WO1996016989A1

WO1996016989A1 - p53 PROTEINS WITH ALTERED TETRAMERIZATION DOMAINS

Info

Publication number: WO1996016989A1
Application number: PCT/US1995/015353
Authority: WO
Inventors: Thanos D. Halazonetis
Original assignee: The Wistar Institute Of Anatomy And Biology
Priority date: 1994-11-28
Filing date: 1995-11-27
Publication date: 1996-06-06
Also published as: EP0799243A1; EP0799243A4; AU4288496A

Abstract

The present invention provides p53 proteins with altered tetramerization domains that retain wild-type p53 function, and the ability to form tetramers and have at least one of the following characteristics: (1) do not hetero-oligomerize with wild-type p53 or tumor-derived p53 mutants, and (2) restricted DNA binding specificity from an alteration in the way that the tetramerization domain orients the DNA binding domains of a p53 tetramer relative to one another. The invention also provides nucleic acids encoding the above proteins and methods of enhancing the cellular response to DNA damaging agents, treating diseases characterized by abnormal cell proliferation, and inducing immune tolerance to facilitate transplants and treatment of autoimmune disease, by administration of proteins of the invention or nucleic acid sequences encoding the proteins of the invention.

Description

p53 PROTEINS WITH ALTERED TETRAMERIZATION DOMAINS

Field of the Invention

The present invention relates to the field of p53 proteins with altered oligo erization domains, polynucleotide sequences encoding them, and their use in therapy.

Background of the Invention

Wild-type (wt) p53 is a sequence-specific DNA binding protein found in humans and other mammals, which has tumor suppressor function [See, e.g., Harris (1993), Science, 262: 1980-1981]. The wild-type p53 protein functions to regulate cell proliferation and cell death (also known as apoptosis) . It also participates in the response of the cell to DNA damaging agents [Harris (1993), cited above]. In more than half of all human tumors p53 is inactivated by mutations and is therefore unable to arrest cell proliferation or induce apoptosis in response to DNA damaging agents, such as radiation and chemotherapeutics commonly used for cancer treatment. The nucleotide and amino acid sequences of human p53 are reported below as SEQ ID NOS: 1 and 2, respectively [Zakut-Houri et al, (1985), EMBO J., 4: 1251-1255; GenBank Code Hsp53]. The amino acid sequence of p53 is conserved across evolution [Soussi et al, (1990) , Oncogene, 5: 945-952], suggesting that its function is also conserved.

At the biochemical level, p53 is a tetrameric DNA sequence-specific transcription factor. Its DNA binding and transcriptional activities are required for p53 to suppress tumor growth [Pietenpol et al, (1994) , Proc. Natl. Acad. Sci. USA, 91: 1998-2002]. p53 forms homotetramers in the absence of DNA and maintains its tetrameric stoichiometry when bound to DNA [Kraiss et al, (1988), J. Virol., 62: 4737-4744; Stenger et al, (1992), Mol. Carcinog., 5: 102-106; Sturzbecher et al, (1992), Oncogene, 7: 1513-1523; Friedman et al, (1993), Proc. Natl. Acad. Sci. USA, 90: 3319-3323; Halazonetis and Kandil (1993), EMBO J. , 12: 5057-5064; and Hainaut et al, (1994), Oncogene, 9: 299-303]. Consistent with the observation that p53 binds DNA as a homotetramer, the known physiologically relevant DNA sites recognized by p53 contain four pentanucleotide repeats [El-Deiry et al, (1993), Cell, 75: 817-825; Wu et al, (1993), Genes Dev. , 7: 1126-1132; Kastan et al, (1992), Cell, 71: 587-597]. Each pentanucleotide repeat is recognized by one subunit of the p53 homotetramer [Halazonetis and Kandil (1993) , cited above; Cho et al, (1994), Science, 265: 346-355]. The ability of p53 to bind DNA in a sequence-specific manner maps to amino acid residues 90-290 of SEQ ID NO: 2 [Halazonetis and Kandil (1993), cited above; Pavletich et al, (1993), Genes Dev., 7: 2556-2564; Wang et al, (1993), Genes Dev., 7: 2575-2586].

Once bound to DNA, p53 activates gene transcription from neighboring promoters. The ability of p53 to activate gene transcription has been mapped to within amino acid residues 1-90 of SEQ ID NO: 2 [Fields et al, (1990), Science, 249: 1046-1049].

The C-terminus of the human p53 tumor suppressor protein (i.e., amino acids 290-393 of human p53, SEQ ID NO: 2) has two functions. It induces p53 oligomerization and it regulates p53 DNA binding by controlling the conformation of p53 tetramers. These two functions map to independent regions. Oligomerization maps to amino acid residues 322-355 of SEQ ID NO: 2 [Wang et al,

(1994), Mol. Cell. Biol., 14: 5182-5191; Clore et al, (1994), Science, 265: 386-391]. Regulation of DNA binding maps to amino acid residues 364-393 of human p53 [SEQ ID NO: 2] or to the corresponding region encompassing residues 361-390 of mouse p53 [SEQ ID NO: 15] [Hupp et al, (1992), Cell, 71: 875-886; Halazonetis et al, (1993), EMBO J. , 12: 1021-1028; Halazonetis and Kandil (1993), cited above; Genbank locus Mmp53r] .

Mutations of the p53 protein in most human tumors involve the sequence-specific DNA binding domain, so that the mutant proteins are unable to bind DNA [Bargonetti et al, (1992), Genes Dev., 6: 1886-1898]. The loss of p53 function is critical for tumor development. Introduction of wild-type p53 into tumor cells leads to arrest of cell proliferation or cell death [Finlay et al, (1989) , Cell, 57: 1083-1093; Eliyahu et al, (1989), Proc. Natl. Acad. Sci. USA, 86: 8763-8767; Baker et al, (1990), Science, 249: 912-915; Mercer et al, (1990), Proc. Natl. Acad. Sci. USA, 87: 6166-6170; Diller et al, (1990), Mol. Cell. Biol., 10: 5772-5781; Isaacs et al, (1991), Cancer Res., 51: 4716-4720; Yonish-Rouach et al, (1993), Mol. Cell. Biol., 13: 1415-1423; Lowe et al, (1993), Cell, 74: 957-967; Fujiwara et al, (1993), Cancer Res., 53: 4129-4133; Fujiwara et al, (1994), Cancer Res., 54: 2287-2291]. Thus, introduction of wild-type p53 into tumor cells has been proposed to be a viable approach to treat human cancer [see, e.g., International Patent Applications WO 9406910 A, WO 9416716 A, WO 9322430 Al, EP 390323, and EP 475623 Al]. However, most tumors express mutant versions of p53 at high levels [Harris (1993), cited above]. Because these p53 mutants have intact oligomerization domains, they form hetero-tetramers with wild-type p53. Such hetero-tetra ers are biochemically inactive or characterized by considerably reduced activity compared to wild-type p53 tetramers [Milner and Medcalf (1991), Cell, 65: 765-774; Bargonetti et al, (1992), cited above; Farmer et al, (1992), Nature, 358: 83-86; Kern et al, (1992), Science, 256: 827-830]. Thus, if one were to treat human cancer by introduction of wild-type p53 in tumor cells, the effectiveness of this therapeutic approach would be limited by the presence of mutant p53 in the cancer cells.

Thus, there is a need in the art for the identification of compositions which are not inhibited by endogenous p53, as well as for methods for the uses of such compositions for therapeutic purposes.

Summary of the Invention

The present invention provides novel modified p53 proteins, including preferably chimeric proteins formed by the association of sequences of p53 and sequences of other selected proteins, which novel proteins have desirable functional characteristics.

In one aspect, the present invention provides p53 proteins with altered tetramerization domains characterized by the ability to form tetramers, bind DNA and activate transcription indistinguishably from wild-type p53, but incapable of forming hetero-tetramers with p53 proteins that have an intact tetramerization domain, such as wild-type p53 or tumor-derived p53 mutants. These p53 proteins of the invention are preferably chimeric proteins, characterized by disruption of the native p53 tetramerization domain and insertion of a heterologous oligomerization domain in a way that preserves tetramerization.

In another aspect, the invention provides p53 proteins characterized by restricted DNA binding specificity from an alteration in the way the tetramerization domain orients the DNA binding domains of a p53 tetramer relative to one another. These proteins are characterized by deletion of all or a significant portion of, or disruption of, the region between the DNA binding domain (amino acid residues 90-289 of human p53 of SEQ ID NO: 2) and tetramerization domain (amino acid residues 322-355 of human p53 of SEQ ID NO: 2) . This region (spanning residues 290-321 of human p53 of SEQ ID NO: 2) is considered an extension of the p53 tetramerization domain. In still another aspect, the invention provides p53 proteins with both of the characteristics described above, namely: (1) ability to form tetramers, but inability to hetero-tetramerize with p53 proteins having an intact tetramerization domain, such as wild-type p53 or tumor-derived p53 mutants; (2) restricted DNA binding specificity from an alteration in the way that the tetramerization domain orients the DNA binding domains of a p53 tetramer relative to one another.

In still another aspect, the invention provides further modifications of the p53 proteins provided above. These modifications include: (l) altered transcription activation sequences (amino acid residues 1-90 of human p53 of SEQ ID NO: 2) ; (2) insertion of one or more nuclear localization signals; and (3) replacement of selected regions of the p53 proteins with homologous regions of non-human p53 protein, as well as conventional modifications such as insertion or deletion or substitution of individual amino acid residues throughout the sequence, and the use of linkers between portions of the chimeric proteins.

Still another aspect of this invention provides p53 proteins having two or more of the above-described modifications.

In yet another aspect, the present invention provides a nucleic acid sequence encoding a protein of the invention. These nucleic acids may be inserted into an appropriate vector for delivery to patients for gene therapy. Alternatively the nucleic acids may be inserted into a vector for in vitro expression of a protein of the invention, which is then introduced into patients. In a further aspect, the invention provides a method of treating an individual having a condition characterized by abnormal cell proliferation by delivering a protein or, preferably, a nucleic acid sequence, of the invention to the patient.

Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

Brief Description of the Drawings Fig. IA schematically illustrates wild-type p53. The amino acid numbering, which is also maintained throughout Figs. 1A-1K, refers to the residues of human p53 as indicated in SEQ ID NO: 2. The entire length of human p53 is 393 amino acids. Symbols for the DNA binding domain (residues 90-290 of SEQ ID NO: 2) (checkerboard bar) and the oligomerization domain (residues 322-355 of SEQ ID NO: 2) (solid bar) are maintained throughout Figs. 1A-1K.

Fig. IB schematically illustrates a heterologous dimerization domain p53 chimeric protein containing residues 1-346 of p53 [SEQ ID NO: 2], a glutamic acid for cloning convenience and a GCN4 dimerization domain corresponding to residues 253-281 of GCN4 of SEQ ID NO: 4 (hatched bar) . Fig. IC schematically illustrates a heterologous dimerization domain p53 chimeric protein containing residues 1-347 of p53 [SEQ ID NO: 2] and residues 253-281 of GCN4 of SEQ ID NO: 4 (hatched bar).

Fig. ID schematically illustrates a heterologous dimerization domain chimeric p53 protein containing an insertion of a glutamic acid and residues 253-281 of GCN4 of SEQ ID NO: 4 (hatched bar) between residues 346 and 347 Of p53 [SEQ ID NO: 2]. Fig. IE schematically illustrates a heterologous dimerization domain chimeric p53 protein containing residues 1-356 of human p53 [SEQ ID NO: 2] with a mutation within the native p53 oligomerization domain (leucine 344 to alanine) linked to the dipeptide glycine-asparagine and residues 253-281 of GCN4 of SEQ ID NO: 4 (hatched bar) .

Fig. IF schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-334 of human p53 [SEQ ID NO: 2] linked to an asparagine and then to a tetrameric variant of GCN4 residues 249-281 of SEQ ID NO: 6 (hatched bar) .

Fig. IG schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-325 of human p53 [SEQ ID NO: 2] linked to the tripeptide arginine-glycine-asparagine [SEQ ID NO: 7] and then to a tetrameric variant of GCN4 residues 249-281 of SEQ ID NO: 6 (hatched bar) .

Fig. 1H schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-323 of human p53 [SEQ ID NO: 2] linked to the hexapeptide arginine-glycine-glycine- asparagine-proline-glutamic acid [SEQ ID NO: 8] and then to a tetrameric variant of GCN4 residues 250-281 of SEQ ID NO: 6 (hatched bar) .

Fig. 1J schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-300 of human p53 [SEQ ID NO: 2] linked to the pentapeptide glycine-glycine-asparagine- glutamine-alanine [SEQ ID NO: 9] and then to a tetrameric variant of GCN4 residues 250-281 of SEQ ID NO: 6 (hatched bar) .

Fig. IK schematically illustrates a heterologous tetramerization domain chimeric p53 protein which contains residues 1-325 of human p53 [SEQ ID NO: 2] linked to the tripeptide arginine-glycine-asparagine [SEQ ID NO: 7], a tetrameric variant of GCN4 residues 249-281 of SEQ ID NO: 6 and an isoleucine (hatched bar) , and then followed by residues 352-393 of human p53 [SEQ ID NO: 2]. Fig. 2A schematically illustrates the chimeric p53 protein of Fig. IF, which serves as a paradigm to indicate the various modifications that can be introduced into any of the p53 proteins of this invention (Figs. 2B-2F) . Symbols for the p53 DNA binding domain (checkerboard bar) , the truncated p53 oligomerization domain (solid bar) and the heterologous tetramerization domain (hatched bar) are maintained in Figs. 2B-2F. Also, the numbering throughout Figs. 2A-2F refers to the residues of human p53 as indicated in SEQ ID NO: 2. Fig. 2B schematically illustrates a deletion of residues 300-327 of human p53 [SEQ ID NO: 2], that confers novel DNA binding specificities.

Fig. 2C schematically illustrates the substitution of the transcription activation domain of p53 with that of the herpes simplex virus protein VP16 (reverse hatched bar) , also known as α trans-inducing factor.

Fig. 2D schematically illustrates the insertion of a nuclear localization signal (NLS) between amino acid residues 80 and 81 of p53 [SEQ ID NO: 2] (horizontal lined bar) . The abbreviation a.a. represents amino acids.

Fig. 2E schematically illustrates the substitution of human p53 residues 3-80 of SEQ ID NO: 2 with the corresponding xenopus sequences (cross-hatched bar) . Fig. 2F schematically illustrates two mutations that enhance function of the p53 proteins [SEQ ID NO: 2] of this invention, such as substitution of Arg 174 with Gin, or Arg 175 with Leu.

Fig. 3A schematically illustrates a wild-type p53 tetramer bound to a DNA site containing four contiguous pentanucleotides repeats. For Figs. 3 -3H, the p53 DNA binding domains are shown as circles, the oligomerization domain as a thin rectangle, the linker between the DNA binding and oligomerization domains as curved lines, the DNA as a thick rectangle and the specific pentanucleotides as arrows.

Fig. 3B schematically illustrates a wild-type p53 tetramer bound to a DNA site containing two pentanucleotide pairs separated by a 20-30 nucleotide insert.

Figs. 3C and 3D schematically illustrate a p53 tetramer with antiparallel alignment of its oligomerization domains and a short linker between the DNA binding and oligomerization domains. Such a p53 tetramer cannot bind to a DNA site containing four contiguous pentanucleotides repeats (Fig. 3C) , but can bind to a DNA site containing two pentanucleotide pairs separated by a 20-30 nucleotide insert (Fig. 3D) .

Fig. 3E schematically illustrates a chimeric p53 tetramer with parallel alignment of its oligomerization domains bound to a DNA site containing four contiguous pentanucleotides repeats.

Fig. 3F schematically illustrates a chimeric p53 tetramer with parallel alignment of its oligomerization domains bound to a DNA site containing two pentanucleotide pairs separated by a 20-30 nucleotide insert.

Figs. 3G and 3H schematically illustrate a chimeric p53 tetramer with parallel alignment of its oligomerization domains and a short linker between the DNA binding and oligomerization domains. Such a p53 tetramer can bind to a DNA site containing four contiguous pentanucleotides repeats (Fig. 3G) , but not to a DNA site containing two pentanucleotide pairs separated by a 20-30 nucleotide insert (Fig. 3H) . Fig. 4 is a bar graph demonstrating the tumor suppressing activities of the proteins encoded by the listed expression plasmids, presented as means + l standard error of G418 resistant colonies per plate. Fig. 5 is a graph charting tumor suppressor activities of p53 proteins in the presence of the p53 tumor-derived mutant tryptophan 248 (W248) . The results are presented relative to activity in the absence of p53W248. Fig. 6A schematically illustrates how a c-Jun modified leucine zipper directs parallel assembly of p53 - c-Jun chimeric proteins. For Figs. 6A-6D, the p53 segment is indicated as a rectangle with rounded edges, the c-Jun zipper as a rectangle with sharp edges, the leucine (Leu) and isoleucine (lie) residues, which mediate oligomerization are indicated. The p53 - c-Jun chimera forms tetramers, but for simplicity only two of the subunits are indicated.

Fig. 6B schematically illustrates how a c-Jun modified leucine zipper can direct antiparallel assembly of p53 - c-Jun chimeric proteins. The number of hydrophobic interactions are the same whether the zippers assemble parallel (Fig. 6A) or antiparallel (Fig. 6B) . Fig. 6C schematically illustrates how substitution of one isoleucine (lie) of the c-Jun modified leucine zipper with asparagine (Asn) compromises one hydrophobic interaction when the p53 - c-Jun chimeric proteins are assembled parallel.

Fig. 6D schematically illustrates how substitution of one isoleucine (lie) of the c-Jun modified leucine zipper with asparagine (Asn) compromises two hydrophobic interactions when the p53 - c-Jun chimeric proteins are assembled antiparallel. Fig. 7 is a graph charting the tumor suppressor activities of p53-zipper chimera in Saos-2 cells using the colony-forming assay.

Detailed Description of the Invention The present invention provides p53 proteins with modifications in the native p53 sequence. These modifications, which do not interfere with its native tumor-suppressor function, provide the protein with at least one of the following functional characteristics: (1) the ability to bind DNA and activate transcription like wild-type p53, but to not hetero-oligo erize with wild-type p53 or tumor-derived p53 mutants; and (2) restricted DNA binding specificity from an alteration in the way that the tetramerization domain orients the DNA binding domains of a p53 tetramer relative to one another. In addition nucleic acids encoding such proteins and methods of using such proteins or nucleic acid sequences therapeutically are provided. All references to human p53 residue numbers refer to the numbering scheme provided by Zakut-Houri et al, (1985) [cited above]. The nucleotide and amino acid sequences of human p53 are reproduced as SEQ ID NOS: 1 and 2, respectively.

A. p53 Proteins with Altered Tetramerization Domains Definitions: A dimerization domain is defined as a domain that allows formation of dimers, while a tetramerization domain is defined as a domain that allows formation of tetramers. An oligomerization domain allows formation of oligomers, which can be of any subunit stoichiometry (of course greater than one). Thus, the term oligomerization domain is more general and encompasses both dimerization and tetramerization domains (which direct formation of oligomers of subunit stoichiometries 2 and 4, respectively) . The term chimeric protein refers to a protein containing sequences from two different proteins, for example from p53 and GCN4.

Al. p53 Associated with a Heterologous Dimerization Domain

In one embodiment, a protein of this invention is comprised of a p53 protein bearing a partial functional inactivation of its tetramerization domain and a heterologous dimerization domain. Thus, certain regions of the p53 tetramerization domain must be maintained (so that the chimeric protein can form tetramers, in spite of containing a heterologous dimerization domain) , while other regions are inactivated (so that tetramerization is dependent on the heterologous dimerization domain) . The p53 tetramerization domain maps to residues 322-355 of SEQ ID NO: 2 [Wang et al, (1994), cited above; Clore et al, (1994), cited above]. According to this invention, a disruption of the p53 tetramerization domain, involving residues 335-348 of SEQ ID NO: 2 or a subset of these residues, sufficiently disrupts the function of this domain, so that it can no longer drive tetramerization with wild-type p53 or tumor-derived p53 mutants. At the same time, however, introduction of a heterologous dimerization domain reestablishes the ability to form tetramers, which is mediated both by the heterologous dimerization domain and by the residual tetramerization domain of p53.

A heterologous dimerization domain is defined herein as a sequence of amino acids heterologous to p53 and capable of forming homodi ers. One example of a dimerization domain is the leucine zipper (LZ) element. A leucine zipper has been defined as stretch of about 35 amino acids containing 4-5 leucine residues separated from each other by six amino acids [Maniatis and Abel (1989), Nature, 341: 24-25]. The leucine zipper occurs in a variety of evikaryotic DNA binding proteins, such as GCN4, C/EBP, c-Fos, c-Jun, c-Myc and c-Max.

Heterologous dimerization domains may also be selected from other proteins, such as the retinoic acid receptor, the thyroid hormone receptor or other nuclear hormone receptors [Kurokawa et al, (1993), Genes Dev. , 7:1423-1435] or from the yeast transcription factors Gal4 and HAPl [Marmonstein et al, (1992), Nature, 356:408-414; Zhang et al, (1993), Proc. Natl. Acad. Sci. USA, 90:2851-2855]. One of skill in the art may identify additional suitable dimerization domains, including artificial dimerization domains [O'Shea et al, (1992), Cell, 68:699-708; Krylov et al, (1994), EMBO J., 13: 2849-2861]. For ease in description, the leucine zipper of the yeast transcription factor GCN4 is used herein as the exemplary dimerization domain. The nucleotide and amino acid sequences of GCN4 are presented as SEQ ID NO: 3 and NO: 4, respectively. The numbering of the GCN4 nucleotide and amino acid residues follows Hinnenbusch (1984) Proc. Natl. Acad. Sci. USA, 81: 6442-6446 and Ellenberger et al, (1992) Cell, 71:1223-1237, respectively. The coding region of GCN4 is encompassed by nucleotide 778-1623 of SEQ ID NO: 3. The nucleotide and amino acid sequence are found in GenBank under the Code Yscgcn4.

Partial functional inactivation of the p53 tetramerization domain can be accomplished by deletions, insertions and/or amino acid substitutions targeting part of this domain. Such mutations should involve residues 335-348 of SEQ ID NO: 2 or a subset of these residues, but need not be confined within the p53 tetramerization domain. For example, a deletion whose N-terminal boundary is within residues 335-348 of SEQ ID NO: 2 may extend as far as the p53 C-terminus. The precise boundaries of the mutations will depend on the nature of the heterologous dimerization domain and the presence, if any, of amino acid sequences introduced for cloning or other purposes between p53 and the heterologous dimerization domain. For example, in one preferred embodiment, residues 1-346 of human p53 [SEQ ID NO: 2] are juxtaposed to the dimerization domain of GCN4 (residues 253-281 of GCN4 SEQ ID NO: 4) through a glutamic acid linker (Fig. IB) . In another embodiment residues 1-347 of human p53 [SEQ ID NO: 2] are juxtaposed to residues 253-281 of GCN4 [SEQ ID NO: 4] (Fig. IC) . Alternatively, the function of the p53 tetramerization domain may be partially disrupted by insertion of the heterologous dimerization domain within the p53 tetramerization domain and preferably between residues 335 and 348 of human p53 [SEQ ID NO: 2]. In a preferred embodiment (Fig. ID) a glutamic acid and residues 253-281 of GCN4 [SEQ ID NO: 4] are inserted between residues 346 and 347 of human p53 [SEQ ID NO:2]. Alternatively, the function of the p53 tetramerization domain may be partially disrupted by insertions, deletions or amino acid substitutions, while the heterologous dimerization domain is inserted outside the boundaries of the p53 tetramerization domain. The mutations should again target residues 335-348 of human p53 [SEQ ID NO: 2], or a subset thereof. In one embodiment, the function of the p53 [SEQ ID NO: 2] tetramerization domain is inactivated by substitution of residue 344 by alanine. This mutation only partially disrupts the function of the p53 tetramerization domain (see Examples section) . A heterologous dimerization domain can then be inserted even outside the p53 tetramerization domain, for example following residue 356 of human p53 [SEQ ID NO: 2], to reestablish tetramer formation (Fig. IE) . At least two novel features characterize the class of proteins described here. First these chimeric proteins form tetramers. This was unexpected because the disruption in the p53 tetramerization domain is of sufficient magnitude to disrupt p53 tetramers into monomers. Yet, when the heterologous dimerization domain is introduced, the chimeric protein forms tetramers, rather than dimers, as would be expected. A second novel feature of these chimeric proteins is that their ability to form tetramers with wild-type p53 or with tumor-derived p53 mutants is greatly reduced. This is surprising, because these proteins must utilize p53 structural determinants to form tetramers (recall that in the invention a heterologous dimerization domain is juxtaposed to the p53 sequence) . For example the chimeric protein of Fig. IB that retains residues 1-346 of human p53 [SEQ ID NO: 2], retains all the critical residues (Gly 334, Arg 337, Phe 341 and Leu 344 of SEQ ID NO:2) that are required to make the inter-subunit contacts for tetramer formation (see Example 3 and Clore et al, (1994) , cited above) . Yet this chimeric protein fails to form tetramers with wild-type p53 or tumor-derived p53 mutants.

The specific embodiments described in this section display the desired functional characteristics, namely of forming tetramers, binding DNA and activating transcription equivalently to wild-type p53, and in addition fail to associate or associate very weakly with tumor-derived p53 mutants (see Examples section) . Additional examples of p53 proteins of this invention may be generated by one of skill in the art given the teachings contained herein. A2 . p53 Associated with a Heterologous Tetramerization Domain

In this embodiment of the invention, the p53 protein bears a partial or preferably a complete functional inactivation of its tetramerization domain and contains a heterologous tetramerization domain.

A heterologous tetramerization domain is defined as a sequence of amino acids heterologous to p53 and capable of forming stable homo-tetramers. Exemplary suitable tetramerization domains include that of the lac repressor, or an artificial tetramerization domain, such as variants of the GCN4 leucine zipper that form tetramers [Alberti et al, (1993), EMBO J., 12: 3227-3236; Harbury et al, (1993), Science, 262: 1401-1407; Krylov et al, (1994), cited above]. One of skill in the art could readily select alternate tetramerization domains. For ease in description, the tetrameric variant of the GCN4 leucine zipper [Harbury et al, (1993) , cited above] is used herein as the exemplary tetramerization domain. This variant has isoleucines at positions d of the coiled coil and leucines at positions a, in contrast to the original zipper which has leucines and valines, respectively [Harbury et al, (1993), cited above]. The nucleotide and amino acid sequences of this tetrameric leucine zipper variant are presented in the context of the full-length sequences, as SEQ ID NO: 5 and NO: 6, respectively. The numbering of the amino acid residues follows Ellenberger et al, (1992) [cited above].

The insertion of the tetramerization domain in the p53 chimeric protein can be quite liberal, provided the functions of the transcription activation (also known as transactivation) and DNA binding domains are not disrupted. Preferably, the heterologous tetramerization domain would be inserted C-terminally to residue 290 of human p53 [SEQ ID NO: 2], since this maintains the integrity of both the transactivation and DNA binding domains. Functional inactivation of the p53 tetramerization domain can be accomplished by deletions, insertions and/or amino acid substitutions. Such mutations should involve residues 322-355 of SEQ ID NO: 2, or a subset of these residues, since the p53 tetramerization domain maps to these residues [Wang et al, (1994), cited above; Clore et al, (1994), cited above]. Desirably, selected mutations target residues 328-348 of human p53 [SEQ ID NO: 2], or a subset thereof. Within this region the most critical residues for tetramer formation are residues 337, 341 and 344 of SEQ ID NO: 2. However, mutation of other residues within the regions indicated above can disrupt tetramer formation. While the mutations should involve residues 322-355 [SEQ ID NO: 2], or a subset thereof, they need not be confined within the p53 tetramerization domain. Thus, they can extend as far N-terminally as residue 290 of human p53 [SEQ ID NO: 2] or as far as the p53 C-terminus (residue 393 of SEQ ID NO: 2) . In addition to the types of mutations just described, functional inactivation of the p53 tetramerization domain can be accomplished by inserting the heterologous tetramerization domain within residues 322-355 of human p53 [SEQ ID NO: 2], and preferably within residues 328-348 of SEQ ID NO: 2.

Embodiments of the invention are presented in Figs. 1F-1K. In one embodiment, the chimeric protein comprises a p53 sequence spanning amino acids 1 to 334 of human p53 [SEQ ID NO:2] fused to an asparagine linker and then to a tetrameric variant of GCN4 residues 249-281 [SEQ ID NO:6] (Fig. IF) . In another embodiment, the chimeric protein comprises a p53 sequence spanning amino acids 1 to 325 of human p53 [SEQ ID NO:2] fused to an arginine-glycine-asparagine linker [SEQ ID NO: 7] and then to a tetrameric variant of GCN4 residues 249-281 [SEQ ID NO:6] (Fig. IG) . In another embodiment, the chimeric protein comprises a p53 sequence spanning amino acids 1 to 323 of human p53 [SEQ ID NO:2] fused to an arginine-glycine-glycine-asparagine-proline-glutamic acid linker [SEQ ID NO: 8] and then to a tetrameric variant of GCN4 residues 250-281 [SEQ ID NO:6] (Fig. 1H) . In yet another embodiment the chimeric protein comprises a p53 sequence spanning from amino acid 1 to 300 of human p53 [SEQ ID NO:2] fused to a glycine-glycine-asparagine- glutamine-alanine linker [SEQ ID NO: 9] and then to a tetrameric variant of GCN4 residues 250-281 [SEQ ID NO:6] (Fig. 1J) . In another embodiment, the chimeric protein comprises a p53 sequence spanning amino acids 1 to 325 of human p53 [SEQ ID NO:2] fused to an arginine-glycine- asparagine linker [SEQ ID NO: 7], a tetrameric variant of GCN4 residues 249-281 [SEQ ID NO:6], an isoleucine linker and then to residues 352-393 of human p53 [SEQ ID NO:2] (Fig. IK) .

One might have expected that a heterologous tetramerization domain would not be able to substitute for the native p53 tetramerization domain, because the function of the tetramerization domain is not only to drive tetramerization, but also to position the subunits appropriately relative to one another, so that the p53 tetramer can align to the DNA site. More specifically, the tetrameric variant of the GCN4 leucine zipper would be expected to be a particularly unsuitable choice for a heterologous tetramerization domain, since it drives parallel subunit assembly [Harbury et al, (1993) , cited above] , while the native p53 tetramerization domain drives antiparallel assembly [Clore et al, (1994), cited above; Sakamoto et al, (1994), Proc. Natl. Acad. Sci. USA, 91: 8974-8978]. Nevertheless, the inventor observed that such chimeric proteins bound DNA as homotetramers with very high efficiency. Without wishing to be bound by theory, the inventor believes that while p53 subunits align antiparallel in the absence of DNA, they adopt a parallel orientation upon DNA binding. Thus a heterologous tetramerization domain that drives parallel assembly of p53 subunits, such as the tetrameric variant of the GCN4 leucine zipper, is compatible with DNA binding.

Because the proteins described in this section form homotetramers and maintain high affinity for the specific p53 DNA sites, but do not maintain the integrity of the native p53 oligomerization domain, they do not form hetero-tetramers with wild-type p53 or tumor-derived p53 mutants, and thus will display tumor suppressing activity even in cancer cells expressing high amounts of mutant p53. Additional p53 proteins of this invention can be generated by one of skill in the art following the teachings herein.

B. Modifications of p53 Proteins with Altered Tetramerization Domains In yet another embodiment, the p53 proteins described herein contain modifications. These modifications can be trivial (defined as having no effect on function) or beneficial (i.e. they improve upon some aspect of the protein) , and can include deletions, insertions, amino acid substitutions and/or replacement of functional domains or regions of functional domains by functionally equivalent domains or regions of other proteins. Various modifications of the p53 proteins encompassed by the invention are illustrated in Figs. 2A through 2F. It is understood that the proteins of the invention may contain more than one of the modifications described below. Bl. Length of Sequence between the DNA Binding and Tetramerization Domains

The following modification may be made in the context of wild-type p53 or in the context of the p53 proteins described in sections Al and A2 above. This modification restricts the DNA binding specificities of the above mentioned proteins and involves a change in the length of the sequence between the p53 DNA binding and tetramerization domains. This modification does not affect the ability of p53 to tetramerize, rather it affects the positioning of the DNA binding domains relative to one another in a p53 tetramer. Since the function of an oligomerization domain in general is not only to induce oligomerization, but also to bring together the subunits in an appropriate orientation, this modification is considered an alteration of the p53 tetramerization domain, as it affects a function of the tetramerization domain and involves sequences that are extensions of the p53 tetramerization domain. Changing the length of the sequences between the DNA binding and tetramerization domains can affect the DNA binding properties of wild-type p53 or of a chimeric p53 protein of this invention both in terms of sequence specificity and affinity for DNA. Such changes can therefore confer desired properties.

Without wishing to be bound by theory, the inventor realizes that the tetramerization domain of p53 is the site at which four p53 subunits contact each other. Thus, the positioning of the four p53 DNA binding domains relative to each other is dependent on the length of the sequence between the C-terminal boundary of the DNA binding domain (residue 289 of human p53, [SEQ ID NO: 2]) and the N-terminal boundary of the tetramerization domain (residue 322 for human wild-type p53, [SEQ ID N0:2]). A long linker, such as the linker present in wild-type p53 (i.e., residues 289-322 of SEQ ID NO: 2) provides freedom in positioning the DNA binding domains relative to one another, which in turn allows p53 to bind to different types of DNA sites. A long linker, however, reduces the affinity for DNA, since it allows p53 to adopt multiple conformations, only one of which is compatible with a specific DNA site. A short linker, on the other hand, allows p53 to bind only to specific types of DNA sites, but the affinity for these sites is increased because p53 can adopt few alternate conformations.

To illustrate the effect of changing the length of the sequence between the p53 DNA binding and tetramerization domains on DNA binding specificity. Figs. 3A-3H illustrate the effect of deletions between these two domains. Fig. 3A shows a schematic of a wild-type p53 tetramer bound to a DNA site with contiguous pentanucleotides. Figure 3B shows the same p53 tetramer bound to a DNA site with a 20-30 nucleotide insert between the 2 pentanucleotide pairs. From Figs. 3A and 3B it is apparent that the naturally-occurring sequence between the tetramerization and DNA binding domains provides the flexibility for wild-type p53 to recognize both types of DNA sites. In Figs. 3C and 3D the sequences (linkers) between the tetramerization and DNA binding domains are shortened. This is performed by deletions within the region spanning residues 290-327 of human p53 [SEQ ID NO: 2], preferably involving more than 22 amino acids. Such deletions in the context of wild-type p53 limit the ability to position one pair of DNA binding domains close enough to the other pair.

Therefore, DNA sites with contiguous pentanucleotides cannot be recognized (Fig. 3C) . However, the same deletions do not limit the ability to recognize DNA sites with a 20-30 nucleotide insert between the two pentanucleotide pairs (Fig. 3D) . For example, wild-type p53, and p53 mutants lacking residues 290-297 of SEQ ID NO: 2 or 300-308 of SEQ ID NO: 2 or 300-317 of SEQ ID NO: 2 or 300-321 of SEQ ID NO: 2 bind to both types of DNA sites. But p53 mutants lacking residues 300-327 of SEQ ID NO: 2 or residues 290-297 of SEQ ID NO: 2 and 300-321 of SEQ ID NO: 2 bind only to DNA sites with a 20 nucleotide insert (see Example 3E) .

This type of modification achieves the same result as described above for wild-type p53 when the modification is made to the chimeric p53 proteins that retain the antiparallel subunit alignment of wild-type p53. Chimeric p53 proteins that contain a heterologous dimerization domain (Section Al above) have their subunits aligned antiparallel. Modifications of the p53 chimeric proteins containing a tetramerization domain driving parallel subunit alignment, such as the p53 proteins containing the exemplary tetrameric variant of the GCN4 leucine zipper (Section A2 above) produce a different effect. A schematic of the chimeric protein of Fig. IF binding to DNA sites of different types is shown in Figs. 3E and 3F. This chimeric protein can bind to both types of DNA sites via flexibility in positioning its DNA binding domains. Deletions within the sequences between the DNA binding and tetramerization domains create the opposite effect than the one observed for wild-type p53. A short linker, preferably by the deletion of 22 or more amino acids between residues 290 and 334 of SEQ ID NO: 2, allows p53 chimeras with parallel tetramerization domains to recognize only the DNA sites with contiguous pentanucleotides (Figs. 3G and 3H) .

While the effect of changing the length of the sequences between the DNA binding and tetramerization domains was illustrated by introducing deletions between these domains, changes in the length can also be introduced by insertions. The inserted sequences are p53 or non-p53 sequences. It is most meaningful to introduce insertions in the context of p53 proteins of this invention with very short sequences (i.e., 0 to about 12 amino acid residues) between the DNA binding and tetramerization domains, for example the protein of Fig. 1J, to expand the range of DNA sites they can recognize. Finally the length of the sequences between the DNA binding and tetramerization domains can be altered by changing the site of insertion of the heterologous oligomerization domain.

The novelty of this type of modification relates both to the realization that wild-type p53 recognizes DNA sites with 20 or more nucleotides between the two pairs of contiguous pentanucleotides, as well as to the observation that changes in the length of the sequences between the p53 DNA binding and tetramerization domains of p53 modulate its ability to bind to the different types of DNA sites. It has not been appreciated before that wild-type p53 can bind DNA sites, where the pairs of contiguous pentanucleotide repeats are separated by as many as 20 or more nucleotides. While it had been established that wild-type p53 can bind to the mdm2 site, it was thought that this site actually contains two DNA sites (each comprising four contiguous pentanucleotides) , as indicated below:

-GGTCA-AGTTG-Gi5A^-c^Ti2ς-ggcgtcggctgtcggag-SΔG^TA-_kS3£C- TGACA-TGTCT- [SEQ ID NO: 11]. The repeats are indicated by capital letters and are separated by hyphens. However, the underlined repeats contain mismatches from the DNA sites recognized by wild-type p53. Such mismatches, even in the context of an otherwise optimal DNA site, completely abrogate DNA binding [Halazonetis et al, (1993), cited above; and the inventor's own additional unpublished observations]. Furthermore, one of the underlined repeats is not even five nucleotides long. In addition, if p53 could recognize the underlined repeats, then two p53 tetramers should be able to bind to the DNA fragment illustrated above, but the inventor has determined experimentally that only one p53 tetramer can bind. Thus, only the non-underlined repeats are recognized by wild-type p53 in the above DNA fragment, and they are separated by more than 30 nucleotides. See the Examples, which also demonstrate that wild-type p53 can bind DNA sites with more than 20 nucleotides between the pairs of pentanucleotide repeats.

The therapeutic significance of altering the DNA binding properties of the p53 chimeric proteins, or of wild-type p53, relates to the biological consequences of activation of the different p53 target genes. More specifically, induction of the wafl gene which contains four contiguous pentanucleotides leads to tumor suppression [El-Deiry et al, (1993), cited above], and is thus desirable for cancer therapy. The nucleotide sequence of this wafl site is: -GAACA-TGTCC-CAACA-TGTTG- [SEQ ID NO: 10]. On the other hand, induction of the mdm2 gene leads to expression of the Mdm2 protein, which in turn downregulates the activity of p53 by masking its transactivation domain [Oliner et al, (1992), Nature, 358: 80-83; Momand et al, (1992), Cell, 69: 1237-1245; Oliner et al, (1993), Nature, 362: 857-860; Wu et al, (1993), cited above], and is thus undesirable for cancer therapy. The mdm2 site contains two pairs of contiguous pentanucleotides separated by more than thirty nucleotides [Wu et al, (1993), cited above]. This site is as follows:

-GGTCA-AGTTG-ggacacgtccggcgtcggctgtcggaggagctaagtcc- TGACA-TGTCT- [SEQ ID NO: 11], with the specific pentanucleotide repeats in capital letters and demarcated by hyphens. Introducing appropriate changes in the lengths of the sequences between the DNA binding and tetramerization domains of wild-type p53 or chimeric p53 proteins of the invention, as described above, permits restriction of their DNA binding specificity. Thus, p53 proteins of this invention are constructed as described above that recognize only DNA sites with contiguous pentanucleotides or only DNA sites with 20 to 30 nucleotide inserts between the two pentanucleotide pairs. Such a p53 protein of this invention that recognizes the wafl DNA site, but not the mdm2 DNA site, has the ability to suppress tumor growth, but is not subject to negative regulatory feedback by Mdm2. One exemplary p53 protein bearing a modification in the length of the sequences between the DNA binding and tetramerization domains is shown in Fig. 2B, i.e., deletion of residues 300-327 of SEQ ID NO: 2.

B2. Substitutions of Functional Domains or Regions Thereof with Equivalent Domains or Regions of Other Proteins As an example of this type of modification, the p53 transactivation (also known as transcription activation) domain, contained within amino acids 1-90 of human p53 [SEQ ID NO: 2], is substituted with that of another protein, e.g., the herpes simplex virus protein VP16, also known as α trans-inducing factor [Pellett et al,

(1985), Proc. Natl. Acad. Sci. USA, 82: 5870-5874]. The nucleotide sequence spanning VP16 nucleotides 2074 - 2307 [Pellett et al, (1985) cited above] is reported as nucleotides 1 - 234 in SEQ ID NO: 12. The amino acid sequence of the VP16 HSV fragment from amino acid 402 through 479 [Pellett et al, (1985) cited above] is reported as amino acid 1 - 78 in SEQ ID NO: 13. See, also, GenBank Code Helcg.

One exemplary modified p53 protein of the invention is illustrated in Fig. 2C, in which amino acid residues 402-479 of VP16 [aa 1-78 of SEQ ID NO: 13] have replaced amino acid residues 3-80 of human p53 [SEQ ID NO:2]. An equivalent substitution has been reported to maintain p53 tumor suppressor function [Pietenpol et al, (1994), cited above] . The advantage of this substitution is that in certain tumors, overexpression of the Mdm2 protein suppresses p53-mediated transcription by masking its transactivation domain [Oliner et al, (1992) , cited above; Momand et al, (1992), cited above; Oliner et al, (1993), cited above]. When the transactivation domain of p53 is replaced by that of VP16, it is no longer inhibited by Mdm2, because Mdm2 does not suppress the transcriptional activity mediated by VP16 [Oliner et al, (1993), cited above]. Substitution of a functional domain can involve part of the domain. For example, amino acid residues 1-52 of p53 [SEQ ID NO: 2] are sufficient to mediate interaction with Mdm2, but residues 18-52 of p53 [SEQ ID NO: 2] are not [Chen et al, (1993), Mol. Cell. Biol., 13: 4107-4114]. Thus, replacement of only residues 1-18 of p53 [SEQ ID NO: 2] with sequences from the VP16 activation domain abolishes the p53-Mdm2 interaction. As another example, since within residues 1-52 of p53 [SEQ ID NO: 2], amino acids Leu 14, Phe 19, Leu 22 and Trp 23, are critical for interaction with Mdm2 [Lin et al, (1994), Genes Dev., 8: 1235-1246], the substitution involves only these residues.

B3. Nuclear Localization Signals

Nuclear localization is required for p53 function [Shaulsky et al, (1991), Oncogene, 6: 2056-2065].

Wild-type p53 contains three nuclear localization signals (NLS) , all of which map to the C-terminus of wild-type p53 and specifically to residues 316-325, 369-375 and 379-384 of p53 [SEQ ID NO: 2] [Shaulsky et al, (1990), Mol. Cell. Biol., 10: 6565-6577]. Some of the p53 proteins of the invention lack one or more of these NLS. Thus, such proteins have an impaired ability to localize to the nucleus and consequently their function is impaired. It is therefore beneficial to reintroduce NLS. Even p53 proteins that maintain all three NLS may benefit from introduction of additional NLS. Therefore an analog of the p53 proteins described above may contain a NLS fused to its N-terminus, or its C-terminus, or at the junction of the transactivation and DNA binding domains or at the junction of the DNA binding and tetramerization domains or elsewhere in the protein, as long as the function of the p53 protein is not disrupted by insertion of the NLS. Fig. 2D demonstrates the insertion of a NLS at the boundary of the transactivation and DNA binding domains. The NLS may be that of p53 or of any other nuclear protein, such as the NLS of SV40 large T antigen which is comprised of amino acids proline-lysine-lysine-lysine- arginine-lysine-valine [SEQ ID NO: 14] [Kalderon et al, (1984), Cell, 39: 499-509]. Additional heterologous NLS are described by Shaulsky et al, (1990); (1991) [cited above] .

B4 . Substitution by Homologous Non-human p53 Sequences

The amino acid sequence of p53 is conserved across species [Soussi et al, (1990), cited above], implying that function is also conserved. Indeed, analysis of xenopus and human p53 proteins has revealed no functional differences [Cox et al, (1994), Oncogene, 9: 2951-2959]. Thus, human p53 sequences of the p53 [SEQ ID NO: 2] proteins of this invention can readily be replaced with the homologous non-human p53 sequences.

The sequences of human p53 [SEQ ID NO: 2] and certain non-human p53 proteins have been aligned by Soussi et al, (1990) , cited above. This alignment permits identification of regions that are homologous across species. For p53 species that are not listed by Soussi et al, (1990), cited above, the alignment to the human p53 [SEQ ID NO: 2] sequences is obtained by computer programs commercially available and known in the art, such as the program BESTFIT of the University of Wisconsin GCG package.

The benefit of substituting human p53 [SEQ ID NO: 2] sequences with equivalent non-human sequences relates to the realization that interactions of p53 with specific cellular or viral proteins are species-specific. For example, human p53 is inactivated by the human Mdm2 protein [Oliner et al, (1993), cited above; Momand et al, (1992), cited above; Wu et al, (1993), cited above]. Non-human p53 sequences have lower or no affinity for the human Mdm2. Thus, p53 proteins of this invention that contain non-human p53 sequences are not susceptible to inhibition by Mdm2.

One of skill in the art with resort to the teaching of this invention may readily select which non-human p53 sequence to use, as well as the extent of the region for substitution. For example, the p53-Mdm2 interaction has been mapped to residues 1-52 of p53 [SEQ ID NO: 2], and more specifically to residues 14, 19, 22 and 23 of SEQ ID NO: 2 [Chen et al, (1993), cited above; Lin et al, (1994), cited above]. Thus, to eliminate the p53-Mdm2 interaction, N-terminal sequences within residues 1-52 of the p53 proteins of this invention are substituted with the homologous non-human p53 sequences.

The species of p53 that can be used to substitute for the human p53 sequences can readily be selected by one of skill in the art. Species, such as xenopus and trout, that diverge most from human p53 [Soussi et al, (1990), cited above] are preferred, although other species may also be selected. As an exemplary modification of this type, residues 3-80 of human p53 [SEQ ID NO: 2] are substituted by the homologous xenopus sequence (Fig. 2E) to produce a modified p53 protein incapable of interacting with Mdm2. B5. Amino Acid Substitutions, Deletions and Insertions

Other modifications of the p53 proteins described in this invention include amino acid substitutions, small deletions and small insertions. (Deletions and insertions within the sequences between the DNA binding and tetramerization domains are discussed in section Bl above.) These modifications involve either the p53 sequences or the heterologous oligomerization domain sequences or both. The modifications may enhance function or introduce a useful property. For example a modification may introduce a tag to optimize protein purification [Scopes (1994) , Protein Purification, Principles and Practice, third edition, Springer-Verlag, New York] , or may enhance expression and/or stability of a p53 protein of the invention when expressed in vitro or in a patient. Modifications in the p53 fragment may enhance DNA binding and growth suppressing activities. Two such modifications have already been described: substitution of arginine 174 with glutamine or of arginine 175 with leucine (the numbering refers to human p53 [SEQ ID NO: 2]; in mouse p53 the corresponding residues are 171 and 172 of SEQ ID NO: 15, respectively) [Halazonetis and Kandil (1993), cited above; Li et al, (1994), Cell Growth Differentiation, 5: 711-721]. Modifications in p53 may also affect interaction with cellular or viral proteins, for example, substitution of leucine 14 of SEQ ID NO: 2 with glutamine and phenylalanine 19 of SEQ ID NO: 2 with serine abolish the p53-Mdm2 interaction [Lin et al, (1994), cited above]. Modifications in the heterologous oligomerization domain may increase the stability of tetramer formation, for example, substitutions that stabilize oligomerization driven by leucine zippers are known [Krylov et al, (1994), cited above; O'Shea et al, (1992), cited above]. As an exemplary modification of this type, residues 174 or 175 of human p53 [SEQ ID NO: 2] are substituted by glutamine or leucine, respectively (Fig. 2F) in a p53 chimeric protein of this invention. B6. Linkers between p53 and the Heterologous Oligomerization Domain

Another modification of the proteins of this invention is the presence of an amino acid or peptide linker between the p53 fragment and the heterologous oligomerization domain. In one embodiment of this invention (Fig. IC) , there is no linker between p53 and the GCN4 leucine zipper. In other embodiments however (Figs. IB, IF and IK) , there are glutamic acid or asparagine or isoleucine linkers, respectively. Linkers may be present for cloning convenience or to confer some useful property. For example, residues that stabilize specific secondary structure elements, such as α-helices, are known [Richardson and Richardson (1988), Science 240: 1648-1652]. Such residues can be introduced in the linkers to stabilize the heterologous oligomerization domains. For example the linkers glycine-asparagine, arginine-glycine-asparagine [SEQ ID NO: 7], arginine- glycine-glycine-asparagine-proline-glutamic acid [SEQ ID NO: 8], glycine-glycine-asparagine-glutamine-alanine [SEQ ID NO: 9] present in the examples shown in Figs. IE, IG and IK, 1H and 1J, respectively, are all designed to stabilize the N-terminus of the α-helical heterologous oligomerization domain. A variety of other amino acid or peptide linkers may be used for the reasons discussed above, provided they do not interfere with the function of the p53 chimeric protein. C. Nucleic Acid Sequences Encoding p53 Proteins of the Invention

The present invention further provides nucleic acid sequences encoding the proteins of this invention, which includes the proteins described in sections A and B above. In addition to the coding strand, the nucleic acid sequences of the invention include the complementary DNA sequence representing the non-coding strand, the messenger RNA sequence, the corresponding cDNA sequence and the RNA sequence complementary to the messenger RNA sequence. Variants of these nucleic acids of the invention include variations due to the degeneracy of the genetic code and are encompassed by this invention. Such variants may be readily identified and/or constructed by one of skill in the art. In certain cases specific codon usage may be employed to optimize expression. The above nucleotide sequences can be included within larger DNA or RNA fragments, or may be interrupted by introns.

In another embodiment the nucleic acids encoding the p53 proteins of the invention are present in the context of vectors suitable for amplification in prokaryotic or eukaryotic cells. Many such vectors are known [Ausubel et al, (1994), cited above] and many of these are commercially available. For example plasmids with bacterial or yeast replication origins allow amplification in bacteria or yeast, respectively. Such vectors allow the production of large quantities of nucleic acids encoding the proteins of the invention, which nucleic acids can be used for gene therapy or for expression of the p53 proteins of the invention.

In yet another embodiment the nucleic acids encoding the proteins of the invention are present in the context of vectors suitable for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells. Many such vectors are known [Ausubel et al, (1994) , cited above] and many of these are commercially available. For example, the vector pGEM4 (Promega, Madison, WI) is suitable for expression of the p53 proteins in cell-free lysates, while the vector pSV2 [Mulligan et al, (1992), cited above] is suitable for expression in mammalian cells. Such vectors allow the production of the proteins of the invention in vitro for analysis of their functional properties or for delivery to patients. D. Gene Therapy The nucleic acid sequences of the invention may be inserted into a vector capable of targeting and infecting a desired cell, either in vivo or ex vivo for gene therapy, and causing the encoded p53 protein of this invention to be expressed by that cell. Many such viral vectors are useful for this purpose, e.g., adenoviruses, retroviruses and adeno-associated viruses (AAV) [Schreiber et al, (1993), Biotechniques, 14: 818-823; Davidson et al, (1993), Nature Genetics, 3: 219-223; Roessler et al, (1993), J. Clin. Invest., 92: 1085-1092; Smythe et al, (1994), Ann. Thorac. Surg. , 57: 1395-1401; Kaplitt et al, (1994), Nature Genetics, 8: 148-154]. There has already been success using viral vectors driving expression of wild-type p53 [Fujiwara et al, (1993), Cancer Res., 53: 4129-4133; Fujiwara et al, (1994), Cancer Res., 54: 2287-2291; Friedmann (1992),

Cancer, 70(6 Suppl) : 1810-1817; Fujiwara et al, (1994b), Curr. Opin. Oncol., 6: 96-105].

For use in gene therapy, these viral vectors containing nucleic acid sequences encoding a p53 protein of the invention, are prepared by one of skill in the art with resort to conventional techniques (see references mentioned above) . For example, a recombinant viral vector, e.g. an adenovirus, of the present invention comprises DNA of at least that portion of the viral genome which is capable of infecting the target cells operatively linked to the nucleic acid sequences of the invention. By "infection" is generally meant the process by which a virus transfers genetic material to its host or target cell. Preferably, the virus used in the construction of a vector of the invention is rendered replication-defective to remove the effects of viral replication on the target cells. In such cases, the replication-defective viral genome can be packaged by a helper virus in association with conventional techniques. Briefly, the vector(s) containing the nucleic acids encoding a protein of the invention is suspended in a pharmaceutically acceptable carrier, such as saline, and administered parenterally (or by other suitable means) in sufficient amounts to infect the desired cells and provide sufficient levels of p53 activity to arrest abnormal cellular proliferation. Other pharmaceutically acceptable carriers are well known to those of skill in the art. A suitable amount of the vector containing the chimeric nucleic acid sequences is between about 10⁶ to 10⁹ infectious particles per mL carrier. The delivery of the vector may be repeated as needed to sustain satisfactory levels of p53 activity, as determined by monitoring clinical symptoms.

As desired, this therapy may be combined with other therapies for the disease or condition being treated. For example, therapy involving the administration of a vector capable of expressing a p53 protein of the invention is well suited for use in conjunction with conventional cancer therapies, including surgery, radiation and chemotherapy.

Nucleic acid sequences driving expression of a p53 protein of the invention may also be introduced by "carriers" other than viral vectors, such as liposomes, nucleic acid-coated gold beads or can simply be injected in situ [Fujiwara et al (1994b) , cited above; Fynan et al, (1993), Proc. Natl. Acad. Sci. USA, 90: 11478-11482; Cohen (1993), Science, 259: 1691-1692; Wolff et al, (1991), Biotechniques, 11: 474-485]. E. Pharmaceutical Compositions The proteins of this invention may also be formulated into pharmaceutical compositions and administered using a therapeutic regimen compatible with the particular formulation. Pharmaceutical compositions within the scope of the present invention include compositions containing a protein of the invention in an effective amount to have the desired physiological effect, e.g. to arrest the growth of cancer cells without causing unacceptable toxicity for the patient.

Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form, e.g. saline. Alternatively, suspensions of the active compounds may be administered in suitable conventional lipophilic carriers or in liposomes. The compositions may be supplemented by active pharmaceutical ingredients, where desired. Optional antibacterial, antiseptic, and antioxidant agents in the compositions can perform their ordinary functions. The pharmaceutical compositions of the invention may further contain any of a number of suitable viscosity enhancers, stabilizers, excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Preferably, these preparations, as well as those preparations discussed below, are designed for parenteral administration. However, compositions designed for oral or rectal administration are also considered to fall within the scope of the present invention.

Those of skill in the pharmaceutical art should be able to derive suitable dosages and schedules of administration. As used herein, the terms "suitable amount" or "effective amount" means an amount which is effective to treat the conditions referred to below. A preferred dose of a pharmaceutical composition containing a protein of this invention is generally effective above about 0.1 mg p53 protein per kg of body weight (mg/kg), and preferably from about 1 mg/kg to about 100 mg/kg. These doses may be administered with a frequency necessary to achieve and maintain satisfactory p53 activity levels. Although a preferred range has been described above, determination of the effective amounts for treatment of each type of tumor or other condition may be determined by those of skill in the art.

Dosage units of such pharmaceutical compositions containing the proteins of this invention preferably contain about 1 mg to 5 g of the protein. F. Therapeutic Indications

The nucleic acids and proteins of the invention can be introduced into human patients for therapeutic benefits in conditions characterized by insufficient wild-type p53 activity. As stated above, the nucleic acids of the invention may be introduced into the patient in the form of a suitable viral vectors (or by direct DNA delivery) to harness the patient's cellular machinery to express the proteins of the invention in vivo.

Alternatively, the proteins of the invention may be introduced into the patient in appropriate pharmaceutical formulations as described above.

As one example, the pharmaceutical compositions of this invention, containing a protein of the invention or a nucleic acid or a viral vector which express a protein of the invention in vivo, may be employed to induce the cellular defence to DNA damaging agents. Examples of DNA damaging agents include sunlight UV irradiation, as well as radiation and chemotherapeutics used for cancer treatment. By administering a suitable amount of a composition of this invention, patients may tolerate higher doses of such DNA damaging agents.

Another therapeutic use of the compositions of this invention is in inducing apoptosis of specific cells, such as proliferating lymphocytes. According to this method of use, a suitable amount of an appropriate pharmaceutical composition of this invention is administered to a subject to enhance the development of immune tolerance. This method may employ both in vivo and ex vivo modes of administration. Preferably, this therapy is useful as the sole treatment or as an accessory treatment to prevent transplant rejection, or to treat autoimmune diseases, e.g., systemic lupus erythrematosis, rheumatoid arthritis and the like.

The pharmaceutical compositions of this invention may also be employed to restore p53 function in tumor cells. Introduction of p53 function in tumor cells leads to arrest of cell proliferation or to cell death [Finlay et al, (1989), cited above; Eliyahu et al, (1989), cited above; Baker et al, (1990) , cited above; Mercer et al, (1990), cited above; Diller et al, (1990), cited above; Isaacs et al, (1991), cited above; Yonish-Rouach et al, (1993), cited above; Fujiwara et al, (1993), cited above] . In addition p53 function primes tumor cells to undergo cell death in response to DNA damaging agents currently used in cancer therapy [Lowe et al, (1993), cited above; Fujiwara et al, (1994) , cited above; Fisher (1994), Cell, 78: 539-542]. Desirably, a suitable amount of the composition of this invention is administered systemically, or locally to the site of the tumor with or without concurrent administration of conventional cancer therapy (i.e. DNA damaging agents).

Additionally, the compositions of this invention may be administered in methods to suppress cell proliferation in diseases other than cancers, which are characterized by aberrant cell proliferation. Among such diseases are included psoriasis, atherosclerosis and arterial restenosis. This method is conducted by administering a suitable amount of the selected composition systemically or locally to the patient.

Specific Examples

The present invention provides p53 proteins with modifications in the native p53 sequence. These modifications, which do not interfere with its native tumor-suppressor function, provide the protein with at least one of the following functional characteristics: (1) the ability to bind DNA and activate transcription like wild-type p53, but to not hetero-oligomerize with wild-type p53 or tumor-derived p53 mutants; and (2) restricted DNA binding specificity from an alteration in the way that the tetramerization domain orients the DNA binding domains of a p53 tetramer relative to one another. Exemplary p53 proteins of this invention, which demonstrate the aforementioned functional characteristics, are described in sections A and B, above. Additional functionally equivalent proteins can be constructed by one of skill in the art with resort to the teachings of sections A and B and the Examples below. The following examples illustrate preferred methods for preparing p53 proteins of the invention and characterizing their functional activities. These examples are illustrative only and do not limit the scope of the invention. As throughout the specification, numbering of the human p53 [SEQ ID NO: 2] amino acid residues in the examples follows Zakut-Houri et al, (1994) [cited above], while numbering of the GCN4 residues follows Ellenberger et al, (1992) [cited above]. Example 1 - Recombinant Plasmids

Standard cloning procedures were used to prepare the plasmids described below [Ausubel et al, (1994) ; Innis et al, (1990)]. A. Plflsmjd pGEMhygpp53wt

Plasmid pGEMhump53wt encodes full-length human wild-type p53 [SEQ ID NOS: 1 and 2]. This plasmid was prepared by PCR [Innis et al, (1990), cited above] using a human p53 cDNA, which is readily available to those practicing the art. The PCR procedure was designed to incorporate unique restriction sites within the coding sequence of human p53 [SEQ ID NO: 1]: Kpn I at codon 218, Sst I at codon 299, Sst II at codon 333, Bst BI at codon 338 and Sal I immediately following the termination codon. An Msc I site at codon 138 was eliminated. These changes did not alter the sequence of the encoded p53, and were only performed to expedite construction of mutant proteins bearing altered tetramerization domains or point mutations associated with human cancer. The PCR product of the human p53 cDNA was digested with Nco I and Sal I and cloned in the vector pGEM4 [Promega, Madison, WI], which was linearized with Eco Rl and Sal I. Synthetic oligonucleotides were used to bridge the Eco Rl site of the vector and the Nco I site at the initiation codon of p53. The entire nucleotide sequence of the Eco Rl-Sal I human p53 insert in plasmid pGEMhump53wt is presented as SEQ ID NO: 20 (the first nucleotide of the EcoRI site is numbered nucleotide 1) . Plasmid, pGEMhump53wt, was used to generate all the p53 mutants described below, as well as for expression of wild-type p53 by in vitro translation. B. Plasmids of the pGEMhump53 Series Encoding p53 Proteins with Altered Tetramerization Domains

Bl. Plasmid pGEMJufflP?3 Z34$E

A fragment encoding amino acids 253-281 [SEQ ID NO: 4] of the yeast transcription factor GCN4 [SEQ ID NO: 4] [Hinnenbusch et al, (1984), cited above] was prepared by PCR of plasmid pSP64-GCN4 [Halazonetis et al, (1988), Cell 55: 917-924]. The sequence of the 5' PCR primer GCAGAGGAGC AAAAGCTTGA AGACAAGGTT [SEQ ID NO: 21] incorporates a HindiII site, while the sequence of the 3' primer CTTCAGGTCG ACTCAGCGTT CGCCAACTAA TTTC [SEQ ID NO: 22] incorporates a termination codon and a Sal I restriction site. The PCR fragment was blunt-ended at the Hind III site and cloned into pGEMhump53wt linearized with Stu I and Sal I. The resultant plasmid, pGEMhump53LZ346E, encodes amino acids 1-346 of human p53 [SEQ ID NO: 2], a glutamic acid, and then amino acids 253-281 of GCN4 [SEQ ID NO: 4].

B2. Plasmi GEHhvwp53 Z347 A fragment encoding amino acids 253-281 [SEQ ID NO: 4] of the yeast transcription factor GCN4 [SEQ ID NO: 4] [Hinnenbusch et al, (1984), cited above] was prepared by PCR of plasmid pSP64-GCN4 [Halazonetis et al, (1988), cited above]. The sequence of the 5' PCR primer ATGAGGCCTT GGAAGACAAG GTTGAAGAAT TG [SEQ ID NO: 23] incorporates a Stu I site, while the sequence of the 3' primer CTTCAGGTCG ACTCAGCGTT CGCCAACTAA TTTC [SEQ ID NO: 22] incorporates a termination codon and a Sal I restriction site. The PCR fragment was cloned into pGEMhump53wt linearized with Stu I and Sal I. The resultant plasmid, pGEMhump53LZ347, encodes amino acids 1-347 of human p53 [SEQ ID NO: 2] and amino acids 253-281 Of GCN4 [SEQ ID NO: 4]. B3. Plasmid PGEMhump53LZ335Q

A fragment encoding amino acids 253-281 of the yeast transcription factor GCN4 [SEQ ID NO: 4] [Hinnenbusch et al, (1984), cited above] was prepared by PCR of plasmid pSP64-GCN4 [Halazonetis et al, (1988), cited above]. The sequence of the 5' PCR primer GCAGAGGAGC AAAAGCTTGA AGACAAGGTT [SEQ ID NO: 21] incorporates a Hind III site, while the sequence of the 3• primer CTTCAGGTCG ACTCAGCGTT CGCCAACTAA TTTC [SEQ ID NO: 22] incorporates a termination codon and a Sal I restriction site. The PCR fragment was blunt-ended at the Hind III site and cloned into pGEMhump53wt linearized with Sst II and Sal I. The Sst II site of the vector and the blunt-ended Hind III site of the PCR product were bridged by annealed synthetic oligonucleotides GGGCGTC [SEQ ID NO: 24] and GACGCCCGC [SEQ ID NO: 25]. The resultant plasmid, pGEMhump53LZ335Q, encodes amino acids 1-335 of human p53 [SEQ ID NO: 2], a glutamine, and then amino acids 253-281 Of GCN4 [SEQ ID NO: 4]. B4. Plasmid pGEMhump53LZ343RMKO

A fragment encoding amino acids 253-281 of the yeast transcription factor GCN4 [SEQ ID NO: 4] [Hinnenbusch et al, (1984), cited above] was prepared by PCR of plasmid pSP64-GCN4 [Halazonetis et al, (1988), cited above]. The sequence of the 5' PCR primer GCAGAGGAGC AAAAGCTTGA

AGACAAGGTT [SEQ ID NO: 21] incorporates a Hind III site, while the sequence of the 3' primer CTTCAGGTCG ACTCAGCGTT CGCCAACTAA TTTC [SEQ ID NO: 22] incorporates a termination codon and a Sal I restriction site. The PCR fragment was blunt-ended at the Hind III site and cloned into pGEMhump53wt linearized with Bst BI and Sal I. The Bst BI site of the vector and the blunt-ended Hind III site of the PCR product were bridged by annealed synthetic oligonucleotides CGAAATGTTC CGAGAGCGAA TGAAAC and GTTTCATTCG CTCTCGGAAC ATTT [SEQ ID NO: 26 and 27]. The resultant plasmid, pGEMhump53LZ343RMKQ, encodes amino acids 1-343 of human p53 [SEQ ID NO: 2] and then amino acids 249-281 of GCN4 [SEQ ID NO: 4]. B5. Plasmid pGEMhump53TZ334NR Synthetic oligonucleotides were used to generate a tetrameric variant of the GCN4 leucine zipper. These oligonucleotides TATCCGCGGT AATCGTCTGA AACAGATCGA AGACAAGTTA GAAGAAATCC TTTCGAAGCT CTATCACATC GAG and TTTGTCGACT CAACGTTCAC CCAATAATTT TTTGATGCGC GCTAACTCAT TCTCGATGTG ATAGAGCTTC G [SEQ ID NO: 28 and 29] were subjected to a PCR cycle in the absence of any additional DNA. The PCR product was digested with restriction endonucleases Sst II and Sal I and cloned into pGEMhump53wt linearized with Sst II and Sal I. The resultant plasmid, pGEMhump53TZ334NR, encodes amino acids 1-334 of human p53 [SEQ ID NO: 2], an asparagine, and then the tetrameric zipper variant corresponding to amino acids 249-281 [SEQ ID NO: 6] of GCN4.

B6. ia-smid P<?EMhUPtp53TZ323RGE A deletion of residues 324-332 of human p53 [SEQ ID NO: 2] was created within the Sst I-Sst II fragment of plasmid pGEMhump53TZ334NR by substituting the wild-type Sst I-Sst II fragment with a PCR fragment generated using the oligonucleotide TTCTCCGCGG AGTGGTTTCT TCTTTGGCTG [SEQ ID NO: 30]. The resultant plasmid, pGEMhump53TZ323RGN, encodes amino acids 1-323 of human p53 [SEQ ID NO: 2], an arginine-glycine-asparagine tripeptide [SEQ ID NO: 7], and then the tetrameric zipper variant corresponding to amino acids 249-281 [SEQ ID NO: 6] of GCN4. B7. Plasmid pGEMhump53TZ334GNPE

This plasmid is a modification of plasmid pGEMhump53TZ334NR. The Sst II-Sal I fragment of pGEMhump53TZ334NR containing the tetrameric zipper was modified by PCR using the primer TATCCGCGGT GGAAATCCTG AACTGaAAACA GATCGAAGAC AAG [SEQ ID NO: 31]. The PCR fragment was cloned using the Sst II-Sal I sites into pGEMhump53TZ334NR, replacing the original Sst II-Sal I fragment. The resultant plasmid, pGEMhump53TZ334GNPE, encodes amino acids 1-334 of human p53 [SEQ ID NO: 2], a glycine-asparagine-proline-glutamic acid tetrapeptide [SEQ ID NO: 32] and the tetrameric zipper variant corresponding to amino acids 250-281 [SEQ ID NO: 6] of GCN4.

B8. Plasmid pGEMhumP53LZ346E352I This plasmid is a modification of plasmid pGEMhump53LZ346E. A Cla I restriction site was introduced just after the last codon of pGEMhump53LZ346E by PCR with the primer GTCATCGATG CGTTCGCCAA CTAATTTCTT [SEQ ID NO: 33]. A PCR fragment encoding residues 352-393 of human p53 [SEQ ID NO:2] containing a Cla I site at its 5' end was also generated using the primer ATGAGGCCTT GGAACTCATC GATGCCCAGG CTGGG of SEQ ID NO: 34. The latter fragment was cloned using the Cla I-Sal I sites into the modified (using the primer of SEQ ID NO: 33) pGEMhump53LZ346E vector. The resultant plasmid, pGEMhump53LZ346E352I, encodes amino acids 1-346 of human p53 [SEQ ID NO: 2], a glutamic acid, the leucine zipper corresponding to amino acids 253-281 of GCN4 [SEQ ID NO: 4], an isoleucine and then residues 352-393 of human p53 [SEQ ID NO: 2].

B9. Plasmid pGEMhumP53TZ334NR/I352 This plasmid is a modification of plasmid pGEMhump53TZ334NR. A Cla I restriction site was introduced just after the last codon of pGEMhump53TZ334NR by PCR with the primer TTTGTCGACT CAATCGATAC GTTCACCCAA TAATTTTTTG [SEQ ID NO: 35]. A PCR fragment encoding residues 352-393 of human p53 [SEQ ID NO:2] containing a Cla I site at its 5' end was also generated using the primer ATGAGGCCTT GGAACTCATC GATGCCCAGG CTGGG of SEQ ID NO: 34. The latter fragment was cloned using the Cla I-Sal I sites into the modified (using the primer of SEQ ID NO: 35) pGEMhump53TZ334NR vector. The resultant plasmid, pGEMhump53TZ334NR/I352, encodes amino acids 1-334 of human p53 [SEQ ID NO: 2], an asparagine, the tetrameric zipper variant corresponding to amino acids 249-281 [SEQ ID NO: 6] of GCN4, an isoleucine and then residues 352-393 of human p53 [SEQ ID NO: 2].

BIO. Plasmids pGEMhump53H175. pGEMhump530334. pGEMhump53L337. PGEMhumP53A341 and PGEMhump53A344 Plasmids pGEMhump53H175, pGEMhump53Q334, pGEMhump53L337, pGEMhump53A3 1 and pGEMhump53A344 encode proteins that differ by one amino acid from wild-type human p53 [SEQ ID NO: 2]. Specifically, they substitute Argl75 with His, Gly334 with Gin, Arg337 with Leu, Phe341 with Ala and Leu344 with Ala of p53 SEQ ID NO: 2, respectively. These single amino acid substitutions were generated using standard recombinant techniques [Ausubel et al, (1994), cited above; Innis et al, (1990), cited above]. Substitution of Argl75 of SEQ ID NO: 2 with His is often associated with human tumors. Bll. Plasmids PGEMhump53D290-297. pGEMhump53D290-297D300-321. PGEMhump53D300-308. pGEMhump53D300-317. PGEMhumo53D300-321. pGEMhump53D300-327. and PGEMhump53D364-393 Plasmids pGEMhump53D290-297, pGEMhump53D290-297D300-321, pGEMhump53D300-308, pGEMhump53D300-317, pGEMhump53D300-321, pGEMhump53D300-327, and pGEMhump53D364-393 encode proteins that contain deletions within wild-type human p53 [SEQ ID NO: 2]. These deletions involve residues

290-297, 290-297 and 300-321, 300-308, 300-317, 300-321, 300-327 and 364-393 of p53 SEQ ID NO: 2, respectively. These deletions were generated using standard recombinant techniques [Ausubel et al, (1994) , cited above; Innis et al, (1990), cited above]. c. piagffiifl pSV2hwBp53wt

Plasmid pSV2hump53wt encodes full-length human wild-type p53 [SEQ ID NO: 2], and directs transcription of this protein in mammalian cells. The pSV2 vector has been previously described [Mulligan et al, (1981), Proc. Natl. Acad. Sci. USA, 78: 2072-2076]. A pSV2 vector containing a human c-jun insert has also been described [Zhang et al, (1990), Proc. Natl. Acad. Sci. USA, 87: 6281-6285]. The c-jun insert was removed from the latter plasmid using Sal I and Bgl II restriction endonucleases, and the ends of the vector were blunted. Into this vector a blunted Eco RI-Hind III p53 insert from pGEMhump53wt was cloned.

D. Plasmids of the pSV2hump53 Series Encoding p53 Proteins with Altered Tetramerization Domains

Because plasmids pGEMhump53wt and pSV2hump53wt contain the same p53 insert, it is possible to use restriction sites that are common within the inserts of these plasmids, to transfer p53 subfragments from plasmids of the pGEMhump53 series to pSV2hump53wt.

Specifically, it is possible to transfer, for example, Sst II-Sal I fragments encoding altered tetramerization domains into the pSV2hump53 vector, and thus allow expression of p53 proteins of the invention in mammalian cells. pSV2 vectors expressing most of the proteins described above have been constructed. The name of the p53 protein with altered tetramerization domain is retained from the pGEM to the pSV2 series. For example, transfer of the Sst II-Sal I fragment of pGEMhump53TZ334NR to pSV2hump53wt, yields pSV2hump53TZ334NR, which allows expression in mammalian cells of a p53 protein containing amino acids 1-334 of human p53 [SEQ ID NO: 2], an asparagine, and then the tetrameric zipper variant corresponding to amino acids 249-281 [SEQ ID NO: 6] of GCN4.

E. Reporter Plasmids To Assay P53-Mediated

Transcriptiona Activity

The reporter plasmids drive expression of alkaline phosphatase in a p53-dependent manner. Plasmids pEwafl-TK-SEAP, pBC.V4A-TK-SEAP and pBC-TK-SEAP have one copy each of double-stranded oligonucleotides Ewafl [SEQ ID NO: 16], BC.V4A [SEQ ID NO: 17] and BC [SEQ ID NO: 18] , respectively cloned into the unique Eco RV site of pTK-SEAP. These oligonucleotides contain p53 binding sites of different affinities.

The sequence of oligonucleotide Ewafl (top strand) is: CCC-GAACA-TGTCC-CAACA-TGTTG-GGG [SEQ ID NO: 16]. This oligonucleotide corresponds to the enhancer that drives p53-dependent transcription of the wafl gene

[El-Deiry et al, (1993), cited above]. The sequence of oligonucleotide BC.V4A (top strand) is: TC-GAGCA-TGTTC- GAGCA-TGTTC-GAGCATGT [SEQ ID NO: 17], and the sequence of oligonucleotide BC (top strand) is: CC-GGGCA-TGTCC- GGGCA-TGTCC-GGGCATGT [SEQ ID NO: 18]. Oligonucleotides BC.V4A [SEQ ID NO: 17] and BC [SEQ ID NO: 18] contain artificial sites recognized by p53. For the three sites indicated above, the specific pentanucleotide repeats recognized by p53 are demarcated by hyphens. Plasmid pTK-SEAP drives expression of a secreted form of alkaline phosphatase under the control of a minimal thymidine kinase promoter [Halazonetis (1992) , Anticancer Res., 12: 285-292]. It contains no p53 binding site, and thus serves as a control. F. Plasmid pSV2crot

Plasmid pSV2gpt [Mulligan et al, (1981) , cited above] drives expression of gpt in mammalian cells. In these studies it only serves to bring the total amount of transfected DNA to 30 μg, when necessary. Expression of gpt does not interfere with p53 function. Example 2 - In Vitro Translation and DNA Binding Assay

Plasmids of the pGEMhump53 series of Example 1 were used to produce in vitro transcribed mRNA according to standard procedures [Halazonetis et al, (1988) , cited above]. The mRNA is subsequently translated in vitro using preferably rabbit reticulocyte lysate (Promega, Madison, WI) [Halazonetis et al (1988) , cited above] . In vitro translated p53 can be used directly for DNA binding, without further purification. Alternate strategies for expression of p53 for DNA binding assays include expression in E. coli or in Sf9 insect cells using appropriate vectors (many are commercially available) for expression in bacterial cells or baculovirus vectors, respectively. Lysates or extracts prepared from bacterial or insect cells are used without purification, or optimally, following partial or complete purification using standard protein purification techniques [Scopes (1994), cited above].

The in vitro translated proteins were assayed for DNA binding, as previously described [Halazonetis et al, (1993), cited above].

Briefly, as exemplified below, the in vitro translated protein is incubated with a radioactively labeled oligonucleotide containing a p53 binding site in the presence of non-specific competitor DNA. The reaction mixture is incubated 20 min. at room temperature and directly loaded on a native 5% polyacrylamide electrophoresis gel. In this type of DNA binding assay free DNA migrates to the bottom of the gel, whereas p53/DNA complexes migrate more slowly. Thus, the presence of slowly migrating DNA, which can be detected by autoradiography, indicates p53 DNA binding [Halazonetis et al (1993), cited above; Halazonetis and Kandil (1993), cited above]. As non-specific competitor DNAs, the following were used: 0.1 μg single-stranded oligonucleotide MI7 [GAGAGCCCCAGTTACCATAACTACTCT, SEQ ID NO: 36] and 0.05 μg double-stranded oligonucleotide TF3 [ATCACGTGATATCACGTGATATCACGTGAT, SEQ ID NO: 37] per reaction.

A number of double-stranded oligonucleotides containing p53 binding sites were radioactively labeled for these experiments. These included oligonucleotides Ewafl, BC.V4A and BC [SEQ ID NOS: 16, 17 and 18, respectively], and oligonucleotide BC.S21. The sequence Of BC.S21 is: TAT-GGGCA-TGTCC-TATATATATGCGTATATATAT- GGGCA-TGTCC-TAT [SEQ ID NO: 19]. The pentanucleotide repeats, which are recognized by p53, are indicated by hyphens. These DNAs were radioactively labeled using

32P-labeled nucleotides, as described [Halazonetis et al, (1988), cited above].

Results using this assay are presented in Example 3, below.

Example 3 - DNA Binding Activities of P53 Proteins with Altered Tetramerization Domains

A. DNA Binding Activities of Wild-type Human P53 The ability of wild-type human p53 to recognize the DNA sites present in oligonucleotides Ewafl, BC.V4A and BC [SEQ ID NOS: 16, 17 and 18, respectively] has been previously demonstrated [El-Deiry et al, (1993), cited above; Halazonetis et al, (1993), cited above]. Using the assay described in Example 2, wild-type p53 recognized all these DNAs. The highest signal was obtained using the BC oligonucleotide [SEQ ID NO: 18], while oligonucleotide Ewafl [SEQ ID NO: 16] gave the weakest signal. The intensity of the signal in this assay reflects the affinity of p53 for the different DNA sites. The intensity of the signal using oligonucleotides BC.V4A [SEQ ID NO: 17] or Ewafl [SEQ ID NO: 16] was enhanced in the presence of 0.1 μg anti-p53 antibody PAb421 [Oncogene Science, Uniondale, NY]. This antibody activates DNA binding of wild-type p53, by switching the conformation of the protein [Halazonetis et al, (1993), cited above; Halazonetis and Kandil (1993), cited above]. Binding to oligonucleotide BC [SEQ ID NO: 18] is quite potent, and very little further enhancement is observed following incubation with antibody PAb421. In addition to antibody PAb421, the conformation of p53 can be switched by a C-terminal truncation that removes residues 364-393 of human p53 [SEQ ID NO: 2] [Halazonetis and Kandil (1993), cited above; Hupp et al (1992), Cell, 71:875-886; Hupp and Lane (1994), Current Biology, 4: 865-875]. The C-terminally truncated p53 protein, p53D364-393, bound all three oligonucleotides with high affinity, comparable to wild-type p53 in the presence of PAb421.

Binding of wild-type p53 to oligonucleotide BC.S21 was also examined. This oligonucleotide [SEQ ID NO: 19] contains two pairs of contiguous pentanucleotide repeats separated by 21 nucleotides. Wild-type p53 bound efficiently to this DNA, as indicated by a strong signal in the DNA binding assay described in Example 2, above. The signal was as strong as with oligonucleotide BC [SEQ ID NO: 18] (which represents the optimal p53 DNA site) and was not further enhanced by antibody PAb421. As discussed in the Section V.B1, oligonucleotide BC.S21 [SEQ ID NO: 19] does not match the consensus p53 DNA site. Thus, the ability of wild-type p53 to bind to oligonucleotide BC.S21 [SEQ ID NO: 19] is a novel finding.

B. DNA Binding Activities of P53LZ346E. P53LZ347. P53TZ334NR and P53TZ323RGN Proteins p53LZ346E, p53LZ347, p53TZ334NR and p53TZ323RGN represent chimeric proteins of the invention, which are encoded by plasmids pGEMhump53LZ346E, pGEMhump53LZ347, pGEMhump53TZ334NR and pGEMhump53TZ323RGN, respectively, described in Example 1. The ability of these proteins to bind oligonucleotides Ewafl, BC.V4A and BC [SEQ ID NOS: 16, 17 and 18, respectively] was examined using the assay described in Example 2. All of them bound to all three oligonucleotides. The signal intensities were overall comparable to those of wild-type p53 bound to the respective oligonucleotides in the presence of PAb421 or to those of p53D364-393 bound to the respective oligonucleotides. p53LZ347, p53TZ334NR and p53TZ323RGN exhibited somewhat higher affinity for DNA, as compared to p53LZ346E.

The DNA complexes of proteins p53LZ346E, p53LZ347, P53TZ334NR and p53TZ323RGN comigrated with the DNA complex of wild-type p53 or the DNA complex of p53D364-393. Since migration on acrylamide gels depends on the molecular size of the migrating species [Hope and Struhl (1987), EMBO J. , 6: 2781-2784] this indicates that the complexes of wild-type p53, p53D364-393, p53LZ346E, P53LZ347, p53TZ334NR and p53TZ323RGN with DNA have similar molecular sizes. Since wild-type p53 and p53D364-393 bind DNA as tetramers [Halazonetis and Kandil (1993), cited above; Cho et al, (1994), cited above; Hupp and Lane (1994), cited above], then p53LZ346E, p53LZ347, P53TZ334NR and p53TZ323RGN also bind DNA as tetramers. C. DNA Binding Activities of P53LZ3350 and P53LZ343KMKQ

Proteins p53LZ335Q and p53LZ343RMKQ are chimeric proteins of p53 with the GCN4 leucine zipper, encoded by plasmids pGEMhump53LZ335Q and pGEMhump53LZ343RMKQ, respectively. Protein p53LZ343RMKQ was first described by Pietenpol et al, (1994) [cited above]. The ability of these proteins to bind oligonucleotides BC.V4A and BC [SEQ ID NOS: 17 and 18, respectively] was examined using the assay described in Example 2. Both bound to oligonucleotide BC [SEQ ID NO: 18] (the optimal p53 DNA site [Halazonetis et al, (1993), cited above], but neither bound oligonucleotide BC.V4A [SEQ ID NO: 17] (a suboptimal site) . Thus, the DNA binding activities of these proteins are compromised relative to wild-type p53. In addition the complexes of p53LZ335Q and p53LZ343RMKQ with oligonucleotide BC [SEQ ID NO: 18] migrate significantly faster than the corresponding complexes of wild-type p53 or p53D364-393. Thus, the molecular sizes of the complexes of p53LZ335Q and p53LZ343RMKQ are smaller than those of wild-type p53 or p53D364-393. Since wild-type p53 and p53D364-393 are tetramers

[Halazonetis and Kandil (1993), cited above; Cho et al, (1994), cited above; Hupp and Lane (1994), cited above], p53LZ335Q and P53LZ343RMKQ are dimers. They cannot be monomers, because monomeric p53 does not bind DNA [Halazonetis and Kandil (1993), cited above].

In conclusion the ability of p53 proteins with altered tetramerization domains to bind to the full panel of p53 DNA sites correlates with their ability to form tetramers. Furthermore, p53LZ335Q and p53LZ343RMKQ are not proteins of this invention, since they fail to form tetramers.

D. DNA Binding Activities of P530334. P53L337. P53A341 and P53A344

Proteins p53Q334, p53L337, p53A341 and p53A344 are encoded by plasmids pGEMhump53Q334, pGEMhump53L337, pGEMhump53A34l and pGEMhump53A344, respectively, described in Example 1. The ability of these proteins to bind oligonucleotide BC [SEQ ID NO: 18] was examined using the assay described in Example 2. Proteins p53Q334, p53L337 and p53A341 bound DNA very weakly, if at all. This finding indicates that these substitutions disrupt completely the function of the p53 tetramerization domain, because they map within the tetramerization domain [Wang et al, (1994) , cited above; Clore et al, (1994), cited above] and DNA binding by p53 requires oligomerization [Halazonetis and Kandil (1993) , cited above].

Protein p53A344 bound oligonucleotide BC [SEQ ID NO: 18]. However, its complex with DNA migrated significantly faster than the DNA complexes of wild-type p53 or p53D364-393. Thus, p53A344 is a dimer, rather than a tetramer. Thus, this single amino acid substitution partially disrupts the function of the p53 tetramerization domain. E. P A Pinging Activities of p5? 29Q-397. p53D290-297D300-321. P53D300-308. P53D300-317. P53D300-321. and P53D300-327

Proteins p53D290-297, p53D290-297D300-321, p53D300-308, p53D300-317, p53D300-321 and p53D300-327 are encoded by plasmids pGEMhump53D290-297, pGEMhump53D290-297D300-321, pGEMhump53D300-308, pGEMhump53D300-317, pGEMhump53D300-321 and pGEMhump53D300-327, respectively, described in Example 1. The ability of these proteins to bind oligonucleotides BC and BC.S21 [SEQ ID NOS: 18 and 19, respectively] was examined using the assay described in Example 2. Proteins p53D290-297, p53D300-308, p53D300-317, and p53D300-321 bound both oligonucleotides BC and BC.S21 [SEQ ID NOS: 18 and 19, respectively] with efficiencies paralleling that of wild-type p53. In addition the complexes of these proteins with DNA comigrated with the complexes of wild-type p53 with DNA. Thus, p53D290-297, p53D300-308, p53D300-317 and p53D300-321 exhibit DNA binding properties similar to wild-type p53. Proteins p53D290-297D300-321 and p53D300-327 formed aberrant complexes with oligonucleotide BC [SEQ ID NO: 18]. These complexes migrated very slowly and the signal intensity was very low. In contrast, the complexes of p53D290-297D300-321 and p53D300-327 with oligonucleotide BC.S21 [SEQ ID NO: 19], were similar to those of wild-type p53. Proteins p53D290-297D300-321 and P53D300-327 have diminished ability to recognize oligonucleotide BC [SEQ ID NO: 18], but their ability to recognize oligonucleotide BC.S21 [SEQ ID NO: 19] is intact.

Example 4 - Immunoprecipitation of P53 Proteins

For immunoprecipitation experiments the p53 proteins were in vitro translated, as described in Example 2, in the presence of 35S-methionine, so that they would be radioactively labeled. Following in vitro translation, 3 μl of the lysate containing the translated protein(s) was incubated in 30 μl DNA binding buffer [Halazonetis et al, (1993) , cited above] for 20 minutes at room temperature. Then 0.6 μg anti-p53 antibody PAb421 [Oncogene Science, Uniondale, NY], 30 μl packed protein G-Sepharose beads [Pharmacia, Piscataway, NJ] and 400 μl high-salt/EDTA immunoprecipitation buffer [Halazonetis et al, (1993) , cited above] were added. After 45 min incubation at room temperature on a rotator, the beads were washed three times with the high-salt/EDTA immunoprecipitation buffer and the proteins absorbed to the beads were eluted with SDS sample buffer and subjected to SDS-PAGE.

The use of this assay is described in Example 5 below. Example 5 - Hetero-Oligomerization Assay

This assay was used to demonstrate that p53LZ346E, one of the proteins of the invention encoded by plasmid pGEMhump53LZ346E (Example 1) , does not hetero-oligomerize with p53 proteins having intact native p53 tetramerization domains, such as wild-type p53 and tumor-derived p53 mutants.

Wild-type p53 and p53LZ346E were cotranslated in the presence of 35S-methionine. Simultaneous translation of the two proteins provides opportunity for the two different subunit types to form hetero-oligomers. After cotranslation was completed, the mixture was immunoprecipitated using antibody PAb421, as described in Example 4 above. The epitope of antibody PAb421 maps to residues

373-381 of human p53 [SEQ ID NO: 2]. Therefore, PAb421 recognizes wild-type p53, but not p53LZ346E. Thus, if the two proteins hetero-oligomerize when cotranslated, then both will be precipitated by PAb421. If they do not hetero-oligomerize, then only wild-type p53 will be precipitated.

Following SDS-polyacrylamide gel electrophoresis, a radioactive band migrating like wild-type p53 was noted. No band corresponding to p53LZ346E was observed. These results indicate that wild-type p53 and p53LZ346E do not hetero-oligomerize. As a positive control the ability of wild-type p53 and p53D364-393 to hetero-oligomerize using this assay was examined. Like p53LZ346E, p53D364-393 lacks the epitope for PAb421. However, p53D364-393 has an intact native p53 tetramerization domain. When cotranslated with wild-type p53, both wild-type p53 and P53D364-393 were precipitated by PAb421, indicating that wild-type p53 and p53D364-393 form hetero-oligomers. Example 6 - Transcriptional Activity Assay

An assay for transcriptional activity entails introducing vectors expressing wild-type p53 or p53 proteins of the invention into cells together with a reporter plasmid that expresses a reporter marker in a p53-dependent manner. Preferably the cells are human tumor cells that do not express endogenous p53, so that the transcriptional activity can be evaluated without interference from endogenous wild-type or mutant p53. [See, e.g., Lin et al, (1994), cited above; Wu et al, (1993), cited above; Chen et al, (1993), Oncogene, 8: 2159-2166; Chumakov et al, (1993), Oncogene, 8: 3005-3011; Unger et al, (1992), EMBO J, 11: 1383-1390; Kern et al, (1992) , Science, 256: 827-830; Pietenpol et al, (1994), cited above].

Transcriptional activity was assayed in Saos-2 human osteosarcoma cells [ATCC HTB 85] . These cells do not contain any endogenous p53, because both p53 alleles are deleted [Diller et al, (1990), Mol. Cell. Biol., 10: 5772-5781]. Thus, any transcriptional activity can be attributed to the transfected p53. Transcriptional activity was assayed as previously described [Halazonetis (1992), Anticancer Res., 12: 285-292]. Briefly, plasmids expressing p53 in mammalian cells (of the pSV2 series described in Example 1) were cotransfected with reporter plasmids (of the pTK-SEAP series described in Example 1) using the calcium phosphate technique [Halazonetis (1992), cited above]. Alkaline phosphatase activity, which reflects p53-mediated transcriptional activity, was assayed as previously described [Halazonetis (1992) , cited above] .

The use of this assay is described in Examples 7 and 8 below. Example 7 - Comparison of Transcriptional Activities of Wild-tVPe P53. P53LZ346E. P53LZ347. P53LZ335Q.

P53LZ343KMK9 and P53TZ334NR

Wild-type p53, p53LZ346E, p53LZ347, p53LZ335Q, p53LZ343RMKQ and p53TZ334NR were expressed in Saos-2 cells [ATCC HTB 85] by transfecting plasmids pSV2hump53wt, pSV2hump53LZ346E, pSV2hump53LZ347, pSV2hump53LZ335Q, pSV2hump53LZ343RMKQ and pSV2hump53TZ334NR, respectively. The transcriptional activities of the expressed proteins were assayed using one or more of the reporter plasmids pBC-TK-SEAP, pBC.V4A-TK-SEAP and pEwafl-TK-SEAP, described in Example 1.

Results of representative experiments are presented in Table 1 below. The units of transcriptional activity are relative, and comparisons should only be made within the same experiment. For each transfection the amounts of transfected plasmid are indicated in μg. Where the total amount was less than 30 μg, then plasmid pSV2gpt (described in Example 1) was used to bring the total to

30 μg.

Table

Experiment 1

Reporter Plasmid: 10 μg pBC.V4A-TK-SEAP p53 Expression Transcriptional Plqsmjd f? μq) Activ ty pSV2hump53wt 1700 pSV2hump53LZ347 524 pSV2hump53LZ335Q 18 pSV2hump53LZ343RMKQ 22 Experiment 2

Reporter Plasmid: 10 μg pEwafl-TK-SEAP p53 Expression Transcriptional Plas-mjd (3 μq) AptjvjtY pSV2hump53wt 1990 pSV2hump53LZ346E 520

Experiment 3 p53 Expression Reporter Transcriptional lasmi (15 μq) Plasmjd (15 μg) Activity pSV2hump53wt pEwafl-TK-SEAP 1640 pSV2hump53TZ334NR pEwaf1-TK-SEAP 1480 pSV2hump53TZ334NR pBC.V4A-TK-SEAP 2750 pSV2hump53TZ334NR pBC-TK-SEAP 2550

From the results of Table 1 it is apparent that wild-type p53 is able to activate transcription from all the reporter plasmids examined, including plasmid pEwaf1-TK-SEAP, which contains the weakest p53 binding site. The tetrameric p53 proteins of the invention: P53LZ346E, p53LZ347 and p53TZ334NR, all exhibit transcriptional activity. In contrast the dimeric p53 chimeric proteins, such as p53LZ335Q and p53LZ343RMKQ, do not exhibit transcriptional activity that is detectably above background in this assay, and are thus clearly inferior to the tetrameric proteins of this invention. It is possible that the dimeric proteins may exhibit higher transcriptional activity when grossly overexpressed, since Pietenpol et al, (1994) [cited above] have reported detectable transcriptional activity using protein p53LZ343RMKQ. The inventor has been unable to reproduce this result, as the Experiments of Table 1 indicate. Even if p53LZ343RMKQ has detectable transcriptional activity when grossly overexpressed, our experiments indicate that the tetrameric p53 proteins of this invention are clearly superior to the dimeric proteins. Example 8 - .Ability of Tumor-Derived P53 Mutants to suppress the Transcriptional Activities of Wild-Type p53 or Tetrameric P53 Proteins of the Invention

Tumor-derived p53 mutants are known to suppress the transcriptional activity of wild-type p53 by forming hetero-tetramers with wild-type p53 [Milner and Medcalf (1991), cited above; Bargonetti et al, (1992), cited above; Farmer et al, (1992), cited above; Kern et al, (1992), cited above]. The tetrameric p53 proteins of this invention do not hetero-tetramerize with tumor-derived p53 mutants, because the native p53 tetramerization domain is partially or completely disrupted (See Example 5) . Consequently, the transcriptional activities of the tetrameric p53 proteins should not be inhibited by tumor-derived p53 mutants.

To establish this, the transcriptional activities of the p53 proteins of this invention in the presence of excess of a tumor-derived p53 mutant were compared to their transcriptional activities in the absence of the tumor-derived mutant. The tumor-derived mutants p53Hisl75 and p53Ser249 have histidine at position 175 or serine at position 249 of human p53 [SEQ ID NO: 2], respectively. Other tumor-derived p53 mutants [Caron de Fromentel and Soussi (1992), Genes Chrom. Cancer, 4: 1-15] can also be used, as long as they potently inhibit the transcriptional activity of wild-type p53.

Results of relevant experiments are presented in Table 2 below. Suppression of transcriptional activity by the tumor-derived p53 mutant is presented as percent of residual transcriptional activity in the presence of the tumor-derived p53 mutant, as compared to the transcriptional activity in the absence of the mutant. For each transfection the amounts of transfected plasmids are indicated in μg. Where the total amount was less than 30 μg (as in the absence of the tumor-derived p53 mutant) , then plasmid pSV2gpt (described in Example 1) was used to bring the total to 30 μg. For these experiments the tumor-derived p53 mutant His 175 was used (described in Example 1) .

Table 2

Experiment 1

Reporter Plasmid: 10 μg pBC-TK-SEAP Plasmid for Tumor-Derived p53 Mutant: 19 μg pSV2hump53H175 Residual Transcriptional p53 Expression Activity in the Presence Plasmid (1 ua) of Tumor-Derived P53 Mutant pSV2hump53wt 23 % pSV2hump53LZ346E 100 % pSV2hump53LZ347 46 %

Experiment 2

Reporter Plasmid: 10 μg pBC.V4A-TK-SEAP Plasmid for Tumor-Derived p53 Mutant: 19 μg pSV2hump53H175 Residual Transcriptional p53 Expression Activity in the Presence Plasmid (1 ua) of Tumor-Derived o53 Mutant pSV2hump53wt 2 % pSV2hump53TZ334NR 80 % From the results of the experiments presented in Table 2 it is apparent that, in contrast to wild-type p53, the transcriptional activities of the p53 tetrameric proteins of the invention are not suppressed, or only minimally suppressed, by gross excess of a tumor-derived p53 mutant.

In another experiment, the transcriptional activity of p53TZ334NR and p53TZ334NR/I352 were examined by transient transfection in Saos-2 osteosarcoma cells, which lack endogenous p53 [L. Diller et al, Mol. Cell. Biol.. 10:5772-5781 (1990)]. Briefly, as above. transcriptional activity was determined by transfecting in quadruplicated Saos-2 cells with 5μg of p53 expression and 25 μg of reporter plasmids.

Alkaline phosphatase activity was determined 48 hrs later [Halazonetis, Anticancer Res.. 12-:285-292 (1992)]. To assay inhibition of transcriptional activities of functional p53 proteins by p53Trp248, Saos-2 cells were cotransfected in triplicate with lμg of p53 expression plasmid, 9 μg of plasmid expressing the mutant of 9 μg of pSV plasmid without insert and 20 μg of the Ep21/TK-seap reporter plasmid. The reporter plasmids, Ep21/TK-seap and pEmdm2/TKseap have one copy of oligonucleotide Ep21 [SEQ ID NO: 45: CCC-GAACA-TGTCC-TGTTG-GGG] or Emdm2 [SEQ ID NO: 46: GGCT-GGTCA-AGTTG-GGACA-CGTCC- GGCGTCGGCTGTCGGAG-GAGCT-A-AGTCC-TGACA-CCAG] , respectively, cloned in the Eco RV site of pTKseap [Halazonetis, cited above] and express secreted alkaline phosphatase in a p53-responsive manner.

Both chimeric proteins of the invention activated transcription from reporter plasmids containing the p21 or mdm2 p53 sites. The dimeric p53-leucine zipper hybrids were less potent transcriptional activators, especially with the reporter plasmid containing the mdm2 site. Transcriptional activity for all p53 proteins examined was sequence-specific, since none of them activated transcription from a reporter plasmid that lacked a p53 site.

Example 9 - Turcqr suppression Activities of p53 fusion proteins in Saos-2 cells. The tumor suppressing activities of p53TZ334NR of

Example 1, part B5 and p53TZ334NR/I352 of Example 1, part B9, as well as other expression plasmids, including p53wt (wild-type p53; see Example 1, part A), p53L2343 (Example 1, part B4), p53LZ335Q (Example 1, part B3) , p53W248 (wild-type p53 with a point mutation associated with human cancer at Trp 248), and p53W248TZ334N (p53T2334N containing the point mutation at Trp 248) were tested in a colony formation assay, by cotransfecting Saos-2 osteosarcoma cells in quadruplicate with 5 μg of expression plasmid directing p53 expression, and 1 μg of pSV7neo, a plasmid conferring neomycin resistance [Zhang et al, Proc. Natl. Acad. Sci.. USA. £2:6281-6285 (1990)], and 24 μg of pBC12/PLseap, a carrier plasmid [Halazonetis, Anticancer Res.. l i:285-292 (1992)]. The transfected cells were selected for G418 resistance. Two weeks later the colonies were stained with crystal violet and counted.

The results, obtained as number of G418 resistant colonies per plate and reported in Fig. 4, demonstrated that the number of G418 resistant colonies is inversely related to tumor suppressing activity. Both p53TZ334NR/I352 and p53TZ334NR suppressed tumor growth almost as efficiently as wild-type p53. Furthermore, a point mutation associated with human cancer (Trp248) in the DNA binding domain of p53TZ334NR, i.e., P53W248T2334N, was sufficient to abrogate tumor- suppressing activity. The same mutation also abrogated the tumor-suppressing activity of wild-type p53, i.e., p53W248, as expected [L. Diller et al, cited above; S. Baker et al, cited above; and C. Finlay et al, Cell. .57:1083 (1989)]. The tumor suppressing activities of P53TZ334NR and p53TZ334NR/I352 correlated with their ability to form tetramers, since the dimeric proteins p53LZ343RMKQ and p53LZ335Q did not significantly suppress colony formation.

To determine if the tetrameric zipper alleviates transdominant inhibition by tumor-derived mutants, the tumor suppressor activities of p53 proteins was examined in the presence of the tumor-derived mutant p53tryptophan248 (p53W248) . For these experiments Saos-2 cells were cotransfected in triplicate with 2.5 μg of p53 expression plasmid, IQ μg of plasmid expressing p53W248 or 10 μg of pSV2 plasmid without insert, l μg of pSV7neo and 16.5 μg of pBC12/PLseap carrier plasmid. Tumor suppressor activity was assayed by the colony forming assay as described above. In contrast to wild-type p53, neither p53TZ334NR, nor p53TZ334NR/I352 were transdominantly inhibited by p53Trp248. Figure 5.

Example 10 - Recombinant Plasmids Using Jun Leucine Zipper

Standard cloning procedures were used to prepare the plasmids described below.

A. Plasmid pGEMhump53 unTZ334N Plasmid pGEMhump53junTZ334N encodes a p53 - modified c-Jun chimeric protein consisting (in an N-terminal to C- erminal direction) of residues 1-334 of human p53 [SEQ ID NO: 2], an asparagine, a modified c-Jun leucine zipper corresponding to residues 276-313 of human c-Jun [SEQ ID NO: 41] and a tripeptide glycine-glutamic acid-arginine. Synthetic oligonucleotides were used to generate a tetrameric variant of the c-Jun leucine zipper.

These oligonucleotides [SEQ ID NO: 42 and 43] were subjected to a PCR cycle in the absence of any additional DNA. The PCR product was digested with restriction endonucleases Sst II and Sal I and cloned into pGEMhump53wt (described in Example IA) linearized with Sst II and Sal I. The DNA and amino acid sequences of native c-Jun are presented as SEQ ID NOs: 38 and 39, respectively, while the DNA and amino acid sequences of modified c-Jun are presented as SEQ ID NOs: 40 and 41, respectively.

Plasmid pGEMhump53junTZ334N was designed to resemble plasmid pGEMhump53TZ334NR as much as possible. The latter plasmid encodes a p53 - modified GCN4 chimeric protein consisting in an N-terminal to C-terminal direction of residues 1-334 of human p53 [SEQ ID NO: 2], an asparagine, and a modified GCN4 leucine zipper corresponding to amino acids 249-281 of GCN4 [SEQ ID NO: 6]. The modifications for both the GCN4 and c-Jun leucine zippers were the same: residues at positions a of the coiled-coil were substituted with leucine and residues at positions d of the coiled-coil were substituted with isoleucine. The GCN4 leucine zipper is at the very c-terminus of the GCN4 protein, for this reason the three C-terminal amino acids of GCN4, glycine-glutamic acid-arginine, which are not part of the GCN4 leucine zipper, were included in pGEMhump53TZ334NR. The c-Jun leucine zipper is not at the very C-terminus of c-Jun. So that plasmid pGEMhump53junTZ334N would resemble as much as possible pGEMhump53TZ334NR, the tripeptide glycine-glutamic acid-arginine was inserted C-terminal to the c-Jun modified leucine zipper. The structures of wild-type p53 and of the protein encoded by plasmid pGEMhump53TZ334NR are represented schematically in Figs. IA and IF, respectively. The protein encoded by plasmid pGEMhumpp53junTZ334N is substantially identical to the schematic diagram of Fig. IF, except that the tetrameric variant of c-Jun residues 276-313 and the above-identified tripeptide are substituted for tetrameric variant of GCN4.

B. Plasmid pGEMhump53iunN287TZ334N

Plasmid pGEMhump53junN287TZ334N encodes a protein that is identical to the protein encoded by pGEMhump53junTZ334N, except that one of the isoleucines at position a of the coiled-coil p53, corresponding to amino acid 287 of human c-Jun [SEQ ID NO: 39], was substituted with asparagine. This plasmid was generated with PCR-directed mutagenesis [Innis et al, (1990)] using the oligonucleotide described in SEQ ID NO: 44 as the PCR primer. See, Figs. 6A through 6D. Oligomerization of the modified zippers is mediated by the hydrophobic amino acids leucine (Leu) and isoleucine (lie) . In principle, zippers may assemble either in parallel (Fig. 6A) or antiparallel (Fig. 6B) orientations. Substitution of an lie with asparagine (Asn) affects the ability of the zipper to form hydrophobic interactions, since Asn is not a hydrophobic residue. For parallel assembly of zippers only one hydrophobic interaction is compromised (Fig. 6C) . In contrast for antiparallel assembly of zippers two hydrophobic interactions are compromised (Fig. 6D) . Thus, substitution of an lie with Asn discriminates against antiparallel assembly. A similar effect would be expected by substitution of an lie with alanine (a small hydrophobic that cannot form strong hydrophobic interactions) or with polar residues other than Asn, such as serine, threonine, glutamine, etc. C. lasmifl pgV3hvm 53lu Tg 4N Plasmid pSV2hump53junTZ334N directs expression of the p53 - c-Jun chimeric protein described above (Example 10A) in mammalian cells. It was constructed by substituting the Sst II-Sal I fragment of pSV2hump53wt, which expresses full-length human wild-type p53 (Example IC) , with the corresponding Sst II-Sal I fragment of pGEMhump53junTZ334N (Example 10A) .

Example 11 - DNA Binding Activities of P53 - c-Jun Chimeric Proteins

Plasmids pGEMhump53junTZ334N and pGEMhump53junN287TZ334N were used to generate in vitro translated proteins as described in Example 2. These proteins were subsequently tested for their ability to bind DNA, again as described in Example 2. Both proteins (hump53junTZ334N and hump53junN287TZ334N) bound DNA as efficiently as wild-type p53. Hump53junTZ334N bound DNA as a tetramer as determined by migration of its DNA complexes on native electrophoretic gels relative to the DNA complexes of wild-type p53. A second complex of hump53junTZ334N with DNA was also observed. This complex migrated more slowly. It might represent p53 - c-Jun chimera assembled in an antiparallel manner (See Fig. 6B) , because hump53junN287TZ334N formed only one type of complex with DNA, which migrated like the DNA complexes of tetrameric wild-type p53.

Example 12 - Tumor Suppression Activities

Tumor suppressor activity was assayed as described in Example 9. The results are illustrated in Figure 7. P53TZ334NR, p53junTZ334N and p53TZ334NR/I352 (Examples 1B5, 10A and 1B9, respectively) all suppressed tumor growth almost as efficiently as wild-type p53 (Fig. 7) .

All references referred to above are incorporated by reference herein. Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: The Wistar Institute of Anatomy and Biology Halazonetis, Thanos D.

(ii) TITLE OF INVENTION: p53 Proteins With Altered

Tetramerization Domains

(iii) NUMBER OF SEQUENCES: 46

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Howson and Howson

(B) STREET: Spring House Corporate Cntr. , PO Box 457

(C) CITY: Spring House

(D) STATE: Pennsylvania

(E) COUNTRY: USA

(F) ZIP: 19477

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/347,792

(B) FILING DATE: 28-NOV-1994

(Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/431,357

(B) FILING DATE: 28-APR-1995

(Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/456,623

(B) FILING DATE: 01-JUN-1995

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bak, Mary E.

(B) REGISTRATION NUMBER: 31,215

(C) REFERENCE/DOCKET NUMBER: WST58CPCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-540-9206

(B) TELEFAX: 215-540-5818 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1317 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 136..1314

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GTCTAGAGCC ACCGTCCAGG GAGCAGGTAG CTGCTGGGCT CCGGGGACAC 50

TTTGCGTTCG GGCTGGGAGC GTGCTTTCCA CGACGGTGAC ACGCTTCCCT 100

GGATTGGCAG CCAGACTGCC TTCCGGGTCA CTGCC ATG GAG GAG CCG 147

Met Glu Glu Pro

1

CAG TCA GAT CCT AGC GTC GAG CCC CCT CTG AGT CAG GAA ACA 189 Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gin Glu Thr 5 10 15

TTT TCA GAC CTA TGG AAA CTA CTT CCT GAA AAC AAC GTT CTG 231 Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30

TCC CCC TTG CCG TCC CAA GCA ATG GAT GAT TTG ATG CTG TCC 273 Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu Ser 35 40 45

CCG GAC GAT ATT GAA CAA TGG TTC ACT GAA GAC CCA GGT CCA 315 Pro Asp Asp lie Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro 50 55 60

GAT GAA GCT CCC AGA ATG CCA GAG GCT GCT CCC CCC GTG GCC 357 Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala

65 70

CCT GCA CCA GCA GCT CCT ACA CCG GCG GCC CCT GCA CCA GCC 399 Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala 75 80 85

CCC TCC TGG CCC CTG TCA TCT TCT GTC CCT TCC CAG AAA ACC 441 Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr 90 95 100 TAC CAG GGC AGC TAC GGT TTC CGT CTG GGC TTC TTG CAT TCT 483 Tyr Gin Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser 105 110 115

GGG ACA GCC AAG TCT GTA ACT TGC ACG TAC TCC CCT GCC CTC 525 Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu 120 125 130

AAC AAG ATG TTT TGC CAA CTG GCC AAG ACC TGC CCT GTG CAG 567 Asn Lys Met Phe Cys Gin Leu Ala Lys Thr Cys Pro Val Gin

135 140

CTG TGG GTT GAT TCC ACA CCC CCG CCC GGC ACC CGC GTC CGC 609 Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg 145 150 155

GCC ATG GCC ATC TAC AAG CAG TCA CAG CAC ATG ACG GAG GTT 651 Ala Met Ala lie Tyr Lys Gin Ser Gin His Met Thr Glu Val 160 165 170

GTG AGG CGC TGC CCC CAC CAT GAG CGC TGC TCA GAT AGC GAT 693 Val Arg Arg Cys Pro His His Glu Arg Cys Ser Asp Ser Asp 175 180 185

GGT CTG GCC CCT CCT CAG CAT CTT ATC CGA GTG GAA GGA AAT 735 Gly Leu Ala Pro Pro Gin His Leu lie Arg Val Glu Gly Asn 190 195 200

TTG CGT GTG GAG TAT TTG GAT GAC AGA AAC ACT TTT CGA CAT 777 Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His

205 210

AGT GTG GTG GTG CCC TAT GAG CCG CCT GAG GTT GGC TCT GAC 819 Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp 215 220 225

TGT ACC ACC ATC CAC TAC AAC TAC ATG TGT AAC AGT TCC TGC 861 Cys Thr Thr lie His Tyr Asn Tyr Met Cys Asn Ser Ser Cys 230 235 240

ATG GGC GGC ATG AAC CGG AGA CCC ATC CTC ACC ATC ATC ACA 903 Met Gly Gly Met Asn Arg Arg Pro lie Leu Thr lie lie Thr 245 250 255

CTG GAA GAC TCC AGT GGT AAT CTA CTG GGA CGG AAC AGC TTT 945 Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe 260 265 270

GAG GTG CGT GTT TGT GCC TGT CCT GGG AGA GAC CGG CGC ACA 987 Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr

275 280 GAG GAA GAG AAT CTC CGC AAG AAA GGG GAG CCT CAC CAC GAG 1029 Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu 285 290 295

CTG CCC CCA GGG AGC ACT AAG CGA GCA CTG CCC AAC AAC ACC 1071 Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr 300 305 310

AGC TCC TCT CCC CAG CCA AAG AAG AAA CCA CTG GAT GGA GAA 1113 Ser Ser Ser Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu 315 320 325

TAT TTC ACC CTT CAG ATC CGT GGG CGT GAG CGC TTC GAG ATG 1155 Tyr Phe Thr Leu Gin lie Arg Gly Arg Glu Arg Phe Glu Met 330 335 340

TTC CGA GAG CTG AAT GAG GCC TTG GAA CTC AAG GAT GCC CAG 1197 Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gin

345 350

GCT GGG AAG GAG CCA GGG GGG AGC AGG GCT CAC TCC AGC CAC 1239 Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365

CTG AAG TCC AAA AAG GGT CAG TCT ACC TCC CGC CAT AAA AAA 1281 Leu Lys Ser Lys Lys Gly Gin Ser Thr Ser Arg His Lys Lys 370 375 380

CTC ATG TTC AAG ACA GAA GGG CCT GAC TCA GAC TGA 1317

Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp 385 390

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 393 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Glu Glu Pro Gin Ser Asp Pro Ser Val Glu Pro Pro Leu Ser 1 5 10 15

Gin Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn

20 25 30 Val Leu Ser Pro Leu Pro Ser Gin Ala Met Asp Asp Leu Met Leu

35 40 45

Ser Pro Asp Asp He Glu Gin Trp Phe Thr Glu Asp Pro Gly Pro

50 55 60

Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro

65 70 75

Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser

80 85 90

Trp Pro Leu Ser Ser Ser Val Pro Ser Gin Lys Thr Tyr Gin Gly

95 100 105

Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys

110 115 120

Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys

125 130 135

Gin Leu Ala Lys Thr Cys Pro Val Gin Leu Trp Val Asp Ser Thr

140 145 150

Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala He Tyr Lys Gin

155 160 165

Ser Gin His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu

170 175 180

Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gin His Leu He

185 190 195

Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn

200 205 210

Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val

215 220 225

Gly Ser Asp Cys Thr Thr He His Tyr Asn Tyr Met Cys Asn Ser

230 235 240

Ser Cys Met Gly Gly Met Asn Arg Arg Pro He Leu Thr He He

245 250 255

Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe

260 265 270

Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu

275 280 285 Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro

290 295 300

Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser

305 310 315

Pro Gin Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu

320 325 330

Gin He Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn

335 340 345

Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly

350 355 360

Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gin

365 370 375

Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro

380 385 390

Asp Ser Asp

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1824 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 778..1620

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

ATCTTCGGGG ATATAAAGTG CATGAGCATA CATCTTGAAA AAAAAAGATG 50

AAAAATTTCC GACTTTAAAT ACGGAAGATA AATACTCCAA CCTTTTTTTC 100

CAATTCCGAA ATTTTAGTCT TCTTTAAAGA AGTTTCGGCT CGCTGTCTTA 150

CCTTTTAAAA TCTTCTACTT CTTGACAGTA CTTATCTTCT TATATAATAG 200

ATATACAAAA CAAAACAAAA CAAAAACTCA CAACACAGGT TACTCTCCCC 250

CCTAAATTCA AATTTTTTTT GCCCATCAGT TTCACTAGCG aAATTATACAA 300 CTCACCAGCC ACACAGCTCA CTCATCTACT TCGCAATCAA AACAAAATAT 350

TTTATTTTAG TTCAGTTTAT TAAGTTATTA TCAGTATCGT ATTAAAAAAT 400

TAAAGATCAT TGAAAAATGG CTTGCTAAAC CGATTATATT TTGTTTTTAA 450

AGTAGATTAT TATTAGAAAA TTATTAAGAG AATTATGTGT TAAATTTATT 500

GAAAGAGAAA ATTTATTTTC CCTTATTAAT TAAAGTCCTT TACTTTTTTT 550

GAAAACTGTC AGTTTTTTGA AGAGTTATTT GTTTTGTTAC CAATTGCTAT 600

CATGTACCCG TAGAATTTTA TTCAAGATGT TTCCGTAACG GTTACCTTTC 650

TGTCAAATTA TCCAGGTTTA CTCGCCAATA AAAATTTCCC TATACTATCA 700

TTAATTAAAT CATTATTATT ACTAAAGTTT TGTTTACCAA TTTGTCTGCT 750

CAAGAAAATA AATTAAATAC AAATAAA ATG TCC GAA TAT CAG CCA 795

Met Ser Glu Tyr Gin Pro 1 5

AGT TTA TTT GCT TTA AAT CCA ATG GGT TTC TCA CCA TTG GAT 837 Ser Leu Phe Ala Leu Asn Pro Met Gly Phe Ser Pro Leu Asp 10 15 20

GGT TCT AAA TCA ACC AAC GAA AAT GTA TCT GCT TCC ACT TCT 879 Gly Ser Lys Ser Thr Asn Glu Asn Val Ser Ala Ser Thr Ser

25 30

ACT GCC AAA CCA ATG GTT GGC CAA TTG ATT TTT GAT AAA TTC 921 Thr Ala Lys Pro Met Val Gly Gin Leu He Phe Asp Lys Phe 35 40 45

ATC AAG ACT GAA GAG GAT CCA ATT ATC AAA CAG GAT ACC CCT 963 He Lys Thr Glu Glu Asp Pro He He Lys Gin Asp Thr Pro 50 55 60

TCG AAC CTT GAT TTT GAT TTT GCT CTT CCA CAA ACG GCA ACT 1005 Ser Asn Leu Asp Phe Asp Phe Ala Leu Pro Gin Thr Ala Thr 65 70 75

GCA CCT GAT GCC AAG ACC GTT TTG CCA ATT CCG GAG CTA GAT 1047 Ala Pro Asp Ala Lys Thr Val Leu Pro He Pro Glu Leu Asp 80 85 90

GAC GCT GTA GTG GAA TCT TTC TTT TCG TCA AGC ACT GAT TCA 1089 Asp Ala Val Val Glu Ser Phe Phe Ser Ser Ser Thr Asp Ser

95 100 ACT CCA ATG TTT GAG TAT GAA AAC CTA GAA GAC AAC TCT AAA 1131 Thr Pro Met Phe Glu Tyr Glu Asn Leu Glu Asp Asn Ser Lys 105 110 115

GAA TGG ACA TCC TTG TTT GAC AAT GAC ATT CCA GTT ACC ACT 1173 Glu Trp Thr Ser Leu Phe Asp Asn Asp He Pro Val Thr Thr 120 125 130

GAC GAT GTT TCA TTG GCT GAT AAG GCA ATT GAA TCC ACT GAA 1215 Asp Asp Val Ser Leu Ala Asp Lys Ala He Glu Ser Thr Glu 135 140 145

GAA GTT TCT CTG GTA CCA TCC AAT CTG GAA GTC TCG ACA ACT 1257 Glu Val Ser Leu Val Pro Ser Asn Leu Glu Val Ser Thr Thr 150 155 160

TCA TTC TTA CCC ACT CCT GTT CTA GAA GAT GCT AAA CTG ACT 1299 Ser Phe Leu Pro Thr Pro Val Leu Glu Asp Ala Lys Leu Thr

165 170

CAA ACA AGA AAG GTT AAG AAA CCA AAT TCA GTC GTT AAG AAG 1341 Gin Thr Arg Lys Val Lys Lys Pro Asn Ser Val Val Lys Lys 175 180 185

TCA CAT CAT GTT GGA AAG GAT GAC GAA TCG AGA CTG GAT CAT 1383 Ser His His Val Gly Lys Asp Asp Glu Ser Arg Leu Asp His 190 195 200

CTA GGT GTT GTT GCT TAC AAC CGC AAA CAG CGT TCG ATT CCA 1425 Leu Gly Val Val Ala Tyr Asn Arg Lys Gin Arg Ser He Pro 205 210 215

CTT TCT CCA ATT GTG CCC GAA TCC AGT GAT CCT GCT GCT CTA 1467 Leu Ser Pro He Val Pro Glu Ser Ser Asp Pro Ala Ala Leu 220 225 230

AAA CGT GCT AGA AAC ACT GAA GCC GCC AGG CGT TCT CGT GCG 1509 Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg Arg Ser Arg Ala

235 240

AGA AAG TTG CAA AGA ATG AAA CAA CTT GAA GAC AAG GTT GAA 1551 Arg Lys Leu Gin Arg Met Lys Gin Leu Glu Asp Lys Val Glu 245 250 255

GAA TTG CTT TCG AAA AAT TAT CAC TTG GAA AAT GAG GTT GCC 1593 Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala 260 265 270

AGA TTA AAG AAA TTA GTT GGC GAA CGC TGATTTCATT TACCTTTTAT 1640 Arg Leu Lys Lys Leu Val Gly Glu Arg 275 280 TTTATATTTT TTATTTCATT CTCGTGTATA ACGAAATAGA TACATTCACT 1690

TAGATAAGAA TTTAATCTTT TTTATGCCAA TTTTCTTAAG TAGAATTTTA 1740

CACCACGCAT TTATAATCTG CCGTATGTTC TGGTATTTAC TGGTTAGGAA 1790

TAGATAAAAA AAACACTCAC GATGGGGGTC GAAC 1824

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 281 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ser Glu Tyr Gin Pro Ser Leu Phe Ala Leu Asn Pro Met Gly

1 5 10 15

Phe Ser Pro Leu Asp Gly Ser Lys Ser Thr Asn Glu Asn Val Ser

20 25 30

Ala Ser Thr Ser Thr Ala Lys Pro Met Val Gly Gin Leu He Phe

35 40 45

Asp Lys Phe He Lys Thr Glu Glu Asp Pro He He Lys Gin Asp

50 55 60

Thr Pro Ser Asn Leu Asp Phe Asp Phe Ala Leu Pro Gin Thr Ala

65 70 75

Thr Ala Pro Asp Ala Lys Thr Val Leu Pro He Pro Glu Leu Asp

80 85 90

Asp Ala Val Val Glu Ser Phe Phe Ser Ser Ser Thr Asp Ser Thr

95 100 105

Pro Met Phe Glu Tyr Glu Asn Leu Glu Asp Asn Ser Lys Glu Trp

110 115 120

Thr Ser Leu Phe Asp Asn Asp He Pro Val Thr Thr Asp Asp Val

125 130 135

Ser Leu Ala Asp Lys Ala He Glu Ser Thr Glu Glu Val Ser Leu

140 145 150 Val Pro Ser Asn Leu Glu Val Ser Thr Thr Ser Phe Leu Pro Thr 155 160 165

Pro Val Leu Glu Asp Ala Lys Leu Thr Gin Thr Arg Lys Val Lys 170 175 180

Lys Pro Asn Ser Val Val Lys Lys Ser His His Val Gly Lys Asp 185 190 195

Asp Glu Ser Arg Leu Asp His Leu Gly Val Val Ala Tyr Asn Arg 200 205 210

Lys Gin Arg Ser He Pro Leu Ser Pro He Val Pro Glu Ser Ser 215 220 225

Asp Pro Ala Ala Leu Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg 230 235 240

Arg Ser Arg Ala Arg Lys Leu Gin Arg Met Lys Gin Leu Glu Asp 245 250 255

Lys Val Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu 260 265 270

Val Ala Arg Leu Lys Lys Leu Val Gly Glu Arg 275 280

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1824 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 778..1620

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ATCTTCGGGG ATATAAAGTG CATGAGCATA CATCTTGAAA AAAAAAGATG 50

AAAAATTTCC GACTTTAAAT ACGGAAGATA AATACTCCAA CCTTTTTTTC 100

CAATTCCGAA ATTTTAGTCT TCTTTAAAGA AGTTTCGGCT CGCTGTCTTA 150

CCTTTTAAAA TCTTCTACTT CTTGACAGTA CTTATCTTCT TATATAATAG 200 ATATACAAAA CAAAACAAAA CAAAAACTCA CAACACAGGT TACTCTCCCC 250

CCTAAATTCA AATTTTTTTT GCCCATCAGT TTCACTAGCG AATTATACAA 300

CTCACCAGCC ACACAGCTCA CTCATCTACT TCGCAATCAA AACAAAATAT 350

TTTATTTTAG TTCAGTTTAT TAAGTTATTA TCAGTATCGT ATTAAAAAAT 400

TAAAGATCAT TGAAAAATGG CTTGCTAAAC CGATTATATT TTGTTTTTAA 450

AGTAGATTAT TATTAGAAAA TTATTAAGAG AATTATGTGT TAAATTTATT 500

GAAAGAGAAA ATTTATTTTC CCTTATTAAT TAAAGTCCTT TACTTTTTTT 550

GAAAACTGTC AGTTTTTTGA AGAGTTATTT GTTTTGTTAC CAATTGCTAT 600

CATGTACCCG TAGAATTTTA TTCAAGATGT TTCCGTAACG GTTACCTTTC 650

TGTCAAATTA TCCAGGTTTA CTCGCCAATA AAAATTTCCC TATACTATCA 700

TTAATTAAAT CATTATTATT ACTAAAGTTT TGTTTACCAA TTTGTCTGCT 750

CAAGAAAATA AATTAAATAC AAATAAA ATG TCC GAA TAT CAG CCA 795

Met Ser Glu Tyr Gin Pro 1 5

25 30

GCA CCT GAT GCC AAG ACC GTT TTG CCA ATT CCG GAG CTA GAT 1047 Ala Pro Asp Ala Lys Thr Val Leu Pro He Pro Glu Leu Asp 80 85 90 GAC GCT GTA GTG GAA TCT TTC TTT TCG TCA AGC ACT GAT TCA 1089 Asp Ala Val Val Glu Ser Phe Phe Ser Ser Ser Thr Asp Ser

95 100

ACT CCA ATG TTT GAG TAT GAA AAC CTA GAA GAC AAC TCT AAA 1131 Thr Pro Met Phe Glu Tyr Glu Asn Leu Glu Asp Asn Ser Lys 105 110 115

165 170

235 240

AGA AAG TTG CAA CGT CTG AAA CAG ATC GAA GAC AAG TTA GAA 1551 Arg Lys Leu Gin Arg Leu Lys Gin He Glu Asp Lys Leu Glu 245 250 255

GAA ATC CTT TCG AAG CTC TAT CAC ATC GAG AAT GAG TTA GCG 1593 Glu He Leu Ser Lys Leu Tyr His He Glu Asn Glu Leu Ala 260 265 270 CGC ATC AAA AAA TTA TTG GGT GAA CGT TGATTTCATT TACCTTTTAT 1640 Arg He Lys Lys Leu Leu Gly Glu Arg 275 280

TTTATATTTT TTATTTCATT CTCGTGTATA ACGAAATAGA TACATTCACT 1690

TAGATAAGAA TTTAATCTTT TTTATGCCAA TTTTCTTAAG TAGAATTTTA 1740

CACCACGCAT TTATAATCTG CCGTATGTTC TGGTATTTAC TGGTTAGGAA 1790

TAGATAAAAA AAACACTCAC GATGGGGGTC GAAC 1824

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 281 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Ser Glu Tyr Gin Pro Ser Leu Phe Ala Leu Asn Pro Met Gly

1 5 10 15

Phe Ser Pro Leu Asp Gly Ser Lys Ser Thr Asn Glu Asn Val Ser

20 25 30

Ala Ser Thr Ser Thr Ala Lys Pro Met Val Gly Gin Leu He Phe

35 40 45

Asp Lys Phe He Lys Thr Glu Glu Asp Pro He He Lys Gin Asp

50 55 60

Thr Pro Ser Asn Leu Asp Phe Asp Phe Ala Leu Pro Gin Thr Ala

65 70 75

Thr Ala Pro Asp Ala Lys Thr Val Leu Pro He Pro Glu Leu Asp

80 85 90

Asp Ala Val Val Glu Ser Phe Phe Ser Ser Ser Thr Asp Ser Thr

95 100 105

Pro Met Phe Glu Tyr Glu Asn Leu Glu Asp Asn Ser Lys Glu Trp

110 115 120

Thr Ser Leu Phe Asp Asn Asp He Pro Val Thr Thr Asp Asp Val

125 130 135 Ser Leu Ala Asp Lys Ala He Glu Ser Thr Glu Glu Val Ser Leu 140 145 150

Val Pro Ser Asn Leu Glu Val Ser Thr Thr Ser Phe Leu Pro Thr 155 160 165

Pro Val Leu Glu Asp Ala Lys Leu Thr Gin Thr Arg Lys Val Lys 170 175 180

Lys Pro Asn Ser Val Val Lys Lys Ser His His Val Gly Lys Asp 185 190 195

Asp Glu Ser Arg Leu Asp His Leu Gly Val Val Ala Tyr Asn Arg 200 205 210

Lys Gin Arg Ser He Pro Leu Ser Pro He Val Pro Glu Ser Ser 215 220 225

Asp Pro Ala Ala Leu Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg 230 235 240

Arg Ser Arg Ala Arg Lys Leu Gin Arg Leu Lys Gin He Glu Asp 245 250 255

Lys Leu Glu Glu He Leu Ser Lys Leu Tyr His He Glu Asn Glu 260 265 270

Leu Ala Arg He Lys Lys Leu Leu Gly Glu Arg 275 280

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Arg Gly Asn

1 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

( i) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Arg Gly Gly Asn Pro Glu 1 5

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Gly Gly Asn Gin Ala 1 5

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GAACATGTCC CAACATGTTG 20 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 58 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGTCAAGTTG GGACACGTCC GGCGTCGGCT GTCGGAGGAG CTAAGTCCTG 50 ACATGTCT 58

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 234 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..234

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

GCC CCC CCG ACC GAT GTC AGC CTG GGG GAC GAG CTC CAC TTA 42 Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His Leu 1 5 10

GAC GGC GAG GAC GTG GCG ATG GCG CAT GCC GAC GCG CTA GAC 84 Asp Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp 15 20 25

GAT TTC GAT CTG GAC ATG TTG GGG GAC GGG GAT TCC CCG GGG 126 Asp Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly 30 35 40

CCG GGA TTT ACC CCC CAC GAC TCC GCC CCC TAC GGC GCT CTG 168 Pro Gly Phe Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu 45 50 55 GAT ATG GCC GAC TTC GAG TTT GAG CAG ATG TTT ACC GAT GCC 210

Asp Met Ala Asp Phe Glu Phe Glu Gin Met Phe Thr Asp Ala

60 65 70

CTT GGA ATT GAC GAG TAC GGT GGG 226 Leu Gly He Asp Glu Tyr Gly Gly

75

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His Leu Asp 1 5 10 15

Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp Phe

20 25 30

Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe

35 40 45

Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp

50 55 60

Phe Glu Phe Glu Gin Met Phe Thr Asp Ala Leu Gly He Asp Glu

65 70 75

Tyr Gly Gly

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Pro Lys Lys Lys Arg Lys Val 1 5

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 390 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Met Thr Ala Met Glu Glu Ser Gin Ser Asp He Ser Leu Glu Leu

1 5 10 15

Pro Leu Ser Gin Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro

20 25 30

Pro Glu Asp He Leu Pro Ser Pro His Cys Met Asp Asp Leu Leu

35 40 45

Leu Pro Gin Asp Val Glu Glu Phe Phe Glu Gly Pro Ser Glu Ala

50 55 60

Leu Arg Val Ser Gly Ala Pro Ala Ala Gin Asp Pro Val Thr Glu

65 70 75

Thr Pro Gly Pro Val Ala Pro Ala Pro Ala Thr Pro Trp Pro Leu

80 85 90

Ser Ser Phe Val Pro Ser Gin Lys Thr Tyr Gin Gly Asn Tyr Gly

95 100 105

Phe His Leu Gly Phe Leu Gin Ser Gly Thr Ala Lys Ser Val Met

110 115 120

Cys Thr Tyr Ser Pro Pro Leu Asn Lys Leu Phe Cys Gin Leu Val

125 130 135

Lys Thr Cys Pro Val Gin Leu Trp Val Ser Ala Thr Pro Pro Ala

140 145 150

Gly Ser Arg Val Arg Ala Met Ala He Tyr Lys Lys Ser Gin His

155 160 165 Met Thr Glu Val Val Arg Arg Cys Pro His His Glu Arg Cys Ser

170 175 180

Asp Gly Asp Gly Leu Ala Pro Pro Gin His Leu He Arg Val Glu

185 190 195

Gly Asn Leu Tyr Pro Glu Tyr Leu Glu Asp Arg Gin Thr Phe Arg

200 205 210

His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Ala Gly Ser Glu

215 220 225

Tyr Thr Thr He His Tyr Lys Tyr Met Cys Asn Ser Ser Cys Met

230 235 240

Gly Gly Met Asn Arg Arg Pro He Leu Thr He He Thr Leu GlU

245 250 255

Asp Ser Ser Gly Asn Leu Leu Gly Arg Asp Ser Phe Glu Val Arg

260 265 270

Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn

275 280 285

Phe Arg Lys Lys Glu Val Leu Cys Pro Glu Leu Pro Pro Gly Ser

290 295 300

Ala Lys Arg Ala Leu Pro Thr Cys Thr Ser Ala Ser Pro Pro Gin

305 310 315

Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Lys He Arg

320 325 330

Gly Arg Lys Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu

335 340 345

Glu Leu Lys Asp Ala His Ala Thr Glu Glu Ser Gly Asp Ser Arg

350 355 360

Ala His Ser Ser Tyr Leu Lys Thr Lys Lys Gly Gin Ser Thr Ser

365 370 375

Arg His Lys Lys Thr Met Val Lys Lys Val Gly Pro Asp Ser Asp

380 385 390 (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CCCGAACATG TCCCAACATG TTGGGG 26

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TCGAGCATGT TCGAGCATGT TCGAGCATGT 30

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CCGGGCATGT CCGGGCATGT CCGGGCATGT 30 (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TATGGGCATG TCCTATATAT ATGCGTATAT ATATGGGCAT GTCCTAT 47

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1215 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GAATTCAACC AGCAGCCTCC CGCGACCATG GAGGAGCCGC AGTCAGATCC 50

TAGCGTCGAG CCCCCTCTGA GTCAGGAAAC ATTTTCAGAC CTATGGAAAC 100

TACTTCCTGA AAACAACGTT CTGTCCCCCT TGCCGTCCCA AGCAATGGAT 150

GATTTGATGC TGTCCCCGGA CGATATTGAA CAATGGTTCA CTGAAGACCC 200

AGGTCCAGAT GAAGCTCCCA GAATGCCAGA GGCTGCTCCC CCCGTGGCCC 250

CTGCACCAGC AGCTCCTACA CCGGCCGCCC CTGCACCAGC CCCCTCCTGG 300

CCCCTGTCAT CTTCTGTCCC TTCCCAGAAA ACCTACCAGG GCAGCTACGG 350

TTTCCGTCTG GGCTTCTTGC ATTCTGGGAC AGCCAAGTCT GTGACTTGCA 400

CGTACTCCCC TGCCCTCAAC AAGATGTTTT GCCAACTGGC GAAGACCTGC 450

CCTGTGCAGC TGTGGGTTGA TTCCACACCC CCGCCCGGCA CCCGCGTCCG 500

CGCCATGGCC ATCTACAAGC AGTCACAGCA CATGACGGAG GTTGTGAGGC 550

GCTGCCCCCA CCATGAGCGC TGCTCAGATA GCGATGGTCT GGCCCCTCCT 600 CAGCATCTTA TCCGAGTGGA AGGAAATTTG CGTGTGGAGT ATTTGGATGA 650

CAGAAACACT TTTCGACATA GTGTGGTGGT ACCCTATGAG CCGCCTGAGG 700

TTGGCTCTGA CTGTACCACC ATCCACTACA ACTACATGTG TAACAGTTCC 750

TGCATGGGCG GCATGAACCG GAGGCCCATC CTCACCATCA TCACACTGGA 800

AGACTCCAGT GGTAATCTAC TGGGACGGAA CAGCTTTGAG GTGCGTGTTT 850

GTGCCTGTCC TGGGAGAGAC CGGCGCACAG AGGAAGAGAA TCTCCGCAAG 900

AAAGGGGAGC CTCACCACGA GCTCCCCCCA GGGAGCACTA AGCGAGCACT 950

GCCCAACAAC ACCAGCTCCT CTCCCCAGCC AAAGAAGAAA CCACTGGATG 1000

GAGAATATTT CACCCTTCAG ATCCGCGGGC GTGAGCGCTT CGAAATGTTC 1050

CGAGAGCTGA ATGAGGCCTT GGAACTCAAG GATGCCCAGG CTGGGAAGGA 1100

GCCAGGGGGG AGCAGGGCTC ACTCCAGCCA CCTGAAGTCC AAAAAGGGTC 1150

AGTCTACCTC CCGCCATAAA AAACTCATGT TCAAGACAGA AGGGCCTGAC 1200

TCAGACTGAG TCGAC 1215

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCAGAGGAGC AAAAGCTTGA AGACAAGGTT 30

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CTTTAATCAA CCGCTTGCGA CTCAGCTGGA CTTC 34

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ATGAGGCCTT GGAAGACAAG GTTGAAGAAT TG 32

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GGGCGTC 7

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GACGCCCGC 9 (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) ( i) SEQUENCE DESCRIPTION: SEQ ID NO:26: CGAAATGTTC CGAGAGCGAA TGAAAC 26

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GTTTCATTCG CTCTCGGAAC ATTT 24

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: TATCCGCGGT AATCGTCTGA AACAGATCGA AGACAAGTTA GAAGAAATCC 50 TTTCGAAGCT CTATCACATC GAG 73 (2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 71 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GCTTCGAGAT AGTGTAGCTC TTACTCAATC GCGCGTAGTT TTTTAATAAC 50 CCACTTGCAA CTCAGCTGTT T 71

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: TTCTCCGCGG AGTGGTTTCT TCTTTGGCTG 30

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TATCCGCGGT GGAAATCCTG AACTGAAACA GATCGAAGAC AAG 43 96/16989 PC17US95/15353

90 (2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Gly Asn Pro Glu

1

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GTCATCGATG CGTTCGCCAA CTAATTTCTT 30

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: ATGAGGCCTT GGAACTCATC GATGCCCAGG CTGGG 35 (2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: TTTGTCGACT CAATCGATAC GTTCACCCAA TAATTTTTTG 40

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GAGAGCCCCA GTTACCATAA CTACTCT 27

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: ATCACGTGAT ATCACGTGAT ATCACGTGAT 30 (2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 996 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..993

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

ATG ACT GCA AAG ATG GAA ACG ACC TTC TAT GAC GAT GCC CTC 42 Met Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu 1 5 10

AAC GCC TCG TTC CTC CCG TCC GAG AGC GGA CCT TAT GGC TAC 84 Asn Ala Ser Phe Leu Pro Ser Glu Ser Gly Pro Tyr Gly Tyr 15 20 25

AGT AAC CCC AAG ATC CTG AAA CAG AGC ATG ACC CTG AAC CTG 126 Ser Asn Pro Lys He Leu Lys Gin Ser Met Thr Leu Asn Leu 30 35 40

GCC GAC CCA GTG GGG AGC CTG AAG CCG CAC CTC CGC GCC AAG 168 Ala Asp Pro Val Gly Ser Leu Lys Pro His Leu Arg Ala Lys 45 50 55

AAC TCG GAC CTC CTC ACC TCG CCC GAC GTG GGG CTG CTC AAG 210 Asn Ser Asp Leu Leu Thr Ser Pro Asp Val Gly Leu Leu Lys 60 65 70

CTG GCG TCG CCC GAG CTG GAG CGC CTG ATA ATC CAG TCC AGC 252 Leu Ala Ser Pro Glu Leu Glu Arg Leu He He Gin Ser Ser

75 80

AAC GGG CAC ATC ACC ACC ACG CCG ACC CCC ACC CAG TTC CTG 294 Asn Gly His He Thr Thr Thr Pro Thr Pro Thr Gin Phe Leu 85 90 95

TGC CCC AAG AAC GTG ACA GAT GAG CAG GAG GGG TTC GCC GAG 336 Cys Pro Lys Asn Val Thr Asp Glu Gin Glu Gly Phe Ala Glu 100 105 110

GGC TTC GTG CGC GCC CTG GCC GAA CTG CAC AGC CAG AAC ACG 378 Gly Phe Val Arg Ala Leu Ala Glu Leu His Ser Gin Asn Thr 115 120 125 CTG CCC AGC GTC ACG TCG GCG GCG CAG CCG GTC AAC GGG GCA 420 Leu Pro Ser Val Thr Ser Ala Ala Gin Pro Val Asn Gly Ala 130 135 140

GGC ATG GTG GCT CCC GCG GTA GCC TCG GTG GCA GGG GGC AGC 462 Gly Met Val Ala Pro Ala Val Ala Ser Val Ala Gly Gly Ser

145 150

GGC AGC GGC GGC TTC AGC GCC AGC CTG CAC AGC GAG CCG CCG 504 Gly Ser Gly Gly Phe Ser Ala Ser Leu His Ser Glu Pro Pro 155 160 165

GTC TAC GCA AAC CTC AGC AAC TTC AAC CCA GGC GCG CTG AGC 546 Val Tyr Ala Asn Leu Ser Asn Phe Asn Pro Gly Ala Leu Ser 170 175 180

AGC GGC GGC GGG GCG CCC TCC TAC GGC GCG GCC GGC CTG GCC 588 Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala Ala Gly Leu Ala 185 190 195

TTT CCC GCG CAA CCC CAG CAG CAG CAG CAG CCG CCG CAC CAC 630 Phe Pro Ala Gin Pro Gin Gin Gin Gin Gin Pro Pro His His 200 205 210

CTG CCC CAG CAG ATG CCC GTG CAG CAC CCG CGG CTG CAG GCC 672 Leu Pro Gin Gin Met Pro Val Gin His Pro Arg Leu Gin Ala

215 220

CTG AAG GAG GAG CCT CAG ACA GTG CCC GAG ATG CCC GGC GAG 714 Leu Lys Glu Glu Pro Gin Thr Val Pro Glu Met Pro Gly Glu 225 230 235

ACA CCG CCC CTG TCC CCC ATC GAC ATG GAG TCC CAG GAG CGG 756 Thr Pro Pro Leu Ser Pro He Asp Met Glu Ser Gin Glu Arg 240 245 250

ATC AAG GCG GAG AGG AAG CGC ATG AGG AAC CGC ATC GCT GCC 798 He Lys Ala Glu Arg Lys Arg Met Arg Asn Arg He Ala Ala 255 260 265

TCC AAG TGC CGA AAA AGG AAG CTG GAG AGA ATC GCC CGG CTG 840 Ser Lys Cys Arg Lys Arg Lys Leu Glu Arg He Ala Arg Leu 270 275 280

GAG GAA AAA GTG AAA ACC TTG AAA GCT CAG AAC TCG GAG CTG 882 Glu Glu Lys Val Lys Thr Leu Lys Ala Gin Asn Ser Glu Leu

285 290

GCG TCC ACG GCC AAC ATG CTC AGG GAA CAG GTG GCA CAG CTT 924 Ala Ser Thr Ala Asn Met Leu Arg Glu Gin Val Ala Gin Leu 295 300 305 AAA CAG AAA GTC ATG AAC CAC GTT AAC AGT GGG TGC CAA CTC 966 Lys Gin Lys Val Met Asn His Val Asn Ser Gly Cys Gin Leu 310 315 320

ATG CTA ACG CAG CAG TTG CAA ACA TTT TGA 996

Met Leu Thr Gin Gin Leu Gin Thr Phe 325 330

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 331 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

Met Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu Asn

1 5 10 15

Ala Ser Phe Leu Pro Ser Glu Ser Gly Pro Tyr Gly Tyr Ser Asn

20 25 30

Pro Lys He Leu Lys Gin Ser Met Thr Leu Asn Leu Ala Asp Pro

35 40 45

Val Gly Ser Leu Lys Pro His Leu Arg Ala Lys Asn Ser Asp Leu

50 55 60

Leu Thr Ser Pro Asp Val Gly Leu Leu Lys Leu Ala Ser Pro Glu

65 70 75

Leu Glu Arg Leu He He Gin Ser Ser Asn Gly His He Thr Thr

80 85 90

Thr Pro Thr Pro Thr Gin Phe Leu Cys Pro Lys Asn Val Thr Asp

95 100 105

Glu Gin Glu Gly Phe Ala Glu Gly Phe Val Arg Ala Leu Ala Glu

110 115 120

Leu His Ser Gin Asn Thr Leu Pro Ser Val Thr Ser Ala Ala Gin

125 130 135

Pro Val Asn Gly Ala Gly Met Val Ala Pro Ala Val Ala Ser Val

140 145 150 Ala Gly Gly Ser Gly Ser Gly Gly Phe Ser Ala Ser Leu His Ser

155 160 165

Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe Asn Pro Gly Ala

170 175 180

Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala Ala Gly Leu

185 190 195

Ala Phe Pro Ala Gin Pro Gin Gin Gin Gin Gin Pro Pro His His

200 205 210

Leu Pro Gin Gin Met Pro Val Gin His Pro Arg Leu Gin Ala Leu

215 220 225

Lys Glu Glu Pro Gin Thr Val Pro Glu Met Pro Gly Glu Thr Pro

230 235 240

Pro Leu Ser Pro He Asp Met Glu Ser Gin Glu Arg He Lys Ala

245 250 255

Glu Arg Lys Arg Met Arg Asn Arg He Ala Ala Ser Lys Cys Arg

260 265 270

Lys Arg Lys Leu Glu Arg He Ala Arg Leu Glu Glu Lys Val Lys

275 280 285

Thr Leu Lys Ala Gin Asn Ser Glu Leu Ala Ser Thr Ala Asn Met

290 295 300

Leu Arg Glu Gin Val Ala Gin Leu Lys Gin Lys Val Met Asn His

305 310 315

Val Asn Ser Gly Cys Gin Leu Met Leu Thr Gin Gin Leu Gin Thr

320 325 330

Phe

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 996 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..993

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

75 80

GGC TTC GTG CGC GCC CTG GCC GAA CTG CAC AGC CAG AAC ACG 378 Gly Phe Val Arg Ala Leu Ala Glu Leu His Ser Gin Asn Thr 115 120 125

CTG CCC AGC GTC ACG TCG GCG GCG CAG CCG GTC AAC GGG GCA 420 Leu Pro Ser Val Thr Ser Ala Ala Gin Pro Val Asn Gly Ala 130 135 140

145 150 GGC AGC GGC GGC TTC AGC GCC AGC CTG CAC AGC GAG CCG CCG 504 Gly Ser Gly Gly Phe Ser Ala Ser Leu His Ser Glu Pro Pro 155 160 165

215 220

TCC AAG TGC CGA AAA AGG AAG CTG GAG CGT TTG GCC CGT ATC 840 Ser Lys Cys Arg Lys Arg Lys Leu Glu Arg Leu Ala Arg He 270 275 280

GAG GAA AAA CTG AAA ACC ATC AAA GCT CAA TTG TCG GAG ATC 882 Glu Glu Lys Leu Lys Thr He Lys Ala Gin Leu Ser Glu He

285 290

GCG TCC ACG TTG .AAC ATG ATC CGT GAA CAG TTG GCA CAG ATC 924 Ala Ser Thr Leu Asn Met He Arg Glu Gin Leu Ala Gin He 295 300 305

AAA CAG AAA CTG ATG AAC CAC GTT AAC AGT GGG TGC CAA CTC 966 Lys Gin Lys Leu Met Asn His Val Asn Ser Gly Cys Gin Leu 310 315 320

ATG CTA ACG CAG CAG TTG CAA ACA TTT TGA 996

Met Leu Thr Gin Gin Leu Gin Thr Phe 325 330 (2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 331 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

Met Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu Asn

1 5 10 15

Ala Ser Phe Leu Pro Ser Glu Ser Gly Pro Tyr Gly Tyr Ser Asn

20 25 30

Pro Lys He Leu Lys Gin Ser Met Thr Leu Asn Leu Ala Asp Pro

35 40 45

Val Gly Ser Leu Lys Pro His Leu Arg Ala Lys Asn Ser Asp Leu

50 55 60

Leu Thr Ser Pro Asp Val Gly Leu Leu Lys Leu Ala Ser Pro Glu

65 70 75

Leu Glu Arg Leu He He Gin Ser Ser Asn Gly His He Thr Thr

80 85 90

Thr Pro Thr Pro Thr Gin Phe Leu Cys Pro Lys Asn Val Thr Asp

95 100 105

Glu Gin Glu Gly Phe Ala Glu Gly Phe Val Arg Ala Leu Ala Glu

110 115 120

Leu His Ser Gin Asn Thr Leu Pro Ser Val Thr Ser Ala Ala Gin

125 130 135

Pro Val Asn Gly Ala Gly Met Val Ala Pro Ala Val Ala Ser Val

140 145 150

Ala Gly Gly Ser Gly Ser Gly Gly Phe Ser Ala Ser Leu His Ser

155 160 165

Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe Asn Pro Gly Ala

170 175 180

Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala Ala Gly Leu

185 190 195 Ala Phe Pro Ala Gin Pro Gin Gin Gin Gin Gin Pro Pro His His 200 205 210

Leu Pro Gin Gin Met Pro Val Gin His Pro Arg Leu Gin Ala Leu 215 220 225

Lys Glu Glu Pro Gin Thr Val Pro Glu Met Pro Gly Glu Thr Pro 230 235 240

Pro Leu Ser Pro He Asp Met Glu Ser Gin Glu Arg He Lys Ala 245 250 255

Glu Arg Lys Arg Met Arg Asn Arg He Ala Ala Ser Lys Cys Arg 260 265 270

Lys Arg Lys Leu Glu Arg Leu Ala Arg He Glu Glu Lys Leu Lys 275 280 285

Thr He Lys Ala Gin Leu Ser Glu He Ala Ser Thr Leu Asn Met 290 295 300

He Arg Glu Gin Leu Ala Gin He Lys Gin Lys Leu Met Asn His 305 310 315

Val Asn Ser Gly Cys Gin Leu Met Leu Thr Gin Gin Leu Gin Thr 320 325 330

Phe

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 88 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: TATCCGCGGT AATCGTTTGG CCCGTATCGA GGAAAAACTG AAAACCATCA 50 AAGCTCAATT GTCGGAGATC GCGTCCACGT TGAACATG 88 (2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: TTTGTCGACT TATTAACGTT CACCCATCAG TTTCTGTTTG ATCTGTGCCA 50 ACTGTTCACG GATCATGTTC AACGTGGACG C 81

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CGACAATTGA GCTTTGTTGG TTTTCAGTTT TTCCTC 36

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: CCCGAACATG TCCTGTTGGG G 21 (2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 61 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GGCTGGTCAA GTTGGGACAC GTCCGGCGTC GGCTGTCGGA GGAGCTAAGT 50 CCTGACACCA G 61

Claims

WHAT IS CLAIMED IS:

1. A modified p53 protein which is a chimeric p53 protein comprising a native p53 sequence and a selected heterologous tetramerization sequence which forms homo¬ tetramers, said modification providing said protein with the inability to hetero-oligomerize with wild-type p53 or tumor-derived mutant p53 proteins and not interfering with the native tumor-suppression function of the protein.

2. The modified p53 protein according to claim 1 wherein the chimeric p53 protein comprises a selected p53 sequence in which the native p53 tetramerization domain is wholly or partially disrupted.

3. The modified p53 protein according to claim 1, having the DNA binding and transcriptional functions characteristic of wild-type p53.

4. The modified p53 protein according to claim 1 wherein said heterologous tetramerization domain is selected from the group consisting of the lac represser tetramerization domain, a modified leucine zipper sequence containing isoleucines at positions d of the coiled coil and leucines at positions a and characterized by the ability to form tetramers, and variants thereof.

5. The modified p53 protein according to claim 4 wherein the leucine zipper is derived from a protein from the group consisting of Jun, Max, GCN4, C-Myc, C-Fos, and C/EBP.

6. The modified p53 protein according to claim 5 wherein the leucine zipper sequence is a modification of amino acid residues 249 to 281 SEQ ID NO: 6 of GCN4 or a fragment thereof.

7. The modified p53 protein according to claim 2 wherein said p53 sequence contains a mutation that interrupts oligomerization activity of the native sequence spanning amino acids 322-355 of SEQ ID NO: 2, said mutation selected from the group consisting of a deletion, insertion and amino acid substitution.

8. The modified p53 protein according to claim 7 wherein said mutation is present within the p53 sequence between amino acids 322-355 of SEQ ID NO:2.

9. The modified p53 protein according to claim 8 wherein said mutation is present within the p53 sequence between amino acids 290-393 of SEQ ID NO:2.

10. The modified p53 protein according to claim 7 wherein said mutation is a deletion selected from the group consisting of amino acids 301-393, amino acids 324-393, amino acids 326-393 and amino acids 335-393 of SEQ ID NO: 2 and fragments thereof.

11. The modified p53 protein according to claim 7 wherein said mutation is an amino acid substitution at one or more of the amino acid residues 334, 337, 341 and 344 of SEQ ID NO: 2.

12. The modified p53 protein according to claim 7 wherein the heterologous tetramerization domain is inserted at a site between amino acids 1-90 or 290-393 of SEQ ID NO:2.

13. The modified p53 protein according to claim 12 wherein said site is located after a p53 amino acid residue selected from the group consisting of residues 300, 323, 325, 334, 346, 347 and 356 of SEQ ID NO: 2.

14. The modified p53 protein according to claim 1, further comprising an amino acid linker between said p53 sequence and said heterologous tetramerization domain.

15. The modified p53 protein according to claim 14 wherein said linker is selected from the group consisting of Glu, Asn, He, Gly-Asn, Asn-Arg, Arg-Gly-Asn SEQ ID NO: 7, Gly-Gly-Asn-Gln-Ala SEQ ID NO: 9, Arg-Gly-Gly-Asn-Pro-Glu SEQ ID NO: 8 and Gly-Asn-Pro-Glu SEQ ID NO: 32.

16. The modified p53 protein according to claim 1 selected from the group consisting of:

(a) a p53 sequence spanning from N-terminus to C- terminus residues 1-334 of SEQ ID NO: 2, an Asn linker, and a heterologous sequence spanning residues 249-281 of SEQ ID NO: 6 containing isoleucines at positions d of the coiled coil and leucines at positions a;

(b) a p53 sequence spanning from N-terminus to C- terminus residues 1-334 of SEQ ID NO: 2, a Gly-Asn-Pro-Glu SEQ ID NO: 32 linker, and a heterologous sequence spanning residues 250-281 of SEQ ID NO: 6 containing isoleucines at positions d of the coiled coil and leucines at positions a;

(c) a p53 sequence spanning from N-terminus to C- terminus residues 1-325 of SEQ ID NO: 2, an Arg-Gly-Asn SEQ ID NO: 7 linker, and the heterologous sequence of (a) above; (d) a p53 sequence spanning from N-terminus to C- terminus residues 1-325 of SEQ ID NO: 2, an Arg-Gly-Gly-Asn-Pro-Glu SEQ ID NO: 8 linker, and the heterologous sequence of (b) above;

(e) a p53 sequence spanning from N-terminus to C- terminus residues 1-323 of SEQ ID NO: 2, an Arg-Gly-Asn SEQ ID NO: 7 linker, and the heterologous sequence of (a) above;

(f) a p53 sequence spanning from N-terminus to C- terminus residues 1-323 of SEQ ID NO: 2, an aArg-Gly-Gly-Asn-Pro-Glu SEQ ID NO: 8 linker, and the heterologous sequence of (b) above; and

(g) a p53 sequence spanning from N-terminus to C- terminus residues 1-300 of SEQ ID NO: 2, a Gly-Gly-Asn-Gln-Ala SEQ ID NO: 9 linker, and the heterologous sequence of (b) above.

17. The modified p53 protein according to claim 1 selected from the group consisting of:

(a) a p53 sequence spanning from N-terminus to C- terminus residues 1-325 of SEQ ID NO: 2, an Arg-Gly-Asn SEQ ID NO: 7 linker, a heterologous sequence spanning residues 249-281 of SEQ ID NO: 6 containing isoleucines at positions d of the coiled coil and leucines at positions a, an He linker, and a p53 sequence spanning residues 352-393 of SEQ ID NO: 2;

(b) a p53 sequence spanning from N-terminus to C- terminus residues 1-325 of SEQ ID NO: 2, an Arg-Gly-Gly-Asn-Pro-Glu SEQ ID NO: 8 linker, a heterologous sequence spanning residues 250-281 of SEQ ID NO: 6 containing isoleucines at positions d of the coiled coil and leucines at positions a, an He linker, and a p53 sequence spanning residues 352-393 of SEQ ID NO: 2;

(c) a p53 sequence spanning from N-terminus to C- terminus residues 1-323 of SEQ ID NO: 2, an Arg-Gly-Asn SEQ ID NO: 7 linker, the heterologous sequence of (a) above, an He linker, and a p53 sequence spanning residues 352-393 of SEQ ID NO: 2;

(d) a p53 sequence spanning from N-terminus to C- terminus residues 1-323 of SEQ ID NO: 2, an Arg-Gly-Gly-Asn-Pro-Glu SEQ ID NO: 8 linker, the heterologous sequence of (b) above, an He linker, and a p53 sequence spanning residues 352-393 of SEQ ID NO: 2;

(e) a p53 sequence spanning from N-terminus to C- terminus residues 252-393 of SEQ ID NO: 2, an He linker, and a heterologous sequence of (a) above, and a p53 sequence spanning residues 352-393 of SEQ ID NO:2; and

(f) a p53 sequence spanning from N-terminus to C- terminus residues 1-334 of SEQ ID NO: 2, a Gly-Asn-Pro-Glu SEQ ID NO: 32 linker, and a heterologous sequence of (b) above, and a p53 sequence spanning residues 352-393 of SEQ ID NO:2.

18. The modified p53 protein according to claim 1, wherein said p53 sequence contains a mutation within amino acids 335-348 of SEQ ID NO:2, said mutation selected from the group consisting of a deletion, insertion and single amino acid substitution.

19. The modified p53 protein according to claim 18 wherein said mutation is present within amino acids 290- 393 of SEQ ID NO:2, provided that the sequence spanning amino acids 328-334 of SEQ ID NO:2 is left intact.

20. The modified p53 protein according to claim 18 wherein said mutation is a deletion selected from the group consisting of amino acids 347-393, 348-393 of SEQ ID NO:2 and fragments thereof. 21. The modified p53 protein according to claim 18 wherein said mutation is an amino acid substitution at amino acid residue 344 of SEQ ID NO:2.

22. The modified p53 protein according to claim 18 wherein the heterologous tetramerization domain is inserted at a site between amino acids 335-393 of SEQ ID NO:2.

23. The modified p53 protein according to claim 22 wherein said site is located after a p53 amino acid residue selected from the group consisting of residues 346 and 347 of SEQ ID NO:2.

24. The modified p53 protein according to claims 1 or 2 having restricted DNA binding specificity, said protein comprising a p53 sequence with a modification between the DNA binding and tetramerization domains, which modification alters the distance between said domains.

25. The modified p53 protein according to claim 24 wherein said p53 modification comprises a deletion or insertion between amino acids 290-327 of SEQ ID NO: 2.

26. The modified p53 protein according to claim 25 wherein said deletion involves more than 22 amino acids.

27. The modified p53 protein according to claim 25 wherein said deletion is selected from the group consisting of a single deletion of amino acids 300-327 of SEQ ID NO: 2 and a double deletion of amino acids 290-297 and 300-321 of SEQ ID NO: 2. 28. The modified p53 protein according to claim 1 or 2 wherein said p53 sequence comprises a modification to the native p53 amino acid region spanning residues 1-90 of SEQ ID NO: 2.

29. The modified p53 protein according to claim 28 wherein said modification comprises the replacement of all or a portion of said region with a heterologous transactivation domain.

30. The modified p53 protein according to claim 29 wherein said heterologous transactivation domain comprises amino acids 402-479 of the herpes simplex virus protein VP16 (amino acids 1-78 of SEQ ID NO: 13) .

31. The modified p53 protein according to claim 1 or 2 wherein said p53 sequence contains additional nuclear localization signals, said signals inserted in any site in said p53 sequence that would not affect tumor suppressor function.

32. The modified p53 protein according to claim 1 or 2 wherein said p53 sequence comprises a selected homologous sequence from a non-human p53 sequence in place of the corresponding human p53 sequence.

33. The modified p53 protein according to claim 31 wherein the non-human p53 sequence replaces amino acids 1-90 of SEQ ID NO: 2, said substitution abrogating inhibitory interactions with the cellular Mdm2 protein.

34. A nucleotide sequence encoding a modified p53 protein of any of claims 1-33. 35. A vector comprising the nucleotide sequence of claim 34 under the control of regulatory sequences capable of targeting to a selected cell and directing expression of the protein encoded by said sequence in said cell.

36. A pharmaceutical composition comprising a modified p53 protein of any of claims 1 to 33 in a pharmaceutically acceptable carrier.

37. A pharmaceutical composition comprising a nucleotide sequence of claim 34 or a vector of claim 35 in a pharmaceutically acceptable carrier.

38. The use of a modified p53 protein of any of claims 1 to 33 in preparation of a medicament.

39. The use of a nucleotide sequence of claim 34 or a vector of claim 35 in preparation of a medicament.

AMENDED CLAIMS

[received by the International Bureau on 13 May 1996 (13.05.96); original claim 17 amended; remaining claims unchanged Opage)]'

SEQ ID NO: 7 linker, the heterologous sequence of (a) above, an He linker, and a p53 sequence spanning residues 352-393 of SEQ ID NO: 2;

(d) a p53 sequence spanning from N-terminus to C- terminus residues 1-323 of SEQ ID NO: 2 , an Arg-Gly-Gly-Asn-Pro-Glu SEQ ID NO: 8 linker, the heterologous sequence of (b) above, an He linker, and a p53 sequence spanning residues 352-393 of SEQ ID NO: 2;

(e) a p53 sequence spanning from N-terminus to c- terminus residues 1-334 of SEQ ID NO: 2, an Asn linker, the heterologous sequence of (a) above, an He linker and a p53 sequence spanning residues 352-393 of SEQ ID NO: 2; and

(f) a p53 sequence spanning from N-terminus to c- terminus residues 1-334 of SEQ ID NO: 2, a Gly-Asn-Pro-Glu SEQ ID NO: 32 linker, the heterologous sequence of (b) above, an He linker and a p53 sequence spanning residues 352-393 of SEQ ID NO: 2.

18. The modified p53 protein according to claim l, wherein said p53 sequence contains a mutation within amino acids 335-348 of SEQ ID NO: 2, said mutation selected from the group consisting of a deletion, insertion and single amino acid substitution.

19. The modified p53 protein according to claim 18 wherein said mutation is present within amino acids 290- 393 of SEQ ID NO: 2, provided that the sequence spanning amino acids 328-334 of SEQ ID NO: 2 is left intact.

20. The modified p53 protein according to claim 18 wherein said mutation is a deletion selected from the group consisting of amino acids 347-393, 348-393 of SEQ ID NO: 2 and fragments thereof.