amino acid sequence shown in SEQ LD NO:2.

3. A fusion protein comprising a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment comprises at least 8 contiguous amino acids of a human FLP protein selected from the amino acid sequence shown in SEQ LD NO: 2.

4. A preparation of antibodies which specifically bind to a human FLP protein.

5. An isolated and purified subgenomic polynucleotide encoding an amino acid sequence of a human FIP protein or a human FLP protein variant, wherein the nucleotide sequence ofthe subgenomic polynucleotide is at least 85% identical to the nucleotide sequence shown in SEQ LD NO: 1.

6. An expression constmct, comprising: a subgenomic polynucleotide, wherein the subgenomic polynucleotide comprises at least 11 contiguous nucleotides selected from the nucleotide sequence shown in SEQ LD NO: 1; and a promoter, wherein the subgenomic polynucleotide is located downstream from the promoter, and wherein transcription ofthe subgenomic polynucleotide initiates at the promoter.

7. A homologously recombinant cell having incorporated therein a new transcription initiation unit, wherein the new transcription initiation unit comprises:

(a) an exogenous regulatory sequence;

(b) an exogenous exon; and

(c) a splice donor site, wherein the transcription initiation unit is located upstream of a coding sequence of a FIP gene, wherein the exogenous regulatory sequence directs transcription ofthe coding sequence ofthe FIP gene.

8. A host cell which comprises an expression constmct, wherein the expression constmct comprises (a) a subgenomic polynucleotide comprising at least 11 contiguous nucleotides selected from the nucleotide sequence shown in SEQ LD

NO:l and (b) a promoter, wherein the subgenomic polynucleotide is located downstream from the promoter, and wherein transcription ofthe subgenomic polynucleotide initiates at the promoter.

9. A method of inducing apoptosis in a cell, comprising the step of: contacting the cell with a polypeptide comprising amino acids 348-727 of SEQ LD NO:2, whereby apoptosis ofthe cell is induced.

10. A method of preventing apoptosis of a cell, comprising the step of: contacting the cell with a composition comprising (a) a polynucleotide encoding a reagent which specifically binds to a wild-type human FIP expression product and (b) a pharmaceutically acceptable carrier, whereby apoptosis ofthe cell is prevented.

11. A composition comprising: a polynucleotide encoding a reagent which specifically binds to a wild- type human FIP expression product; and a pharmaceutically acceptable carrier.

12. A composition, comprising: all or a portion of a protein having the amino acid sequence shown in SEQ LD NO:2 or all or a portion of a gene having the nucleotide sequence shown in SEQ LD NO:l; and a pharmaceutically acceptable carrier.

13. A method of screening test compounds for the ability to induce or prevent apoptosis, comprising the steps of:

(a) contacting a cell with a test compound, wherein the cell comprises (i) a first expression constmct comprising a subgenomic polynucleotide encoding at least a portion of FADD comprising amino acids 1-110 of the amino acid sequence shown in SEQ LD NO:4, wherein the portion of FADD binds to a portion of FLP and (ii) a second expression constmct comprising a subgenomic polynucleotide encoding at least a portion of FLP, wherein the portion of FLP comprises amino acids 348-727 of SEQ LD NO:2 and wherein the portion of FLP binds to the portion of FADD; and (b) measuring the ability ofthe test compound to decrease or increase binding ofthe portion of FLP to the portion of FADD, a test compound which increases the binding being a potential dmg for inducing apoptosis, and a test compound which decreases the binding being a potential dmg for preventing apoptosis.

14. A method of screening test compounds for the ability to induce or prevent apoptosis, comprising the steps of:

(a) contacting a cell with a test compound, wherein the cell comprises: i) a first fusion protein comprising (1) at least a portion of a FLP protein comprising amino acids 348-727 of SEQ LD NO:2 and (2) either a DNA binding domain or a transcriptional activating domain; ii) a second fusion protein comprising at least a portion of a FADD protein comprising amino acids 1-110 of SEQ LD NO:4, wherein the portion ofthe FLP protein binds to the portion ofthe FADD protein, wherein if the first fusion protein comprises a DNA binding domain, then the second fusion protein comprises a transcriptional activating domain, wherein if the first fusion protein comprises a transcriptional activating domain, then the second fusion protein comprises a DNA binding domain, wherein the interaction ofthe first and second fusion proteins reconstitutes a sequence-specific transcription activating factor; and iii) a reporter gene comprising a DNA sequence to which the DNA binding domain specifically binds; and

(b) measuring the expression ofthe reporter gene, wherein a test compound which decreases the expression ofthe reporter gene is identified as a potential agent for preventing apoptosis in the cell, and wherein a test compound which increases the expression ofthe reporter gene is identified as a potential agent for inducing apoptosis in the cell.

15. A method of screening test compounds for the ability to induce or prevent apoptosis, comprising the steps of:

(a) contacting a test compound with (i) a first polypeptide comprising a FADD-binding site, wherein the FADD-binding site comprises amino acids 348-727 of SEQ LD NO:2; and (ii) a second polypeptide comprising a FLP-binding site, wherein the FLP-binding site comprises amino acids 1-110 of SEQ LD NO:4, wherein the FADD-binding site binds to the FLP-binding site; and

(b) measuring the amount of binding between the first and second polypeptides in the presence ofthe test compound, wherein a test compound which increases the amount of binding between the first and second polypeptides is identified as a potential dmg for inducing apoptosis, and a test compound which decreases the amount of binding between the first and second polypeptides is identified as a potential dmg for preventing apoptosis.

16. A method of determining the tissue source of a body sample of a human, comprising the step of: assaying the body sample for the presence of a polypeptide consisting of amino acids 348-727 of SEQ LD NO:2 or a 2.5 kb FIP mRNA encoding the FLP polypeptide, wherein the presence ofthe FLP polypeptide or the 2.5 kb FIP mRNA indicates that the body sample originates from a tissue selected from the group consisting of brain, pancreas, spleen, and peripheral blood lymphocytes.

17. A method of screening for test compounds which are useful for enhancing transfer of FIP subgenomic polynucleotides to a cell, comprising the steps of: delivering aPTP subgenomic polynucleotide to a cell; contacting the cell with a test compound; and monitoring transfer ofthe FIP subgenomic polynucleotide to the cell or monitoring a biological effect ofthe FIP subgenomic polynucleotide within the cell, wherein a test compound which enhances the transfer to the cell or the biological effect within the cell ofthe FIP subgenomic polynucleotide identifies a compound which is useful for enhancing transfer ofthe FIP subgenomic polynucleotide to the cell.

18. A method of expressing a FIP subgenomic polynucleotide in a cell, comprising the step of: deUvering the FIP subgenomic polynucleotide to the cell, whereby the FIP subgenomic polynucleotide is expressed.