AU1161497A - Vertebrate deltex proteins, nucleic acids, and antibodies, and related methods and compositions - Google Patents

Description

VERTEBRATE DELTEX PROTEINS, NUCLEIC ACIDS, AND ANTIBODmS, AND RELATED METHODS AND COMPOSITIONS

1. INTRODUCTION

⁵ The present invention relates to vertebrate deltex genes and their encoded protein products, as well as derivatives and analogs thereof. The invention further relates to production of vertebrate Deltex proteins, derivatives and antibodies. Related therapeutic compositions and methods of therapy and diagnosis are also provided. 0

2. BACKGROUND OF THE INVENTION

In Drosophila melanogaster , the so called "Notch group" of genes has been implicated in events crucial for the correct developmental choices of a wide variety of precursor cells (for review, see Fortini and Artavams-Tsakonas, 1993, Cell 75: 1245-1247;

Artavams-Tsakonas and Simpson, 1991 , Trends Genet. 7:403-408). The accumulated genetic and molecular studies suggest that these genes encode elements of a cell communication mechanism which includes cell surface, cytoplasmic, and nuclear components. _Q Very little is known about the mechanisms underlying cell fate choices in higher organisms such as vertebrates; a knowledge of such mechanisms could provide insights into pathologies associated with abnormal differentiation events Thus, a need exists in the art to obtain and characterize the human members of the "Notch group" of genes, including deltex. since these genes appear to play crucial roles in the determination of cell fate

Numerous developmental genetic studies in recent years have shown that the Notch locus plays a central role in regulative events influencing cell fate decisions in Drosophila in a very broad spectrum of developing tissues (reviewed in Artavams-Tsakonas 0 and Simpson, 1991 , Trends Genet. 7:403-408; and in Artavams-Tsakonas et al. , 1991 , Ann.

Rev. Cell Biol. 7.427-452). This pleiotropy of Notch function is revealed by mutations affecting all stages of development and a variety of tissues (e.g. , Welshons, 1965. Science

150: 1122-1229, Welshons, 1971 , Genetics 68:259-268; Shellenbarger and Mohler. 1978,

Dev Biol 62:432-446). A dramatic illustration of Notch function is seen in the development 5 of the embryonic nervous system, whereby loss of function mutations cause the misroutmg of epithelial precursor cells into a neural developmental pathway and result in what has been termed a 'neurogenic' phenotype (Poulson, 1937, Proc. Natl. Acad. Sci. USA, 23: 133-137; Lehman et al., 1983, Roux's Arch. Dev. Biol. 192:62-74).

In attempts to understand the molecular contexts by which the Notch protein communicates signals from the cell surface to the nucleus to effect changes in cell fate,

5 genetic means have been used to identify loci that interact phenotypically with various Notch alleles. These genetic studies led to the definition of a small group of interacting loci, which has been operationally termed the 'Notch group' (Artavams-Tsakonas and Simpson, 1991 , Trends Genet. 7:403-408). The other members of the Notch group are deltex (Xu and 0 Artavanis-Tsakonas, 1991 , Genetics 126:665-677), Enhancer of (split) [E(spl)] (Knust et al. , 1987, EMBO J. 6:4113-4123; Hartley et al. , 1988, Cell 55:785-795; Preiss et al. , 1988, EMBO J. 7:3917-3927. Klambt et al. , 1989, EMBO J. 8:203-210), and mastermind (mam) (Smoller et al., 1990, Genes Dev. 4: 1688-1700). mastermind, Hairless (H). the Enhancer of (split), and Suppressor of Hairless (Su(H)) encode nuclear proteins (Smoller et al.. 1990, Genes Dev. 4: 1688-1700, Bang et al. , 1992 Genes Dev. 6: 1752-1769; Maier et al. , 1992, Mech Dev. 38: 143-156; Delidakis et al. , 1991 , Genetics 129:803-823; Schrons et al. , 1992, Genetics 132:481 -503; Furukawa et al. , 1991 , J. Biol Chem. 266:23334-23340, Furukawa et al, 1992, Cell 69: 1 191-1197; Schweisguth et al. , 1992, Cell 69: , 1 199-1212). deltex ° mutations suppress the pupal lethality conferred by certain heteroallelic combinations of the Abruptex class of Notch alleles (Xu et al. , 1990, Genes Dev. 4:464-475) From this same genetic screen, the genes Delta and mastermind were also identified, both of which belong to the same 'neurogenic' class of genes as Notch because of the similar mutant phenotypes _c they produce Moreover, subsequent analysis has shown that alleles of deltex exhibit genetic interactions with those of Delta, mastermind, Hairless, and Su(H), a further suggestion of functional links among these loci (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665- 677).

The manner by which Notch is thought to influence determinative events is 0 indirect, that is, not through the direct specification of cellular fates. Instead, recent experimental studies (Coffman et al, 1993, Cell 73:659-671 ; Fortini et al, Nature, in press) indicate that Notch activity delays differentiation, and in this manner renders precursor cells competent to receive and/or interpret any number of specific developmental cues (Cagan and 5 Ready, 1989, Genes Dev. 3: 1099-1112). In loss of function mutants, this inhibition is lost and cells assume default pathways of differentiation. For example, during the development of the Drosophila nervous system, cells that normally would become epidermis instead adopt a neural fate in the absence of Notch function However, a saiient feature of Notch activity is its pleiotropy Notch is required for the proper specification of many other cell types, including those of the compound eye (Cagan and Ready, 1989, Genes Dev. 3.1099-1 112),

5 ovary (Ruohola et al., 1991, Cell 66:433-449; Xu et al., 1992, Development 115.913-922), and mesoderm (Corb et al, 1991, Cell 67:311-323). Similarly, the widespread expression patterns exhibited by vertebrate Notch cognates suggest also a broad-based functional role in these species (Coffrnan et al, 1993, Cell 73:659-671; Coffman et al., 1990, Science

10 249 1438-1441; Weinmaster et al., 1991, Development 113: 199-205, Weinmaster et al., 1992, Development 116:931-941; Kopan and Weintraub, 1993, J Cell Biol. 121 :631-641; Franco del Amo et al., 1992, Development 115:737-744, Ellisen et al., 1991 , Cell 66.649- 661; Stifani et al., 1992, Nature Genetics 2- 119-127).

Notch homologs have been isolated from a variety of vertebrate species and have been shown to be remarkably similar to their Drosophila counterpart in terms of structure, expression pattern and ligand binding properties (Rebay et al., 1991, Cell 67:687-699; Coffrnan et al., 1990, Science 249: 1438-1441, Ellisen et al, 1991 , Cell 66-649-661 ; Weinmaster et al. , 1991 , Development 113- 199-205) Two human Notch

²⁰ homologs have been isolated (PCT Publication No. WO 92/19737 dated November 12, 1992), termed hN and TAN-1. A human Notch (TAN-1) malfunction has been associated with a lymphatic cancer (Ellisen et al., 1991, Cell 66-649-661).

Notch encodes a large, structurally-complex transmembrane protein, _rr consistent with an involvement in cell-cell communication (Wharton et al , 1985, Cell 43:567-581 ; Kidd et al., 1986, Mol. Cell. Biol. 6:3094-3108). Notch has an extracellular domain containing 36 tandem EGF-like repeats and 3 Notch/hnll repeats The intracellular domain bears several common structural motifs including 6 cdclO/SW16/ankyrιn repeats ("ANK" repeats) Lux et al., 1990, Nature 344:36-42; Breeden and Nasmyth, 1987, Nature

³⁰ 329:651-654; Michaely and Bennett, 1992, Trends Cell Biol 2.127-129, Blank et al . 1992, Trends Biochem. Sci. 17: 135-140; Bennett, 1992, J. Biol Chem. 267.8703-8706)), a polyglutamine stretch known as 'opa', and a PEST motif (Stifani et al , 1992, Nature Genetics 2: 119-127). The remarkable degree to which these motifs have been conserved in

35 homologs isolated from mice (Weinmaster et al., 1991, Development 1 13.199-205,

Weinmaster et al , 1992, Development 1 16:931-941, Kopan and Weintraub, 1993, J Cell Biol. 121 :631-641), rats (Kopan and Weintraub, 1993, J. Cell Biol. 121 :631-641 ; Franco del Amo et al., 1993, Genomics 15:259-264), humans (Ellisen et al., 1991, Cell 66:649- 661; Stifani et al., 1992, Nature Genetics 2: 119-127; PCT Publication No WO 92/19737 dated November 12, 1992), and Xenopus (Coffrnan et al. 1993, Cell 73:659-671 ; Coffrnan

5 et al., 1990, Science 249: 1438-1441) implies that they will have a common biochemical mode of action. In particular, ANK repeats, which constitute the most conserved region

( - 70% ammo acid identity) between Notch and its vertebrate counterparts (Stifani et al.,

1992, Nature Genetics 2: 119-127), are thought to mediate protem-protein interactions among 0 diverse groups of proteins, including those involved in signal transduction processes and cytoskeletal interactions (Lux et al., 1990, Nature 344:36-42; Breeden and Nasmyth. 1987,

Nature 329:651-654; Michaely and Bennett, 1992, Trends Cell Biol. 2: 127-129, Blank et al. , 1992, Trends Biochem. Sci. 17: 135-140, Bennett, 1992, J. Biol. Chem. 267:8703-

8706). Indeed, Rebay et al. (1993, Cell 74.319-329) have recently demonstrated that the 15

ANK repeats are crucial for Notch-mediated signaling events. Both EGF- ke repeats and ankyrtn motifs are found in a variety of proteins known to mteract with other protein molecules Indeed, evidence has shown a direct interaction between Notch and the products of the Delta and Serrate loci, which also encode transmembrane proteins containing EGF- 0 hke repeats (Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699)

In Drosophila, it has been demonstrated mat dominant 'activated' phenorypes result from in vivo overexpression of a Notch protein lacking most extracellular, ligand- binding sequences, while 'dominant-negative' phenorypes result from overexpression of a

» _ protein lacking most intracellular sequences (Rebay et al , 1993, Cell 74:319-329)

25

In Drosophila, Deltex has been demonstrated to play a critical role in development and other physiological processes, in particular, in the signaling pathway of Notch which is involved in cell fate (differentiation) determination We have demonstrated through expression studies conducted in cultured Drosophila cells, in yeast, and m the

30 imaginal wing disc that Drosophila Deltex mediates the intracellular portion of the signal transduction cascade involved in Notch function (Diedeπch et al., 1994, Development 120.473-481) These studies show that Drosophila Deltex is localized within the cytoplasm, that it is a protein of unique sequence, that it displays homotypic interactions, and that it 35 directly physically interacts with the Drosophila Notch intracellular ANK repeats. Additionally, we have demonstrated that Drosophila Deltex directly interacts with the ANK repeats of human Notch.

The ANK repeat motif is shared by many proteins and has been implicated in protein-protein interactions (Lux et al., 1990, Nature 344:36-42, Thompson et al., 1991,

Science 253:762-768, reviewed in Bennett, 1992, J. Biol. Chem. 267:8703-8706. Blank et al., 1992, Trends Biochem. Sci. 17: 135-140, Rebay et al , 1993, Cell 74:319-329).

Moreover, an in vivo functional analysis of various truncated forms of Notch has implicated these ANK repeats in downstream signaling events and that dominant 'activated' phenotypes result from in vivo overexpression of a Notch protein lacking most extracellular, ligand binding sequences, while 'dominant negative' phenotypes result from overexpression of a protein lacking most intracellular sequences (Rebay et al. , 1993, Cell, 74:319-329). Furthermore, deltex displays genetic interactions with Notch and Delta, both transmembrane proteins, and with mastermind, a nuclear localized protein (Smoller et al., 1990, Genes Dev. 4: 1688-1700). This makes deltex the first identified cytoplasmic component of the Notch group of interacting loci.

We have also subsequently demonstrated that a fragment mostly composed of the ankyrin repeats of the Notch protein mediate molecular interactions between Notch and the Su(H) protein (Fortini et al., 1994, Cell 79:273-282). The Drosophila Su(H) gene encodes a protein of 594 amino acids and binds to the promoters of several viral and cellular genes and interacts directly with a viral transactivator protein termed Epstein-Barr virus nuclear antigen 2 (EBNA2), which enables a virus to subvert the normal program of B cell differentiation (Schweisguth, F. , et al, 1992, Cell 69: 1 199; Furukawa T. , et al.. 1991 , J. Biol. Chem. 266:23334). Genetic and molecular studies suggest that Deltex and Delta may act in concert to multimerize Notch proteins and to interfere with the cytoplasmic retention of Su(H) by Notch, thus activating the Notch signaling pathway (Diederich, J. , et al. , 1994, Development 120:473; Fortini, M., et al. , 1994, Cell, 79:273; Matsuno, K. , et al., unpublished, Artavanis-Tsakonas et al. , 1995, Science, 268:225-232). This pathway is believed to control nuclear events in order to influence the progression of uncommitted cells to a more differentiated state. Three loci encoding putative nuclear proteins Hairless, Enhancer of split, and mastermind, have been implicated in these nuclear events. Despite the cloning of a Drosophila deltex gene (See PCT Publication WO 95/19770 published July 27, 1995), no vertebrate deltex gene had been obtained prior to the present mvenuon.

Citation of a reference herein shall not be construed as an admission that

5 such reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention relates to nucleotide sequences of vertebrate deltex

10 genes, and ammo acid sequences of the encoded vertebrate Deltex protems. The mvennon further relates to fragments and other deπvatives, and analogs, of vertebrate Deltex proteins, as well as antibodies thereto Nucleic acids encoding such fragments or derivatives are also within the scope of the invention Production of the foregoing proteins and derivatives. e.g , bv recombinant methods, is provided. 15

In a specific embodiment, the invention relates to human deltex nucleic acids and proteins

In another specific embodiment, the invention relates to mammalian deltex nucleic acids and proteins

20 In specific embodiments, the invention relates to vertebrate Deltex protein derivatives and analogs of the invention which are functionally active, or which comprise one or more domains of a vertebrate Deltex protein, including but not limited to the SH3- bmding domains, πng-H2-Zmc fingers, domains which mediate binding to Notch or to a _ ,. Notch dem ative containing Notch cdcl0/SW16/ankyπn ("ANK") repeats, or combination of the foregoing.

The present invention also relates to therapeutic and diagnostic methods and compositions based on vertebrate Deltex proteins and nucleic acids The invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic

3 0 compound of the invention. Such therapeutic compounds (termed herein "Therapeutics") include vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof, antibodies thereto; nucleic acids encoding the vertebrate Deltex proteins, analogs, or derivatives, and vertebrate deltex antisense nucleic acids In a preferred embodiment, a 3 Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state. In other specific embodiments, a Therapeutic of the invention is administered to treat a nervous system disorder or to promote tissue regeneration and repair.

In one embodiment, Therapeutics which antagonize, or inhibit, vertebrate

Notch and/or Deltex function (hereinafter "Antagonist Therapeutics") are administered for therapeutic effect. In another embodiment, Therapeutics which promote vertebrate Notch and/or Deltex function (hereinafter "Agonist Therapeutics") are administered for therapeutic effect.

Disorders of cell fate, in particular hyperproliferative (e.g. , cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of vertebrate Notch and/or Deltex protein can be diagnosed by detecting such levels, as described more fully infra.

In a preferred aspect, a Therapeutic of the invention is a protein consisting of at least a fragment (termed herein "adhesive fragment") of vertebrate Deltex which mediates binding to a Notch protein or a fragment thereof.

The invention also provides methods of inactivating Notch function in a cell, methods of identifying a compound that inhibits or reduces the binding of a vertebrate

Deltex protein to a Notch protein, and methods of expanding non-terminally differentiated cells.

4. DESCRIPTION OF THE FIGURES

Figure 1A-F. Nucleotide sequence (SEQ ID NO: l) and deduced amino acid sequence (SEQ ID NO:2) of Drosophila deltex cDNA.

Figure 2A-C. Composite nucleotide sequence (SEQ ID NO: 11) derived from the cDNA (nucleotide 1 to 2547), and deduced amino acid sequence (SEQ ID NO: 12) of the human deltex locus. The predicted amino acid sequence is depicted below the DNA sequence. The symbol: * designates the start of T05200 and $ the end of T05200. Core H and C residues in Rtng-H2-zinc finger are shown by underlining. PCR primers hdx-1 to 4

(SEQ ID NO:26), (SEQ ID NO:27), (SEQ ID NO:28), and (SEQ ID NO:29), respectively, are indicated in bold. X and N represent amino acid residues and nucleotides, respectively, not yet determined. Figure 3. Aligned amino acid sequences of human Deltex (SEQ ID NO: 12) and Drosophila Deltex (SEQ ID NO:2) proteins. Those positions at which residues are identical are shaded. Sites in which amino acids are chemically similar are boxed.

Figure 4A-B. Amino acid sequence ol Drosophila Deltex (SEQ ID NO:2) 5 and designated fragments implicated in protein-protein interactions. Fragments A-D

(SEQ ID NOS: 13-16, respectively) are shown.

Figure 5. Schematic diagram of Deltex fragments mediating Deltex-Deltex interactions. 0 Figure 6. Schematic diagram of the Deltex and Notch fragments mediating

Deltex-Notch interactions.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleotide sequences of vertebrate deltex genes, and amino acid sequences of their encoded Deltex proteins. The invention further relates to fragments and other derivatives, and analogs, of vertebrate Deltex proteins. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, e.g., by recombinant ⁰ methods, is provided.

In a specific embodiment, the invention relates to a human deltex gene and protein.

In a another specific embodiment, the invention relates to a mammalian _? _ deltex gene and protein.

The invention also relates to vertebrate Deltex protein derivatives and analogs of the invention which are functionally active, i.e. , they are capable of displaying one or more known functional activities associated with a full-length (wild-type) vertebrate Deltex protein. Such functional activities include but are not limited to antigenicity [ability to bind

30 (or compete with a vertebrate Deltex protein for binding) to an anti-vertebrate Deltex protein antibody], immunogenicity (ability to generate antibody which binds to a vertebrate Deltex protein), ability to bind (or compete with a vertebrate Deltex protein for binding) to Notch or a second Deltex protein or other proteins or fragments thereof, ability to bind (or

3₅ compete with a vertebrate Deltex protein for binding) to a receptor or ligand for a vertebrate

Deltex protein. The mvenuon further relates to fragments (and deπvauves and analogs thereof) of a vertebrate Deltex protein which comprise one or more domains of a vertebrate Deltex protem (see infra), including but not limited to the SH3-bindιng domains, rιng-H2- zmc fingers, domains which mediate bmdmg to Notch (or a derivauve thereof contaimng the

Notch ANK repeats) or to a second Deltex molecule or fragment thereof, or any combinauon of the foregoing

Anubodies to vertebrate Deltex proteins, their deπvatives and analogs, are additionally provided Our prior attempts to clone human, zebrafish, and Xenopus deltex using

Drosophila deltex as a probe were unsuccessful In contrast to such prior failures, the present invention is based on the successful cloning of human deltex As described by way of example below, we have used an innovative methodology to clone the transcription unit corresponding to human deltex As described therein (see Section 6), our results show a significant structural conservation of Deltex in humans, indicative of functional conservation Moreover, we demonstrate that human Deltex displays direct molecular interaction with both human and Drosophila Notch intracellular ANK repeats (see Section 7) Knowledge of the sequence of human deltex allows the identification of regions strongl) conserved between Drosophila and human deltex, and provides a method for readily isolating other vertebrate deltex genes by use of such strongly conserved regions (see Sections 5 6 and 8 infra).

The vertebrate deltex nucleic acid and ammo acid sequences and antibodies thereto of the invention can be used for the detection and quantitation of vertebrate deltex mRNA and protem, to study expression thereof, to produce vertebrate Deltex protems, fragments and other derivatives, and analogs thereof, m the study, assay, and manipulation of differentiation and other physiological processes, and are of therapeutic and diagnostic use, as described infra The agomsts and antagomsts of Deltex function can be used to alter the ability of a cell to differentiate The vertebrate deltex nucleic acids and antibodies can also be used to clone vertebrate deltex homologs of other species, as described infra Such vertebrate deltex homologs are expected to exhibit significant homology to each other, and encode protems which exhibit the ability to bind to a Notch protein The present invention also relates to therapeutic and diagnostic methods and compositions based on vertebrate Deltex protems and nucleic acids The mvention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the invention. Such therapeutic compounds (termed herein "Therapeutics") include: vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the vertebrate Deltex protems, analogs, or

5 derivatives; and vertebrate deltex antisense nucleic acids. In a preferred embodiment, a

Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state. In other specific embodiments, a Therapeutic of the invention is administered to treat 0 a nervous system disorder or to promote tissue regeneration and repair.

In one embodiment, Therapeutics which antagonize, or inhibit, Notch and/or vertebrate Deltex function (hereinafter "Antagonist Therapeutics") are administered for therapeutic effect. In another embodiment, Therapeutics which promote Notch and/or vertebrate Deltex function (hereinafter "Aεorust Therapeutics") are administered for 5 therapeutic effect.

Disorders of cell fate, in particular hyperproliferative (e.g. , cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of Notch and/or vertebrate Deltex protein can be diagnosed by detecting such 0 levels, as described more fully infra.

In a preferred aspect, a Therapeutic of the invention is a protem consisting of at least a fragment (termed herein "adhesive fragment") of vertebrate Deltex that mediates binding to a Notch protein, a second Deltex protein, or a fragment of Notch or Deltex.

The mvenuon is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of human deltex, and the identification of regions of human Deltex which are predicted to bind to the ANK repeats of Notch, or which are predicted to bind to regions of human Deltex.

For clarity of disclosure, and not by way of limitation, the detailed ° description of the invention is divided into the subsections set forth below.

5.1. ISOLATION OF THE VERTEBRATE DELTEX NUCLEIC ACIDS

The invention relates to the nucleotide sequences of vertebrate deltex nucleic 35 acids. In specific embodiments, human deltex nucleic acids comprise the cDNA sequence shown in Figure 2A-C (SEQ ID NO: 11), or the coding region thereof (nucleotide numbers 504-2363), or nucleic acids encoding a human Deltex protem (e.g. , having the sequence of

SEQ ID NO.12). The mvention provides nucleic acids consisung of at least 8 nucleotides

(i.e , a hybπdizable porOon) of a vertebrate deltex sequence; m other embodiments, the nucleic acids consist of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides,

150 nucleotides, or 200 nucleotides of a vertebrate deltex sequence, or a full-length vertebrate deltex coding sequence. The mvention also relates to nucleic acids hybπdizable to or complementary to the foregoing sequences. In specific aspects, nucleic acids are provided which compπse a sequence complementary to at least 10, 25, 50, 100, or 200 nucleoudes or the entire codmg region of a vertebrate deltex gene In a specific embodiment, the sequence is naturally occurring.

In other specific embodiments, the mvention provides nucleic acids comprising at least 110, 150, or 200 continuous nucleotides of the sequence of SEQ ID NO 11 In other embodiments, the mvention provides a nucleic acid compπsmg the first 25, 50, 100, 150, 200, or 230 ammo acids of SEQ ID NO.12

In a specific embodiment, vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encoding the ammo terminal 180 ammo acids (encoded, e.g., by nucleotide numbers 504-1044 of SEQ ID: 11) of human deltex, or fragments thereof In one embodiment, the vertebrate deltex nucleic acid has at least 50% identity over the correspondmg nucleotide sequence of an identically sized human deltex In another embodiment this identity is greater than 55 % In a preferred embodiment, the nucleotide sequence identity of the vertebrate deltex is greater than 60% In a more preferred embodiment this identity is greater than 65 % In a most preferred embodiment, the nucleotide sequence identity of the vertebrate deltex is greater than 70% over that of the correspondmg nucleotide sequence of identically sized human deltex

In another specific embodiment, vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encodmg the central region ammo acids of human deltex (e.g. , nucleotide numbers 1045-1821 of SEQ ID NO 11) or fragments thereof In one embodiment, the nucleic acids encodmg the central ammo acids of the vertebrate Deltex protein has at least 50% nucleotide sequence identity with the correspondmg human deltex sequence of identical size In another embodiment this identity is greater than 55%. In a preferred embodiment, this nucleotide sequence identity is greater than 60% . In a more preferred embodiment this identity is greater than 65%. In a most preferred embodiment, the homology of the nucleic acids encoding the central region amino acids of the vertebrate deltex has a nucleotide sequence identity that is greater than

65% over that of the corresponding nucleotide sequence of identically sized human deltex 5

In another specific embodiment, vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encoding the 180 carboxy terminal amino acids of human deltex (nucleotide numbers 1822-2366), or fragments thereof. In one embodiment, the nucleic acids encoding the carboxy terminal 0 region of the vertebrate Deltex protein has at least 50% nucleotide sequence identity over the corresponding human deltex sequence of identical size. In another embodiment this identity is greater than 55%. In a preferred embodiment, this identity is greater than 60%. In a more preferred embodiment this identity is greater than 65 % . In a most preferred embodiment, the identity of the nucleotides encoding the amino terminal amino acids of the vertebrate deltex is greater than 70% over that of the corresponding nucleotide sequence of identically sized human deltex.

In a specific embodiment, a nucleic acid which is hybridizable to a vertebrate deltex nucleic acid (e.g. , having sequence SEQ ID NO: 11), or to a nucleic acid encoding a ⁰ vertebrate deltex derivative, under conditions of low stringency is provided. By way of example and not limitation, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA are pretreated for 6 h at 40°C in a solution containing 35% ₅ formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 M EDTA, 0.1 % PVP, 0.1 % Ficoll, 1 % BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10⁶ cpm ³²P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at

^{3 0} 40° C, and then washed for 1.5 h at 55° C in a solution containing 2X SSC, 25 mM

Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1 % SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and reexposed

35 to film. Other conditions of low stringency which may be used are well known in the art (e.g. , as employed for cross-species hybridizations). In another specific embodiment, a nucleic acid which is hybridizable to a vertebrate deltex nucleic acid under conditions of high stringency is provided. By way of example and not limitation, procedures using such conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at

5

65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02%

PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65 °C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 10⁶ cpm of ³²P-labeled probe. Washing of filters is done at

10 37°C for 1 h in a solution containing 2X SSC, 0.01 % PVP, 0.01 % Ficoll, and 0.01 % BSA.

This is followed by a wash in 0.1X SSC at 50°C for 45 min before autoradiography. Other conditions of high stringency which may be used are well known in the art.

Nucleic acids encoding derivatives (e.g. , fragments) of vertebrate Deltex proteins (see Section 5.6), and vertebrate deltex antisense nucleic acids (see Section 5.11) are additionally provided. As is readily apparent, as used herein, a "nucleic acid encoding a fragment or portion of a vertebrate Deltex protein" shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the vertebrate Deltex protein and not the other contiguous portions of the vertebrate Deltex protein as a continuous

20 sequence.

Specific embodiments for the cloning of a vertebrate deltex gene, e.g. , a human deltex gene, presented as a particular example but not by way of limitation, follows:

For expression cloning (a technique commonly known in the art), an _ expression library is obtained or is constructed by methods known in the art. For example. mRNA (e.g. , human) is isolated, cDNA is made and ligated into an expression vector (e.g. , a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced. Various screening assays can then be used to select for the expressed vertebrate Deltex product. In a preferred aspect, anti-human Deltex antibodies

30 can be used to select the recombinant host cell expressing a cloned vertebrate deltex gene.

In a specific embodiment, PCR is used to amplify the desired vertebrate deltex sequence in a genomic or cDNA library, prior to selection (see, by way of example Section 8, infra). Oligonucleotide primers representing known vertebrate deltex sequences, 35 preferably regions known to be conserved between Drosophila and human, can be used as primers in PCR. The synthetic oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA), preferably a cDNA library, of potential interest. PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp"). The DNA being amplified can include human mRNA or cDNA or genomic DNA. One can choose to synthesize several different degenerate primers, for use in the PCR reactions. It is also possible to vary the stringency of hybridization conditions used in priming the PCR reactions, to allow for greater or lesser degrees of nucleotide sequence similarity between the known vertebrate deltex nucleotide sequence and the nucleic acid homolog being isolated. For cross species hybridization, low stringency conditions are preferred (see supra). For same species hybridization, moderately stringent or highly stringent conditions are preferred (see supra). After successful amplification of a segment of a vertebrate deltex gene homolog, that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra. In a preferred aspect, human genes encoding Deltex proteins may be identified in this fashion. Alternatively to selection by hybridization, the PCR-amplified DNA can be inserted into an expression vector for expression cloning as described above.

In the event that it is desired to isolate a vertebrate deltex gene by cross- species hybridization (either by direct hybridization to a vertebrate deltex probe representing all or a part of a vertebrate deltex gene of a different species, or by PCR using oligonucleotide primers derived from the sequence of a vertebrate deltex gene of a different species), the desired vertebrate deltex gene can be isolated as set forth in Example 8, by screening with a probe, or priming for PCR with an oligonucleotide, containing deltex sequences encoding regions highly conserved between human and Drosophila. For example, the human Deltex amino acid stretches 414-419 (SEQ ID NO:30), 475-480 (SEQ ID NO:31), 504-511 (SEQ ID NO: 32), 531-539 (SEQ ID NO:33) and 557-564 (SEQ ID NO:34) are conserved in Drosophila Deltex amino acid stretches 549-555 (SEQ ID NO: 35), 603-608 (SEQ ID NO:36), 632-639 (SEQ ID NO:37), 659-667 (SEQ ID NO:38) and 685- 692 (SEQ ID NO:39), respectively. In a preferred embodiment, a pair of oligonucleotides comprising sequences separated by a length in the range from 50-500 nucleotides is used as primers in PCR. The invention envisions the use of nucleic acids encoding conserved regions of the Deltex protein in combination to isolate the Deltex encoding nucleic acids of other organisms, by use in PCR to amplify the desired sequence or less preferably, without PCR, as a probe in selection by virtue of direct colony hybridization (e.g. , Grunstein, M. and Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961).

In the event that it is desired to isolate a deltex gene by cross-species hybridization (either by direct hybridization to a deltex probe representing all or a part of a deltex gene of an evolutionarily distant, different species, or by PCR using oligonucleotide primers derived from the sequence of a deltex gene of a different, evolutionarily distant species), the desired deltex gene can be isolated by a more gradual method of evolutionary walking via first isolating a deltex gene from a more closely related species, identifying the portions of deltex which are conserved cross-species, and then screening with a probe or priming for PCR with a nucleic acid containing the conserved sequence. This method, while more cumbersome, is straightforward and can be readily carried out by routine methods. For example, if it is desired to proceed further down the evolutionary tree, one may first isolate a murine deltex gene using nucleic acids encoding human Deltex as a probe. A conserved portion of the murine deltex sequence is then used to screen or amplify deltex in an avian library; a conserved portion of the avian clone is used to screen an amphibian library, a conserved portion of the amphibian clone is used to screen a fish library, etc. If desired, the species to be selected in the next round of screening can be selected from among various species by hybridizing the deltex probe to a Southern blot containing genomic DNA from each species, and selecting a species to which hybridization occurs.

The above-methods are not meant to limit the following general description of methods by which clones of vertebrate deltex may be obtained.

Any eukaryotic cell can potentially serve as the nucleic acid source for the molecular cloning of the vertebrate deltex gene. The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g. , a DNA "library"), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired human cell (see, for example Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2d. Ed., Cold Spring

Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, π.) Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways For example, if an amount of a portion of a vertebrate deltex (of any species) gene or its specific RNA, or a fragment thereof e.g., the adhesive domain, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, R., 1977, Science 196, 180; Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961). Those DNA fragments with substantial homology to the probe will hybridize. For cross species hybridization, low stringency conditions are preferred (see supra). For same species hybridization, moderately stringent conditions are preferred (see supra). It is also possible to identify the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map if such is available. Further selection can be carried out on the basis of the properties of the gene. Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, e.g. , has similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, binding to Notch, or antigenic properties as known for vertebrate Deltex. If an antibody to vertebrate Deltex is available, the vertebrate Deltex protein may be identified by binding of labeled antibody to the putatively vertebrate Deltex synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay )-type procedure. The vertebrate deltex gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified vertebrate deltex DNA of another species (e.g., human).

Immunoprecipitation analysis or functional assays (e.g., ability to bind Notch) of the in vitro translation products of the isolated products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences. In addition, specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against vertebrate Deltex protein. A radiolabelled vertebrate deltex cDNA can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. The radiolabelled mRNA or cDNA may then be used as a probe to identify the vertebrate deltex DNA fragments from among other genomic DNA fragments.

Alternatives to isolating the vertebrate deltex genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence or making cDNA to the mRNA which encodes the vertebrate deltex gene. For example, RNA for cDNA cloning of the vertebrate deltex gene can be isolated from cells which express vertebrate Deltex. Other methods are possible and within the scope of the invention. The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as PBR322, pUC, or Blue script (Stratagene) plasmid derivatives. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and vertebrate deltex gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced mto host cells via transformauon, transfecuon, infection, electroporation, etc., so that many copies of the gene sequence are generated.

In an alternauve method, the desired gene may be identified and isolated after insernon mto a suitable cloning vector in a "shot gun" approach Enrichment for the desired gene, for example, by size fractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the isolated vertebrate deltex gene, cDNA, or synthesized DNA sequence enables generauon of muluple copies of the gene Thus, the gene may be obtamed in large quanuties by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.

The vertebrate deltex sequences provided by the instant mvention include those nucleotide sequences encodmg substannally the same ammo acid sequences as found in native vertebrate Deltex protem, and those encoded amino acid sequences with functionally equivalent ammo acids, all as described in Section 5.6 infra for vertebrate Deltex derivatives

5.2. EXPRESSION OF VERTEBRATE DELTEX NUCLEIC ACIDS

The nucleic acid coding for a vertebrate Deltex protem or a functionally active fragment or other derivative thereof can be inserted mto an appropriate expression vector, i e , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence The necessary transcπptional and translational signals can also be supplied by the native vertebrate deltex gene and/or its flanking regions. A variety of host-vector systems may be utilized to express the protein- coding sequence. These include but are not limited to vertebrate cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc ), insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast contaimng yeast vectors, or bacteria transformed with bacteπophage, DNA, plasmid DNA, or cosmid DNA The expression elements of vectors vary m their strengths and specificities Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used

In a specific embodiment, a molecule comprising a portion of a vertebrate deltex gene which encodes a protem that b ds to Notch or to a molecule compnsmg the Notch ANK repeats is expressed. In another embodiment, a molecule compnsmg a poruon of a vertebrate deltex gene which encodes a protem that binds to a fragment of a Deltex protem is expressed In other specific embodiments, mammalian deltex gene is expressed, or a sequence encodmg a

5 funcuonally active portion of mammalian Deltex. In other specific embodiments, the human deltex gene is expressed, or a sequence encodmg a functionally active portion of human

Deltex In a specific embodiment, a chimeric protein comprising a Notch-binding domain of a vertebrate Deltex protem is expressed In other specific embodiments, a full-length

10 vertebrate deltex cDNA is expressed, or a sequence encodmg a functionally active portion of a vertebrate Deltex protem In yet another embodiment, a fragment of a vertebrate Deltex protem compnsmg a domain of the protem, or other deπvative, or analog of a vertebrate Deltex protem is expressed

Any of the methods previously described for the insertion of DNA fragments mto a vector may be used to construct expression vectors containing a chimeric gene consisting of appropπate transcnptional/translauonal control signals and the protem codmg sequences These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombinauon). Expression of a nucleic acid sequence

2 encodmg a vertebrate Deltex protem or pepude fragment may be regulated by a second nucleic acid sequence so that the vertebrate Deltex protem or pepude is expressed in a host transformed with the recombinant DNA molecule For example, expression of a vertebrate Deltex protem may be controlled by any promoter/enhancer element known m the art Promoters which may be used to control vertebrate deltex gene expression mclude, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981 , Nature 290 304-310), the promoter contained m the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al , 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al , 1981, Proc. Natl Acad. Sci. U.S.A. 78: 1441-1445), the regulatory sequences of the metallothionein gene (Bπnster et al., 1982, Nature 296.39-42); prokaryotic expression vectors such as the 3-lactamase (Villa-Kamaroff et al., 1978, Proc Natl Acad Sci U S A 75.3727-3731), tec (DeBoer et al , 1983, Proc Natl Acad Sci U S A 80.21-25), λP_L, or trc promoters, see also "Useful protems from recombinant bacteria" in Scientific American,

35 1980, 242 74-94; plant expression vectors comprising the nopa ne synthetase promoter region or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring

Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature

315:115-122), immunoglobulin gene control region which is active in lymphoid cells

(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;

Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444), mouse mammary tumor vims control region which is active in testicular, breast, lymphoid and mast cells (Leder et al. ,

1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al.,

1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1 : 161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-

94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al.,

1986, Science 234: 1372-1378).

Expression vectors containing vertebrate deltex gene inserts can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted vertebrate deltex gene. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g. , thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formauon in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if the vertebrate deltex gene is inserted within the marker gene sequence of the vector, recombinants containing the vertebrate deltex insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the vertebrate deltex gene product in in vitro assay systems, e.g. , binding to

Notch, bindmg with antibody. Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared m quantity. As previously expla ed, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives, human or animal viruses such as vaccmia virus or adenovirus; msect viruses such as baculovirus, yeast vectors; bacteπophage vectors (e.g. , lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences , or modifies and processes the gene product in the specific fashion desired Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engmeered vertebrate Deltex protein may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e g , phosphorylation, cleavage) of protems. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.

In other specific embodiments, the vertebrate Deltex protein, fragment, analog, or derivative may be expressed as a fusion, or chimeric protein product (comprising the protem, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art Alternatively, such a chimeric product may be made by protem synthetic techniques, e g. , by use of a peptide synthesizer Both cDNA and genomic sequences can be cloned and expressed.

In other embodiments, a vertebrate deltex cDNA sequence may be chromosomally integrated and expressed. Homologous recombination procedures known in the art may be used.

5.3. IDENTIFICATION AND PURIFICATION OF THE VERTEBRATE DELTEX GENE PRODUCTS

In parucular aspects, the mvention provides amino acid sequences of vertebrate Deltex, preferably human Deltex, and fragments and derivatives thereof which comprise an antigenic determinant (i.e., can be recognized by an antibody) or which are functionally acuve, as well as nucleic acid sequences encoding the foregoing. "Functionally active" material as used herein refers to that material display mg one or more known functional activities associated with the full-length (wild-type) vertebrate Deltex protem product, e.g. , bmdmg to Notch or a portion thereof, bmdmg to another Deltex molecule or portion thereof, bmdmg to any other Deltex ligand, antigenicity (bindmg to an anu- vertebrate Deltex antibody), lmmunogemcity (generating anti-Deltex antibody), Notch intracellular signal transduction, etc. In specific embodiments, the mvention provides fragments of a vertebrate

Deltex protein consistmg of at least 6 ammo acids, 10 ammo acids, 50 amino acids, or of at least 75 ammo acids In other embodiments, the proteins comprise, or alternatively, consist essentially of; one or more of the SH3-bιndιng domams (e.g. , SEQ ID NOS: 17-21 of Table

III): one or more πng-H2-zιnc finger domains (e.g., SEQ ID NO:25), or a portion which bmds to Notch (e.g. , compnsmg the first approximately 230 ammo acids of vertebrate

Deltex), or any combination of the foregoing, of a vertebrate Deltex protein. Fragments, or protems compnsmg fragments, lacking some or all of the foregoing regions of vertebrate

Deltex are also provided. Molecules comprising more than one copy of the foregoing regions are also provided. Nucleic acids encodmg the foregoing are provided

Once a recombinant which expresses a vertebrate deltex gene sequence is identified, the gene product can be analyzed This is achieved by assays based on the physical or functional properties of the product, including radioactive labelling of the product followed by analysis by gel electrophoresis, lmmunoassay, etc. Chemically synthesized proteins, derivatives, and analogs can be similarly analyzed. Once a vertebrate Deltex protein is identified, it may be isolated and purified by standard methods including chromatography (e.g. , ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. The functional properues may be evaluated using any suitable assay (see Secuon 5.7).

Alternatively, the amino acid sequence of a vertebrate Deltex protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant. Once the amino acid sequence is thus known, the protein can be synthesized by standard chemical methods known in the art (e.g. , see Hunkapiller et al., 1984, Nature 310:105- 111).

By way of example, the deduced amino acid sequence (SEQ ID NO: 12) of a human Deltex protem is presented in Figure 2A-C.

5.4. STRUCTURE OFTHEVERTEBRATE DELTEXGENES AND PROTEINS

The structure of the vertebrate deltex genes and proteins can be analyzed by various methods known in the art.

5.4.1. GENETIC ANALYSIS

The cloned DNA or cDNA corresponding to the vertebrate deltex gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975,

J. Mol. Biol. 98:503-517), Northern hybridization (see e.g. , Freeman et al., 1983, Proc.

Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction endonuclease mapping (Maniatis, 1982,

Molecular Cloning, A Laboratory, Cold Spring Harbor, New York), and DNA sequence analysis. Polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7652-7656; Ochman et al., 1988, Genetics 120:621-623; Loh et al., 1989, Science 243:217-220) followed by Southern hybridization with a vertebrate deltex-specific probe can allow the detecuon of the vertebrate deltex genes in DNA from various cell types. In one embodiment, Southern hybridization can be used to determine the genetic linkage of vertebrate deltex. Northern hybridization analysis can be used to determine the expression of the vertebrate deltex genes. Various cell types, at various states of development or activity can be tested for vertebrate deltex gene expression. The stringency of the hybridization conditions for both Southern and Northern hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific vertebrate deltex probe used.

Restriction endonuclease mapping can be used to roughly determine the genetic structure of the vertebrate deltex gene. Restriction maps derived by restriction 5 endonuclease cleavage can be confirmed by DNA sequence analysis. Alternatively, restriction maps can be deduced, once d e nucleotide sequence is known.

DNA sequence analysis can be performed by any techniques known in the an, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol.

10 65:499-560), the Sanger dideoxy method (Sanger et al, 1977, Proc. Natl. Acad. Sci.

U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No.

4,795,699; Sequenase, U.S. Biochemical Corp.), or Taq polymerase, or use of an automated DNA sequenator (e.g.. Applied Biosystems, Foster City, CA). The cDNA sequence of a human deltex gene is shown in Figure 2A-C (SEQ ID NO.1 1) and is described in Section 6, infra.

5.4.2. PROTEIN ANALYSIS

The amino acid sequence of a vertebrate Deltex protem can be deπved by ²⁰ deduction from the DNA sequence, or alternatively, by direct sequencing of the protem, e.g. , with an automated amino acid sequencer. The ammo acid sequence of a representative vertebrate Deltex protem comprises the sequence substantially as depicted in Figure 2A-C (SEQ ID NO: 12), and detailed in Section 6, infra. . The vertebrate Deltex protem sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S. A 78.3824) A hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of a vertebrate Deltex protein and the corresponding regions of the gene sequence which encode such regions. Hydrophilic regions are predicted to be lmmunogenic.

Secondary, structural analysis (Chou and Fasman, 1974, Biochemistry 13:222) can also be done, to identify regions of a vertebrate Deltex protein that assume specific secondary structures.

Manipulation, translation, and secondary structure prediction, as well as open 3 readmg frame prediction and plotting, can also be accomplished using computer software programs available in the art. Other methods of structural analysis can also be employed These mclude but are not limited to X-ray crystallography (Engstom, 1974, Biochem. Exp. Biol. 11:7-13) and computer modelmg (Fletteπck and Zoller (eds.), 1986, Computer Graphics and

Molecular Modelmg, in Current Commumcations m Molecular Biology, Cold Sprmg Harbor

5

Laboratory, Cold Sprmg Harbor, New York).

5.5. GENERATION OFANTIBODIES TOVERTEBRATE DELTEX PROTEINS AND DERIVATIVES THEREOF

Accordmg to the mvention, a vertebrate Deltex protem, its fragments or other derivatives, or analogs thereof, may be used as an lmmunogen to generate antibodies which recognize such an immunogen Such antibodies mclude but are not limited to polyclonal, monoclonal, chimeric, smgle chain, Fab fragments, and an Fab expression library In a preferred embodiment, antibodies which specifically bmd to vertebrate Deltex ⁵ proteins are produced In a more preferred embodiment, an antibody which bmds to a vertebrate Deltex protem (e g , mammalian, preferably human) but does not bmd to (full length) Drosophila Deltex protem, is produced In a preferred embodiment, such an antibody is produced by us g as immunogen, regions least conserved between Drosophila o melanogaster and the vertebrate Deltex protem

In another embodiment, antibodies to a particular domam of a vertebrate Deltex protem are produced. In a specific embodiment, an antibody is produced which bmds to a fragment of vertebrate Deltex which bmds to Notch; m another embodiment, an antibody bmds to a molecule compnsmg the first 230 amino-terminal ammo acids of 5 vertebrate Deltex In another embodiment the antibody binds to an ammo-terminal fragment of vertebrate Deltex containing not more than the first 200 ammo acids of vertebrate Deltex In yet another embodiment, an antibody bmds to a fragment of vertebrate Deltex which bmds to a second Deltex molecule. o Various procedures known in the art may be used for the production of polyclonal antibodies to a vertebrate Deltex protem or deπvative or analog In a particular embodiment, rabbit polyclonal antibodies to an epitope of the vertebrate Deltex protem having a sequence depicted in Figure 2A-C or a subsequence thereof, can be obtamed. For the production of antibody, various host animals can be immunized by injection with a 5 native vertebrate Deltex protein, or a synthetic version, or derivative (e.g , fragment) thereof, mcludmg but not limited to rabbits, mice, rats, etc Various adjuvants may be used to mcrease the lmmunological response, depending on the host species, and mcludmg but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithm, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dmitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Gueπn) and corynebacteπum parvum.

In a preferred embodiment, polyclonal or monoclonal antibodies are produced by use of a hydrophilic portion of a vertebrate Deltex peptide (e.g., identified by the procedure of Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S. A 78:3824)) For preparation of monoclonal antibodies directed toward a vertebrate Deltex protem sequence or analog thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used For example, the hybπdoma technique originally developed by Kohler and Milstein (1975, Nature 256 495-

497), as well as the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al ,

1983, Immunology Today 4:72), and the EBV-hybπdoma technique to produce human monoclonal antibodies (Cole et al., 1985, m Monoclonal Antibodies and Cancer Therapy,

Alan R. Liss, Inc., pp. 77-96) can be used. In an additional embodiment of the mvention, monoclonal antibodies can be produced in germ-free animals (PCT Publication No. WO 89/12690 dated December 28, 1989) Accordmg to the invention, human antibodies may be used and can be obtained by usmg human hybπdomas (Cote et al , 1983, Proc

Natl Acad Sci U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al , 1985, m Monoclonal Antibodies and Cancer Therapy, Alan R Liss, pp 77-96), or by other methods known in the art In fact, accordmg to the mvention, techniques developed for the production of "chimeric antibodies" (Morrison et al , 1984,

Proc Natl. Acad. Sci U.S.A. 81:6851-6855, Neuberger et al., 1984, Nature 312:604-608;

Takeda et al , 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for a vertebrate Deltex protein together with genes from a human antibody molecule of appropriate biological activity can be used, such antibodies are within the scope of this mvention Non-human antibodies can be humanized by the method of Winter (see

U.S Patent No 5,225,539)

Accordmg to the invention, techniques described for the production of smgle cham antibodies (U.S. Patent 4,946,778) can be adapted to produce vertebrate Deltex protem-specific smgle chain antibodies An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for vertebrate Deltex proteins, derivatives, or analogs.

Antibody fragments and other derivatives which contain the idiotype (binding

5 domam) of the molecule can be generated by known techniques. For example, such fragments mclude but are not limited to: the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragment, and the Fab fragments which can be

10 generated by treating the antibody molecule with papam and a reducing agent.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies which recognize a specific domam of a vertebrate Deltex protem, one may assay generated hybπdomas for a product which bmds to a vertebrate Deltex fragment contaming such domam. For selection of an antibody specific to human Deltex protein(s), one can select on the basis of positive bmdmg to a human Deltex protein and a lack of binding to Drosophila Deltex protem.

In a specific embodiment, antibodies specific to a phosphorylated epitope of

20 vertebrate Deltex are produced.

The foregoing antibodies can be used in methods known m the art relatmg to the localization and activity of the protein sequences of the mvention e.g. , for imagmg these protems, measuring levels thereof in appropriate physiological samples, etc. Antibodies to

_ vertebrate Deltex (since it normally colocalizes with Notch) can be used to determine the

25 intracellular distribution of Notch and/or vertebrate Deltex, m diagnostic methods such as described infra. The antibodies also have use m immunoassays. In another embodunent of the invention (see infra), anti-vertebrate Deltex antibodies and fragments thereof contaimng the bmdmg domam are Therapeutics. 30

5.6. VERTEBRATE DELTEX PROTEINS, DERIVATIVES AND ANALOGS

The invention further provides vertebrate Deltex protems, and derivatives

(including but not limited to fragments) and analogs of vertebrate Deltex proteins Nucleic 35 acids encoding vertebrate Deltex protein deπvatives and protein analogs are also provided.

In one embodiment, the vertebrate Deltex proteins are encoded by me vertebrate deltex nucleic acids described m Section 5.1 supra In particular aspects, the proteins, derivatives, or analogs are of mouse or rat; agricultural stock such as cow, sheep, horse, goat, pig and the like; pets such as cats, dogs; or other domesticated mammals, or primate Deltex proteins. 5

The production and use of derivatives and analogs related to vertebrate

Deltex are within the scope of the present mvention. In a specific embodunent, the derivative or analog is functionally active, i e. , capable of exhibitmg one or more functional activities associated with a full-length, wild-type vertebrate Deltex protem. 0 In particular, vertebrate Deltex derivatives can be made by altering vertebrate deltex sequences by substitutions, additions or deletions that provide for functionally equivalent molecules Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same ammo acid sequence as a vertebrate deltex gene may be used m the practice of the present mvention These mclude but are not limited to nucleotide sequences compnsmg all or portions of vertebrate deltex genes which are altered by the substitution of different codons that encode a functionally equivalent ammo acid residue within the sequence, thus producmg a silent change. Likewise, the vertebrate

Deltex deπvatives of the mvention mclude, but are not limited to, those contaimng, as a 0 primary ammo acid sequence, all or part of the ammo acid sequence of a vertebrate Deltex protem mcludmg altered sequences in which functionally equivalent ammo acid residues are substituted for residues within the sequence resulting a silent change For example, one or more am o acid residues within the sequence can be substituted by another ammo acid _ _j. of a similar polarity which acts as a functional equivalent, resulting in a silent alteration Substitutes for an ammo acid within the sequence may be selected from other members of the class to which the ammo acid belongs For example, the nonpolar (hydrophobic) ammo acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral ammo acids include glycine, seπne, threomne, cysteine,

30 tyrosine, asparagme, and glutamine The positively charged (basic) ammo acids mclude argimne, lysme and histidine The negatively charged (acidic) ammo acids mclude aspartic acid and glutamic acid

In a specific embodiment of the invention, proteins consisting of or compnsmg a fragment of a vertebrate Deltex protein consisting of at least 10 (contmuous) amino acids of the vertebrate Deltex protein is provided In other embodiments, the fragment consists of at least 20 or 50 ammo acids of the vertebrate Deltex protem In specific embodiments, such fragments are not larger than 35, 100 or 200 ammo acids. Derivatives or analogs of vertebrate Deltex mclude but are not limited to those peptides which are substantially homologous to human Deltex or fragments thereof

In a specific embodiment, derivatives or analogs of vertebrate Deltex mclude those peptides which are substantially homologous to the ammo terminal 180 ammo acids (1- 180) of human Deltex In one embodiment, the ammo terminal region of the vertebrate Deltex protem has at least 30% identity over the ammo terminal ammo acid sequence of identically sized human Deltex In another embodiment this identity is greater than 35% In a preferred embodiment, the ammo terminal ammo acid identity of the vertebrate Deltex is greater than 45 % In a more preferred embodiment this identity is greater than 55 % In a most preferred embodiment, the homology of the ammo terminal ammo acids of the vertebrate Deltex is greater than 65 % over the correspondmg human Deltex ammo terminal ammo acid sequence of identical size

In another specific embodunent, derivatives or analogs of vertebrate Deltex mclude those peptides which are substantially homologous to the central region (ammo acids 181-441) of human Deltex, or fragments thereof In one embodiment, the central region of the vertebrate Deltex protem has at least 30% identity with the correspondmg human Deltex sequence of identical size In another embodiment this identity is greater than 35 % In a preferred embodiment, the ammo acid identity of the central region of vertebrate Deltex and human Deltex is greater than 45 % In a more preferred embodiment this identm is greater than 55 % In a most preferred embodiment, the homology of the central amino acids of the vertebrate Deltex to corresponding human Deltex ammo acids of identical size is greater than 65%

Additionally, derivatives or analogs of vertebrate Deltex mclude but are not limited to those peptides which are substantially homologous to the carboxy terminal ammo acids of human Deltex or fragments thereof In one embodiment, the carboxy terminal region of the vertebrate Deltex protem (the carboxy terminal 180 ammo acids) has at least 45% identity over the ammo acid sequence of identical size In another embodiment this identity is greater than 50% In a preferred embodiment, the amino terminal ammo acid identity of the vertebrate Deltex is greater than 55% In a more preferred embodiment this identity is greater than 60%. In a most preferred embodiment, the homology of the ammo terminal ammo acids of the vertebrate Deltex is greater than 65 % In another preferred embodunent, derivatives or analogs of vertebrate Deltex comprise regions conserved between Drosophila and human Deltex (see Section 8).

The vertebrate Deltex protem derivatives and analogs of the mvention can be produced by various methods known in the art The manipulations which result m their production can occur at the gene or protem level. For example, the cloned vertebrate deltex gene sequence can be modified by any of numerous strategies known m the art (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Sprmg Harbor

Laboratory, Cold Sprmg Harbor, New York). The sequence can be cleaved at appropπate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro In the production of the gene encodmg a derivative or analog of a vertebrate Deltex protem, care should be taken to ensure that the modified gene remains within the same translational readmg frame as the vertebrate deltex gene, uninterrupted by translational stop signals, in the gene region where the desired vertebrate Deltex activity is encoded.

Additionally, the vertebrate Deltex-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, imtiation, and/or termination sequences, or to create variations coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchmson et al., 1978, J. Biol. Chem 253:6551), use of TAB^® linkers (Pharmacia), etc. Manipulations of the vertebrate deltex sequence may also be made at the protem level Included within the scope of the invention are vertebrate Deltex protein fragments or other derivatives or analogs which are differentially modified durmg or after translation, e.g. , by acetylation, phosphorylation, carboxylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, mcludmg but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papam, V8 protease, NaBH₄, acetylation, formylation, oxidation, reduction, etc. In a preferred aspect, phosphorylation or, alternatively, dephosphorylation is carried out, which can be to various extents, on the purified vertebrate Deltex protein or derivative thereof. The phosphorylation state of the molecule may be important to its role in intracellular signal transduction of Notch function. Phosphorylation can be carried out by reaction with an appropriate kinase (e.g. , possibly cdc2 or CK II). Dephosphorylation can be carried out by reaction with an appropriate phosphatase.

5

In addition, analogs and derivatives of vertebrate Deltex proteins can be chemically synthesized. For example, a peptide corresponding to a portion of a vertebrate

Deltex protein which comprises the desired domain, or which mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, 0 nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the vertebrate Deltex protein sequence. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, α-amino isobutyric acid,

4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, 3-alanine, designer amino acids such as β- 5 methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids.

In a specific embodiment, the vertebrate Deltex derivative is a chimeric, or fusion, protein comprising a vertebrate Deltex protein or fragment thereof (preferably consisting of at least a domain or motif of the vertebrate Deltex protein, or at least 10 amino 0 acids of the vertebrate Deltex protein) joined at its amino or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein

(comprising a vertebrate Deltex-coding sequence joined in-frame to a coding sequence for a different protein). Such a chimeric product can be made by ligating the appropriate nucleic 5 acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. A specific embodiment ° relates to a chimeric protein comprising a fragment of a vertebrate Deltex protein which comprises a domain or motif of the vertebrate Deltex protein, e.g., a portion which binds to a Notch protein or to a second Deltex protein, an SH-3 binding domain, a ring-H2-zinc finger domain, etc. In a particular embodiment, a chimeric nucleic acid can be constructed, ₅ encoding a fusion protein consisting of a vertebrate Deltex Notch-binding fragment joined to a non-Deltex protein. As another example, and not by way of limitation, a recombinant molecule can be constructed according to the invention, comprising coding portions of both a vertebrate deltex gene and another gene which is a member of the "Notch group."

Another specific embodiment relates to a chimeric protein comprising a fragment of a vertebrate Deltex protein of at least six amino acids. Particular examples of the construction and expression of fusion proteins comprising human Deltex or various Notch fragments, are described in Section 7.

Other specific embodiments of derivatives and analogs are described in the subsection below and examples sections infra.

5.6.1. DERIVATIVES OF VERTEBRATE DELTEX CONTAINING ONE OR MORE DOMAINS OF THE PROTEIN

In a specific embodiment, the invention provides vertebrate Deltex derivatives and analogs, in particular vertebrate Deltex fragments and derivatives of such fragments, that comprise or consist of one or more domains of the vertebrate Deltex protein, including but not limited to a region which binds to a Notch protein (or a molecule comprising the ANK repeats thereof), a region which binds to a second Deltex protein or portion thereof, an SH3-binding domain, or a ring-H2-zinc finger domain. In specific embodiments, the vertebrate Deltex derivative may lack all or a portion of one or more of the foregoing domains.

In specific embodiments directed to the domains of the human Deltex protein, the aforesaid domains consist of approximately the following amino-acid sequences (see

Section 6.1.1 infra): SH3 binding domains: SEQ ID NOS: 17-21

Ring-H2-zinc finger domain: SEQ ID NO:25

Other binding fragments, e.g., smaller than those set forth above, can be identified by routine methods, e.g. , by construction of nucleic acids encoding such fragments and assays for binding (e.g. , via the interaction trap method described in Section 7 infra.

In a specific embodiment, relating to a vertebrate Deltex protein of a species other than human, fragments comprising specific domains of vertebrate Deltex are those comprising domains in the respective vertebrate Deltex protein most homologous to the specific domain of the human Deltex protein. We have demonstrated that Drosophila Deltex binds to human Notch- 1 and

2, suggesting evolutionary conservation of biochemical activity between human and

Drosophila Deltex. We have also demonstrated that human Deltex binds to human Notch- 1 and 2, and that human Deltex binds to Drosophila Notch. Using the interaction trap system 5

(described infra) as our assay we systematically examined, by deletion analysis, the domains of Notch and Deltex which are responsible for protein-protein interactions. Both Deltex-

Deltex as well as Deltex-Notch interactions were detected. Deletion constructs encoding various fragments (described below) of Drosophila Deltex, Drosophila Notch and human 0 Notch were expressed as fusion constructs (LexA or ACT fusions), and assayed.

The sequences of fragments A-D (SEQ ID NOS: 13-16, respectively) of Drosophila Deltex which were expressed are shown in Fig. 4A-B.

Figure 5 summarizes the Deltex-Deltex interactions we have detected. Fragment A interacts with Fragment A (homotypic interactions). Fragment B interacts with Fragment B (homotypic interactions). Fragment C interacts with Fragment C (homotypic interactions). In addition, we detected interactions between fragments C and B. However, we can only detect the fragment C-B interaction if fragment C is tested as the "bait" (i.e. , as the LexA fusion). If Fragment B is the bait, this interaction is not detected. All the other

²0 aforesaid interactions occur irrespective of which fragment is used as the bait. Fragment A consists of amino acids 1-303. Fragment B consists of amino acids 306-486. Fragment C consists of amino acids 514-737.

The heterotypic interaction between Notch and Deltex is occurred between

_ _ς the ANK repeat region of Notch and fragment D of Deltex (which is part of fragment A and includes amino acids 1-204). Drosophila Notch ANK repeats as well as the ANK repeats of both human Notch proteins (encoded by TAN-1 and hN) were tested in this interaction assay and showed positive binding to fragment D. The following fragments containing the ANK repeat region were used: Drosophila Notch amino acids: 1889-2076 (numbering per

^{3 0} Wharton et al., 1985, Cell 43:567-581); Human Notch TAN-1 amino acids: 1826-2146; Human Notch hN amino acids: 1772-2093. All displayed interactions with fragment D. Figure 6 summarizes schematically this interaction.

In specific embodiments, vertebrate Deltex regions are provided that are most

35 homologous to Drosophila fragment A (SEQ ID NO: 13), fragment B (SEQ ID NO: 14), fragment C (SEQ ID NO: 15), and fragment D (SEQ ID NO: 16), shown in Figure 4A-B. Bmdmg interactions between fragments are indicated by arrows in Figures 5 and 6. Such regions homologous to A-D are predicted also to display the bmdmg interactions shown m Figures 5 and 6 Thus, ammo acids 1-237, 238-391, 392-620, and 1-175 of SEQ ID NO:12 correspond to Drosophila fragments A-D, respectively Molecules compnsmg one or more

5 of the foregomg regions are provided. Accordmgly, by way of example, a molecule compnsmg amino acid numbers 1-237 of SEQ ID NO.12 is predicted to bmd the Notch ankyπn repeats.

Also provided are inhibitors (e.g. , peptide inhibitors) of the foregomg protem 0 interactions with Notch or with a second Deltex protem

The ability to bmd to a Notch protem or a Deltex protem (or derivative thereof) can be demonstrated by m vitro assays such as the interaction trap technique (Section 7, infra)

The nucleic acid sequences encodmg Notch or vertebrate Deltex proteins or fragments thereof, for use m such assays, can be isolated from porcine, bovme, equine, felme, canine, as well as primate sources and any other mammals in which homologs of known genes can be identified For example, me Notch protem or portion thereof compnsmg the ANK repeats which can be expressed and assayed for bmdmg to Deltex or a Deltex derivative can be derived from any of the Notch homologs human hN, human TAN-1, Xenopus, and Drosophila.

Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same ammo acid sequence as the aforesaid domains may be

_ _c used in the practice of the present mvention These mclude but are not lunited to nucleotide sequences compnsmg all or portions of the vertebrate deltex genes which are altered by the substitution of different codons that encode a functionally equivalent ammo acid residue within the sequence, thus producing a silent change Likewise, the vertebrate Deltex proteins, fragments or derivatives thereof, of the mvention include, but are not limited to,

30 those contaimng, as a primary ammo acid sequence, all or part of the ammo acid sequence of the domains mcludmg altered sequences in which functionally equivalent ammo acid residues are substituted for residues within the sequence ("conservative" changes)

The derivatives, analogs, and peptides of the mvenuon can be produced by various methods known in the art The manipulations which result in their production can occur at the gene or protem level Additionally, the nucleic acid sequence can be mutated in vitro or in vivo; and manipulations of the sequence may also be made at the protein level.

In addition, analogs and peptides can be chemically synthesized.

5

5.7. IN VITRO ASSAYS OF VERTEBRATE DELTEX

PROTEINS. DERIVATIVES AND ANALOGS

The functional activity of vertebrate Deltex proteins, derivatives and analogs, can be assayed in vitro by various methods.

For example, in one embodiment, where one is assaying for the ability to bind or compete with wild-type vertebrate Deltex for binding to anti-vertebrate Deltex antibody, various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g. , gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein _Q A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the 5 present invention.

In another embodiment, where one is assaying for the ability to mediate binding to Notch or portion thereof (e.g. , Notch ankyrin repeats) to a second Deltex protein or portion thereof, one can carry out assays such as that described infra in Section 7. o Other methods will be known to the skilled artisan and are within the scope of the invention.

In another embodiment, a method of identifying a molecule that inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein is provided. In this manner, agonists and antagonists of Deltex can be identified. Such a method comprises the 5 steps of contacting a Notch protein and a vertebrate Delta protein such that binding between the Notch protein and the Deltex protein can occur, in the presence of one or more molecules which are desired to be tested for the ability to inhibit or reduce bindmg between the Notch protem and the Deltex protein, and identifying the molecules that mhibit or reduce the bindmg of the Deltex protem to the Notch protein. Any of various binding assays known in the art can be used to carry out such a method, including but not limited to yeast interaction trap assays, cell culture in vitro aggregation assays, and soluble bmdmg assays usmg purified Notch and Deltex protems. A specific embodiment is as follows: Cultured cells are cotransfected with plasmid expression constructs that place Notch and deltex under distinct mducible promoters. Notch expression in these cells is first induced to ensure proper cell surface localization; Deltex expression is then induced. These cells are then aggregated with cells expressing Delta, to produce mutual cappmg of Notch and Delta at the point of mutual contact (see Singer J., 1992, Science, 255.1671-1677; Fehon et al., 1990,

Cell, 61 :523-534, Heitzler, et al., 1991, Cell, 64: 1083-1092) Under these conditions,

Deltex colocalizes with the capped Notch by virtue of its bmdmg to Notch The cells are then incubated m the presence of one or more molecules (preferably, purified molecules) which are desired to be tested for the ability to mhibit bmdmg between Notch and Deltex.

Molecules which inhibit or reduce die bindmg of Deltex to Notch will result m an increased localization of Deltex throughout the cell cytoplasm. This mcreased localization can be determmed accordmg to methods known in the art (e.g. , immunofluorescent staining with antibody to Deltex). The method can also be carried out usmg derivatives of Notch and Deltex that mediate bindmg to Deltex and to Notch, respectively.

5.8. THERAPEUTIC USES

The invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the mvention. Such therapeutic compounds (termed herein "Therapeutics") mclude vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof (e.g., as described hereinabove); antibodies thereto (as described hereinabove); nucleic acids encodmg the vertebrate Deltex proteins, analogs, or derivatives (e.g. , as described hereinabove); and vertebrate deltex antisense nucleic acids. As stated supra, the Antagonist Therapeutics of the invention are those Therapeutics which antagonize, or inhibit, a vertebrate Deltex function and/or Notch function Such Antagonist Therapeutics are most preferably identified by use of known convenient in vitro assays, e.g., based on their ability to inhibit bmdmg of vertebrate Deltex to another protein (e.g., a Notch protein), or inhibit any known Notch or vertebrate Deltex function as preferably assayed in vitro or in cell culture, although genetic assays (e.g., in

Drosophila or mouse) may also be employed. In a preferred embodiment, the Antagonist

Therapeutic is a protein or derivative thereof comprising a functionally active fragment such 5 as a fragment of vertebrate Deltex which mediates bindmg to Notch, or an antibody thereto.

In other specific embodiments, such an Antagonist Therapeutic is a nucleic acid capable of expressing a molecule comprising a fragment of vertebrate Deltex which binds to Notch, or a vertebrate deltex antisense nucleic acid (see Section 5.11 herein). It should be noted that 0 preferably, suitable in vitro or in vivo assays, as described infra, should be utilized to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue, since the developmental history of the tissue may determine whether an Antagonist or Agonist Therapeutic is desired.

In another embodiment of the invention, a nucleic acid containing a portion 5 of a vertebrate deltex gene is used, as an Antagonist Therapeutic, to promote vertebrate deltex inactivation by homologous recombination (Koller and Smithies, 1989, Proc. Natl.

Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

The Agonist Therapeutics of the invention, as described supra, promote ° vertebrate Deltex function. Such Agonist Therapeutics mclude but are not limited to proteins and derivatives comprising the portions of Notch that mediate binding to vertebrate

Deltex, i.e. , the ANK repeats, and nucleic acids encoding the foregoing (which can be administered to express their encoded products in vivo). _ς Further descriptions and sources of Therapeutics of the inventions are found in Sections 5.1 through 5.7 herein.

Molecules which retain, or alternatively inhibit, a desired vertebrate Deltex property, e.g. , binding to Notch, binding to an intracellular ligand, can be used therapeutically as inducers, or inhibitors, respectively, of such property and its physiological 0 coπelates. In a specific embodiment, a peptide (e.g. , m the range of 6-50 or 15-25 amino acids; and particularly of about 10, 15, 20 or 25 amino acids) containing the sequence of a portion of vertebrate Deltex which binds to Notch is used to antagonize Notch function. In a specific embodiment, such an Antagonist Therapeutic is used to treat or prevent human or 5 other malignancies associated with increased Notch expression (e.g. , cervical cancer, colon cancer, breast cancer, squamous adenocarcinomas (see infra)). Derivatives or analogs of vertebrate Deltex can be tested for the desired activity by procedures known in the art, mcludmg but not limited to the assays described m the examples infra. For example, molecules compnsmg Deltex fragments which bind to Notch ANK repeats (see Section 7), can be obtamed and selected by expressing deletion mutants of human Deltex (or of a 5 nucleotide sequence of another species and assaymg for bmdmg of die expressed product to

Notch by any of several methods, such as the interaction trap system described m the

Examples Sections infra In one specific embodiment, peptide libraries can be screened to select a peptide with the desired activity; such screening can be earned out by assaymg,

10 e g. , for bmdmg to Notch or a molecule contaimng the Notch ANK repeats.

The Agonist and Antagonist Therapeutics of the mvention have therapeutic utility for disorders of cell fate The Agomst Therapeutics are administered therapeutically

(mcludmg prophylactically) (1) m diseases or disorders mvolvmg an absence or decreased

(relative to normal, or desired) levels of Notch or vertebrate Deltex function, for example, 15 m patients where Notch or vertebrate Deltex protem is lacking, genetically defective, biologically inactive or underactive, or underexpressed, and (2) m diseases or disorders wherein in vitro (or in vivo) assays (see infra) indicate the utility of vertebrate Deltex agomst administration The absence or decreased levels m Notch or vertebrate Deltex

20 function can be readily detected, e g., by obtaining a patient tissue sample (e g , from biopsy tissue) and assaying it in vitro for protem levels, structure and/or activity of the expressed Notch or vertebrate Deltex protem Many methods standard m the art can be thus employed, mcludmg but not limited to immunoassays to detect and/or visualize Notch or

» .. vertebrate Deltex protem (e g , Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry, etc ) and/or hybridization assays to detect Notch or vertebrate Deltex expression by detecting and/or visualizing respectively Notch or vertebrate deltex mRNA (e.g.. Northern assays, dot blots, in situ hybridization, etc )

30 In vitro assays which can be used to determine whether administration of a specific Agomst Therapeutic or Antagonist Therapeutic is mdicated, mclude in vitro cell culture assays m which a patient tissue sample is grown m culture, and exposed to or otherwise administered a Therapeutic, and the effect of such Therapeutic upon the tissue

35 sample is observed. In one embodiment, where the patient has a malignancy, a sample of cells from such malignancy is plated out or grown m culture, and the cells are then exposed to a Therapeutic. A Therapeutic which inhibits survival or growth of the malignant cells (e.g., by promoting terminal differentiation) is selected for therapeutic use in vivo. Many assays standard in the art can be used to assess such survival and/or growtii; for example, ceil proliferation can be assayed by measuring ³H-thymιdιne incorporation, by direct cell

5 count, by detectmg changes m transcπptional activity of known genes such as proto- oncogenes (e.g., fos, myc) or cell cycle markers; cell viability can be assessed by trypan blue staining, differentiation can be assessed visually based on changes m morphology, etc.

In a specific aspect, the malignant cell cultures are separately exposed to (1) an Agonist

10 Therapeutic, and (2) an Antagomst Therapeutic; the result of the assay can mdicate which type of Therapeutic has therapeutic efficacy.

In another embodiment, a Therapeutic is indicated for v_ae which exhibits the desired effect, inhibition or promotion of cell growth, upon a patient cell sample from tissue havmg or suspected of havmg a hyper- or hypoproliferative disorder, respectivel) Such hyper- or hypoproliferative disorders mclude but are not limited to those described in Sections 5.8.1 through 5 8.3 infra

In another specific embodiment, a Therapeutic is indicated for use in treating nerve mjury or a nervous system degenerative disorder (see Section 5.8.2) which exhibits in

²0 _Vιtro promotion of nerve regeneration/neuπte extension from nerve cells of the affected patient type

In addition, administration of an Antagomst Therapeutic of the mvention is also indicated in diseases or disorders determmed or known to involve a Notch or Deltex dominant activated phenotype ("gam of function" mutations ) Administration of an Agonist

-fa 3

Therapeutic is indicated in diseases or disorders determmed or known to mvolve a Notch or Deltex dommant negative phenotype ("loss of function" mutations) The functions of various structural domains of die Notch protein have been investigated in vivo, by ectopically expressmg a series of Drosophila Notch deletion mutants under the hsp70 heat-

30 shock promoter, as well as eye-specific promoters (see Rebay et al., 1993, Cell

74:319-329) Two classes of dominant phenotypes were observed, one suggestive of Notch loss-of function mutations and the other of Notch gam-of-function mutations Dommant "activated" phenotypes resulted from overexpression of a protein lacking most extracellular 3 sequences, while dommant "negative" phenotypes resulted from overexpression of a protem lackmg most intracellular sequences The results indicated that Notch functions as a receptor whose extracellular domain mediates ligand-binding, resulting in the transmission of developmental signals by the cytoplasmic domain. The phenotypes observed also suggested that the ANK repeat region within the intracellular domain plays an essential role in Notch mediated signal transduction events (intracellular function). We have shown that Drosophila 5

Deltex binds to the Notch ANK repeat region.

In various specific embodiments, in vitro assays can be carried out with representative cells of cell types involved in a patient's disorder, to determine if a

Therapeutic has a desired effect upon such cell types. 0 In another embodiment, cells of a patient tissue sample suspected of being pre-neoplastic are similarly plated out or grown in vitro, and exposed to a Therapeutic. The Therapeutic which results in a cell phenotype diat is more normal (i.e., less representative of a pre-neoplastic state, neoplastic state, malignant state, or transformed phenotype) is selected for therapeutic use. Many assays standard in the art can be used to assess whether a pre-neoplastic state, neoplastic state, or a transformed or malignant phenotype, is present. For example, characteristics associated with a transformed phenotype (a set of in vitro characteristics associated with a tumorigenic ability in vivo) include a more rounded cell moφhology, looser substratum attachment, loss of contact inhibition, loss of anchorage 0 dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton surface protein, etc. (see Luria et al., 1978, General Virology, 3d Ed., John Wiley & Sons, New York pp. 436-446).

_> _ In other specific embodiments, the in vitro assays described supra can be carried out using a cell line, rather than a cell sample derived from the specific patient to be treated, in which the cell line is derived from or displays characteristic(s) associated with the malignant, neoplastic or pre-neoplastic disorder desired to be treated or prevented, or is derived from the neural or other cell type upon which an effect is desired, according to the

30 present mvention.

In a specific embodiment, an antagonist of Notch and/or Deltex function that can be used as an Antagonist Therapeutic is a molecule comprising a Deltex protein or portion thereof that mediates binding to Notch, covalently bound to a protease or

35 proteolytically active fragment thereof. Such protease preferably is able to cleave a Notch protein. The molecule is preferably a fusion protein (i.e. , the covalent bond is a peptide bond). The Deltex protein is preferably a vertebrate protein, most preferably human.

Accordingly, the invention provides a method of targeting or inactivating proteins to which

Deltex binds (e.g. , Notch) in a cell. According to this method, the molecule comprising the

Deltex protein or portion thereof and the protease sequences is produced through chemical 5 or via molecular biological techniques. This molecule (e.g. , fusion protein) is introduced into the cell by techniques known in the art (e.g., transfection of the cell with a nucleic acid encoding the molecule such that its expression occurs intracellularly). Inside the cell, the molecule can bind to Notch and/or ouier Deltex binding partners. Upon such binding, the

10 protease portion of the molecule cleaves the protein to which the molecule is bound, thus inactivating it. For example, a fusion protein contaming domain I of human Deltex and the protease thermolysin, when introduced into the cell would bind to and cleave Notch, thereby inactivating the Notch signaling pathway. Molecules which would inactivate protein function e.g. , by binding thereto, can be used as an alternative to proteases.

The Antagonist Therapeutics are administered therapeutically (including prophylactically): (1) in diseases or disorders involving increased (relative to normal, or desired) levels of Notch or vertebrate Deltex function, for example, where the Notch or vertebrate Deltex protein is overexpressed or overactive; and (2) in diseases or disorders

²0 wherein in vitro (or in vivo) assays indicate the utility of vertebrate Deltex antagonist administration. The increased levels of Notch or vertebrate Deltex function can be readily detected by methods such as those described above, by quantifying protein and/or RNA. In vitro assays with cells of patient tissue sample or the appropriate cell line or cell type, to „ determine therapeutic utility, can be carried out as described above.

5.8.1. MALIGNANCIES

Malignant and pre-neoplastic conditions which can be tested as described supra for efficacy of intervention with Antagonist or Agonist Therapeutics, and which can

30 be treated upon dius observing an indication of therapeutic utility, mclude but are not limited to those described below in Sections 5.8.1 and 5.9.1.

Malignancies and related disorders, cells of which type can be tested in vitro (and/or in vivo), and upon observing the appropriate assay result, treated according to the

35 present invention, include but are not limited to those listed in Table 1 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed. , J.B. Lippincott Co., Philadelphia): TABLE 1 MALIGNANCIES AND RELATED DISORDERS Leukemia acute leukemia acute lymphocytic leukemia acute myelocytic leukemia myeloblastic promyelocytic myelomonocytic monocytic erythroleukemia chronic leukemia chronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemia Polycythemia vera Lymphoma

Hodgkin's disease non-Hodgkin's disease Multiple myeloma Waldenstrom's macroglobulinemia Heavy chain disease Solid tumors sarcomas and carcinomas fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendomeliosarcoma synovioma mesothelioma

Ewing's tumor leiomyosarcoma rhabdomyosarcoma colon carcinoma pancreatic cancer breast cancer ovarian cancer prostate cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma

Wilms' tumor cervical cancer testicular tumor lung carcinoma small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma menangioma melanoma neuroblastoma retinobiastoma

In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias) are treated or prevented in epithelial tissues such as those in the cervix, esophagus, and lung.

Malignancies of the colon and cervix can exhibit increased expression of human Notch relative to such non-malignant tissue (see PCT Publication WO 94/07474 published April 14, 1 94, incorporated by reference herein in its entirety). Thus, in specific embodiments, malignancies of the colon or cervix are treated or prevented by administering an effective amount of an Antagonist Therapeutic of the invention. The presence of increased Notch expression in colon, and cervical cancer suggests that many more cancerous and hyperproliferative conditions exhibit upregulated Notch. Thus, in specific embodiments, various cancers, e.g. , breast cancer, squamous adenocarcinoma, seminoma, melanoma, and lung cancer, as well as other hyperproliferative disorders, can be treated or prevented by

5 administration of an Antagonist Therapeutic.

5.8.2. NERVOUS SYSTEM DISORDERS

Nervous system disorders, involving cell types which can be tested as 0 described supra for efficacy of intervention with Antagonist or Agonist Therapeutics, and which can be treated upon thus observing an indication of merapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelinauon. Nervous system lesions which may be treated in a patient (including human and non-human vertebrate patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of 0 the nervous system, or compression injuries;

(ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) malignant lesions, in which a portion of the nervous system is

.fa o destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non- nervous system tissue; (iv) infectious lesions, in which a portion of the nervous system is

™ destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;

₃₅ (v) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;

(vi) lesions associated with nutritional diseases or disorders, in which a portion of die nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of die corpus callosum), and alcoholic cerebellar degeneration;

(vii) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropa y, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;

(viii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and

(ix) demyelinated lesions in which a portion of die nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus- associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopafhy, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons (see also Section 5.8). For example, and not by way of limitation, Therapeutics which elicit any of die following effects may be useful according to the invention: (i) increased survival time of neurons in culture;

(ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g. , choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or

(iv) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any metiiod known in ie art. In prefened, non-limiting embodiments, increased survival of neurons may be measured by die mediod set forth m

Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by mediods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4: 17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody bindmg, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessmg the physical manifestation of motor neuron disorder, e.g. , weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treated accordmg to the invention mclude but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy mat may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and mcludmg but not lunited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and me post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Maπe-Tooth Disease).

5.8.3. TISSUE REPAIR AND REGENERATION

In another embodiment of the invention, a Therapeutic of the mvention is used for promotion of tissue regeneration and repair, mcludmg but not lunited to treatment of benign dysprohferative disorders Specific embodiments are directed to treatment of cirrhosis of the liver (a condition m which scarring has overtaken normal liver regeneration processes) , treatment of keloid (hypertrophic scar) formation (disfiguring of die skm in which die scarring process interferes with normal renewal), psoriasis (a common skm condition characterized by excessive proliferation of the skm and delay in proper cell fate determination), and baldness (a condition in which terminally differentiated hair follicles (a tissue rich in Notch) fail to function properly).

Deltex agonists and antagonists can also be used to manipulate the differentiation state of non-terminally differentiated (e.g , stem and progenitor) cells For example, a stem cell can be exposed to such an agonist to inhibit its differentiation and achieve expansion of the stem cell population by incubation in vitro under growth conditions. Such stem cells have use in transplantation for in vivo repopulation of their progeny cells and tissue regeneration. (For mediods mat can be used in die foregoing, see

United States patent application Serial No. 08/537,210 filed September 29, 1995 by

5

Artavanis-Tsakonas et al., entitled "Manipulation of Non-Terminally Differentiated Cells

Using the Notch Pati way," which is incorporated by reference herein in its entirety.) For example, a method for d e expansion of a precursor cell (e.g., a human stem or progenitor cell) comprises contacting die cell with an amount of a vertebrate (e.g. , human) Deltex

10 protein or functionally active portion thereof effective to inhibit differentiation of the cell, and exposing die cell to cell growdi conditions such diat die cell proliferates. In various embodiments, die precursor cell can be but is not limited to a hematopoietic precursor cell, epithelial precursor cell, kidney precursor cell, neural precursor cell, skin precursor cell, osetoblast precursor cell, chondrocyte precursor cell, liver precursor cell, and muscle cell.

5.9. PROPHYLACTIC USES 5.9.1. MALIGNANCIES

The Therapeutics of the invention can be administered to prevent progression

20 to a neoplastic or malignant state, including but not limited to those disorders listed in Table

1. Such administration is indicated where the Therapeutic is shown in assays, as described supra, to have utility for treatment or prevention of such disorder. Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, 2 _,b _ in particular, where non-neoplastic cell growth consisting of hyperplasia. metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, witi out significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growdi in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. 3 Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growdi, involving a loss in individual cell uniformity and in die architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism Dysplasia characteristically occurs where tiiere exists chronic irritation or inflammation, and is often found in die cervix, respiratory passages, oral cavity, and gall bladder.

Alternatively or m addition to the presence of abnormal cell growth characterized as hyperplasia, metaplasia, or dysplasia, the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient, can indicate die desirability of prophylactic/dierapeutic administration of a Therapeutic of die invention. As mentioned supra, such characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protem, etc (see also id , at pp 84-90 for characteristics associated with a transformed or malignant phenotype)

In a specific embodiment, leukoplakia, a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are pre- neoplastic lesions indicative of the desirability of prophylactic intervention in another embodiment, fibrocystic disease (cystic hyperplasia, mammary dysplasia, particularly adenosis (bemgn epidielial hyperplasia)) is indicative of the desirability of prophylactic intervention.

In other embodiments, a patient which exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of a

Therapeutic a chromosomal translocation associated wirh a malignancy (e.g. , the

Philadelphia chromosome for chrome myelogenous leukemia, t(14;18) for folhcular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), bemgn monoclonal gammopathy (a possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mende an (genetic) inheritance pattern (e.g. , familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocπne adenomatosis, medullary diyroid carcmoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen, retmoblastoma, carotid body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., PhUadelphia, pp. 112-113) etc.)

In another specific embodiment, an Antagonist Therapeutic of die invention is administered to a human patient to prevent progression to breast, colon, or cervical cancer.

5.9.2. OTHER DISORDERS

In other embodiments, a Therapeutic of die invention can be administered to prevent a nervous system disorder described in Section 5.8.2, or other disorder (e.g. , liver cirrhosis, psoriasis, keloids, baldness) described in Section 5.8.3.

5.10. DEMONSTRATION OF THERAPEUTIC OR PROPHYLACTIC UTILITY

The Therapeutics of die invention can be tested in vivo for die desired dierapeutic or prophylactic activity. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used.

5.11. ANTISENSE REGULATION OF VERTEBRATE DELTEX EXPRESSION

The present invention provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding vertebrate

Deltex or a portion thereof. "Antisense" as used herein refers to a nucleic acid capable of hybridizing to a portion of a vertebrate deltex RNA (preferably mRNA) by virtue of some sequence complementarity. Such antisense nucleic acids have utility as Antagonist

Therapeutics of die invention, and can be used in me treatment or prevention of disorders as described supra in Section 5.8 and its subsections.

The antisense nucleic acids of die invention can be oligonucleotides diat are double -stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be direcdy administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences. In a specific embodiment, me vertebrate deltex antisense nucleic acids provided by die instant invention can be used for the treatment of tumors or other disorders, the cells of which tumor type or disorder can be demonstrated (in vitro or in vivo) to express a vertebrate deltex gene or a Notch gene. Such demonstration can be by detection 5 of RNA or of protein.

The invention further provides pharmaceutical compositions comprising an effective amount of die vertebrate deltex antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra in Section 5.12. Mediods for 0 treatment and prevention of disorders (such as tiiose described in Sections 5.8 and 5.9) comprising administering the pharmaceutical compositions of die invention are also provided.

In anodier embodiment, die invention is directed to methods for inhibiting the expression of a vertebrate deltex nucleic acid sequence in a prokaryotic or eukaryotic cell comprising providing die cell with an effective amount of a composition compnsmg an antisense vertebrate deltex nucleic acid of the invention.

Vertebrate deltex antisense nucleic acids and tiieir uses are described in detail below. 0

5.11.1. VERTEBRATE DELTEX ANTISENSE NUCLEIC ACIDS

The vertebrate deltex antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 oligonucleotides). In ₅ specific aspects, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include odier appending groups such as

30 peptides, or agents facilitating transport across the cell membrane (see, e.g. , Letsinger et al., 1989, Proc. Nad. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl.

Acad. Sci. 84:648-652; PCT Publication No. WO 88/09810, published December 15, 1988) or blood-brain barrier (see, e.g. , PCT Publication No. WO 89/10134, published April 25,

35 1988), hybridization-triggered cleavage agents (see, e.g. , Krol et al., 1988, BioTechniques

6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549) In a preferred aspect of the invention, a vertebrate deltex antisense oligonucleotide is provided, preferably of single-stranded DNA. In a most preferred aspect, such an oligonucleotide comprises a sequence antisense to me sequence encoding an SH3- binding domain or a Notch-binding domain of vertebrate deltex or zinc finger domain, most preferably, of human deltex. The oligonucleotide may be modified at any position on its structure witii substituents generally known in die art.

The vertebrate deltex antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracd, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,

4-acetylcytosine, 5 -(carboxy hydroxy lmediyl) uracil, 5-carboxymethylaminomethyl-

2-thiouridine, 5-carboxymedιylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-medιylguanine, l-methylinosine, 2,2-dimedιylguanine,

2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,

7-medιylguanine, 5-med ylaminomethyluracil, 5-methoxyaminomed yl-2-diiouracil, beta- D-mannosylqueosine, 5'-medιoxycarboxymedιyluracil, 5-methoxyuracil, 2-methyldιio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-dιiocytosine, 5-methyl-2-thiouracil, 2-thiouracd, 4-dιiouracil, 5-methyluracil, uracil- 5-oxyacetic acid mediylester, uracil-5-oxyacetic acid (v), 5-metiιyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

In anouier embodiment, the oligonucleotide comprises at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidodiioate, a phosphoramidate, a phosphordiamidate, a me iylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof. In yet another embodiment, die oligonucleotide is an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual /3-units, the strands run parallel to each other (Ga ier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide may be conjugated to another molecule, e.g. , a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

Oligonucleotides of the invention may be synthesized by standard mediods known in die art, e.g., by use of an automated DNA syndiesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by die method of Stein et al. (1988, Nucl. Acids Res.

16:3209), mediylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Nad. Acad. Sci. U.S.A. 85:7448-7451), etc.

In a specific embodiment, die vertebrate deltex antisense oligonucleotide comprises catalytic RNA, or a ribozyme (see, e.g. , PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al., 1990, Science 247: 1222-1225). In another embodiment, die oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

In an alternative embodiment, the vertebrate deltex antisense nucleic acid of the invention is produced intracellular ly by transcription from an exogenous sequence. For example, a vector can be introduced in vivo such that it is taken up by a cell, within which cell die vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the vertebrate deltex antisense nucleic acid Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA Such vectors can be constructed by recombinant DNA technology mediods standard m the art. Vectors can be plasmid, viral, or others known in die art, used for replication and expression in vertebrate cells. Expression of the sequence encoding die vertebrate deltex antisense RNA can be by any promoter known in die art to act in vertebrate, preferably human, cells. Such promoters can be inducible or constitutive. Such promoters mclude but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981 , Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. , 1980, Cell 22:787-797), die herpes thymidine kinase promoter (Wagner et al. , 1981 , Proc. Nad. Acad. Sci. U.S.A. 78:1441-1445), die regulatory sequences of the metallodiionein gene (Brinster et al., 1982, Nature 296:39-42), etc.

The antisense nucleic acids of die invention comprise a sequence complementary to at least a portion of an RNA transcript of a vertebrate deltex gene, preferably a human deltex gene. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA," as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with die

RNA, forming a stable duplex; in the case of double-stranded vertebrate deltex antisense nucleic acids, a single strand of the duplex DNA may tiius be tested, or triplex formation may be assayed. The ability to hybridize will depend on both die degree of complementarity and the lengtii of the antisense nucleic acid. Generally, die longer the hybridizing nucleic acid, die more base mismatches with a vertebrate deltex RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in die art can ascertain a tolerable degree of mismatch by use of standard procedures to determine die melting point of the hybridized complex.

5.11.2. THERAPEUTIC UTILITY OF VERTEBRATE DELTEX ANTISENSE NUCLEIC ACIDS

The vertebrate deltex antisense nucleic acids can be used to treat (or prevent) malignancies or other disorders, of a cell type which has been shown to express vertebrate deltex or Notch. In specific embodiments, die malignancy is cervical, breast, or colon cancer, or squamous adenocarcinoma. Malignant, neoplastic, and pre-neoplastic cells which can be tested for such expression include but are not limited to ose described supra in

Sections 5.8. 1 and 5.9.1. In a preferred embodiment, a single-stranded DNA antisense vertebrate deltex oligonucleotide is used.

Malignant (particularly, tumor) cell types which express vertebrate deltex or Notch RNA can be identified by various methods known in die art. Such methods include but are not limited to hybridization with a vertebrate deltex or ΛtotcΛ-specific nucleic acid

(e.g., by Northern hybridization, dot blot hybridization, in situ hybridization), observing the ability of RNA from the cell type to be translated in vitro into Notch or vertebrate Deltex, immunoassay, etc. In a preferred aspect, primary tumor tissue from a patient can be assayed for Notch or vertebrate Deltex expression prior to treatment, e.g. , by immunocytochemistry or in situ hybridization. Pharmaceutical compositions of die invention (see Section 5.12), compπsing an effective amount of a vertebrate deltex antisense nucleic acid m a pharmaceutically acceptable carrier, can be administered to a patient havmg a malignancy which is of a type that expresses Notch or vertebrate deltex RNA or protem.

The amount of vertebrate deltex antisense nucleic acid which wdl be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques Where possible, it is desirable to determine die antisense cytotoxicity of the tumor type to be treated in vitro, and tiien m useful animal model systems prior to testing and use m humans.

In a specific embodunent, pharmaceutical compositions comprising vertebrate deltex antisense nucleic acids are administered via hposomes, microparticles, or microcapsules In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the vertebrate deltex antisense nucleic acids In a specific embodiment, it may be desirable to utilize hposomes targeted via antibodies to specific identifiable tumor antigens (Leonetti et al , 1990, Proc. Nad Acad. Sci U.S. A

87:2448-2451 , Renneisen et al., 1990, J. Biol. Chem. 265: 16337-16342).

5.12. THERAPEUTIC/PROPHYLACTIC

ADMINISTRATION AND COMPOSITIONS

The invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the mvention In a preferred aspect, the Therapeutic is substantially purified The subject is preferably an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.

Various delivery systems are known and can be used to administer a

Therapeutic of the invention, e.g. , encapsulation in hposomes, microparticles, microcapsules, expression by recombinant cells, receptor mediated endocytosis (see, e.g. ,

Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to mtradermal, intramuscular, lntrapeπtoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together witii other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce die pharmaceutical compositions of die invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer die pharmaceutical compositions of the invention locally to die area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g. , in conjunction with a wound dressing after surgery, by injection, by means of a cadieter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.

In another embodiment, die Therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid. , pp. 317-327; see generally ibid.)

In yet another embodiment, die Therapeutic can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC

Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and

Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al. , Science 228: 190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71 : 105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of die therapeutic target, i.e. , the brain, ti us requiring only a fraction of the systemic dose (see, e.g. , Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer

(Science 249: 1527-1533 (1990)).

In a specific embodiment, administration of a Therapeutic into a Notch- expressing cell is accomplished by linkage of the Therapeutic to a Delta (or other toporydimic) protein or portion thereof capable of mediating binding to Notch. Contact of a Notch-expressing cell wi i die linked Therapeutic results in binding of die linked Therapeutic via its Delta portion to Notch on the surface of die cell, followed by uptake of the linked Therapeutic into the Notch-expressing cell.

In a specific embodiment, die Therapeutic is delivered intracellularly (e.g., by expression from a nucleic acid vector, or by linkage to a Delta protein capable of binding to Notch followed by binding and internalization, or by receptor-mediated or diffusion mechanisms).

In a specific embodiment where the Therapeutic is a nucleic acid encoding a protein Therapeutic, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so diat it becomes intracellular, e.g. , by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g. , a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g. , Joliot et al., 1991 , Proc. Natl. Acad. Sci. USA 88: 1864-1868), etc. Alternatively, a nucleic acid Therapeutic can be introduced intracellularly and incorporated witiiin host cell DNA for expression, by homologous recombination.

In specific embodiments directed to treatment or prevention of particular disorders, preferably the following forms of administration are used:

Disorder Preferred Forms of Administration

Cervical cancer Topical Gastrointestinal cancer Oral; intravenous Lung cancer Inhaled; intravenous Leukemia Intravenous; extracorporeal

Metastatic carcinomas Intravenous; oral

Brain cancer Targeted; intravenous; intrathecal

Liver cirrhosis Oral; intravenous

Psoriasis Topical

Keloids Topical

Baldness Topical

Spinal cord injury Targeted; intravenous; intrathecal

10 Parkinson's disease Targeted; intravenous; intrathecal

Motor neuron disease Targeted; intravenous; intrathecal

Alzheimer's disease Targeted; intravenous; intradiecal

The present invention also provides pharmaceutical compositions. Such

15 compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of die Federal or a state government or listed in the U.S. Pharmacopeia or otiier generally recognized pharmacopeia for use in 0 animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which die dierapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mmeral oil, sesame oil and die Id e. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk,

³⁰ glycerol, propylene, glycol, water, ethanol and die like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pdls, capsules, powders, sustained-release formulations and the like. The composition can be

, _ς formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's

Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of ie Therapeutic, preferably in purified form, togetiier widi a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, die composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. In anodier preferred embodiment, me composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to mammals. Typically, compositions for intravenous administration are solutions in sterϋe isotonic aqueous buffer. Where necessary, the composition may also include a solubdizing agent and a local anesmetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating die quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the 0 composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The Therapeutics of die invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ed ylamino eti anol, histidine, procaine, etc.

The amount of the Therapeutic of die invention which will be effective in the ° treatment of a particular disorder or condition wdl depend on die nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on die route of administration, and ₅ the seriousness of die disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active mgredient in die range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of die ingredients of d e pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in e form prescribed by a governmental agency regulating die manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by die agency of manufacture, use or sale for human adniinistration.

5.13. DIAGNOSTIC UTILITY

Vertebrate Deltex proteins, analogues, derivatives, and subsequences diereof, vertebrate deltex nucleic acids (and sequences complementary diereto), anti-vertebrate Deltex antibodies, have uses in diagnostics. Such molecules can be used in assays, such as 0 immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting vertebrate Deltex expression, or monitor e treatment diereof. In particular, such an immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-vertebrate Deltex antibody under conditions such diat immunospecific binding can occur, and detecting or measuring d e amount of any immunospecific binding by the antibody. In a specific aspect, such binding of antibody, in tissue sections, preferably in conjunction with binding of anti-Notch can be used to detect aberrant Notch and/or vertebrate Deltex localization or aberrant levels of Notch-vertebrate Deltex colocalization in a disease state. In a specific embodiment, antibody to vertebrate " Deltex can be used to assay in a patient tissue or serum sample for the presence of vertebrate Deltex where an aberrant level of vertebrate Deltex is an indication of a diseased condition. Aberrant levels of vertebrate Deltex binding abUity in an endogenous Notch protein, or aberrant levels of binding ability to Notch (or otiier vertebrate Deltex ligand) in 5 an endogenous vertebrate Deltex protein may be indicative of a disorder of cell fate (e.g. , cancer, etc.) By "aberrant levels," is meant increased or decreased levels relative to that present, or a standard level representing diat present, in an analogous sample from a subject not having the disorder.

The immunoassays which can be used include but are not lunited to competitive and non-competitive assay systems using techniques such as western blots. 5 radioi munoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitm reactions, lmmunodiffusion assays, agglutination assays, complement-fixation assays, lmmunoradiometπc assays, fluorescent immunoassays, protein A immunoassays, to name 0 but a few.

Vertebrate deltex genes and related nucleic acid sequences and subsequences, mcludmg complementary sequences, and odier toporydiπuc gene sequences, can also be used hybridization assays Vertebrate deltex nucleic acid sequences, or subsequences thereof compnsmg about at least 8 nucleotides, can be used as hybridization probes

Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with aberrant changes in vertebrate Deltex expression and/or activity as described supra. In particular, such a hybridization assay is carried out by a mediod comprising contacting a sample containing nucleic acid with a nucleic acid

²0 probe capable of hybridizing to vertebrate deltex DNA or RNA, under conditions such that hybridization can occur, and detecting or measuring any resultmg hybridization

6. EXAMPLE: CLONING AND CHARACTERIZATION OF HUMAN DELTEX

_ 2 b _ As described herein, we have accomplished die isolation and molecular characterization of human deltex. We report the cloning and sequencing of human deltex

Human deltex encodes five putative SH3 domain binding sites and a πng-H2-zmc finger in similar locations to the corresponding motifs found m Drosophila Deltex

^{3 0} 6.1. RESULTS

6.1.1. MOLECULAR CLONING OF THE HUMAN DELTEX LOCUS

Human deltex was isolated dirough a combination of computer and biochemical screens. Initially, a human expressed sequence tag database was screened for

35 homology against the ammo acid sequence of Drosophila Deltex The critical part of this search involved die assumption at stop codons in a particular readmg frame of the database are die result of sequencmg mistakes. Accordingly, stop codons were ignored and the open readmg frame was extended in a different frame. The predicted ammo acid sequence encoded by die hypodietical open readmg frames were then compared widi die protem product of die Drosophila deltex transcription unit.

We previously identified die Drosophila deltex transcription unit by showing via germline-mediated transformation experiments that a genomic fragment containing this transcription umt is capable of complementing most deltex mutant defects Moreover, mis genomic fragment rescues die normally lethal genetic interaction diat results when flies are doubly mutant for deltex and nd Fmally, Northern analysis mdicates a maternal loadmg of deltex transcripts mto die developmg oocyte, a finding diat is consistent widi die maternal effect observed upon embryogenesis m eggs laid by homozygous mutant mothers (Xu and Artavanis-Tsakonas, 1990 Genetics 126:665-677) cDNA clones homologous to the transcription umt were isolated from an embryonic cDNA library, the complete nucleotide sequence (SEQ ID NO 1) and predicted protem product were then determmed (SEQ ID NO:2)

Comparison of the ammo acid sequence of Drosophila Deltex widi diat predicted for what we deduced to be hypothetical open reading frames in the database identified a sequence: gnl I dbest I 24254 T05200 widi significant homology to Drosophila Deltex Within T05200, five conserved stretches of amino acids were found in different readmg frames, at residues 7-39 (SEQ ID NO-4), 102-149 (SEQ ID NO 6), 138-245 (SEQ ID NO 8), and 200-310 (SEQ ID NO.10) corresponding to Drosophila Deltex residues 545- 555 (SEQ ID NO 3), 565-580 (SEQ ID NO 5), 581-616 (SEQ ID NO 7) and 602-638 (SEQ ID NO 9) respectively These sequences are shown in Table II, identical ammo acids are shown in bold

TABLE II

A series of two 5' primers (hdx-1 (SEQ ID NO:26) and hdx-2 (SEQ ID NO:27)) and two 3' primers (hdx-3 (SEQ ID NO:28) and hdx-4 (SEQ ID NO:29 )) were synthesized based on die DNA sequence of gnl I dbest I 24254 T05200. PCR reactions were performed using die four different primer combinations and a human fetal brain cDNA library (Invitrogene) as the template The PCR product was sequenced and found to have the same DNA sequence as gnl I dbest I 24254 T05200.

The PCR product generated using die hdx-1 and hdx-4 primers was dien labeled and used to screen another human fetal brain cDNA library The isolate was sequenced (SEQ ID NO: 11) and die predicted protein determined (SEQ ID NO.12) (Figure 2A-C). Not greater than 107 contmuous nucleotides of SEQ ID NO: 11 were present in T05200 Applying standard techniques, the cDNA isolate obtamed using die PCR product as probe was then labeled and itself used as a probe to screen a northern blot containing poly(A)⁺ mRNA isolated from various human tissue samples This probe was observed to hybridize to a 5.4-kb RNA in heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. When this probe was used to screen a zoo blot (a blot containing genomic EcoRI-digested

DNA of various species, obtained from Clontech) by Southern hybridization, hybridization was observed in genomic human, monkey, rat, mice, dog, cow, and 5 yeast DNA. Hybridization was not observed in die rabbit and chicken genomic

DNA.

Structural analysis of the human Deltex protein:

10 The predicted human Deltex product has 720 amino acids and an estimated molecular mass of approximately 80 kDa. The 180 amino terminal residues of human Deltex have an approximate identity of 33% widi die coπesponding amino acid residues of Drosophila Deltex and the nucleic acids encoding tiiese amino acids have an approximate 52% identity. The 180 carboxy terminal amino acids of human Deltex have an approximate 48 % identity widi die coπesponding amino acid residues of Drosophila Deltex and the nucleic acids encoding diese carboxy terminal amino acids have an approximate 49% identity. A structural analysis of human Deltex protein revealed a conserved 0 structure among Deltex proteins (see Figure 3). Like Drosophila Deltex, human Deltex has both ring-H2-zinc finger (amino acids 411-471) (SEQ ID NO:25) and putative SH3-binding domains. Noticeably absent from the human Deltex are the two opa repeats that subdivide die primary structure of the Drosophila Deltex into

- _j. three domains. Each of the Drosophila Deltex domains I, II, and III, has been found using die yeast "interaction trap assay" to be capable of mediating homotypic interactions (see infra). i) Domain I:

Domain I corresponds to the N-terminal 303 amino acids of

30 Drosophila and the first 237 amino acids of human Deltex. In Drosophila, we have demonstrated that die region of human Deltex corresponding to the first 175 amino acids of domain I is essential and sufficient to bind the Notch ANK repeats and overexpression of this domain can rescue loss-of-function phenotypes of 35 Deltex. Since Drosophila Deltex can bind to human Notch- 1 and 2, conservation of bmdmg activity between human Deltex and Drosophila domain I is suggested

Furthermore, this has been demonstrated. See infra. ii) Putative SH3 binding domams

Domam II of Drosophila Deltex contains a putative SH3-bindιng site

(amino acids 476-484) (Matsuno et al., 1995, Development 121(8):2633-2644)

Five putative SH3-bιndιng sites (SEQ ID NOS: 17-21) are found in human Deltex

(within amino acids 226-377) in a position correspondmg to the SH3-bιndιng site m domain II of Drosophila Deltex (Table TS) SH2 and SH3 domams are conserved protem modules so named based on tiieir homology to the oncogene Src (Src Homology) These motifs have been implicated in mediating protein-protein interactions in a number of signal transduction patiiways (reviewed in Cell 71:359-362;Science 252:668-674; Trends Cell Biol 3-8-13, FEBS 307:55-61) Recently, a complementary motif mat bmds to die SH3 domam has been identified and called simply an ^*SH3-bιndιng domam' ("SH3-BD") (Science 259: 1157-1161) The core bmdmg region of SH3-BD is prolme-rich and approximately ten residues in length As shown m Table III, tins motif, as defined from a mouse protem that experimentally bound an SH3 domam (SEQ ID NO:23), is shown aligned to die putative SH3-bιndιng site m Drosophila Deltex (SEQ ID NO.22) and the five regions (SEQ ID NOS: 17-21) that may represent human versions of this motif These regions are located centrally m die Deltex protem For reference, regions of the protem encoded by die Drosophila Son of sevenless (SOS) protein (SEQ ID NO 24), which may aiso contain SH3-BD, is shown The Son of sevenless encoded protein, a putative guanine nucleotide exchange factor (GNEF), has been shown to bind to an 'adaptor¹ protem (drk) containing only SH2 and SH3 modules, aldiough die actual residues diat mediate bmdmg have not been accurately defined (Sunon M , et al., 1993, Cell 73.169-177 and Olivier J., et al , 1993, Cell 73 179-191) TABLE OI

There are currently only six SH3-containing proteins identified in Drosophila, any one of which may be a direct binding parmer of Deltex, and thus an indirect parmer of Notch.

The functional requirement of the Deltex domain II-III in Drosophila which contains die putative SH3 domain binding site and ring-H2-zinc fmger has been suggested from deletion analyses, in which die deletion of these domains resulted in a significant reduction of the ability to activate the Notch signaling pathway. These results indicate diat domains II and III are not redundant, iii) Ring-H2-zinc finger:

Human deltex (nucleotides 1734-1916 of SEQ ID NO: 11) encodes a ring-H2-zinc finger (SEQ ID NO: 25), appearing as amino acids 411-471 of SEQ ID NO: 12 in mat part of human Deltex which corresponds to domain III of Drosophila Deltex. This type of zinc finger is believed to be involved in protein protein interactions. 6.2. MATERIAL AND METHODS 6.2.1. SEQUENCE DETERMINATION AND ANALYSIS

The EcoRI-cDNA insert was subcloned directiy bo orientations mto Bluescπpt KS. Overlappmg deletions were produced on the insert usmg die

DNAse I method to generate bidirectional deletions (Eberle et al., 1993,

Biotechniques 14:408). The resulting deletions were analyzed usmg an IBI automatic sequencer.

DNA sequence manipulations were performed usmg Intelligenetic's PC-GENE software Open readmg frame prediction and plotting were performed usmg the University of Wisconsin program CODONPREFERENCE (Gribshov et al., 1984, Nucl Acids Res. 12:539-549) The GenPept and SWISS-PROT databases were searched with all or part of the deduced ammo acid sequence usmg the FASTA program (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA,

85:2444-2448) available by the GenBank FASTA server through BITNET.

7. EXAMPLE: HUMAN DELTEX BINDS

HUMAN AND DROSOPHILA NOTCH We have demonstrated in Drosophila a specific and direct physical interaction between Deltex and Notch ANK repeats (Diedeπch et al., 1994, Development 120:473-481) To study protem-protein interactions between human Deltex and various cytoplasmic domams of human and Drosophila Notch receptors, we conducted expression studies in yeast usmg the so-called 'interaction trap' assay technique (Zervos et al., 1993, Cell 72.223-232).

In tins assay, one protem segment is fused to the DNA-bmdmg domam of the LexA protem, which in mrn bmds to me promoter of a LexAop-lacZ reporter construct without activating transcription. These constructs are referred as pEG. A second foreign protem segment is fused to an acidic transcriptional activation domain that does not bmd DNA on its own. These constructs are referred to as pJG Coexpression of these two proteins in yeast cells results in the functional reconstruction of an active LexA "hybrid" transcription factor if the foreign proteins physically interact with one another. Activity of the hybrid transcription factor is monitored by transcription of the /3-galactosιdase reporter gene Expression of fusion proteins from the pJG construct is induceu when yeast cells are cultured in die galactose media but not in die glucose media. Therefore, positive interaction should be observed only in galactose media.

The constructs examined using die yeast interaction were as follows: die pEGhDeltex construct contains die entire coding region of human Deltex; pJGhNotch-1 encodes die ankyrin repeats region of human Notch- 1 from amino acids 1826-2147; pJGhNotch-2 encodes die ankyrin repeats region of human Notch- 2 from amino acids 1772-2084; pJGhNotch encodes die ankyrin repeats of Drosophila Notch from amino acids 1827-2259; and JGfHairless contains the entire coding region of Drosophila Hairless.

As presented in Table IV, significant induction of /3-galactosidase activity was observed when yeast cells cotransfected widi pEGhDeltex and pJGhNotch-1, pJGHNotch-2 or pJGHfNotch, were cultured in galactose media (Table IV). These results indicate mat human Deltex binds to the ankyrin repeats human Notch- 1 , human Notch-2 as well as diat of Drosophila Notch. Standard deviation is presented in the parentheses.

TABLE IV

8. EXAMPLE: CLONING OFVERTEBRATE DELTEX GENES

The evolution of humans and Drosophila diverged about 600 million years ago. As discussed supra, Deltex protein demonstrates a conserved structure m tiiese two evolutionary distant species. Knowledge of die conserved regions of the protein allows one to design synthetic degenerate primers for use m hybridization and PCR reactions which enable the cloning of Deltex encodmg nucleic acids in otiier organisms.

Five regions of high conservation between human and Drosophila are found m ammo acid stretches of human Deltex ammo acid numbers 414-419 (SEQ ID NO:30), 475-480 (SEQ ID NO:31), 504-511 (SEQ ID NO:32), 531-539 (SEQ ID NO:33) and 557-564 (SEQ ID NO:34). These sequences are conserved in Drosophila Deltex ammo acid stretches 549-555 (SEQ ID NO: 35), 603-608 (SEQ ID NO:36), 632-639 (SEQ ID NO:37), 659-667 (SEQ ID NO 38) and 685- 692 (SEQ ID NO:39), respectively. Conserved ammo acid stretches may be used alone or m combination to isolate the deltex encodmg nucleic acids of other organisms.

By way of example, a murme deltex gene is obtamed as follows: Standard techmques are utilized to synthesize a series of degenerate primers encoding amino acids 414-419 in Drosophila (SEQ ID NO 30) and 549-555 in human (SEQ ID NO: 35) m a 5' to 3' orientation A second series of degenerate primers correspondmg to die antisense strand of the nucleic acids encoding ammo acids 475-480 in Drosophila (SEQ ID NO:31) and 603-608 in human (SEQ ID NO.36) is also syntiiesized. The two series of primers are added to a mixture contaming mouse embryomc cDNA as template for the PCR amplification PCR is carried out at a range of stringencies, accordmg to mediods commonly known, to allow for varymg degrees of nucleotide similarity between the known deltex sequences and die mouse nucleic acid homolog bemg isolated

After successful PCR amplification, the segment of mouse deltex gene is molecularly cloned and sequenced through techniques known in the art. This segment is used as a probe to isolate a complete cDNA and genomic clone The complete nucleotide sequence of the mouse deltex homolog is determined by sequence analysis. 9. DEPOSIT OF MICROORGANISMS

Plasmid pBS hdx containing a cDNA insert encoding a full-length human deltex as a EcoRI insert in Bluescript vector (Stratagene) was deposited by S. Leslie Misrock, of Pennie & Edmonds, 1155 Avenue of the Americas, New York, New York 10036 on behalf of Yale University on November 17, 1995, with the American Type Culture Collection, 1201 Parklawn Drive, Rockville, Maryland 20852, under d e provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures, and assigned accession number 97341.

The present invention is not to be limited in scope by die microorganism deposited or the specific embodiments described herein. Indeed, various modifications of d e invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are incorporated in tiieir entireties.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Artavanis-Tsakonas, Spyridon MatBuno, Kenji

(ii) TITLE OF INVENTION: VERTEBRATE DELTEX PROTEINS, NUCLEIC ACIDS, AND ANTIBODIES, AND RELATED METHODS AND COMPOSITIONS

(iii) NUMBER OF SEQUENCES: 39

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pennie & Edmonds

(B) STREET: 1155 Avenue of the Americas

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: U.S.A.

(F) ZIP: 10036-2711

(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: To Be Assigned

(B) FILING DATE: 22-NOV-1995

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Misrock, S. Leslie

(B) REGISTRATION NUMBER: 18,872

(C) REFERENCE/DOCKET NUMBER: 7326-036

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 790-9090

(B) TELEFAX: (212) 869-9741/8864

(C) TELEX: 66141 PENNIE

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3771 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 345..2555

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

AAATGCTAGA AAAACCGTTT TTACCATCAA ACGTGAATTC TTAAGCTGCG CCTAAACGAA 60

ACCGAGTGAC TAAAGAACCA GAACGAAAAC TTCGGGAAAA TGGAAGCCAG GGAAAATCAG 120

GGATAACTAA CGCTGGCAGC GGGTCCACCA TTTTTAATTT CTTTGTTTAT TTTGTGCCCA 180

TCTTCGCGAG CGAGCGAGAT AGCGCGACAG CAACAGCAAG AGAGAGCGAG AGAGA AGTG 240 AGTGAGTGAG AGCTAGTGAA GAGAGCGCAG GAGGAGTTGG ATATGGAAAT GCGCATGGAT 300

ATGGCAATGG GCTCACTCCA CGGATAACGG ATCAACTGCA AGCA ATG GCC AGC AGC 356

Met Ala Ser Ser 1

GCC GGA AGT GCG GCA TCC GGA TCC GTT GTT CCC GGT GGC GGA GGT AGC 404

Ala Gly Ser Ala Ala Ser Gly Ser Val Val Pro Gly Gly Gly Gly Ser

5 10 15 20

GCC GCC TCC AGT TGT GCC ACC ATG GCC CTG TCC ACC GCC GGA TCC GGT 452

Ala Ala Ser Ser Cys Ala Thr Met Ala Leu Ser Thr Ala Gly Ser Gly

25 30 35

GGG CCG CCC GTG AAC CAC GCC CAC GCC GTC TGC GTG TGG GAG TTC GAG 500

Gly Pro Pro Val Aεn His Ala His Ala Val Cys Val Trp Glu Phe Glu

40 45 50

TCG CGC GGC AAG TGG CTG CCC TAT TCG CCG GCG GTG TCG CAG CAC TTG 548

Ser Arg Gly Lys Trp Leu Pro Tyr Ser Pro Ala Val Ser Gin His Leu 55 60 65

GAA CGC GCC CAC GCC AAG AAA CTG ACG CGC GTC ATG CTG AGC GAT GCG 596

Glu Arg Ala His Ala Lys Lys Leu Thr Arg Val Met Leu Ser Asp Ala 70 75 80

GAT CCC AGC CTG GAG CAG TAC TAC GTC AAC GTG CGC ACA ATG ACC CAG 644

Asp Pro Ser Leu Glu Gin Tyr Tyr Val Asn Val Arg Thr Met Thr Gin

85 90 95 100

GAA TCG GAG GCG GAA ACG CGC TCC GGC CTG CTG ACC ATC GGT GTT CGG 692

Glu Ser Glu Ala Glu Thr Arg Ser Gly Leu Leu Thr lie Gly Val Arg

105 110 115

CGC ATG TTA TAC GCA CCC AGC TCG CCG GCG GGC AAG GGC ACC AAG TGG 740

Arg Met Leu Tyr Ala Pro Ser Ser Pro Ala Gly Lys Gly Thr Lys Trp

120 125 130

GAG TGG TCG GGC GGC AGT GCC GAT AGC AAC AAC GAC TGG CGG CCC TAC 788

Glu Trp Ser Gly Gly Ser Ala Asp Ser Asn Aεn Asp Trp Arg Pro Tyr 135 140 145

AAC ATG CAC GTC CAG TGC ATC ATC GAG GAC GCC TGG GCG AGG GGC GAA 836

Asn Met His Val Gin Cys He He Glu Asp Ala Trp Ala Arg Gly Glu 150 155 160

CAA ACC TTG GAC CTG TGC AAC ACC CAC ATC GGC CTG CCG TAC ACC ATT 884

Gin Thr Leu Asp Leu Cys Asn Thr H s He Gly Leu Pro Tyr Thr He

165 170 175 180

AAT TTT TGC AAT CTC ACC CAC GTG CGC CAA CCC AGC GGA CCC ATG CGC 932

Asn Phe Cys Asn Leu Thr His Val Arg Gin Pro Ser Gly Pro Met Arg

185 190 195

AGC ATT CGG CGT ACC CAA CAG GCG CCG TAT CCC TTG GTG AAA CTA ACG 980

Ser He Arg Arg Thr Gin Gin Ala Pro Tyr Pro Leu Val Lys Leu Thr

200 205 210

CCA CAA CAG GCC AAC CAA CTC AAG TCG AAT TCC GCC AGC GTG AGC AGC 1028

Pro Gin Gin Ala Asn Gin Leu Lys Ser Asn Ser Ala Ser Val Ser Ser 215 220 225

CAG TAC AAC ACT CTA CCC AAA CTG GGC GAC ACC AAG AGC CTG CAC AGA 1076

Gin Tyr Asn Thr Leu Pro Lys Leu Gly Asp Thr Lys Ser Leu His Arg 230 235 240

GTG CCC ATG ACC AGG CAA CAG-CAC CCA TTG CCC ACC AGC CAT CAA GTG 1124

Val Pro Met Thr Arg Gin Gin His Pro Leu Pro Thr Ser His Gin Val

245 250 255 260 CAG CAG CAG CAG CAT CAG CTC CAG CAT CAA CAG CAG CAG CAG CAG CAA 1172 Gin Gin Gin Gin His Gin Leu Gin His Gin Gin Gin Gin Gin Gin Gin 265 270 275

CAT CAT CAC CAG CAT CAG CAA CAA CAG CAT CAG CAA CAG CAG CAA CAT 1220 His His His Gin His Gin Gin Gin Gin His Gin Gin Gin Gin Gin Hiε 280 285 290

CAG ATG CAG CAC CAT CAG ATC CAT CAT CAG ACG GCG CCC AGG AAG CCG 1268 Gin Met Gin His Hiε Gin He His His Gin Thr Ala Pro Arg Lys Pro 295 300 305

CCC AAG AAG CAC AGC GAG ATC TCC ACC ACC AAT CTA CGC CAG ATA CTC 1316 Pro Lys Lys His Ser Glu He Ser Thr Thr Asn Leu Arg Gin He Leu 310 315 320

AAC AAC CTA AAC ATC TTC AGC AGC AGC ACT AAG CAC CAA TCG AAC ATG 1364 Asn Asn Leu Asn He Phe Ser Ser Ser Thr Lys His Gin Ser Aεn Met 325 330 335 340

TCG ACG GCG GCC AGT GCC AGT TCA TCC TCC TCA TCG GCC TCG CTG CAC 1412 Ser Thr Ala Ala Ser Ala Ser Ser Ser Ser Ser Ser Ala Ser Leu Hiε 345 350 355

CAT GCC AAC CAT CTG TCG CAT GCG CAC TTT TCG CAC GCC AAG AAC ATG 1460 His Ala Asn His Leu Ser His Ala His Phe Ser His Ala Lys Asn Met 360 365 370

CTG ACT GCC TCG ATG AAC AGT CAT CAT AGT CGC TGC TCG GAG GGA TCG 1508 Leu Thr Ala Ser Met Asn Ser His His Ser Arg Cyε Ser Glu Gly Ser 375 380 385

CTG CAG TCG CAA AGG AGC AGC CGG ATG GGC TCG CAT CGC TCG AGA TCG 1556 Leu Gin Ser Gin Arg Ser Ser Arg Met Gly Ser His Arg Ser Arg Ser 390 395 400

CGA ACG CGG ACC TCG GAC ACG GAC ACG AAC AGT GTG AAA TCG CAT CGG 1604 Arg Thr Arg Thr Ser Asp Thr Asp Thr Asn Ser Val Lys Ser His Arg 405 410 415 420

CGG AGA CCC AGT GTG GAC ACC GTG TCC ACT TAC CTC AGC CAC GAG AGC 1652 Arg Arg Pro Ser Val Asp Thr Val Ser Thr Tyr Leu Ser His Glu Ser 425 430 435

AAG GAG AGC CTG CGC AGC AGG AAC TTT GCC ATT TCG GTC AAT GAT CTG 1700 Lys Glu Ser Leu Arg Ser Arg Asn Phe Ala He Ser Val Aεn Asp Leu 440 445 450

CTG GAC TGC TCG CTT GGC AGC GAT GAA GTT TTT GTG CCC TCC GTG CCG 1748 Leu Asp Cyε Ser Leu Gly Ser Aεp Glu Val Phe Val Pro Ser Val Pro 455 460 465

CCA TCG TCG CTG GGC GAA AGG GCG CCG GTG CCG CCG CCA TTA CCA CTG 1796 Pro Ser Ser Leu Gly Glu Arg Ala Pro Val Pro Pro Pro Leu Pro Leu 470 475 480

CAT CCG CGA CAG CAA CAG CAG CAG CAA CAA CAG CAG CAA CAG CTG CAG 1844 Hiε Pro Arg Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Leu Gin 485 490 495 500

ATG CAA CAG CAG CAA CAG GCG CAG CAG CAG CAG CAG CAA TCA ATC GCC 1892 Met Gin Gin Gin Gin Gin Ala Gin Gin Gin Gin Gin Gin Ser He Ala 505 510 515

GGT TCG ATT GTG GGC GTG GAC CCG GCC AGC GAT ATG ATA TCG CGT TTT 1940 Gly Ser He Val Gly Val Asp Pro Ala Ser Aεp Met He Ser Arg Phe 520 525 530

GTC AAG GTG GTG GAG CCA CCG CTG TGG CCC AAT GCC CAG CCC TGi CCC 1988 Val Lys Val Val Glu Pro Pro Leu Trp Pro Asn Ala Gin Pro Cys Pro 535 540 545

ATG TGC ATG GAG GAG CTG GTG CAC TCC GCC CAG AAT CCG GCC ATT TCG 2036 Met Cys Met Glu Glu Leu Val His Ser Ala Gin Asn Pro Ala He Ser 550 555 560

CTG AGT CGC TGC CAG CAT CTC ATG CAT TTG CAG TGC CTC AAT GGG ATG 2084 Leu Ser Arg Cys Gin His Leu Met His Leu Gin Cys Leu Asn Gly Met 565 570 575 580

ATA ATT GCC CAG CAA AAC GAA ATG AAC AAG AAC CTT TTC ATC GAG TGC 2132 He He Ala Gin Gin Asn Glu Met Aεn Lyε Aεn Leu Phe He Glu Cyε 585 590 595

CCT GTA TGC GGC ATC GTT TAC GGC GAG AAG GTC GGC AAT CAG CCC ATT 2180 Pro Val Cys Gly He Val Tyr Gly Glu Lys Val Gly Asn Gin Pro He 600 605 610

GGC AGC ATG TCG TGG AGC ATA ATT AGC AAG AAT CTG CCA GGA CAC GAG 2228 Gly Ser Met Ser Trp Ser He He Ser Lys Asn Leu Pro Gly His Glu 615 620 625

GGT CAG AAC ACC ATA CAG ATT GTT TAC GAC ATT GCA TCG GGA CTG CAG 2276 Gly Gin Asn Thr He Gin He Val Tyr Asp He Ala Ser Gly Leu Gin 630 635 640

ACG GAG GAG CAT CCG CAT CCA GGT CGT GCC TTC TTC GCC GTG GGA TTC 2324 Thr Glu Glu His Pro His Pro Gly Arg Ala Phe Phe Ala Val Gly Phe 645 650 655 660

CCG CGG ATC TGC TAC TTG CCG GAC TGC CCG CTG GGG CGA AAG GTT TTG 2372 Pro Arg He Cys Tyr Leu Pro Asp Cys Pro Leu Gly Arg Lys Val Leu 665 670 675

CGC TTC CTC AAG ATT GCA TTC GAT CGT CGG CTG CTT TTC TCG ATC GGA 2420 Arg Phe Leu Lys He Ala Phe Aεp Arg Arg Leu Leu Phe Ser He Gly 680 685 690

CGA TCG GTG ACC ACC GGA CGC GAG GAT GTG GTG ATC TGG AAC AGT GTG 2468 Arg Ser Val Thr Thr Gly Arg Glu Aεp Val Val He Trp Asn Ser Val 695 700 705

GAT CAC AAG ACG CAG TTC AAT ATG TTT CCG GAT CCC ACC TAT TTG CAG 2516 Asp His Lys Thr Gin Phe Asn Met Phe Pro Asp Pro Thr Tyr Leu Gin 710 715 720

CGA ACC ATG CAA CAG CTG GTG CAC CTG GGC GTG ACG GAT TAAGGATTAG 2565 Arg Tnr Met Gin Gin Leu Val His Leu Gly Val Thr Asp 725 730 735

TTCCCTGTCC CCAAGTAGAA CTACCAACCA ACCAATCAAC CACCCACCCA CCGAAGTCCC 2625

CTCGATCATT CTCTTCCATT CGTCGTTAAG TTACTTTCTA CATAATCTCA GTGTGTGTGC 2685

AATCCTCGTT TACTATGATA TATTTTTTTT ATAGATATAT TGTAATAGCG TTCGAGCTGC 2745

TCGAACCCTA AAACAACAGC AAACCACAAT TGCAATTGTA GCTTCCTTTC CGCTCTTCCA 2805

ATTCGTATTT GTACGCACAT ACGCAATAAG TTGGCGTACA TCATATGTAT TAGCTAGTTA 2865

GTTAGTTAGT TAGTTAGTTG TAGCTGTAGT TCCCAAGAGA ATCTTGACCC AAGACACCTA 2925

CTAGTATTAG GCATTATCCT GATTCTTGAT TCCTGATTCG ATTCAAGCCA AGCCAAGCCA 2985

CGCCATTCGA GTGCAAGCTG TGCCAAAATC GTAGCGCTCC CGTTTATAGG ATATGTATAT 3045

TGTTGATATA GCTAGCTATA ACCATTGCCC ATCTCTCCAT CTCTCTCGGT TTCGAATTTG 3105

TCTCTTTCAT CAGATCCATG TGAATTTTCT TTATATCGGA TTTATATAGG ATTΛMAATAG 3165 TATTTTGAGA GAGGAAATGG AGATGGGTAA ATTCGATAGA CTTGTCTCAC TTGTCTTGGC 3225

CATTTAATCT CTTTCATTCA GCGAATTTGA TGTGATTTTA ATTTGAATTA TTCATTATTA 3285

AACGGAGCAT TTAGGAAGCA TAGTTGTAAC GCAGCCAGAT ATTCCATTAC GCATATACAT 3345

ATACATATAC ATATACATAC ATACATAAAC ATATTTTAAC ATAGCCCCAT AGCCATACGA 3405

CATAACAATA ATTTTTTTTA TCGAATCCCT TGCATACATT TGATGAATTG TTGCTTTCAT 3465

ATTGATATCA TCGAGCATCG AACGAACTAT CGTATACATC GCCAATATAT AGCATATATA 3525

GCATATAGTA TGTAGAGATC GTACGGACAG CTAGCGGCTA CTGACCGCGC CACCATATTT 3585

GATATGATAT GATATGATTT TACTAAGTTG TATTTAGCAC TGATTAGTTA TTAAAGTTCA 3645

TTTGACGAAT ATTCCACAAC AAATTCCACA CCATTTATGT ATGCATATTA CGCATATATA 3705

ATACAGTACA TTTATATATA GTTCAAATAA AGTAACTTCA TTCATGTTCA AAAAAAAAAA 3765

AAAAAA 3771

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 737 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Ala Ser Ser Ala Gly Ser Ala Ala Ser Gly Ser Val Val Pro Gly 1 5 10 15

Gly Gly Gly Ser Ala Ala Ser Ser Cys Ala Thr Met Ala Leu Ser Thr 20 25 30

Ala Gly Ser Gly Gly Pro Pro Val Asn His Ala His Ala Val Cys Val 35 40 45

Trp Glu Phe Glu Ser Arg Gly Lys Trp Leu Pro Tyr Ser Pro Ala Val 50 55 60

Ser Gin His Leu Glu Arg Ala His Ala Lys Lys Leu Thr Arg Val Met 65 70 75 80

Leu Ser Asp Ala Asp Pro Ser Leu Glu Gin Tyr Tyr Val Asn Val Arg 85 90 95

Thr Met Thr Gin Glu Ser Glu Ala Glu Thr Arg Ser Gly Leu Leu Thr 100 105 110

He Gly Val Arg Arg Met Leu Tyr Ala Pro Ser Ser Pro Ala Gly Lyε 115 120 125

Gly Thr Lyε Trp Glu Trp Ser Gly Gly Ser Ala Aεp Ser Asn Asn Asp 130 135 140

Trp Arg Pro Tyr Asn Met His Val Gin Cys He He Glu Asp Ala Trp 145 150 155 160

Ala Arg Gly Glu Gin Thr Leu Asp Leu Cys Asn Thr His He Gly Leu 165 170 175

Pro Tyr Thr He Asn Phe Cys Asn Leu Thr His Val Arg Gin Pro Ser 180 185 190 Gly Pro Met Arg Ser He Arg Arg Thr Gin Gin Ala Pro Tyr Pro Leu 195 200 205

Val Lys Leu Thr Pro Gin Gin Ala Asn Gin Leu Lys Ser Asn Ser Ala 210 215 220

Ser Val Ser Ser Gin Tyr Asn Thr Leu Pro Lys Leu Gly Asp Thr Lys 225 230 235 240

Ser Leu His Arg Val Pro Met Thr Arg Gin Gin His Pro Leu Pro Thr 245 250 255

Ser Hiε Gin Val Gin Gin Gin Gin Hiε Gin Leu Gin His Gin Gin Gin 260 265 270

Gin Gin Gin Gin Hiε Hiε His Gin His Gin Gin Gin Gin Hiε Gin Gin 275 280 285

Gin Gin Gin Hiε Gin Met Gin His His Gin He Hiε His Gin Thr Ala 290 295 300

Pro Arg Lys Pro Pro Lys Lyε Hiε Ser Glu He Ser Thr Thr Asn Leu 305 310 315 320

Arg Gin He Leu Asn Asn Leu Asn He Phe Ser Ser Ser Thr Lyε His 325 330 335

Gin Ser Asn Met Ser Thr Ala Ala Ser Ala Ser Ser Ser Ser Ser Ser 340 345 350

Ala Ser Leu His His Ala Asn His Leu Ser His Ala His Phe Ser His 355 360 365

Ala Lyε Asn Met Leu Thr Ala Ser Met Asn Ser His His Ser Arg Cyε 370 375 380

Ser Glu Gly Ser Leu Gin Ser Gin Arg Ser Ser Arg Met Gly Ser Hiε 385 390 395 400

Arg Ser Arg Ser Arg Thr Arg Thr Ser Asp Thr Asp Thr Aεn Ser Val 405 410 415

Lyε Ser Hiε Arg Arg Arg Pro Ser Val Aεp Thr Val Ser Thr Tyr Leu 420 425 430

Ser His Glu Ser Lys Glu Ser Leu Arg Ser Arg Asn Phe Ala He Ser 435 440 445

Val Asn Asp Leu Leu Aεp Cys Ser Leu Gly Ser Asp Glu Val Pne Val 450 455 460

Pro Ser Val Pro Pro Ser Ser Leu Gly Glu Arg Ala Pro Val Pro Pro 465 470 475 480

Pro Leu Pro Leu His Pro Arg Gin Gin Gin Gin Gin Gin Gin Gin Gin 485 490 495

Gin Gin Leu Gin Met Gin Gin Gin Gin Gin Ala Gin Gin Gin Gin Gin 500 505 510

Gin Ser He Ala Gly Ser He Val Gly Val Aεp Pro Ala Ser Asp Met 515 520 525

He Ser Arg Phe Val Lyε Val Val Glu Pro Pro Leu Trp Pro Aεn Ala 530 535 540

Gin Pro Cyε Pro Met Cys Met- Glu Glu Leu Val His Ser Ala Gin Aεn 545 550 555 560

Pro Ala He Ser Leu Ser Arg Cys Gin His Leu Met His Leu Gin Cys 565 570 575

Leu Aεn Gly Met He He Ala Gin Gin Aεn Glu Met Asn Lys Asn Leu 580 585 590

Phe He Glu Cys Pro Val Cyε Gly He Val Tyr Gly Glu Lys Val Gly 595 600 605

Asn Gin Pro He Gly Ser Met Ser Trp Ser He He Ser Lys Asn Leu 610 615 620

Pro Gly His Glu Gly Gin Asn Thr He Gin He Val Tyr Asp He Ala 625 630 635 640

Ser Gly Leu Gin Thr Glu Glu His Pro His Pro Gly Arg Ala Phe Phe 645 650 655

Ala Val Gly Phe Pro Arg He Cys Tyr Leu Pro Asp Cys Pro Leu Gly 660 665 670

Arg Lys Val Leu Arg Phe Leu Lyε He Ala Phe Aεp Arg Arg Leu Leu 675 680 685

Phe Ser He Gly Arg Ser Val Thr Thr Gly Arg Glu Asp Val Val He 690 695 700

Trp Asn Ser Val Aεp Hiε Lys Thr Gin Phe Asn Met Phe Pro Asp Pro 705 710 715 720

Thr Tyr Leu Gin Arg Thr Met Gin Gin Leu Val Hiε Leu Gly Val Thr 725 730 735

Asp

(2) INFORMATION FOR SEQ ID Nθ:3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

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

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

Gin Pro Cys Pro Met Cys Met Glu Glu Leu Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

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

(ii) MOLECULE TYPE: peptide

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

Glu Asp Cys Thr He Cys Met Glu Arg Leu Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 ammo acids

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

(ii) MOLECULE TYPE: peptide

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

Leu Ser Arg Cys Gin Hiε Leu Met His Leu Gin Cys Leu Asn Gly Met 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:6:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: ammo acid (D) TOPOLOGY: unknown

(n) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6

Leu Gly Arg Cyε Gly Hiε Met Tyr Hiε Leu Leu Cys Leu Val Ala Met 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:7:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 ammo acids

(B) TYPE: ammo acid (D) TOPOLOGY, unknown

(ll) MOLECULE TYPE, peptide

(Xl) SEQUENCE DESCRIPTION SEQ ID NO: 7

He He Ala Gin Gin Asn Glu Met Asn Lys Asn Leu Phe He Glu Cys 1 5 10 15

Pro Val Cys Gly He Val Tyr Gly Glu Lys Val Gly Aεn Gin Pro He 20 25 30

Gly Ser Met Ser 35

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid (D) TOPOLOGY, unknown

(ii) MOLECULE TYPE: peptide

( i) SEQUENCE DESCRIPTION: SEQ ID NO:8- Leu Val Ala Met Tyr Ser Asn Gly Asn Lys Asp Gly Ser Leu Gin Cys

1 5 10 15

Pro Thr Cyε Lyε Pro Ser Met Gly Arg Arg Arg Val Arg Ser Arg Leu 20 25 30

Gly Arg Trp Ser 35

(2) INFORMATION FOR SEQ ID NO:9:

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 37 amino acids

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

(ii) MOLECULE TYPE: peptide

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

Val Tyr Gly Glu Lys Val Gly Val Gin Pro He Gly Ser Met Ser Trp 1 5 10 15

Ser He He Ser Lys Aεn Leu Pro Gly His Glu Gly Gin Asn Tnr He 20 25 30

Gin He Val Tyr Asp 35

INFORMATION FOR SEQ ID NO.10.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 amino acids

(B) TYPE amino acid (D) TOPOLOGY, unknown

(n) MOLECULE TYPE peptide

(xi) SEQUENCE DESCRIPTION SEQ ID NO:10

He Tyr Gly Glu Lys Thr Gly Thr Gin Pro Pro Gly Lys Met Glu Phe

1 5 10 15

Hiε Leu He Pro His Ser Leu Xaa Phe Gly Pro Aεp Thr Gin Thr Xaa 20 25 30

Arg He Val Tyr Asp 35

(2) INFORMATION FOR SEQ ID NO:11.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 2547 base pairs

(B) TYPE- nucleic acid

(C) STRANDEDNESS double

(D) TOPOLOGY, unknown

(ll) MOLECULE TYPE cDNA

(ix) FEATURE

(A) NAME/KEY- CDS

(B) LOCATION 504..2363 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

GCGAGAAGCC CCACTGAAGC CGGGCGCAGG GTCTGGGACG CAGTTGGGAG TGCAAAGGGC 60

TGGCTGAGAG CCGCAGGAGC AGCAGGCTGT GGCCCAGGCC TCCTGGGTGA CAGGCCCTGT 120

CTGGCGGGGA ACAGGGACCA AGAGACAACA CAGAAGAGGC TGGACCTCGA ACAGGGGCGG 180

CTGCCTCACT CCCTACCTGA GCCAGCCGAG GGGGCCAAGG ACTTTAGAGC TGTTTCCTCC 240

GGCATAAGAG AGACACTTGC TTTCCAGGGC AGCACCCTTT ATCGGAGAAG GCTCTACAGG 300

GAAGGGGTCT TTGCAGCCTG GATGGCCATC CCACATTCCT TTAACGGAGG TCTCTAGGCC 360

TCAGAGAGAA CCCAGAGTTA GAAAGGAGGC CAGACGGTCC TTGCTGTCCC CCTGGGGAGA 420

GAGGAAGTTG CCGCCTGCTG CCAGGCCCAG GAGGAGCTGG GCCTGCAATA GTGGGGGACC 480

TGGCCCCTGA GGCAGTGGCG GCC ATG TCA CGG CCA GGC CAC GGT GGG CTG 530

Met Ser Arg Pro Gly Hiε Gly Gly Leu 1 5

ATG CCT GTG AAT GGT CTG GGC TTC CCA CCG CAG AAC GTG GCC CGG GTG 578 Met Pro Val Asn Gly Leu Gly Phe Pro Pro Gin Aεn Val Ala Arg Val 10 15 20 25

GTG GTG TGG GAG TGT CTG AAT GAG CAC AGC CGC TGG CGG CCC TAC ACG 626 Val Val Trp Glu Cyε Leu Asn Glu His Ser Arg Trp Arg Pro Tyr Thr 30 35 40

GCC ACC GTG TGC CAC CAC ATT GAG AAC GTG CTG AAG GAG GAC GCT CGC 674 Ala Thr Val Cys His His He Glu Asn Val Leu Lys Glu Asp Ala Arg 45 50 55

GGT TCC GTG GTC CTG GGG CAG GTG GAC GCC CAG CTT GTG CCC TAC ATC 722 Gly Ser Val Val Leu Gly Gin Val Asp Ala Gin Leu Val Pro Tyr He 60 65 70

ATC GAC CTG CAG TCC ATG CAC CAG TTT CGC CAG GAC ACA GGC ACC ATG 770 He Asp Leu Gin Ser Met Hiε Gin Phe Arg Gin Asp Thr Gly Thr Met 75 80 85

CGG CCC GTG CGG CGC AAC TTC TAC GAC CCG TCG TCG GCG CCG GGC AAG 618 Arg Pro Val Arg Arg Asn Phe Tyr Asp Pro Ser Ser Ala Pro Gly Lys 90 95 100 105

GGC ATC GTG TGG GAG TGG GAG AAC GAC GGC GGC GCA TGG ACG GCC TAC 866 Gly He Val Trp Glu Trp Glu Asn Asp Gly Gly Ala Trp Thr Ala Tyr 110 115 120

GAT ATG GAC ATC TGC ATC ACC ATC CAG AAC GCC TAC GAG AAG CAG CAC 914 Asp Met Aεp He Cys He Thr He Gin Asn Ala Tyr Glu Lyε Gin His 125 130 135

CCG TGG CTC GAC CTC TCA TCG CTA GGC TTC TGC TAC CTC ATC TAC TTC 962 Pro Trp Leu Asp Leu Ser Ser Leu Gly Phe Cys Tyr Leu He Tyr Phe 140 145 150

AAC AGC ATG TCG CAG ATG ARC CGC CAG ACG CGC CGG CGC CGC CGT CTG 1010 Asn Ser Met Ser Gin Met Xaa Arg Gin Thr Arg Arg Arg Arg Arg Leu 155 160 165

CGC CGC CGC CTG GAC CTC GCC TAC CCG CTC ACC GTG GGC TCC ATC CCT 1058 Arg Arg Arg Leu Asp Leu Ala Tyr Pro Leu Thr Val Gly Ser He Pro 170 175 180 185

AAG TCG CAG TCG TGG CCC GTG- GGT GBC AGC TCG GGH CAG CCC TGC TCC 1106 Lys Ser Gin Ser Trp Pro Val Gly Xaa Ser Ser Gly Gin Pro Cys Ser 190 195 20: iw. i-Ai-, I_A_. TGC CTG YTG GTC AAC AGC ACG CGC GCC GTC TCC AAC GTC 1154 Xaa Gin Gin Cyε Leu Leu Val Asn Ser Thr Arg Ala Val Ser Asn Val 205 210 215

ATC CTG GYC TCG CAG CGT CGT AAG GTG MCC CCC GCG CCC CCG CTG TCG 1202 He Leu Xaa Ser Gin Arg Arg Lys Val Xaa Pro Ala Pro Pro Leu Ser 220 225 230

YCG CCG YCG MCA CCT GGA GGG CCT CCA GGC GCG CTT GGC GTG CGC CCC 1250 Xaa Pro Xaa Xaa Pro Gly Gly Pro Pro Gly Ala Leu Gly Val Arg Pro 235 240 245

AGY GTC ACC TTC ACA GGC GNC GNG CTC TGN GAA GTG NNN TTC NAC GGT 1298 Ser Val Thr Phe Thr Gly Xaa Xaa Leu Xaa Glu Val Xaa Phe Xaa Gly 250 255 260 265

CCC GTC GAG CCC GMG YCG TCT CCC GGG GYG CCC CCA CGG AGC CCG GGC 1346 Pro Val Glu Pro Xaa Xaa Ser Pro Gly Xaa Pro Pro Arg Ser Pro Gly 270 275 2B0

GCC CCC GGC GGA GCG CGC ACC CCG GGG CAG AAC AAC CTC AAC CGG BCC 1394 Ala Pro Gly Gly Ala Arg Thr Pro Gly Gin Asn Asn Leu Asn Arg Xaa 285 290 295

GGG CCC CAG CGC ACC ACC AGH GTG AGC GCG CGC GCC TCC ATC CCG CCG 1442 Gly Pro Gin Arg Thr Thr Xaa Val Ser Ala Arg Ala Ser He Pro Pro 300 305 310

GGG GTC CCC GCA CTC CCG GTG AAG AAC TTG AAT GGT ACT GGG CCG GTC 1490 Gly Val Pro Ala Leu Pro Val Lys Asn Leu Aεn Gly Thr Gly Pro Val 315 320 325

CAT CCG GCC CTG GCA GGG ATG ACC GGG ATA CTG CTG TGC GCG GCC GGG 1538 His Pro Ala Leu Ala Gly Met Thr Gly He Leu Leu Cys Ala Ala Gly 330 335 340 345

CTG CCC GTG TGC CTG ACG CGG GCC CCC AAG CCC ATC CTG CAC CCG CCG 1586 Leu Pro Val Cys Leu Thr Arg Ala Pro Lyε Pro He Leu Hiε Pro Pro 350 355 360

CCC GTG AGC AAG AGC GAC GTG AAG CCC GTG CCT GGC GTG CCC GGG GTG 1634 Pro Val Ser Lys Ser Asp Val Lys Pro Val Pro Gly Val Pro Gly Val 365 370 375

TGC CGC AAG ACC AAG AAG AAG CAC CTT AAA AAG AGT AAG AAT CCC GAG 1682 Cys Arg Lys Thr Lys Lys Lyε His Leu Lyε Lys Ser Lys Asn Pro Glu 380 385 390

GAT GTG GTT CGA AGA TAC ATG CAG AAG GTG AAA AAC CCA CCT GAT GAG 1730 Aεp Val Val Arg Arg Tyr Met Gin Lyε Val Lyε Asn Pro Pro Aεp Glu 395 400 405

GAC TGC ACC ATC TGC ATG GAG CGA CTG GTC ACA GCA TCA GGC TAC GAG 1778 Asp Cyε Thr He Cys Met Glu Arg Leu Val Thr Ala Ser Gly Tyr Glu 410 415 420 425

GGC GTG CTT CGG CAC AAG GGC GTG CGG CCT GAG CTC GTG GGC CGC CTG 1826 Gly Val Leu Arg His Lys Gly Val Arg Pro Glu Leu Val Gly Arg Leu 430 435 440

GGC CGC TGT GGC CAC ATG TAC CAC CTG CTG TGC CTC GTG GCC ATG TAC 1B74 Gly Arg Cys Gly His Met Tyr His Leu Leu Cys Leu Val Ala Met Tyr 445 450 455

TCC AAT GGC AAC AAG GAT GGC AGC CTG CAG TGC CCC ACC TGC AAG GCC 1922 Ser Asn Gly Asn Lys Aεp Gly Ser Leu Gin Cys Pro Thr Cys Lys Ala 460 465 470

ATC TAC GGG GAG AAG ACG GGT ACG CAG CCG CCT GGG AAG ATG GAC TTC 1970 He Tyr Gly Glu Lys Thr Gly Thr Gin Pro Pro Gly Lys Met Glu Phe 4/b 480 485

CAC CTC ATC CCC CAC TCG CTG CCC GGC TTC CCT GAT ACC CAG ACC ATC 2018 His Leu He Pro Hiε Ser Leu Pro Gly Phe Pro Aεp Thr Gin Thr He 490 495 500 505

CGC ATC GTC TAT GAC ATC CCC ACA GGC ATC CAG GGC CCT GAG CAC CCC 2066 Arg He Val Tyr Asp He Pro Thr Gly He Gin Gly Pro Glu His Pro 510 515 520

AAC CCC GGG AAG AAG TTC ACC GCA AGA GGA TTC CCT CGC CAC TGC TAT 2114 Asn Pro Gly Lys Lyε Phe Thr Ala Arg Gly Phe Pro Arg Hiε Cys Tyr 525 530 535

CTA CCC AAC AAC GAG AAA GGC CGG AAG GTG CTG CGG CTG CTC ATC ACG 2162 Leu Pro Asn Asn Glu Lys Gly Arg Lys Val Leu Arg Leu Leu He Thr 540 545 550

GCC TGG GAG AGA AGA CTC ATC TTC ACT ATC GGC ACG TCC AAC ACC ACG 2210 Ala Trp Glu Arg Arg Leu He Phe Thr He Gly Thr Ser Aεn Thr Thr 555 560 565

GGC GAG TCG GAC ACC GTG GTG TGG AAC GAG ATC CAC CAC AAG ACC GAG 2258 Gly Glu Ser Asp Thr Val Val Trp Aεn Glu He His His Lyε Thr Glu 570 575 580 585

TTT GGA TCC AAC CTC ACG GGA CAC GGC TAC CCG GAC GCT AGC TAC CTA 2306 Phe Gly Ser Asn Leu Thr Gly Hiε Gly Tyr Pro Asp Ala Ser Tyr Leu 590 595 600

GAC AAC GTG CTG GCT GAG CTC ACA GSC CAG GGC GTA TCC GAG GCT GCA 2354 Asp Asn Val Leu Ala Glu Leu Thr Xaa Gin Gly Val Ser Glu Ala Ala 605 610 615

GGC AAG GCT TGAGGSCCAA GGCTGCCCAC CTTCCCTCCT GSTTTGGCCC 2403

Gly Lys Ala 620

TGGTCCGGCA AATGCCTCCT TCGCCAGGTG TGTCCTGGTA GCCCAGGTTC AGGGCTGGGG 2463

AGGAGCCTGC GGAAGGGGCC GCAGCCATTC AGGGGACTGN CTGGNGGAAG TTGGATGAGG 2523

AGAGNTGGAT TTNAGGTTGG CCCC 2547

(2) INFORMATION FOR SEQ ID NO 12

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 620 ammo acids (D) TOPOLOGY unknown di) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO.12

Met Ser Arg Pro Gly His Gly Gly Leu Met Pro Val Asn Gly Leu Gly 1 5 10 15

Phe Pro Pro Gin Asn Val Ala Arg Val Val Val Trp Glu Cys Leu Asn 20 25 30

Glu His Ser Arg Trp Arg Pro Tyr Thr Ala Thr Val Cys His His He 35 40 45

Glu Asn Val Leu Lys Glu Asp Ala Arg Gly Ser Val Val Leu Gly Gin 50 55 60

Val Aεp Ala Gin Leu Val Pro Tyr He He Aεp Leu Gin Ser Met His 65 70 75 80 Gin Phe Arg Gin Aεp Thr Gly Thr Met Arg Pro Val Arg Arg Asn Phe 85 90 95

Tyr Asp Pro Ser Ser Ala Pro Gly Lys Gly He Val Trp Glu Trp Glu 100 105 110

Asn Asp Gly Gly Ala Trp Thr Ala Tyr Asp Met Asp He Cyε He Thr 115 120 125

He Gin Asn Ala Tyr Glu Lyε Gin His Pro Trp Leu Asp Leu Ser Ser 130 135 140

Leu Gly Phe Cys Tyr Leu He Tyr Phe Asn Ser Met Ser Gin Met Xaa 145 150 155 160

Arg Gin Thr Arg Arg Arg Arg Arg Leu Arg Arg Arg Leu Asp Leu Ala 165 170 175

Tyr Pro Leu Thr Val Gly Ser He Pro Lys Ser Gin Ser Trp Pro Val 180 185 190

Gly Xaa Ser Ser Gly Gin Pro Cys Ser Xaa Gin Gin Cys Leu Leu Val 195 200 205

Asn Ser Thr Arg Ala Val Ser Asn Val He Leu Xaa Ser Gin Arg Arg 210 215 220

Lys Val Xaa Pro Ala Pro Pro Leu Ser Xaa Pro Xaa Xaa Pro Gly Gly 225 230 235 240

Pro Pro Gly Ala Leu Gly Val Arg Pro Ser Val Thr Phe Thr Gly Xaa 245 250 255

Xaa Leu Xaa Glu Val Xaa Phe Xaa Gly Pro Val Glu Pro Xaa Xaa Ser 260 265 270

Pro Gly Xaa Pro Pro Arg Ser Pro Gly Ala Pro Gly Gly Ala Arg Thr 275 280 285

Pro Gly Gin Asn Asn Leu Aεn Arg Xaa Gly Pro Gin Arg Thr Thr Xaa 290 295 300

Val Ser Ala Arg Ala Ser He Pro Pro Gly Val Pro Ala Leu Pro Val 305 310 315 320

Lys Aεn Leu Aεn Gly Thr Gly Pro Val Hiε Pro Ala Leu Ala Gly Met 325 330 335

Thr Gly He Leu Leu Cyε Ala Ala Gly Leu Pro Val Cyε Leu Thr Arg 340 345 350

Ala Pro Lyε Pro He Leu Hiε Pro Pro Pro Val Ser Lyε Ser Aεp Val 355 360 365

Lys Pro Val Pro Gly Val Pro Gly Val Cys Arg Lys Thr Lys Lys Lyε 370 375 380

His Leu Lys Lys Ser Lys Asn Pro Glu Asp Val Val Arg Arg Tyr Met 385 390 395 400

Gin Lys Val Lyε Aεn Pro Pro Asp Glu Asp Cys Thr He Cys Met Glu 405 410 415

Arg Leu Val Thr Ala Ser Gly Tyr Glu Gly Val Leu Arg His Lys Gly 420 425 430

Val Arg Pro Glu Leu Val Gly- Arg Leu Gly Arg Cyε Gly His Met Tyr 435 440 445

His Leu Leu Cys Leu Val Ala Met Tyr Ser Asn Gly Aεn Lys Asp Gly 4 -> U 55 460

Ser Leu Gin Cys Pro Thr Cyε Lyε Ala He Tyr Gly Glu Lyε Thr Gly 465 470 475 480

Thr Gin Pro Pro Gly Lys Met Glu Phe His Leu He Pro His Ser Leu 485 490 495

Pro Gly Phe Pro Asp Thr Gin Thr He Arg He Val Tyr Aεp He Pro 500 505 510

Thr Gly He Gin Gly Pro Glu His Pro Asn Pro Gly Lys Lys Phe Thr 515 520 525

Ala Arg Gly Phe Pro Arg His Cys Tyr Leu Pro Asn Asn Glu Lys Gly 530 535 540

Arg Lys Val Leu Arg Leu Leu He Thr Ala Trp Glu Arg Arg Leu He 545 550 555 560

Phe Thr He Gly Thr Ser Asn Thr Thr Gly Glu Ser Asp Thr Val Val 565 570 575

Trp Asn Glu He Hiε Hiε Lys Thr Glu Phe Gly Ser Asn Leu Thr Gly 580 585 590

Hiε Gly Tyr Pro Aεp Ala Ser Tyr Leu Asp Asn Val Leu Ala Glu Leu 595 600 605

Thr Xaa Gin Gly Val Ser Glu Ala Ala Gly Lys Ala 610 615 620

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 303 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Ala Ser Ser Ala Gly Ser Ala Ala Ser Gly Ser Val Val Pro Gly 1 5 10 15

Gly Gly Gly Ser Ala Ala Ser Ser Cys Ala Thr Met Ala Leu Ser Thr 20 25 30

Ala Gly Ser Gly Gly Pro Pro Val Asn His Ala His Ala Val Cys Val 35 40 45

Trp Glu Phe Glu Ser Arg Gly Lys Trp Leu Pro Tyr Ser Pro Ala Val 50 55 60

Ser Gin Hiε Leu Glu Arg Ala His Ala Lys Lys Leu Thr Arg Val Met 65 70 75 80

Leu Ser Asp Ala Asp Pro Ser Leu Glu Gin Tyr Tyr Val Asn Val Arg 85 90 95

Thr Met Thr Gin Glu Ser Glu Ala Glu Thr Arg Ser Gly Leu Leu Thr 100 105 110

He Gly Val Arg Arg Met Leu Tyr Ala Pro Ser Ser Pro Ala Gly Lys 115 120 125 Gly Thr Lys Trp Glu Trp Ser Gly Gly Ser Ala Asp Ser Asn Asn Asp 130 135 140

Trp Arg Pro Tyr Asn Met His Val Gin Cys He He Glu Aεp Ala Trp 145 150 155 160

Ala Arg Gly Glu Gin Thr Leu Asp Leu Cys Asn Thr His He Gly Leu 165 170 175

Pro Tyr Thr He Asn Phe Cys Asn Leu Thr His Val Arg Gin Pro Ser 180 185 190

Gly Pro Met Arg Ser He Arg Arg Thr Gin Gin Ala Pro Tyr Pro Leu 195 200 205

Val Lys Leu Thr Pro Gin Gin Ala Asn Gin Leu Lys Ser Asn Ser Ala 210 215 220

Ser Val Ser Ser Gin Tyr Asn Thr Leu Pro Lys Leu Gly Asp Thr Lys 225 230 235 240

Ser Leu His Arg Val Pro Met Thr Arg Gin Gin Hiε Pro Leu Pro Thr 245 250 255

Ser His Gin Val Gin Gin Gin Gin His Gin Leu Gin His Gin Gin Gin 260 265 270

Gin Gin Gin Gin His Hiε Hiε Gin His Gin Gin Gin Gin His Gin Gin 275 280 285

Gin Gin Gin His Gin Met Gin His Hiε Gin He His His Gin Thr 290 295 300

(2) INFORMATION FOR SEQ ID NO:14:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 181 amino acids

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

(ii) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 14

Arg Lys Pro Pro Lys Lys His Ser Glu He Ser Thr Thr Asn Leu Arg 1 5 10 15

Gin He Leu Asn Asn Leu Asn He Phe Ser Ser Ser Thr Lys His Gin 20 25 30

Ser Asn Met Ser Thr Ala Ala Ser Ala Ser Ser Ser Ser Ser Ser Ala 35 40 45

Ser Leu His His Ala Asn His Leu Ser His Ala Hiε Phe Ser His Ala 50 55 60

Lys Asn Met Leu Thr Ala Ser Met Asn Ser His His Ser Arg Cys Ser 65 70 75 80

Glu Gly Ser Leu Gin Ser Gin Arg Ser Ser Arg Met Gly Ser His Arg 85 90 95

Ser Arg Ser Arg Thr Arg Thr Ser Asp Thr Asp Thr Asn Ser Val Lys 100 105 110

Ser His Arg Arg Arg Pro Ser Val Asp Thr Val Ser Thr Tyr Leu Ser 115 120 125 Hiε Glu Ser Lys Glu Ser Leu Arg Ser Arg Asn Phe Ala He Ser Val 130 135 140

Asn Asp Leu Leu Aεp Cys Ser Leu Gly Ser Asp Glu Val Phe Val Pro 145 150 155 160

Ser Val Pro Pro Ser Ser Leu Gly Glu Arg Ala Pro Val Pro Pro Pro 165 170 175

Leu Pro Leu Hiε Pro 180

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 224 amino acids

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

(ii) MOLECULE TYPE: protein

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

Ser He Ala Gly Ser He Val Gly Val Aεp Pro Ala Ser Aεp Met He 1 5 10 15

Ser Arg Phe Val Lys Val Val Glu Pro Pro Leu Trp Pro Asn Ala Gin 20 25 30

Pro Cys Pro Met Cys Met Glu Glu Leu Val Hiε Ser Ala Gin Asn Pro 35 40 45

Ala He Ser Leu Ser Arg Cys Gin His Leu Met His Leu Gin Cys Leu 50 55 60

Asn Gly Met He He Ala Gin Gin Aεn Glu Met Aεn Lys Aεn Leu Phe 65 70 75 80

He Glu Cyε Pro Val Cyε Gly He Val Tyr Gly Glu Lys Val Gly Asn 85 90 95

Gin Pro He Gly Ser Met Ser Trp Ser He He Ser Lys Aεn Leu Pro 100 105 110

Gly His Glu Gly Gin Asn Thr He Gin He Val Tyr Asp He Ala Ser 115 120 125

Gly Leu Gin Thr Glu Glu His Pro His Pro Gly Arg Ala Phe Phe Ala 130 135 140

Val Gly Phe Pro Arg He Cys Tyr Leu Pro Asp Cys Pro Leu Gly Arg 145 150 155 160

Lys Val Leu Arg Phe Leu Lys He Ala Phe Asp Arg Arg Leu Leu Phe 165 170 175

Ser He Gly Arg Ser Val Thr Thr Gly Arg Glu Asp Val Val He Trp 180 185 190

Asn Ser Val Asp His Lyε Thr Gin Phe Aεn Met Phe Pro Asp Pro Thr 195 200 205

Tyr Leu Gin Arg Thr Met Gin Gin Leu Val Hiε Leu Gly Val Thr Aεp 210 215 220

(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 204 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:16:

Met Ala Ser Ser Ala Gly Ser Ala Ala Ser Gly Ser Val Val Pro Gly 1 5 10 15

Gly Gly Gly Ser Ala Ala Ser Ser Cyε Ala Thr Met Ala Leu Ser Thr 20 25 30

Ala Gly Ser Gly Gly Pro Pro Val Asn His Ala His Ala Val Cyε Val 35 40 45

Trp Glu Phe Glu Ser Arg Gly Lyε Trp Leu Pro Tyr Ser Pro Ala Val 50 55 60

Ser Gin His Leu Glu Arg Ala His Ala Lys Lys Leu Thr Arg Val Met 65 70 75 B0

Leu Ser Aεp Ala Aεp Pro Ser Leu Glu Gin Tyr Tyr Val Asn Val Arg B5 90 95

Thr Met Thr Gin Glu Ser Glu Ala Glu Thr Arg Ser Gly Leu Leu Thr 100 105 110

He Gly Val Arg Arg Met Leu Tyr Ala Pro Ser Ser Pro Ala Gly Lyε 115 120 125

Gly Thr Lys Trp Glu Trp Ser Gly Gly Ser Ala Asp Ser Asn Asn Asp 130 135 140

Trp Arg Pro Tyr Asn Met His Val Gin Cys He He Glu Asp Ala Trp 145 150 155 160

Ala Arg Gly Glu Gin Thr Leu Aεp Leu Cys Asn Thr His He Gly Leu 165 170 175

Pro Tyr Thr He Aεn Phe Cyε Aεn Leu Thr His Val Arg Gin Pro Ser 180 185 190

Gly Pro Met Arg Ser He Arg Arg Thr Gin Gin Ala 195 200

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acidε

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

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Val Xaa Pro Ala Pro Pro Leu Ser Xaa Pro Xaa Xaa Pro Gly Gly Pro

1 5 10 15

Pro Gly Ala (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

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

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:18:

Xaa Ser Pro Gly Xaa Pro Pro Arg Ser Pro Gly Ala Pro Gly Gly 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

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

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Ser He Pro Pro Gly Val Pro Ala Leu Pro Val Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

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

(ii) MOLECULE TYPE: peptide

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

Arg Ala Pro Lys Pro He Leu His Pro Pro Pro Val Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

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

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Val Lyε Pro Val Pro Gly Val Pro Gly Val

1 5 10

(2) INFORMATION FOR SEQ ID NO:22: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 ammo acids

(B) TYPE- amino acid (D) TOPOLOGY: unknown

(11) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION. SEQ ID NO:22.

Arg Ala Pro Val Pro Pro Pro Leu Pro Leu His Pro Arg Gin Gin 1 5 10 15

INFORMATION FOR SEQ ID NO:23-

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 ammo acids

(B) TYPE: amino acid <D) TOPOLOGY: unknown

(n) MOLECULE TYPE peptide

(xi) SEQUENCE DESCRIPTION SEQ ID NO 23

Arg Ala Pro Thr Met Pro Pro Pro Leu Pro Pro Val Pro Pro Gin Pro 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:24

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 14 ammo acids

(B) TYPE: ammo acid (D) TOPOLOGY: unknown

(n) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION SEQ ID NO 24

Arg Ala Val Pro Pro Pro Leu Pro Pro Arg Arg Lys Glu Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO:25

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 61 ammo acids

(B) TYPE: ammo acid (D) TOPOLOGY: unknown

(n) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION. SEQ ID NO.25

Cys Thr He Cyε Met Glu Arg Leu Val Thr Ala Ser Gly Tyr Glu Gly

1 5 10 15

Val Leu Arg Hiε Lyε Gly Val Arg Pro Glu Leu Val Gly Arg Leu Gly 20 25 30 Arg Cys Gly His Met Tyr His Leu Leu Cyε Leu Val Ala Met Tyr Ser 35 40 45

Aεn Gly Asn Lys Asp Gly Ser Leu Gin Cyε Pro Thr Cyε 50 55 60

(2) INFORMATION FOR SEQ ID NO:26-

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 24 base pairε

(B) TYPE nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY linear

(ll) MOLECULE TYPE. DNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO:26 CACCATCTGC ATGGAGCGAC TGGT 24

(2) INFORMATION FOR SEQ ID NO:27

(i) SEQUENCE CHARACTERISTICS-

(A) LENGTH 24 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS εingle

(D) TOPOLOGY linear

(ll) MOLECULE TYPE DNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO.27 TGTGGCCACA TGTACCACCT GCTG 24

(2) INFORMATION FOR SEQ ID NO.28-

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH. 24 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ll) MOLECULE TYPE DNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO.28. TACGGGGAGA AGACGGGTAC GCAG 24

(2) INFORMATION FOR SEQ ID NO:29

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 23 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY, linear

(ll) MOLECULE TYPE- DNA 1X1) SEQUENCE DESCRIPTION: SEQ ID NO:29: AAGATGGAGT TCCACCTCAT CCC 23

(2) INFORMATION FOR SEQ ID NO:30:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

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

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Cys Met Glu Arg Leu Val 1 5

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: ammo acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE, peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Tyr Gly Glu Lys Thr Gly

1 5

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: ammo acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE, peptide

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

Thr He Arg He Val Tyr Asp He

1 5

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 am o acids

(B) TYPE: ammo acid (D) TOPOLOGY: unknown di) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33 Gly Phe Pro Arg His Cys Tyr Leu Pro 1 5

(2) INFORMATION FOR SEQ ID NO 34

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 8 amino acids

(B) TYPE amino acid (D) TOPOLOGY unknown

(ll) MOLECULE TYPE peptide

(Xi) SEQUENCE DESCRIPTION SEQ ID NO 34

Arg Arg Leu He Phe Thr He Gly

1 5

(2) INFORMATION FOR SEQ ID NO 35 d) SEQUENCE CHARACTERISTICS

(A) LENGTH 7 ammo acids (D) TOPOLOGY unknown ii MOLECULE TYPE peptide

(xi) SEQUENCE DESCRIPTION SEQ ID NO 35

Met Cys Met Glu Glu Leu Val

1 5

INFORMATION FOR SEQ ID NO 36 d) SEQUENCE CHARACTERISTICS

(A) LENGTH 6 ammo acidε (D) TOPOLOGY unknown in) MOLECULE TYPE peptide

(xi) SEQUENCE DESCRIPTION SEQ ID NO 36

Tyr Gly Glu Lys Val Gly

1 5

(2) INFORMATION FOR SEQ ID NO 37

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 8 ammo acids (D) TOPOLOGY unknown

(ii) MOLECULE TYPE peptide

(Xi) SEQUENCE DESCRIPTION SEQ ID NO 37

Thr He Gin He Val Tyr Aεp He 1 5 (2) INFORMATION FOR SEQ ID NO:36

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 9 amino acidε

(B) TYPE, ammo acid (D) TOPOLOGY, unknown

(ii) MOLECULE TYPE, peptide

(xi) SEQUENCE DESCRIPTION. SEQ ID NO.38

Gly Phe Pro Arg He Cyε Tyr Leu Pro 1 5

(2) INFORMATION FOR SEQ ID NO:39

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 8 ammo acids

(B) TYPE amino acid (D) TOPOLOGY unknown

(ll) MOLECULE TYPE peptide

(xi) SEQUENCE DESCRIPTION SEQ ID NO 39.

Arg Arg Leu Leu Phe Ser He Gly

1 5

International Application No: PCT/

MICROORGANISMS

Optional Sheet in connection wtth the microorganism referred to on page 69 . iines 1- Q of the description

A. IDENTIFICATION OF DEPOSIT '

Further deposits are identified on an additional sheet

Name of depositary institution ' American Type Culture Collection

Address of depositary institution (including postal code and country) '

12301 Parklawn Drive Rockville, MD 20852 US

Date of deposit ' November 17, 995 Accession Number ' 97341

B. ADDITIONAL INDICATIONS (leave blink if not applicable) Thu itiforπaiion is connnu-d on a lepara-: attached iheei

C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ' .....

D. SEPARATE FURNISHING OF INDICATIONS ' (leave blank if not applicable)

The indications listed below will be submitted to tne International Bureau later ' ISpecity t e general nature of the indications e g "Accession Number of Deposit "1

filed (to be checked by the receiving Office)

D The date of receipt (from the applicant) by the International Bureau '

was

(Authorized Officer) Form PCT/RO/134 (January 1981 )

-92.1 -

Claims (1)

1. WHAT IS CLAIMED IS:

   1. A purified vertebrate Deltex protem.

   5

   2. The protem of claim 1 which is a mammalian protem.

   3. The protem of claim 1 which is a human protem.

   0 4. The protein of claim 1 havmg the ammo acid sequence depicted in Figure 2A-C (SEQ ID NO: 12).

   5 A purified protein compnsmg a fragment of a vertebrate

   Deltex protein, said fragment consistmg of at least 10 continuous amino acids of 5 the vertebrate Deltex protem.

   6. A purified protem compnsmg a fragment of a vertebrate Deltex protein, said fragment consistmg of at least 20 contmuous ammo acids of 0 the vertebrate Deltex protem

   7. A purified fragment of a vertebrate Deltex protein consistmg of at least 10 continuous ammo acids of a vertebrate Deltex protein, which displays one or more functional activities associated with a full-length vertebrate Deltex 5 protem

   8. The fragment of claim 7 which consists of at least 20 continuous ammo acids of the Deltex protem.

   30

   9. The protem of claim 5 m which the protein is able to be bound by an antibody to a Deltex protem

   - ₅ 10 A purified protein compnsmg a fragment of a vertebrate

   Deltex protem, which fragment binds to a Notch protein or to a molecule comprising the cdcl0/SW16/ankyπn repeats of a Notch protein

   - 93 - 11. A purified protein comprising a fragment of a first vertebrate Deltex protein, which fragment binds to a second Deltex protein or to a molecule comprising a fragment of a second Deltex protein.

   12. A purified protein comprising a fragment of a vertebrate

   Deltex protein, which fragment comprises a SH3-binding domain of the vertebrate Deltex protein.

   13. A chimeric protein comprising a functionally active fragment of a vertebrate Deltex protein joined via a peptide bond to an amino acid sequence of a protein other than a vertebrate Deltex protein.

   14. The protein of claim 13 in which the fragment binds to a Notch protein or to a molecule comprising the cdclO/SW16/an yrin repeats of a Notch protein.

   15. The protein of claim 13 in which the fragment comprises an SH3-binding domain.

   16. The protein of claim 13 in which the fragment comprises a zinc fmger domain.

   17. A purified derivative of the protein of claim 1 , which is characterized by the ability to be bound by antibody to die protein of claim 1 , which derivative has one or more insertions, deletions, or substitutions relative to the protein.

   18. A purified peptide having an amino acid sequence in the range of 10-35 amino acids, said sequence being a portion of a vertebrate Deltex protein sequence.

   19. A purified derivative of the protein of claim 1 , which is able to display one or more functional activities of the protein of claim 1.

   - 94 - 20. A molecule compnsmg the sequence of a human Deltex protem.

   21. An antibody which binds to a vertebrate Deltex protem, and which does not bmd to a Drosophila Deltex protem

   22. The antibody of claim 21 which binds to a human Deltex protein.

   23. The antibody of claim 21 which is monoclonal.

   24. A fragment or derivative of the antibody of claim 23 contaming the bmdmg domain of the antibody.

   25. A puπfied nucleic acid encodmg a vertebrate Deltex protein

   26. The nucleic acid of claim 25 which lacks mtrons

   27. The nucleic acid of claim 25 which encodes a protein havmg the ammo acid sequence depicted in Figure 2A-C (SEQ ID NO.12)

   28. The nucleic acid of claim 25 which comprises the coding region of the nucleotide sequence depicted in Figure 2A-C (part of SEQ ID NO: 11).

   29. A purified nucleic acid complementary to the nucleic acid of ciaim 25.

   30. The nucleic acid of claim 25 which encodes a human Deltex protein.

   - 95 31. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of low stringency, said second nucleic acid comprising the nucleotide sequence depicted in Figure 2A-C (SEQ ID NO: 11), said first nucleic acid comprising at least 110 continuous nucleotides of SEQ ID NO: 11.

   32. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of high stringency, said second nucleic acid comprising the nucleotide sequence depicted in Figure 2A-C (SEQ ID NO: 11), said first nucleic acid comprising at least 110 continuous nucleotides of SEQ ID NO: 11.

   33. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of low stringency, said second nucleic acid encoding a protein comprising the amino acid sequence depicted in Figure 2A-C

   (SEQ ID NO: 12), said first nucleic acid encoding a protein comprising the first 50 amino acids of SEQ ID NO: 12.

   34. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of high stringency, said second nucleic acid encoding a protein comprising the amino acid sequence depicted in Figure 2A-C (SEQ ID NO: 12), said first nucleic acid encoding a protein comprising the first 50 amino acids of SEQ ID NO: 12.

   35. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of high stringency, said second nucleic acid comprising nucleotide numbers 500-1044, 1045-1821 , or 1822-2380 of SEQ ID NO. l l.

   36. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of low stringency, said second nucleic acid comprising nucleic acids 500-1044, 1045-1821, or 1822-2370 of SEQ ID NO: 11.

   37. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 25 amino acids of SEQ ID NO: 12.

   - 96 - 38. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 50 amino acids of SEQ ID NO: 12.

   39. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 100 amino acids of SEQ ID NO: 12.

   40. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 150 amino acids of SEQ ID NO: 12.

   41. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 230 amino acids of SEQ ID NO: 12.

   42. A purified nucleic acid comprising 110 continuous nucleotides of SEQ ID NO:l l.

   43. A purified nucleic acid encoding the protein of claim 5.

   44. A purified nucleic acid encodmg the protein of claim 10.

   45. A purified nucleic acid encoding the protein of claim 11.

   46. A purified nucleic acid encoding the protein of claim 12

   47. A nucleic acid encoding the chimeric protein of claim 13.

   48. The nucleic acid of claim 25 as contained in plasmid pBS hdx as deposited with the ATCC and assigned accession number 97341.

   49. A nucleic acid vector comprising the nucleic acid of claim 25.

   50. A nucleic acid vector comprising the nucleic acid of claim 26.

   - 97 - 51 A recombmant cell contammg the nucleic acid vector of claim 49.

   52 A recombmant cell contaimng the nucleic acid vector of

   5 claun 50.

   53 A method for producmg a vertebrate Deltex protem compπsing growmg the recombinant cell of claim 51 , such that the vertebrate

   10 Deltex protem is expressed by die cell; and recovermg the expressed vertebrate Deltex protem

   54 A method for producmg a protem compnsmg growmg a cell contammg a recombmant nucleic acid compnsmg the nucleic acid of claim 33, such 15 that the protem is expressed by the cell, and recovermg the expressed protem

   55 A pharmaceutical composition compnsmg a therapeutically effective amount of a vertebrate Deltex protem, and a pharmaceutically acceptable

   20 carrier

   56 The composition of claim 55 in which the vertebrate Deltex protem is a human Deltex protem

   25

   57 A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 7, and a pharmaceutically acceptable carrier

   ^{3 0} 58 A pharmaceutical composition compnsmg a therapeutically effective amount of the protein of claun 10, and a pharmaceutically acceptable carrier

   , _ς 59 A pharmaceutical composition compnsmg a therapeutically effective amount of the protem of claim 1 1 , and a pharmaceutically acceptable carrier

   - 98 - 60. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 12; and a pharmaceutically acceptable carrier.

   61. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 13; and a pharmaceutically acceptable carrier.

   62. A pharmaceutical composition comprising a therapeutically effective amount of a derivative of a vertebrate Deltex protein, which derivative is characterized by die ability to bind to a Notch protein or to a molecule comprising the cdclO/SW16/ankyrin repeats of a Notch protein.

   63. A pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid encoding a vertebrate Deltex protein; and a pharmaceutically acceptable carrier.

   64. The pharmaceutical composition of claim 63 in which the

   Deltex protein is a human Deltex protein.

   65. A pharmaceutical composition comprising a therapeutically effective amount of an antibody which binds to a vertebrate Deltex protein but not to a Drosophila Deltex protein, or a fragment or derivative of the antibody containing the binding domain iereof; and a pharmaceutically acceptable carrier.

   66. A method of treating or preventing a disease or disorder in a ° subject comprising administering to a subject in need of such treatment or prevention a erapeutically effective amount of a molecule which antagonizes the function of a vertebrate Deltex protein.

   ₅ 67. The method according to claim 66 in which the disease or disorder is a malignancy characterized by increased Notch activity or increased

   99 expression of a Notch protem or of a Notch derivative capable of bemg bound by an anti-Notch anπbody, relanve to said Notch activity or expression m an analogous non-malignant sample.

   68. The memod according to claim 66 in which the disease or disorder is cervical cancer.

   69. The method according to claim 66 in which the disease or disorder is breast cancer.

   70 The method accordmg to claun 66 m which the disease or disorder is colon cancer.

   71. The method accordmg to claim 66 in which me malignancy is selected from the group consisting of melanoma, seminoma, and lung cancer.

   72. The memod accordmg to claim 67 in which the subject is a human.

   73. The method accordmg to claim 66 m which me molecule is an antibody to vertebrate Deltex or a derivative of said antibody contammg the bmdmg domam thereof, which antibody does not bind to Drosophila Deltex.

   74. The method accordmg to claim 66 in which the molecule is a protein compnsmg a portion of a vertebrate Deltex protem capable of bmdmg to a Notch protem or to a second molecule comprismg me cdclO/SW16/ankyrm repeats of a Notch protem

   75 The method according to claim 66 m which the molecule is a protem compnsmg the SH3 binding domam of a vertebrate Deltex protein.

   100 76. The memod according to claim 66 in which the molecule is a protein comprising the zinc fmger domain of a vertebrate Deltex protein.

   77. The method according to claim 66 in which the molecule is an oligonucleotide which (a) consists of at least six nucleotides; (b) comprises a sequence complementary to at least a portion of an RNA transcript of a vertebrate deltex gene; and (c) is hybridizable to the RNA transcript.

   78. A method of treating or preventing a disease or disorder in a subject in need of such treatment or prevention comprising adrrimistering to the subject a therapeutically effective amount of a molecule which promotes me function of a vertebrate Deltex protein.

   79. A method of treating or preventing a malignancy in a subject comprising administering to a subject in need of such treatment or prevention an effective amount of a vertebrate Deltex protein.

   80. The method according to claim 79 in which the Deltex protein is a human Deltex protein.

   81. A method of treating or preventing a malignancy in a subject compnsmg administermg to a subject in need of such treatment or prevention an effective amount of the nucleic acid of claim 25.

   82. A method of treating or preventing a malignancy in a subject comprising administering to a subject in need of such treatment or prevention an effective amount of the antibody of claim 21.

   83. A method for treating a patient with a tumor, of a tumor type characterized by expression of a Notch or vertebrate deltex gene, comprising administering to the patient an effective amount of an oligonucleotide, which oligonucleotide (a) consists of at least six nucleotides; (b) comprises a sequence

   - 101 complementary to at least a portion of an RNA transcript of the vertebrate deltex gene; and (c) is hybridizable to the RNA transcript.

   84. An isolated oligonucleotide consistmg of at least six nucleotides, and comprising a sequence complementary to at least a portion of an

   RNA transcript of a vertebrate deltex gene, which oligonucleotide is hybridizable to die RNA transcript.

   85. A pharmaceutical composition comprising me oligonucleotide of claun 84; and a pharmaceutically acceptable carrier.

   86. A method of inhibiting the expression of a nucleic acid sequence encoding a vertebrate Deltex protein m a cell compnsmg providmg the cell with an effective amount of the oligonucleotide of claim 84.

   87. A method of diagnosing a disease or disorder characterized by an aberrant level of Notch-vertebrate Deltex protein binding activity in a patient, 0 comprising measurmg the ability of a Notch protein in a sample derived from me patient to bmd to a vertebrate Deltex protein, in which an increase or decrease m the ability of the Notch protein to bind to the vertebrate Deltex protem, relative to the ability found in an analogous sample from a normal individual, indicates the presence of the disease or disorder in the patient.

   88. A method of identifying a molecule mat inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein, comprismg:

   (a) contacting (i) a Notch protein or fragment diereof that ° mediates binding to a Deltex protem, and (ii) a vertebrate

   Deltex protein or fragment diereof that mediates binding to a Notch protein, such that binding between the Notch protein or fragment and me Deltex protein or fragment can occur, in ₅ die presence of one or more molecules which are desired to be tested for the ability to inhibit or reduce binding between

   102 - the Notch protein or fragment and die Deltex protein or fragment; and (b) identifying die one or more molecules mat inhibit or reduce the binding of me Deltex protein or fragment to me Notch protein or fragment.

   89. The mediod of claim 88 in which the Deltex protein is a human Deltex protein.

   90. A memod of inactivating Notch function in a cell comprising introducing into the cell a molecule, said molecule comprising (a) a Deltex protein or portion thereof that mediates binding to a Notch protein; and (b) a protease or proteolytically active portion diereof.

   91. A method for the expansion of a precursor cell comprising contacting the cell with an amount of a vertebrate Deltex portion or functionally active portion diereof effective to inhibit differentiation of ie cell, and exposing the cell to cell growth conditions such diat the cell proliferates.

   103 -