US 5,750,652 AGrant
Deltex Proteins
Issue Date:1998-05-12
•27 Claims
•40 Drawing Sheets
Abstract
The present invention relates to amino acid sequences of the encoded deltex protein. The invention further relates to fragments and other derivatives, and analogs, of deltex proteins. In specific embodiments, the invention relates to deltex protein derivatives and analogs of the invention which are functionally active, or which comprise one or more domains of a deltex protein, including but not limited to the Gln-rich clusters, SH3 binding domains, domains which mediate binding to Notch or to a Notch derivative containing Notch cdc10/SW16/ankyrin ("ANK") repeats, domains which mediate binding to a second deltex protein, or any combination of the foregoing. The present invention also relates to compositions based on deltex proteins.
Metadata
Assignee
- Yale University
Inventors
- Spyridon Artavanis-Tsakonas
- Isabelle Busseau
- Robert J. Diederich
- Tian Xu
- Kenji Matsuno
Application Information
Application Number:US 1854321
Filing Date:1994-01-21
Priority Date:1994-01-21
Art Unit:182
Classifications
IPC:
C07K 14705
Field of Search:
530930514350;326;328102
Patent Drawings (40 sheets)
Description
TABLE OF CONTENTS
1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
4. DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. ISOLATION OF THE DELTEX NUCLEIC ACIDS
5.2. EXPRESSION OF DELTEX NUCLEIC ACIDS
5.3. IDENTIFICATION AND PURIFICATION THE DELTEX GENE PRODUCTS
5.4. STRUCTURE OF THE DELTEX GENES AND PROTEINS
5.4.1. GENETIC ANALYSIS
5.4.2. PROTEIN ANALYSIS
5.5. GENERATION OF ANTIBODIES TO DELTEX PROTEINS AND DERIVATIVES THEREOF
5.6. DELTEX PROTEINS, DERIVATIVES AND ANALOGS
5.6.1. DERIVATIVES OF DELTEX CONTAINING ONE OR MORE DOMAINS OF THE PROTEIN
5.7. IN VITRO ASSAYS OF DELTEX PROTEINS, DERIVATIVES AND ANALOGS
5.8. THERAPEUTIC USES
5.8.1. MALIGNANCIES
5.8.2. NERVOUS SYSTEM DISORDERS
5.8.3. TISSUE REPAIR AND REGENERATION
5.9. PROPHYLACTIC USES
5.9.1. MALIGNANCIES
5.9.2. OTHER DISORDERS
5.10. DEMONSTRATION OF THERAPEUTIC OR PROPHYLACTIC UTILITY
5.11. ANTISENSE REGULATION OF DELTEX EXPRESSION
5.11.1. DELTEX ANTISENSE NUCLEIC ACIDS
5.11.2. THERAPEUTIC UTILITY OF DELTEX ANTISENSE NUCLEIC ACIDS
5.12. THERAPEUTIC/PROPHYLACTIC ADMINISTRATION AND COMPOSITIONS
5.13. DIAGNOSTIC UTILITY
6. EXAMPLE: A MEMBER OF THE NOTCH GROUP OF INTERACTING LOCI, DELTEX ENCODES A CYTOPLASMIC BASIC PROTEIN
6.1. RESULTS
6.1.1. MOLECULAR CLONING OF THE DELTEX LOCUS
6.1.2. TRANSCRIPTIONAL ACTIVITY OF THE 6A REGION
6.1.3. ALL DELTEX ADULT PHENOTYPES CAN BE RESCUED BY A 10-KB GENOMIC FRAGMENT
6.1.4. DELTEX ENCODES A BASIC PROTEIN RICH IN GLUTAMINE, HISTIDINE, AND SERINE RESIDUES
6.1.5. THE DELTEX PROTEIN IS LOCALIZED WITHIN THE CYTOPLASM
6.1.6. THE GENETIC SUPPRESSION OF DELTEX
6.1.7. SUPPRESSION OF THE PHENOTYPIC INTERACTIONS BETWEEN DELTEX AND NOTCH, DELTA AND MASTERMIND
6.2. DISCUSSION
6.3. MATERIAL AND METHODS
6.3.1. ISOLATION OF NUCLEIC ACIDS
6.3.2. P-MEDIATED TRANSFORMATION
6.3.3. SEQUENCE DETERMINATION AND ANALYSIS
6.3.4. EXPRESSION CONSTRUCTS AND IMMUNOLOGICAL PROCEDURES
6.3.5. STRAINS AND CROSSES
6.3.6. PHENOTYPIC REVERSION OF DX.sup.P
6.3.7. COMPLEMENTATION OF DELTEX ALLELES BY THE DELTEX TRANSPOSONS TXO5B AND TXO12G
6.3.8. ORIGIN OF SU(DX).sup.SP
6.3.9. CROSSES BETWEEN SU(DX) MUTATIONS AND DELTEX ALLELES
6.3.10. CROSSES BETWEEN SU(DX) AND MUTATIONS OF THE NOTCH LOCUS
6.3.11. CROSSES INVOLVING SU(DX), DELTEX, DELTA AND MASTERMIND
6.3.12. CROSSES COMPARING SU(DX) MUTATIONS
6.3.13. CROSSES INVOLVING DUPLICATIONS OF THE DELTEX LOCUS
7. DELTEX IMPLICATED IN NOTCH-MEDIATED SIGNAL TRANSDUCTION: EVIDENCE FOR CYTOSOLIC INTERACTION WITH NOTCH CDC10/SWI6/ANKYRIN REPEATS
7.1. DELTEX INTERACTS WITH NOTCH INTRACELLULAR DOMAIN
7.2. NOTCH AND DELTEX CO-LOCALIZATION IN VIVO
7.3. NOTCH ANKYRIN REPEATS ARE BOTH NECESSARY AND SUFFICIENT FOR DELTEX ASSOCIATION
7.4. DIRECT NOTCH/DELTEX INTERACTION INDICATED BY YEAST EXPRESSION STUDIES
7.5. INTRAGENIC SUPPRESSOR MUTATION MAPS WITHIN ANK REPEAT
7.6. DISCUSSION
8. EXAMPLE: PORTIONS OF DELTEX WHICH MEDIATE HETEROTYPIC AND HOMOTYPIC BINDING
9. EXAMPLE: DELTEX CONTAINS A PUTATIVE SH3-BINDING DOMAIN
10. DEPOSIT OF MICROORGANISMS
1. INTRODUCTION
The present invention relates to deltex genes and their encoded protein products, as well as derivatives and analogs thereof. The invention further relates to production of deltex proteins, derivatives and antibodies. Related therapeutic compositions and methods of therapy and diagnosis are also provided.
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 Artavanis-Tsakonas, 1993, Cell 75:1245-1247; Artavanis-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.
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 Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408; and in Artavanis-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 of the embryonic nervous system, whereby loss of function mutations cause the misrouting 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, 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` (Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408). The other members of the Notch group are deltex (Xu and 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 and Enhancer of (split) encode nuclear proteins (Smoller et al., 1990, Genes Dev. 4:1688-1700; Delidakis et al., 1991, Genetics 129:803-823). 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 they produce. Moreover, subsequent analysis has shown that alleles of deltex exhibit genetic interactions with those of Delta and mastermind, 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 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 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 salient 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. 3:1099-1112), ovary (Ruohola et al, 1991, Cell 66:433-449; Xu et al., 1992, Development 115:913-922), and mesoderm (Corbin 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 (Coffman et al, 1993, Cell 73:659-671; Coffman et al., 1990, Science 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; Coffman 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 homologs have been isolated (PCT Publication No. WO 92/19737 dated Nov. 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, 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/lin12 repeats. The intracellular domain bears several common structural motifs including 6 cdc10SW16/ankyrin repeats ("ANK" repeats) (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)), 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 homologs isolated from mice (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), 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 Nov. 12, 1992), and Xenopus (Coffman et al, 1993, Cell 73:659-671; Coffman 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 (.about.70% amino acid identity) between Notch and its vertebrate counterparts (Stifani et al., 1992, Nature Genetics 2:119-127), are thought to mediate protein-protein interactions among 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 ANK repeats are crucial for Notch-mediated signaling events. Both EGF-like repeats and ankyrin motifs are found in a variety of proteins known to interact 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-like repeats (Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699). Despite the evidence to suggest that Notch functions as a cell surface receptor, few biochemical details of Notch function are known.
It has been demonstrated 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). Despite this evidence further implicating Notch as a cell surface receptor of extracellular signals, the intracellular nature of the signal transduction cascade is unknown.
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention relates to nucleotide sequences of deltex genes, and amino acid sequences of the encoded deltex proteins. The invention further relates to fragments and other derivatives, and analogs, of 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., by recombinant methods, is provided.
In a specific embodiment, the invention relates to Drosophila deltex nucleic acids and proteins. In another embodiment, the invention relates to human deltex nucleic acids and proteins.
In specific embodiments, the invention relates to deltex protein derivatives and analogs of the invention which are functionally active, or which comprise one or more domains of a deltex protein, including but not limited to the Gln-rich clusters, SH3 binding domains, domains which mediate binding to Notch or to a Notch derivative containing Notch cdc10/SW16/ankyrin ("ANK") repeats, or any combination of the foregoing.
The present invention also relates to therapeutic and diagnostic methods and compositions based on deltex proteins and nucleic acids. The invention 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: deltex proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the deltex proteins, analogs, or derivatives; and 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 a nervous system disorder or to promote tissue regeneration and repair.
In one embodiment, Therapeutics which antagonize, or inhibit, Notch and/or deltex function (hereinafter "Antagonist Therapeutics") are administered for therapeutic effect. In another embodiment, Therapeutics which promote 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 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 deltex which mediates binding to a Notch protein or a fragment thereof.
4. DESCRIPTION OF THE FIGURES
FIG. 1. Map of cloned genomic sequences and alignment of deltex cDNA clones. A). Coordinates are indicated in kilobasepairs, 0 corresponding to the P element insertion site of dx.sup.P. The positions of EcoRI (R) and SalI (S) restriction sites are shown. Recombinant phages .lambda.A25 and .lambda.A28 derive from dx.sup.P genomic DNA library (see Section 6.3). Wild-type Canton S clones are represented by .lambda.I1, .lambda.I2 and .lambda.I3. DNA fragments cloned into the Bluescript plasmid vector are denoted with the prefix pd. B). Detail of genomic interval -5 to +6.5. XbaI (X) restriction sites are also shown. Below the map, open bars represent the cDNAs cBE30 and cBE8; thin lines delineate intronic sequences. deltex mRNA sequences represented by the cDNAs are shown as a solid line; broken line depicts the remaining 5' end of the mRNA as inferred from genomic sequencing. Within schematic of the predicted protein, black boxes denote relative position of opa repeats. Stippled bar to left of deltex transcription unit indicates genomic fragments that hybridize to partial cDNAs representing a ribosomal protein gene.
FIG. 2. Developmental Northern blot. Each lane contains 5 .mu.g size-fractionated poly (A).sup.+ RNA isolated from developmentally staged animals. Left: embryonic mRNA, hours (h) after egg laying; (L1-3) first, second and third larval instars. Right: early-(E) and late (L)-staged pupae (P) and adult male and female mRNA. Length of deltex mRNA is indicated in kilobases (kb). Band of deltex hybridization present in adult male lane is due to contaminating female flies. Pattern of actin 5c mRNA accumulation is shown as control.
FIG. 3. Composite nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of the deltex locus. DNA sequences in uppercase letters are derived from the cDNAs cBE30 (nucleotide 1 to 1299) and cBE8 (nucleotides 1116 to 3764); lowercase letters refer to genomic sequence from the dx.sup.P strain. The predicted amino acid sequence is depicted below the DNA sequence, with the opa repeats shown in bold letters. Numbers along left margin refer to the cDNA sequence; numbers on the right refer to the predicted amino acid sequence. Duplicated nucleotides resulting from insertion of the P element of dx.sup.P are shown boxed. A potential polyadenylation signal is underlined twice; a stretch of 17 A's is found in cDNA cBE8 just after nucleotide 3764 (the last uppercase letter). 5' to the cDNA sequence, two groups of seven nucleotides (underlined) are homologous to a transcription initiation consensus sequence.
FIG. 4. Subcellular pattern of induced deltex protein expression. Confocal microscope images of a stage-10 embryo (lateral view: anterior is to the left; dorsal is up) after immunofluorescent staining with monoclonal antibody C645.17A. Embryo contains a transposon (line 142A.sup.D) of deltex coding sequences under the control of the Drosophila Hsp70 promoter and was stained after heat shock induction (see Section 6.3). Upper right: whole embryo; lower left: high magnification of same image detailing ventral region.
FIG. 5. The effects of the Su(dx) mutations on various deltex alleles are summarized. The top row indicates the genetic constitution of the second chromosome and the left column indicates the genetic makeup of the X chromosome in the male. (+++): the deltex wing and ocellar phenotypes are completely suppressed. (++): the deltex wing and ocellar phenotypes are almost completely suppressed; only a trace of extra wing vein material is present at the margin of the fifth longitudinal veins. (+): the deltex wing and ocellar phenotypes are partially suppressed.
FIG. 6. Suppression of deltex wing phenotypes by the Su(dx) mutations. a). Wing of y dx.sup.ENU sn.sup.3 /Y male: note notches and extra vein material. b). Wing of y dx.sup.ENU sn.sup.3 /Y. Su(dx).sup.sp /+ male: the dx.sup.ENU wing phenotype is completely suppressed by one copy of the Su(dx).sup.sp mutation. c). Wing of dx.sup.SM t.sup.2 v/Y male: notches and extra vein material are evident along the veins. d). Wing of dx.sup.SM t.sup.2 v/Y; Su(dx).sup.sp + male: the phenotype of this strong dx allele is completely suppressed by one copy of the Su(dx).sup.sp mutation, but is only partially suppressed by the Su(dx).sup.2 mutation. e). Wing of dx.sup.P sn.sup.3 /Y male: this weak allele displays a mild extra-vein-material phenotype on the ends of the longitudinal veins. f). Wing of dx.sup.P sn.sup.3 /Y, Su(dx).sup.sp + male: the phenotype of the dx.sup.P mutation is only partially suppressed by Su(dx).sup.sp (and also by the other two Su(dx) mutations).
FIG. 7. a). Wing of nd dx.sup.ENU /Y fly. Note the severe deltex wing-notching and the suppressed Abruptex gapped-vein phenotype. b). Wing of Ax.sup.E2 dx.sup.ENU /Y; Su(dx)/+ fly. Note the suppression of the deltex phenotype and the reemergence of the Abruptex gapped-vein phenotype. c). Dorsal view of Ax.sup.E2 sn.sup.3 /Y, Su(dx).sup.sp /+ fly. d). Dorsal view of Ax.sup.E2 dx.sup.ENU /Y, Su(dx).sup.sp /+ fly. Arrow points to region exhibiting a severe loss of micro- and macro-chaetae.
FIG. 8. deltex interacts with Notch cdc10/SW16/ankyrin ("ANK") repeats. Confocal microscope images of Drosophila S2 cells (a-d and f-l) and distal portion of imaginal wing disc (e) are presented as split images (except k) with Notch expression shown in green and deltex in red. For S2 cells, each panel represents a co-transfection experiment involving the deltex expression plasmid pCaSpeR hs-dx (see Section 6) and each of the expression constructs depicted in FIG. 9 or described elsewhere: a and b, pMtNcDNA (full-length Notch; Rebay et al., 1993, Cell 74:319-329); c, pMTD11 (Delta; Fehon et al., 1990, Cell 61:523-534); d, Ser-mtn (Serrate; Rebay et al., 1991, Cell 67:687-699); f, pMT.DELTA.B-S; g, pMT.DELTA.S-S; h, pMTECN; i, pMT.DELTA.cdc10; j and k, pMTDl/NANK; l, pMTDl/CANK. Cell surface `capping` was induced (except in b) as described (Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699). Immunofluorescent labeling of cells was performed as described (Fehon et al., 1990, Cell 61:523-534) using mouse anti-Notch monoclonal antibody (line C458.2H) specific for the extracellular domain and rat anti-deltex polyclonal or monoclonal (line C645.17A) antibodies (see Section 6) directed against the entire coding region. Extraneous cytoplasmic Notch expression was minimized by reacting cells with anti-Notch antibody prior to cell permeabilization and incubation with anti-deltex antibodies. Delta and Serrate expression (c and d) are viewed indirectly through their co-capping with Notch. In some instances, aggregated cell partners were torn apart during manipulation. The diameter of S2 cells is 7-10 microns.
FIG. 9. Plasmid expression constructs used in S2 cell transfections. a, Notch intracellular domain (top; drawn to scale); extracellular domain is not shown but is intact (TM, transmembrane region). Structural motifs and restriction sites used to generate deletion constructs (black bars) are indicated. Deleted amino acids are presented in parentheses (numbering from Wharton et al., 1985, Cell 43:567-581). b, Intracellular domains of Delta-based expression constructs (same scale as in a). NdeI site adjacent to translational stop codon was used as insertion site for Notch-(pMTDl/NANK) and cactus-(pMTDl/CANK) ANK repeat coding sequences. Results of deltex binding are summarized. c, Immunoblot analysis of 52 cell lysates after transfection and expression of plasmid constructs depicted in b (NT, non-transfected cells). Integrity of other expression constructs (a) was also confirmed by immunoblot analysis (not shown).
FIG. 10. Direct Notch/deltex interaction in yeast. Each column lists Drosophila genes that were fused to sequences encoding a LexA DNA-binding domain or an acidic transcription activation domain. These were co-transformed into yeast containing a LexA operator-lacZ reporter gene plasmid (pSH18-34; S. Hanes and R. Brent, unpublished) and were plated onto glucose- and galactose-Ura.sup.- His.sup.- Trp.sup.- X-gal-indicator plates. Three independent yeast isolates are shown for each experiment. Only transformations involving LexA-Notch ICN1 and deltex-ACT resulted in detectable .beta.-galactosidase activity.
FIG. 11. Intragenic suppressor mutation maps within fifth Notch ANK repeat. a, Diagram of Notch protein. The su42c mutation was induced in flies containing the Ax.sup.9B2 mutation (Xu et al., 1990, Genes Dev. 4:464-475). b, DNA sequencing gels showing C to T transition in Ax.sup.9B2 su42c genomic DNA. This residue is unaffected (i.e., wild-type) in the parental Ax.sup.9B2 DNA. c, Amino acid sequence (single letter code) (SEQ ID NO:22) of fifth ANK repeat showing alanine to valine substitution resulting from the su42c mutation (numbering based on Wharton et al., 1985 Cell 43:567-581).
FIG. 12. Nucleotide sequence (SEQ ID NO:3), complimentary strand (SEQ ID NO:23), and deduced amino acid sequence (SEQ ID NO:4) of deltex cDNA.
FIG. 13. Schematic diagram of deltex fragments mediating deltex--deltex interactions.
FIG. 14. Amino acid sequence of Drosophila deltex (SEQ ID NO:2; SEQ ID NO:4) and designated fragments implicated in protein--protein interactions. Fragments A-D (SEQ ID NOS:5-8, respectively) are shown.
FIG. 15. Schematic diagram of the deltex and Notch fragments mediating deltex-Notch interactions.
FIG. 16. Putative SH3-binding domains in Drosophila (fly). The sequence of the mouse GAP SH3-binding domain (SEQ ID NO:9) is shown, with the putative SH3 binding domains of the Drosophila proteins deltex (SEQ ID NO:10 and SEQ ID NO:11), son of Sevenless (SEQ ID NO:12 and SEQ ID NO:13), hairless (SEQ ID NO:14), and disabled (SEQ ID NO:15).
FIG. 17. Aligned amino acid sequences of Notch proteins of various species. humN: the human Notch protein encoded by the hN homolog (SEQ ID NO:16). TAN-1: the human Notch protein encoded by the TAN-1 homolog (SEQ ID NO:17) (the sequence shown is derived partly from work by Artavanis-Tsakonas et al. (see PCT Publication No. WO 92/19737 dated Nov. 12, 1992) and partly from the TAN-1 sequence as published by Ellisen et al., 1991, Cell 66:649-661); Xen N: Xenopus Notch protein (Coffman et al., 1990, Science 249:1438-1441 (SEQ ID NO:18)). Dros N: Drosophila Notch protein (Wharton et al., 1985, Cell 43:567-581 (SEQ ID NO:19)). Structural domains are indicated.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to nucleotide sequences of deltex genes, and amino acid sequences of their encoded deltex proteins. The invention further relates to fragments and other derivatives, and analogs, of 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 methods, is provided.
In particular, the invention relates to the Drosophila deltex gene and protein. In another embodiment, the invention relates to the human deltex gene and protein.
The invention also relates to 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) deltex protein. Such functional activities include but are not limited to antigenicity ability to bind (or compete with a deltex protein for binding) to an anti-deltex protein antibody!, immunogenicity (ability to generate antibody which binds to a deltex protein), ability to bind (or compete with a deltex protein for binding) to Notch or a second deltex protein or other proteins or fragments thereof, ability to bind (or compete with a deltex protein for binding) to a receptor or ligand for a deltex protein.
The invention further relates to fragments (and derivatives and analogs thereof) of a deltex protein which comprise one or more domains of a deltex protein (see infra), including but not limited to the Gln-rich clusters, SH3 binding domains, domains which mediate binding to Notch (or a derivative thereof containing the Notch ANK repeats) or to a second deltex molecule or fragment thereof, or any combination of the foregoing.
Antibodies to deltex proteins, their derivatives and analogs, are additionally provided.
deltex plays 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. Our results presented by way of example below indicate that deltex mediates the intracellular portion of the signal transduction cascade involved in Notch function. As described therein (see Section 6), our results show that deltex is localized within the cytoplasm, that it is a protein of unique sequence, and that it displays direct molecular interaction with the Notch intracellular ANK repeats, motifs shared by many proteins and 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 (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 further examined the functional characteristics of deltex by analyzing its genetic interactions with another locus, Suppressor of deltex Su(dx)!. This analysis implicates Suppressor of deltex in the genetic circuitry of Notch and suggests a three way interaction between Su(dx), deltex and Notch.
The deltex nucleic acid and amino acid sequences and antibodies thereto of the invention can be used for the detection and quantitation of deltex mRNA and protein, to study expression thereof, to produce deltex proteins, fragments and other derivatives, and analogs thereof, in the study, assay, and manipulation of differentiation and other physiological processes, and are of therapeutic and diagnostic use, as described infra. The deltex nucleic acids and antibodies can also be used to clone deltex homologs of other species, as described infra. Such deltex homologs are expected to exhibit significant homology to each other, and encode proteins which exhibit the ability to bind to a Notch protein.
The present invention also relates to therapeutic and diagnostic methods and compositions based on deltex proteins and nucleic acids. The invention 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: deltex proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the deltex proteins, analogs, or derivatives; and 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 a nervous system disorder or to promote tissue regeneration and repair.
In one embodiment, Therapeutics which antagonize, or inhibit, Notch and/or deltex function (hereinafter "Antagonist Therapeutics") are administered for therapeutic effect. In another embodiment, Therapeutics which promote 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 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 deltex that mediates binding to a Notch protein, a second deltex protein, or a fragment of Notch or deltex.
The invention is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of D. melanogaster deltex; the construction and recombinant expression of deltex chimeric/fusion derivatives, production of anti-deltex antibodies, and the identification of regions of deltex which bind to the ANK repeats of Notch, or which bind to regions of 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 Deltex Nucleic Acids
The invention relates to the nucleotide sequences of deltex nucleic acids. In specific embodiments, Drosophila deltex nucleic acids comprise the genomic sequence shown in FIG. 3 (SEQ ID NO:1), or the cDNA sequence shown in FIG. 12 (SEQ ID NO:3), or the coding regions thereof, or nucleic acids encoding a deltex protein (e.g., having the sequence of SEQ ID NO:4). The invention provides nucleic acids consisting of at least 8 nucleotides (i.e., a hybridizable portion) of a deltex sequence; in other embodiments, the nucleic acids consist of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a deltex sequence, or a full-length deltex coding sequence. The invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences. In specific aspects, nucleic acids are provided which comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a deltex gene. In a specific embodiment, a nucleic acid which is hybridizable to a deltex nucleic acid (e.g., having sequence SEQ ID NO:3), or to a nucleic acid encoding a 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.degree. C. in a solution containing 35% formamide, 5.times. SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/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 .mu.g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20.times.10.sup.6 cpm .sup.32 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40.degree. C., and then washed for 1.5 h at 55.degree. C. in a solution containing 2.times. 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.degree. C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65.degree.-68.degree. C. and reexposed 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 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 65.degree. C. in buffer composed of 6.times. SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 .mu.g/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65.degree. C. in prehybridization mixture containing 100 .mu.g/ml denatured salmon sperm DNA and 5-20.times.10.sup.6 cpm of .sup.32 P-labeled probe. Washing of filters is done at 37.degree. C. for 1 h in a solution containing 2.times. SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1.times. SSC at 50.degree. C. for 45 min before autoradiography. Other conditions of high stringency which may be used are well known in the art.
Nucleic acids encoding fragments and derivatives of deltex proteins (see Section 5.6), and 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 deltex protein" shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the deltex protein and not the other contiguous portions of the deltex protein as a continuous sequence.
Specific embodiments for the cloning of a 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 deltex product. In a preferred aspect, anti-deltex antibodies can be used to select the recombinant host cell expressing a cloned deltex gene.
In another specific embodiment, PCR is used to amplify the desired deltex sequence in a genomic or cDNA library, prior to selection. Oligonucleotide primers representing known deltex sequences 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.TM.). 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 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 conditions are preferred (see supra). After successful amplification of a segment of a 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 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), and no hybridizing deltex genes are detected or amplified when hybridization is carried out, the desired deltex gene can be isolated by a more gradual method 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, in a specific aspect directed toward isolating a human deltex gene, first a gene from another species of fly (e.g., Drosophila hidei, Drosophila pseudoobscura, mosquito, or housefly) can be isolated, and the conserved portions of the sequence identified, and used to screen or amplify a human cDNA library. One may also, prior to screening or amplifying the human library, isolate "intermediate" deltex genes, e.g., from fish, rat, cow, or other species, to further define regions of sequence conserved across species.
The above-methods are not meant to limit the following general description of methods by which clones of deltex may be obtained.
Any eukaryotic cell can potentially serve as the nucleic acid source for the molecular cloning of the 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, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) 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 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, isolectric focusing behavior, proteolytic digestion maps, in vitro aggregation activity ("adhesiveness") or antigenic properties as known for deltex. If an antibody to deltex is available, the deltex protein may be identified by binding of labeled antibody to the putatively deltex synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay)-type procedure.
The 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 deltex DNA of another species (e.g., Drosophila). Immunoprecipitation analysis or functional assays (e.g., aggregation ability in vitro; see examples infra) 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 deltex protein. A radiolabelled 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 deltex DNA fragments from among other genomic DNA fragments.
Alternatives to isolating the 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 deltex gene. For example, RNA for cDNA cloning of the deltex gene can be isolated from cells which express 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 or pUC 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 deltex gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
In an alternative method, the desired gene may be identified and isolated after insertion into 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 deltex gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
The deltex sequences provided by the instant invention include those nucleotide sequences encoding substantially the same amino acid sequences as found in native deltex protein, and those encoded amino acid sequences with functionally equivalent amino acids, all as described in Section 5.6 infra for deltex derivatives.
5.2. Expression of Deltex Nucleic Acids
The nucleic acid coding for a deltex protein or a functionally active fragment or other derivative thereof can be inserted into 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 transcriptional and translational signals can also be supplied by the native 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 mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in 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 deltex gene which encodes a protein that binds to Notch or a molecule comprising the Notch ANK repeats is expressed. In another embodiment, a molecule comprising a portion of a deltex gene which encodes a protein that binds to a fragment of a deltex protein is expressed. In other specific embodiments, the human deltex gene is expressed, or a sequence encoding a functionally active portion of human deltex. In a specific embodiment, a chimeric protein comprising a Notch-binding domain of a deltex protein is expressed. In other specific embodiments, a full-length deltex cDNA is expressed, or a sequence encoding a functionally active portion of a deltex protein. In yet another embodiment, a fragment of a deltex protein comprising a domain of the protein, or other derivative, or analog of a deltex protein is expressed.
Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of a nucleic acid sequence encoding a deltex protein or peptide fragment may be regulated by a second nucleic acid sequence so that the deltex protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a deltex protein may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control deltex gene expression include, 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), 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 (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the .beta.-lactamase (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), tac (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25), .lambda.P.sub.L, or trc promoters; see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline 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 virus 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 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 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 formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if the deltex gene is inserted within the marker gene sequence of the vector, recombinants containing the 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 deltex gene product in in vitro assay systems, e.g., binding to Notch, binding 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 in quantity. As previously explained, 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 vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage 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 engineered 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 proteins. 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 deltex protein, fragment, analog, or derivative may be expressed as a fusion, or chimeric protein product (comprising the protein, 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 protein synthetic techniques, e.g., by use of a peptide synthesizer.
Both cDNA and genomic sequences can be cloned and expressed.
In other embodiments, a 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 Deltex Gene Products
In particular aspects, the invention provides amino acid sequences of 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 active, as well as nucleic acid sequences encoding the foregoing. "Functionally active" material as used herein refers to that material displaying one or more known functional activities associated with the full-length (wild-type) deltex protein product, e.g., binding to Notch or a portion thereof, binding to another deltex molecule or portion thereof, binding to any other deltex ligand, antigenicity (binding to an anti-deltex antibody), Notch intracellular signal transduction, etc.
In specific embodiments, the invention provides fragments of a deltex protein consisting of at least 6 amino acids, 10 amino acids, 50 amino acids, or of at least 75 amino acids. In other embodiments, the proteins comprise or consist essentially of one or more Gln-rich clusters (e.g., amino acids 261-302 and amino acids 488-513 of SEQ ID NO:4); one or more of the SH3 binding domains (e.g., SEQ ID NOS:11 and 12 of FIG. 16), or a portion which binds to Notch (e.g., comprising the first approximately 200 amino acids of deltex), or any combination of the foregoing, of a deltex protein. Fragments, or proteins comprising fragments, lacking some or all of the foregoing regions of deltex are also provided. Nucleic acids encoding the foregoing are provided.
Once a recombinant which expresses a 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, immunoassay, etc. Chemically synthesized proteins, derivatives, and analogs can be similarly analyzed.
Once a 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 properties may be evaluated using any suitable assay (see Section 5.7).
Alternatively, the amino acid sequence of a 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 NOS:2 or 4 (which are identical to each other) of a Drosophila deltex protein is presented in FIG. 12.
5.4. Structure of the Deltex Genes and Proteins
The structure of the 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 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, and Section 6.1.3, infra), restriction endonuclease mapping (Maniatis, 1982, Molecular Cloning, A Laboratory, Cold Spring Harbor, N.Y.), and DNA sequence analysis (see Section 6.3.1 and FIGS. 1-4). Polymerase chain reaction (PCR; U.S. Pat. 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 deltex-specific probe can allow the detection of the deltex genes in DNA from various cell types. In one embodiment, Southern hybridization can be used to determine the genetic linkage of deltex. Northern hybridization analysis can be used to determine the expression of the deltex genes. Various cell types, at various states of development or activity can be tested for 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 deltex probe used.
Restriction endonuclease mapping can be used to roughly determine the genetic structure of the deltex gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis. Alternatively, restriction maps can be deduced, once the nucleotide sequence is known.
DNA sequence analysis can be performed by any techniques known in the art, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol. 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. Pat. 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, Calif.). The cDNA sequence of a Drosophila melanogaster deltex gene is shown in FIG. 12 (SEQ ID NO:3) and is described in Section 6, infra.
5.4.2. Protein Analysis
The amino acid sequence of a deltex protein can be derived by deduction from the DNA sequence, or alternatively, by direct sequencing of the protein, e.g., with an automated amino acid sequencer. The amino acid sequence of a representative deltex protein comprises the sequence substantially as depicted in FIG. 12 (SEQ ID NO:4), and detailed in Section 6, infra.
The deltex protein 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 deltex protein and the corresponding regions of the gene sequence which encode such regions.
Secondary, structural analysis (Chou and Fasman, 1974, Biochemistry 13:222) can also be done, to identify regions of a deltex protein that assume specific secondary structures.
Manipulation, translation, and secondary structure prediction, as well as open reading 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 include but are not limited to X-ray crystallography (Engstom, 1974, Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick and Zoller (eds.), 1986, Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
5.5. Generation of Antibodies to Deltex Proteins and Derivatives Thereof
According to the invention, a deltex protein, its fragments or other derivatives, or analogs thereof, may be used as an immunogen to generate antibodies which recognize such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. In a preferred embodiment, antibodies which specifically bind to deltex proteins are produced. In another embodiment, antibodies to a particular domain of a deltex protein are produced. In a specific embodiment, an antibody is produced which binds to a fragment of deltex which binds to Notch; in another embodiment, an antibody binds to a molecule comprising the first 204 amino-terminal amino acids of deltex. In another embodiment the antibody binds to an amino-terminal fragment of deltex containing not more than the first 200 amino acids of deltex. In yet another embodiment, an antibody binds to a fragment of deltex which binds to a second deltex molecule.
Various procedures known in the art may be used for the production of polyclonal antibodies to a deltex protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies to an epitope of the deltex protein having a sequence depicted in FIG. 12 or a subsequence thereof, can be obtained. For the production of antibody, various host animals can be immunized by injection with a native deltex protein, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
In a preferred embodiment, polyclonal or monoclonal antibodies are produced by use of a hydrophilic portion of a 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 deltex protein 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 hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) can be used. In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (PCT Publication No. WO 89/12690 dated Dec. 28, 1989). According to the invention, human antibodies may be used and can be obtained by using human hybridomas (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, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96), or by other methods known in the art. In fact, according to the invention, 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 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 invention.
According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce deltex protein-specific single 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 deltex proteins, derivatives, or analogs.
Antibody fragments and other derivatives which contain the idiotype (binding domain) of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab').sub.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').sub.2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain 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 domain of a deltex protein, one may assay generated hybridomas for a product which binds to a deltex fragment containing such domain. For selection of an antibody specific to human deltex protein(s), one can select on the basis of positive binding to a human deltex protein and a lack of binding to Drosophila deltex protein.
In a specific embodiment, antibodies specific to a phosphorylated epitope of deltex are produced.
The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the protein sequences of the invention e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, etc. Antibodies to deltex (since it normally colocalizes with Notch) can be used to determine the intracellular distribution of Notch and/or deltex, in diagnostic methods such as described infra. The antibodies also have use in immunoassays. In another embodiment of the invention (see infra), anti-deltex antibodies and fragments thereof containing the binding domain are Therapeutics.
5.6. Deltex Proteins, Derivatives and Analogs
The invention further provides deltex proteins, and derivatives (including but not limited to fragments) and analogs of deltex proteins. Nucleic acids encoding deltex protein derivatives and protein analogs are also provided. In one embodiment, the deltex proteins are encoded by the deltex nucleic acids described in Section 5.1 supra. In particular aspects, the proteins, derivatives, or analogs are of fly, frog, mouse, rat, pig, cow, dog, monkey, or human deltex proteins.
The production and use of derivatives and analogs related to deltex are within the scope of the present invention. In a specific embodiment, the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type deltex protein.
In particular, deltex derivatives can be made by altering 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 amino acid sequence as a deltex gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of deltex genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. Likewise, the deltex derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a deltex protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
In a specific embodiment of the invention, proteins consisting of or comprising a fragment of a deltex protein consisting of at least 10 (continuous) amino acids of the deltex protein is provided. In other embodiments, the fragment consists of at least 20 or 50 amino acids of the deltex protein. In specific embodiments, such fragments are not larger than 35, 100 or 200 amino acids. Derivatives or analogs of deltex include but are not limited to those peptides which are substantially homologous to deltex or fragments thereof (e.g., at least 30% identity over an amino acid sequence of identical size) or whose encoding nucleic acid is capable of hybridizing to a coding deltex sequence.
The deltex protein derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned deltex gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of a deltex protein, care should be taken to ensure that the modified gene remains within the same translational reading frame as the deltex gene, uninterrupted by translational stop signals, in the gene region where the desired deltex activity is encoded.
Additionally, the deltex-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in 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 (Hutchinson et al., 1978, J. Biol. Chem 253:6551), use of TAB.RTM. linkers (Pharmacia), etc.
Manipulations of the deltex sequence may also be made at the protein level. Included within the scope of the invention are deltex protein fragments or other derivatives or analogs which are differentially modified during 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, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH.sub.4, 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 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.
In addition, analogs and derivatives of deltex proteins can be chemically synthesized. For example, a peptide corresponding to a portion of a 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, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the deltex protein sequence. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, .alpha.-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, .beta.-alanine, designer amino acids such as .beta.-methyl amino acids, C.alpha.-methyl amino acids, and N.alpha.-methyl amino acids.
In a specific embodiment, the deltex derivative is a chimeric, or fusion, protein comprising a deltex protein or fragment thereof (preferably consisting of at least a domain or motif of the deltex protein, or at least 10 amino acids of the 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 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 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 deltex protein which comprises a domain or motif of the deltex protein, e.g., a Gln-rich cluster, portion which binds to a Notch protein or to a second deltex protein, an SH3 binding domain, etc. In a particular embodiment, a chimeric nucleic acid can be constructed, encoding a fusion protein consisting of a 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 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 deltex protein of at least six amino acids. Particular examples of the construction and expression of fusion proteins comprising deltex or various deltex fragments, are described in Sections 7 and 8 hereof.
Other specific embodiments of derivatives and analogs are described in the subsection below and examples sections infra.
5.6.1. Derivatives of Deltex Containing One or More Domains of the Protein
In a specific embodiment, the invention provides deltex derivatives and analogs, in particular deltex fragments and derivatives of such fragments, that comprise or consist of one or more domains of the deltex protein, including but not limited to a Gln-rich cluster, 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, or an SH3-binding domain. In specific embodiments, the 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 D. melanogaster deltex protein, the aforesaid domains consist of approximately the following amino-acid sequences:
Gln-rich clusters
amino acids 261-302 of FIG. 12 (part of SEQ ID NO:4);
amino acids 488-513 of FIG. 12 (part of SEQ ID NO:4);
SH3 binding domain
SEQ ID NO:10 of FIG. 16
SEQ ID NO:11 of FIG. 16
Notch-binding fragment
Fragment D (SEQ ID NO:8) of FIG. 14
Deltex-binding fragments
Fragment A (SEQ ID NO:5) of FIG. 14
Fragment B (SEQ ID NO:6) of FIG. 14
Fragment C (SEQ ID NO:7) of FIG. 14
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, S2 cell expression assay) such as described in Sections 7 and 8 infra.
In a specific embodiment, relating to a deltex protein of a species other than D. melanogaster, preferably human, fragments comprising specific domains of deltex are those comprising domains in the respective deltex protein most homologous to the specific domain of the Drosophila melanogaster deltex protein.
In specific embodiments, deltex fragments which homotypically bind to an identical deltex fragment (e.g. fragment A (SEQ ID NO:5), fragment B (SEQ ID NO:6), and fragment C (SEQ ID NO:7) shown in FIG. 14) or to a distinct deltex fragment (e.g., fragment B (SEQ ID NO:6) and fragment C (SEQ ID NO:7) shown in FIG. 14) are provided. Examples of such fragments and assays for their selection are described in Section 8 infra.
Also provided are inhibitors (e.g., peptide inhibitors) of the foregoing protein interactions with Notch or with a second deltex protein.
The ability to bind to a Notch protein or a deltex protein (or derivative thereof) can be demonstrated by in vitro assays such as described in Sections 7 and 8 infra, e.g., by S2 cell expression assay (Section 7.3), interaction trap technique (Sections 7.4, 8), etc.
The nucleic acid sequences encoding Notch or deltex proteins or fragments thereof, for use in such assays, can be isolated from human, porcine, bovine, feline, avian, equine, canine, or insect, as well as primate sources and any other species in which homologs of known genes can be identified. For example, the Notch protein or portion thereof comprising the ANK repeats which can be expressed and assayed for binding to deltex or a deltex derivative can be or be derived from any of the Notch homologs shown in FIG. 17 (human hN, human TAN-1, Xenopus, and Drosophila; SEQ ID NOS:16-19, respectively).
Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as the aforesaid domains may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of the deltex genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. Likewise, the deltex proteins, fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of the domains including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence ("conservative" changes).
The derivatives, analogs, and peptides of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein 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.
5.7. In Vitro Assays of Deltex Proteins, Derivatives and Analogs
The functional activity of 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 deltex for binding to anti-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 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 present invention.
In another embodiment, where one is assaying for the ability to mediate binding to Notch or to a second deltex protein or portions thereof, one can carry out assays such as described infra in Section 7 or 8.
Other methods will be known to the skilled artisan and are within the scope of the invention.
5.8. Therapeutic Uses
The invention 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: deltex proteins and analogs and derivatives (including fragments) thereof (e.g., as described hereinabove); antibodies thereto (as described hereinabove); nucleic acids encoding the deltex proteins, analogs, or derivatives (e.g., as described hereinabove); and deltex antisense nucleic acids. As stated supra, the Antagonist Therapeutics of the invention are those Therapeutics which antagonize, or inhibit, a deltex function and/or Notch function (since the data (see Sections 6-9) indicates that deltex functions in intracellular signal transduction from Notch). Such Antagonist Therapeutics are most preferably identified by use of known convenient in vitro assays, e.g., based on their ability to inhibit binding of deltex to another protein (e.g., a Notch protein), or inhibit any known Notch or deltex function as preferably assayed in vitro or in cell culture, although genetic assays (e.g., in Drosophila) may also be employed. In a preferred embodiment, the Antagonist Therapeutic is a protein or derivative thereof comprising a functionally active fragment such as a fragment of deltex which mediates binding 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 deltex which binds to Notch, or a deltex antisense nucleic acid (see Section 5.11 herein). It should be noted that 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 of a deltex gene is used, as an Antagonist Therapeutic, to promote 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 deltex function. Such Agonist Therapeutics include but are not limited to proteins and derivatives comprising the portions of Notch that mediate binding to deltex, i.e., the ANK repeats, and nucleic acids encoding the foregoing (which can be administered to express their encoded products in vivo).
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 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 correlates. In a specific embodiment, a peptide (e.g., in 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 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 other malignancies associated with increased Notch expression (e.g., cervical cancer, colon cancer, breast cancer, squamous adenocarcimas (see infra)). Derivatives or analogs of deltex can be tested for the desired activity by procedures known in the art, including but not limited to the assays described in the examples infra. For example, molecules comprising deltex fragments which bind to Notch ANK repeats and which are smaller than deltex fragment D (see FIG. 14 and Section 8), can be obtained and selected by expressing deletion mutants of fragment D (or of a nucleotide sequence of another species encoding the amino-terminal .about.204 amino acids of deltex) and assaying for binding of the expressed product to Notch by any of the several methods (e.g., interaction trap system) described in 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 carried out by assaying, e.g., for binding to Notch or a molecule containing the Notch ANK repeats.
The Agonist and Antagonist Therapeutics of the invention have therapeutic utility for disorders of cell fate. The Agonist Therapeutics are administered therapeutically (including prophylactically): (1) in diseases or disorders involving an absence or decreased (relative to normal, or desired) levels of Notch or deltex function, for example, in patients where Notch or deltex protein is lacking, genetically defective, biologically inactive or underactive, or underexpressed; and (2) in diseases or disorders wherein in vitro (or in vivo) assays (see infra) indicate the utility of deltex agonist administration. The absence or decreased levels in Notch or deltex function can be readily detected, e.g., by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for protein levels, structure and/or activity of the expressed Notch or deltex protein. Many methods standard in the art can be thus employed, including but not limited to immunoassays to detect and/or visualize Notch or deltex protein (e.g., Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect Notch or deltex expression by detecting and/or visualizing respectively Notch or deltex mRNA (e.g., Northern assays, dot blots, in situ hybridization, etc.)
In vitro assays which can be used to determine whether administration of a specific Agonist Therapeutic or Antagonist Therapeutic is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a Therapeutic, and the effect of such Therapeutic upon the tissue sample is observed. In one embodiment, where the patient has a malignancy, a sample of cells from such malignancy is plated out or grown in 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 growth; for example, cell proliferation can be assayed by measuring .sup.3 H-thymidine incorporation, by direct cell count, by detecting changes in transcriptional 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 in morphology, etc. In a specific aspect, the malignant cell cultures are separately exposed to (1) an Agonist Therapeutic, and (2) an Antagonist Therapeutic; the result of the assay can indicate which type of Therapeutic has therapeutic efficacy.
In another embodiment, a Therapeutic is indicated for use which exhibits the desired effect, inhibition or promotion of cell growth, upon a patient cell sample from tissue having or suspected of having a hyper- or hypoproliferative disorder, respectively. Such hyper- or hypoproliferative disorders include 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 injury or a nervous system degenerative disorder (see Section 5.8.2) which exhibits in vitro promotion of nerve regeneration/neurite extension from nerve cells of the affected patient type.
In addition, administration of an Antagonist Therapeutic of the invention is also indicated in diseases or disorders determined or known to involve a Notch or deltex dominant activated phenotype ("gain of function" mutations.) Administration of an Agonist Therapeutic is indicated in diseases or disorders determined or known to involve a Notch or deltex dominant negative phenotype ("loss of function" mutations). The functions of various structural domains of the Notch protein have been investigated in vivo, by ectopically expressing a series of Drosophila Notch deletion mutants under the hsp70 heat-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 gain-of-function mutations. Dominant "activated" phenotypes resulted from overexpression of a protein lacking most extracellular sequences, while dominant "negative" phenotypes resulted from overexpression of a protein lacking 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 deltex binds to the Notch ANK repeat region (see Sections 6-8).
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.
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 that 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 morphology, looser substratum attachment, loss of contact inhibition, loss of anchorage 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 present invention.
The Antagonist Therapeutics are administered therapeutically (including prophylactically): (1) in diseases or disorders involving increased (relative to normal, or desired) levels of Notch or deltex function, for example, where the Notch or deltex protein is overexpressed or overactive; and (2) in diseases or disorders wherein in vitro (or in vivo) assays indicate the utility of deltex antagonist administration. The increased levels of Notch or 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 be treated upon thus observing an indication of therapeutic utility, include 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 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):
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 exhibit increased expression of human Notch relative to such non-malignant tissue (see copending U.S. application Ser. No. 08/083,590 filed Jun. 25, 1993, incorporated by reference herein in its entirety; PCT application no. PCT/US93/09338 filed Sep. 30, 1993, 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 administration of an Antagonist Therapeutic.
5.8.2. Nervous System Disorders
Nervous system disorders, involving cell types which can be tested as described supra for efficacy of intervention with Antagonist or Agonist Therapeutics, and which can be treated upon thus observing an indication of therapeutic 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 demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian 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 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 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;
(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 the 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 the corpus callosum), and alcoholic cerebellar degeneration;
(vii) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, 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 the 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 leukoencephalopathy, 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 the 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 method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods 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 binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
In a specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that 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 including but not limited 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 the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
5.8.3. Tissue Repair and Regeneration
In another embodiment of the invention, a Therapeutic of the invention is used for promotion of tissue regeneration and repair, including but not limited to treatment of benign dysproliferative disorders. Specific embodiments are directed to treatment of cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin 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).
5.9. Prophylactic Uses
5.9.1. Malignancies
The Therapeutics of the invention can be administered to prevent progression 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, 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, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth 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. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
Alternatively or in 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 the desirability of prophylactic/therapeutic administration of a Therapeutic of the 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 protein, 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 (benign epithelial 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 with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen, retinoblastoma, 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., Philadelphia, pp. 112-113) etc.) In another specific embodiment, an Antagonist Therapeutic of the 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 the 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 the invention can be tested in vivo for the desired therapeutic 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 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 deltex or a portion thereof. "Antisense" as used herein refers to a nucleic acid capable of hybridizing to a portion of a deltex RNA (preferably mRNA) by virtue of some sequence complementarity. Such antisense nucleic acids have utility as Antagonist Therapeutics of the invention, and can be used in the treatment or prevention of disorders as described supra in Section 5.8 and its subsections.
The antisense nucleic acids of the invention can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
In a specific embodiment, the deltex antisense nucleic acids provided by the 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 deltex gene or a Notch gene. Such demonstration can be by detection of RNA or of protein.
The invention further provides pharmaceutical compositions comprising an effective amount of the deltex antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra in Section 5.12. Methods for treatment and prevention of disorders (such as those described in Sections 5.8 and 5.9) comprising administering the pharmaceutical compositions of the invention are also provided.
In another embodiment, the invention is directed to methods for inhibiting the expression of a deltex nucleic acid sequence in a prokaryotic or eukaryotic cell comprising providing the cell with an effective amount of a composition comprising an antisense deltex nucleic acid of the invention.
deltex antisense nucleic acids and their uses are described in detail below.
5.11.1. Deltex Antisense Nucleic Acids
The deltex antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 oligonucleotides). In 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 other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. 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 Dec. 15, 1988) or blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134, published Apr. 25, 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 deltex antisense oligonucleotide is provided, preferably of single-stranded DNA. In a most preferred aspect, such an oligonucleotide comprises a sequence antisense to the sequence encoding an SH3 binding domain or a Notch-binding domain of deltex, most preferably, of human deltex. The oligonucleotide may be modified at any position on its structure with substituents generally known in the art.
The deltex antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
In another 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 phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the oligonucleotide is an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual .beta.-units, the strands run parallel to each other (Gautier 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 methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
In a specific embodiment, the deltex antisense oligonucleotide comprises catalytic RNA, or a ribozyme (see, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225). In another embodiment, the oligonucleotide is a 2'-O-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 deltex antisense nucleic acid of the invention is produced intracellularly 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 the 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 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 methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the deltex antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Such promoters include 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), 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 (Brinster et al., 1982, Nature 296:39-42), etc.
The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a 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 the RNA, forming a stable duplex; in the case of double-stranded deltex antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a deltex RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
5.11.2. Therapeutic Utility of Deltex Antisense Nucleic Acids
The 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 deltex or Notch. In specific embodiments, the 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 those described supra in Sections 5.8.1 and 5.9.1. In a preferred embodiment, a single-stranded DNA antisense deltex oligonucleotide is used.
Malignant (particularly, tumor) cell types which express deltex or Notch RNA can be identified by various methods known in the art. Such methods include but are not limited to hybridization with a deltex or Notch-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 Deltex, immunoassay, etc. In a preferred aspect, primary tumor tissue from a patient can be assayed for Notch or deltex expression prior to treatment, e.g., by immunocytochemistry or in situ hybridization.
Pharmaceutical compositions of the invention (see Section 5.12), comprising an effective amount of a deltex antisense nucleic acid in a pharmaceutically acceptable carrier, can be administered to a patient having a malignancy which is of a type that expresses Notch or deltex RNA or protein.
The amount of deltex antisense nucleic acid which will 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 the antisense cytotoxicity of the tumor type to be treated in vitro, and then in useful animal model systems prior to testing and use in humans.
In a specific embodiment, pharmaceutical compositions comprising deltex antisense nucleic acids are administered via liposomes, microparticles, or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the deltex antisense nucleic acids. In a specific embodiment, it may be desirable to utilize liposomes targeted via antibodies to specific identifiable tumor antigens (Leonetti et al., 1990, Proc. Natl. 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 invention. 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 liposomes, 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 intradermal, intramuscular, intraperitoneal, 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 with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the 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 the pharmaceutical compositions of the invention locally to the 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 catheter, 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, the 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, the 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 the therapeutic target, i.e., the brain, thus 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 toporythmic) protein or portion thereof capable of mediating binding to Notch. Contact of a Notch-expressing cell with the linked Therapeutic results in binding of the linked Therapeutic via its Delta portion to Notch on the surface of the cell, followed by uptake of the linked Therapeutic into the Notch-expressing cell.
In a specific embodiment, the 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 that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. 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 within 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:
The present invention also provides pharmaceutical compositions. Such 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 the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic 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, mineral oil, sesame oil and the like. 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, glycerol, propylene, glycol, water, ethanol and the 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, pills, capsules, powders, sustained-release formulations and the like. The composition can be 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 the Therapeutic, preferably in purified form, together with 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, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic 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 the 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 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 the 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-ethylamino ethanol, histidine, procaine, etc.
The amount of the Therapeutic of the invention which will 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. 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 the route of administration, and the seriousness of the 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 ingredient in the 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 the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
5.13. Diagnostic Utility
deltex proteins, analogues, derivatives, and subsequences thereof, deltex nucleic acids (and sequences complementary thereto), anti-deltex antibodies, have uses in diagnostics. Such molecules can be used in assays, such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting deltex expression, or monitor the treatment thereof. In particular, such an immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-deltex antibody under conditions such that immunospecific binding can occur, and detecting or measuring the 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 deltex localization or aberrant levels of Notch-deltex colocalization in a disease state. In a specific embodiment, antibody to deltex can be used to assay in a patient tissue or serum sample for the presence of deltex where an aberrant level of deltex is an indication of a diseased condition. Aberrant levels of deltex binding ability in an endogenous Notch protein, or aberrant levels of binding ability to Notch (or other deltex ligand) in an endogenous 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 that present, in an analogous sample from a subject not having the disorder.
The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
deltex genes and related nucleic acid sequences and subsequences, including complementary sequences, and other toporythmic gene sequences, can also be used in hybridization assays. deltex nucleic acid sequences, or subsequences thereof comprising 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 deltex expression and/or activity as described supra. In particular, such a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to deltex DNA or RNA, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.
6. EXAMPLE: A MEMBER OF THE NOTCH GROUP OF INTERACTING LOCI, DELTEX ENCODES A CYTOPLASMIC BASIC PROTEIN
As described herein, we have proceeded with a molecular characterization of Drosophila deltex. We report the cloning and sequencing of deltex, and that deltex encodes a maternally and zygotically expressed transcript that results in the production of a basic protein of novel sequence. Expression of the deltex protein appears ubiquitous and at low levels within the cytoplasm. By ectopically expressing deltex under the control of a heatshock promoter, we confirm the cytoplasmic subcellular localization of deltex. Moreover, this overexpression does not result in any obvious phenotypic abnormalities in the fly. Finally, we examine genetically the functional effects of several Suppressor of deltex mutations upon the various deltex interactions. We demonstrate that in addition to suppressing all adult morphological defects of deltex alleles, these suppressors also are capable of suppressing most synergistic effects involving deltex and Notch, Delta, and mastermind.
6.1. Results
6.1.1. Molecular Cloning of the Deltex Locus
The deltex allele dx.sup.P was isolated during a P element-induced mutagenesis screen (Golubovsky, 1983, Dros. Inf. Ser. 59:42-43; Maine et al., 1985, Cell 43:521-529). In situ hybridization of P element sequences to polytene chromosomes revealed that the stock carrying the dx.sup.P mutation (dx.sup.P sn3) contained four P elements in regions 2A, 5C, 5D and 6A, respectively, on the X chromosome (data not shown). Because the cytological position of the deltex locus was previously assigned within the polytene interval 6A3,4 and 6F10,11 (Demerec et al., 1942, Yearbook-Carnegie Institution 41:191; Gorman and Girton, 1992, Genetics 131:99-112), these observations suggested that the deltex phenotype of dx.sup.P was associated with the P element insertion at 6A.
We proceeded to isolate genomic DNA clones representing the 6A chromosomal region by constructing a genomic library of partial Sau3A digests of dx.sup.P DNA and screening it with a P element DNA probe. The overlapping clones, lA25 and lA28, both of which carry P element homologous sequences (FIG. 1A), were shown by in situ hybridization on polytene chromosomes to derive from the 6A region (data not shown). Using DNA fragments from these clones to screen a wild-type genomic library, we obtained three wild-type clones (lI1, lI2, lI3) that covered .about.35 kb around the P element insertion site (FIG. 1A). That the P element in 6A is causative of the deltex phenotype is suggested by an examination of genomic DNA from seven phenotypic revertants of dx.sup.P, which were generated by genetic mobilization of the P element: In all cases the P elements were excised from the cloned region (data not shown).
6.1.2. Transcriptional Activity of the 6A Region
The transcriptional activity within the 35-kb cloned region was examined by Northern analysis: RNA samples from various developmental stages were probed with DNA fragments covering the entire cloned region. Only two transcription units were revealed by this analysis. The first one encoded a single size-class RNA of 1.1 kb that was detectable with both probes pdA8.5 and pdA703 (FIG. 1A). This RNA accumulated at high levels during all stages of development. Limited sequencing of a cDNA clone representing this transcription unit revealed homology to mammalian ribosomal protein L7a (not shown). The second transcription unit, detectable with probes pdA4.8 and pdA702 but not pdA703, encoded a single-sized RNA species of .about.4 kb. Its accumulation profile appeared to be regulated throughout development (FIG. 2): RNA was most abundant in adult females and during the first half of embryogenesis and accumulated at lower levels during the larval and pupal stages. The fact that the P element associated with the dx.sup.P mutation was localized within this 4-kb transcript (see below) provided further evidence that this was the deltex transcription unit.
6.1.3. All Deltex Adult Phenotypes Can Be Rescued by a 10-KB Genomic Fragment
In order to confirm that this transcription unit encoded deltex sequences, we transformed Drosophila embryos with cloned DNA spanning coordinates .about.+6 to .about.-4 (FIG. 1) and tested the ability of the introduced DNA to complement deltex mutations (see Section 6.3). The 10-kb XbaI genomic fragment used for this study contained the putative deltex transcription unit but interrupted the neighboring ribosomal protein transcription unit (FIG. 1B). We obtained two independent transformant lines, denoted TX05B and TX012G, both of which carried insertions of the transposon on the second chromosome. In genetic complementation tests using all four deltex alleles, both TXO05B and TX012G rescued all the adult mutant phenotypes, including the wing, ocellar and rough eye defects (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). This rescue was complete for TXO5B, whereas in the case of TX012G, a small percentage of flies displayed a weak delta at the tip of the fifth longitudinal wing vein for all four deltex alleles (data not shown).
We previously reported that although deltex mutations are viable, about 40% of the eggs laid by homozygous deltex females (dx.sup.ENU failed to hatch (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). During the course of the present work it was noticed that this particular phenotype appeared more variable than previously encountered: between 12 and 48% of dx.sup.ENU embryos produced by homozygous dx.sup.ENU females failed to hatch. Neither TX05B nor TXO12G improved this statistic: the exact same percentage of eggs failed to hatch whether or not their mutant mothers contained one copy of the transposon (see also below), suggesting that the observed lethality is not solely linked to deltex function.
Double mutant combinations between dx.sup.ENU and the Notch allele notchoid (nd) are lethal (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). TXO5B and TXO12G are capable of rescuing this lethality: nd dx.sup.ENU homozygous or hemizygous animals are rescued by one copy of the 10-kb XbaI transposon, although the transformant females are sterile (eggs are laid but never hatch). Two copies of the transposon do not improve this sterility. Additionally, the rescued animals display a wing phenotype (very thick veins ending in deltas and large nicks in the wing margin) very similar to that previously described for nd dx.sup.ENU /nd+ females (see Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677), though it is less severe with two copies of the transposon. On the one hand, these results clearly indicate that the 10-kb XbaI genomic fragment contains sequences encoding deltex information. On the other hand, they suggest that these transformant lines are failing to provide the entire adult complement of deltex function and/or that activity necessary for normal maternal function. It should be emphasized, however, that the semi-lethal phenotype associated with what appears to be deltex maternal function is not understood and the possibility that it reflects some aspect of the genetic background has not been excluded.
6.1.4. Deltex Encodes a Basic Protein Rich in Glutamine, Histidine, and Serine Residues
Two cDNA clones homologous to the 4-kb deltex transcript were isolated from an embryonic cDNA library (see Section 6.3) and their complete nucleotide sequence determined. These cDNAs, denoted cBE30 and cBE8, are 1299 bp and 2648 bp long, respectively, and overlap by 183 bp (FIGS. 1B,3). The composite nucleotide sequence (SEQ ID NO:1) is 3763 bp, which is a few hundred base pairs shorter than the length of the RNA (.about.4 kb) as measured on RNA blots. Because a poly A tail and a poly A addition signal appear in the cDNA sequence, the 5' extent of the deltex transcript appears not to be represented by the cDNAs. From limited sequencing of corresponding dx.sup.P genomic DNA, we detect two short sequences approximately 150 bp and 300 bp upstream of the cDNA sequence that are highly homologous to a conserved sequence found at the cap site of other insect genes (Hultmark et al., 1986, Cell 44:429-438; FIG. 3). There is no obvious TATA box, although parts of this region are A/T rich. Genomic sequencing of dx.sup.P DNA also revealed two small introns of 594 and 68 bp, as well as the location of the P element insertion site to within the 5' untranslated region (see FIG. 1B).
Within the 3763-bp cDNA sequence there is a single open reading frame (ORF) of 2211 bp. The first AUG codon is within a sequence context that is consistent with the translation initiation consensus sequence determined for Drosophila (Cavener, 1987, Nucl. Acids Res. 15:1353-1361). Assuming that translation starts at that AUG, the deltex mRNA contains an untranslated leader sequence of at least 344 bp and an untranslated 3' sequence of 1200 bp (FIG. 3). The predicted protein product (SEQ ID NO:2) has 737 amino acids and an estimated molecular mass of 82 kDa (FIG. 3). Computer database searches (see Section 6.3) failed to provide significant homologies to any known proteins.
The predicted deltex protein is rather basic, with an estimated pI of 9.8. One feature of this protein is the high percentage of the amino acids glutamine, histidine and serine: 11.26%, 5.97% and 11.94% respectively, compared to 5.0%, 2.8%, and 7.6% for the average Drosophila protein (Smoller et al., 1990, Genes Dev. 4:1688-1700). Most of the glutamine residues are concentrated in two clusters: the first one, extending between residues 261 and 302, is rich both in glutamine and histidine. The second one, starting at residue 488 and ending at residue 513, is composed mainly of glutamine. Such glutamine rich stretches are found in many proteins and are often referred to as opa or M repeats (Wharton et al., 1985, Cell 40:55-62; McGinnis et al., 1984, Nature 308:428-433). Their significance is unknown. Hydropathy plot analysis of the protein sequence failed to identify a putative signal sequence or transmembrane domain (Kyte and Doolittle, 1982, J. Mol. Biol. 157:105-132).
6.1.5. The Deltex Protein Is Localized within the Cytoplasm
To assess the spatial distribution and subcellular localization of the deltex protein, we raised mono- and polyclonal antibodies against bacterially expressed deltex fusion proteins (see Section 6.3). The specificity of the antibodies was routinely tested by immunoblot analysis and by immunofluorescent observation of Schneider 2 (S2) tissue culture cells (Schneider, 1972, J. Embryol. Exp. Morph. 27:353-365) transfected with deltex expression constructs. On immunoblots (not shown) the antibodies recognized an .about.80 kDa protein, in agreement with that predicted from the DNA sequence.
The immunological staining of embryos and imaginal tissues with these antibodies revealed an apparently ubiquitous non-nuclear distribution.
However, because this generalized pattern was also of low intensity, it presented difficulties in interpretation. To increase our confidence in this apparently cytoplasmic localization, we overexpressed the deltex protein in flies containing a P element transposon of deltex coding sequences under the control of the Hsp70 promoter (see Section 6.3). Using the antisera to compare the staining patterns of heat-shocked embryos containing or lacking the transposon, we observed a dramatic increase in staining intensity in embryos containing the transposon.
FIG. 4 shows that deltex is localized within the cytoplasm. However, because the Hsp70 promoter is ubiquitously expressed, we are not able to confidently assess the normal overall spatial pattern of deltex expression.
We examined further whether overexpression of deltex from this transposon was capable of rescuing deltex mutant defects and also whether additional phenotypic consequences might result from this over--and possibly ectopic--expression of deltex (see Section 6.3). Under the conditions we have tested, the 124A.sup.D transposon is capable of completely rescuing, albeit with variable penetrance, the defects associated with deltex mutations. Additionally, various heat shock regimens of wild-type animals carrying the transposon revealed no obvious phenotypic consequences of overexpression compared to control animals.
6.1.6. The Genetic Suppression of Deltex
During the course of characterizing deltex we recovered a spontaneous suppressor of deltex, which we denote Su(dx).sup.sp. Meiotic mapping placed this mutation on the second chromosome. This prompted us to examine two other second chromosome mutations, Su(dx) and Su(dx).sup.2, which were described by Morgan et al., 1931, Year book--Carnegie Institution 30:410 to suppress the adult phenotypes of deltex mutations.
FIG. 5 summarizes the effects of these mutations on four deltex mutations, dx.sup.ENU, dx.sup.SM, dx, dx.sup.P. All three Su(dx) mutations suppress in a dominant fashion the adult mutant phenotypes (wing, ocelli and eye) of these deltex alleles, although with differences in expressivity. Su(dx) and Su(dx).sup.sp are the most efficient suppressors, suppressing completely the wing and ocellar defects of dx.sup.ENU and dx.sup.SM, and partially suppressing their rough eye phenotype. Su(dx).sup.2 has a weaker effect, suppressing only partially the wing phenotype of dx.sup.SM (FIG. 6).
Although the genetic behavior of the three second chromosome suppressors is similar with regard to deltex, the fact that they act in a dominant fashion and as homozygotes display no obvious mutant phenotype by themselves precludes us from concluding they are allelic. However, one line of genetic evidence to suggest that they may be allelic makes use of the observation that homozygous Su(dx) or Su(dx).sup.sp animals in an Ax.sup.E2 dx.sup.ENU background do not survive. This contrasts with flies of the genotype Ax.sup.E2 dx.sup.ENU ; Su(dx)/+, which are viable (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). We find that any heterozygous combination of the suppressor mutations in combination with Ax.sup.E2 dx.sup.ENU results in lethality. Thus, these results are consistent with the notion that the three suppressor mutations are allelic.
6.1.7. Suppression of the Phenotypic Interactions between Deltex and Notch, Delta and Mastermind
Characteristic of the genetic behavior of deltex is its interactions with Notch, Delta, and mastermind (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). We were therefore interested in examining further the effects of the Su(dx) loci upon these interactions.
The deltex locus first caught our attention by virtue of its ability to suppress, in a dominant fashion, the lethality conferred by certain transheterozygous combinations of the Abruptex alleles of the Notch locus (e.g., Ax.sup.E2 /Ax.sup.9B2 ; Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). This lethality is restored in the presence of Su(dx), that is, animals that are Ax.sup.E2 dx.sup.ENU /AX.sup.9B2 +; Su(dx)/+ are no longer viable. Similarly, in the presence of Su(dx), the normally lethal nd dx.sup.ENU double mutation (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677) now results in a viable combination, although these animals exhibit a severe loss of wing blade material such that only a narrow strip of blade remains (not shown).
A characteristic pupal lethality is displayed by deltex mutants bearing one copy of N, Dl, or mam (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). With Notch this lethality is complete, whereas with Dl and mam the condition is semi-lethal (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). We found that one copy of Su(dx) can partially suppress these interactions: a few animals of the genotype N.sup.5419 dx.sup.ENU /+ dx.sup.ENU ; Su(dx)/+ are able to escape lethality, although they display the same small, rough-eye defect as their dead siblings (see also Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677); animals that are dx.sup.ENU /Y; Su(dx)+/+mam.sup.IL115 or dx.sup.ENU /Y; Su(dx)/+; Dl.sup.9P39 /+ are perfectly viable, but display clear deltex mutant phenotypes (ocelli and wing).
Previously, we reported that in the wing of Ax.sup.E2 dx.sup.ENU homozygous or hemizygous animals there is a suppression of the Abruptex mutant phenotype concomitant with an enhancement of the deltex mutant phenotype (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). As indicated above, animals homozygous for Su(dx) in this background do not survive. However, with one copy of Su(dx) the wing defects are similar to those of Ax.sup.E2 single mutants. That is, with the addition of a single copy of Su(dx) there is a suppression of the effect of deltex upon AX.sup.E2. In addition, the triple mutants display a novel phenotype never observed in animals carrying any two of these mutations: many micro- and macrochaetae on the thoracic region are missing (FIG. 7).
6.2. Discussion
Genes that individually share similar mutant phenotypes or that combinatorially show a synergistic enhancement of a mutant phenotype are often regarded as integrated within a common developmental pathway. While not constituting proof of such integration, these observations nonetheless provide a powerful means for identifying potentially interacting components of complex developmental processes. This premise has formed the basis of genetic screens we have performed and has led to the identification of deltex as an interacting partner of Notch. Moreover, this finding has been strengthened by the demonstration that deltex also interacts with Delta and mastermind, two genes that not only share similar null phenotypes with those of Notch, but also display phenotypic interactions with Notch (Xu et al., 1990, Genes Dev. 4:464-475; Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677).
In this paper, we present our initial molecular characterization of the deltex locus. We have ultimately identified the deltex transcription unit by showing via germline-mediated transformation experiments that a 10-kb genomic fragment containing a .about.5 kb transcription unit is capable of complementing most deltex mutant defects. Moreover, this genomic fragment rescues the normally lethal genetic interaction that results when flies are doubly mutant for deltex and nd. Finally, Northern analysis indicates a maternal loading of deltex transcripts into the developing oocyte, a finding that is consistent with the maternal effect observed upon embryogenesis in eggs laid by homozygous mutant mothers (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677).
The phenotypic consequences of null mutations of deltex are not known. The only known deficiency (Df(1)G4e.sup.L H24i.sup.R) of the chromosomal region in which deltex resides was generated synthetically (Stephenson and Mahowald, 1987, Dev. Biol. 124:1-8; see also Gorman and Girton, 1992, Genetics 131:99-112) and because of its size uncovers multiple loci. Moreover, the placement of a Minute locus within this deficiency has complicated the genetic analysis of deltex. Minutes exhibit a dominant haplo-insufficient phenotype of reduced fertility, long generation times and shortened and thin bristles. Numerous Minute loci are found within the Drosophila genome and in at least one case has been shown to encode a ribosomal protein gene (Kongsuwan et al., 1985, Nature 317:555-558; see also Burns et al., 1984, Mol. Cell. Biol. 4:2643-2652). In the course of our molecular characterization of deltex, we discovered a ribosomal protein (rp) transcription unit that resides .about.2 kb from the 5' end of the deltex transcription unit. We have shown that P element transformation of a genomic fragment containing this rp transcription unit rescues fully the Minute phenotype of Df(1)G4e.sup.L H24i.sup.R. Thus, the proximity of the rp gene to deltex appears likely to have contributed to the difficulty in recovering deltex deficiencies (see also Lefevre, 1974, Cold Spring Harbor Symp. Quant. Biol. 38 591-599).
We have extended the genetic analysis of deltex by examining three second chromosome mutations that are effective suppressors of the deltex phenotype, though their allelism has not been established. That the relationship of Su(dx) to deltex and Notch reflects not a simple suppression of deltex is suggested by the synergistic effects that are displayed in triple mutants i.e., Ax.sup.E2 dx.sup.ENU; Su(dx)!: a novel phenotype of missing bristles is observed (FIG. 7). Because homozygous Ax.sup.E2 dx.sup.ENU animals carrying a duplication of the deltex locus do not display this novel phenotype (not shown), the Su(dx) loci cannot be mere duplications of the deltex locus. Moreover, whereas animals homozygous for all three mutations are not viable, homozygotes for any two mutations are viable. Thus, there is the suggestion that the effects of Su(dx) mutations are the result of a three-way interaction between Notch, deltex and Su(dx).
We have established by several criteria the specificity of the antibodies for the deltex protein, although immunological staining of tissues has been complicated by the low levels and apparently ubiquitous distribution of the protein product. These results have been consistent with our efforts to detect the embryonic distribution of deltex mRNA in situ using appropriate nucleic acid probes. However, the use of a transposon to overexpress deltex in embryos has afforded us a view of deltex in vivo and to demonstrate a cytoplasmic distribution of the protein, in agreement with conceptual translation of the gene. Additionally, the ability of this transposon to rescue deltex mutant phenotypes without producing overt mutant consequences in wild-type animals is consistent with the evidence showing a generalized distribution of the gene product.
Two observations raise the possibility that the in vivo level of deltex protein accumulation is under translational control. First, in the 5' non-coding region preceding the long ORF, there is an unusual arrangement of five ATG codons that are part of a short ORF of 14 codons (FIG. 3). This organization is reminiscent of genes known to be under translational control (Abastado et al., 1991, Mol. Cell. Biol. 11:486-496; see also Kozak, 1986, Cell 44(2):283-292). Second, we have shown that in plasmid expression constructs, removal of these upstream initiator codons significantly increases the level of deltex protein accumulation in transfected S2 cells, as detected by immunofluorescence. Thus, this may account for the low levels of protein accumulation we detect in vivo, although artificially overexpressing deltex appears not to have any deleterious consequences (see above).
Biochemical and genetic studies have identified Delta and Serrate as ligands of Notch and have provided insights into the nature of their interactions with the Notch extracellular domain (Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699; PCT Publication No. WO 92/19737 dated Nov. 12, 1992).
6.3. Material and Methods
6.3.1. Isolation of Nucleic Acids
Isolation of Drosophila genomic DNA was performed as described by Preiss et al. (1988, EMBO J. 7:3917-3927). Total embryonic and larval RNAs from a Canton S strain were prepared according to the procedure of Chirgwin et al. (1979, Biochemistry 18(24)5294-5299). Pupal and adult RNAs were generously provided by A. Preiss (Preiss et al., 1988, EMBO J. 7:3917-3927). Poly (A).sup.+ RNA was selected by serial passage over oligo(dT)-cellulose (Stratagene) according to Maniatis et al. (1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Laboratory, N.Y.). .sup.32 P-labelled DNA probes were prepared by random oligonucleotide priming (Feinberg and Vogelstein, 1984, Anal. Biochem. 137:266-267). Enzymes were used as instructed by the manufacturers. All other procedures were carried out according to standard protocols (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
The genomic library of dx.sup.P DNA was prepared as follows: 60 mg of DNA from dx.sup.P flies were digested with 2 units of Sau3A in a reaction volume of 320 ml for 1 h, and then fractionated by centrifugation on a continuous 10-40% sucrose gradient for 22 h at 22,000 rpm (rotor SW40, Beckman). Fractions containing DNA of 15 kb or more were collected, dialysed, precipitated with ethanol, and ligated to 1 mg of BamHI cut arms of .lambda.EMBL3 (Frischauf et al., 1983, J. Mol. Biol. 170:827-842) in a reaction volume of 10 ml. Half of the ligation mixture was used in a standard in vitro lambda packaging reaction (kit from Amersham), yielding a total of 2.times.10.sup.6 recombinant phages. The Drosophila wild-type genomic library in .lambda.EMBL3 was provided by J. Tamkum (Tamkun et al., 1992, Cell 68:561-572). The cDNAs cBE8 and cBE30 were recovered from a .lambda.gt10 cDNA library made from 3-12 h embryonic mRNA and donated by Larry Kauvar (Poole et al., 1985, Cell 40:37-43).
6.3.2. P-MEDIATED TRANSFORMATION
For the genomic rescue experiments, a 10-kb XbaI genomic fragment (FIG. 1) encompassing the .about.5-kb deltex transcription unit was cloned into the XbaI site of the P transformation vector pDM23, which carries the rosy gene as a selection marker (G. Rubin, unpublished). This DNA was co-injected with the .DELTA.2-3 helper plasmid (to provide transposase; Robertson et al., 1988, Genetics 118:461-470) into cn; ry.sup.506 embryos using essentially the procedure described by Spradling (1986, in Glover, D. M. (ed.), Drosophila--A Practical Approach, IRL Press, Oxford, pp. 175-197). Three phenotypically cn and ry.sup.+ G1 females were recovered and mated to cn; ry.sup.506 males. One of the insertions was X-linked and male lethal and not used further. The other two insertions were localized to chromosome 2 and single lines were established using the balancer strain. Bc Elp/CyO, ry506.
For experiments involving overexpression of deltex in vivo, we used the pCaSpeR-hs transformation vector (Thummel and Pirrotta, 1991, DIS 71:150), which was kindly provided by C. Thummel. cDNA sequences extending from 33 nucleotides 5' of the translational start site to a sole SpeI site within the 3' non-coding region were ligated into the EcoRI-XbaI digested vector. DNAs were injected into W.sup.1118 embryos; 9 w.sup.+ transformant fly lines were recovered, one of which, 124A.sup.D, was chosen for this study. When crossed to deltex mutants, this transgenic line significantly improved deltex mutant defects in the absence of heat shock and produced wild-type appearing flies with the application of daily 1 hour heat shocks at 37.degree. C. For the immunofluorescent staining pictured in FIG. 4 (see also below), we applied the following heat shock regimen to an overnight collection of embryos produced from homozygous 124A.sup.D flies: 50 min at 37.degree. C., 50 min at 25.degree. C., 50 min at 37.degree. C. After a 4 hour recovery at 25.degree. C., the embryos were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) solution.
6.3.3. SEQUENCE DETERMINATION AND ANALYSIS
The EcoRI-cDNA insert from cBE8 was subcloned directly in both orientations into Bluescript KS-, M13mp18 and M13mp19 vectors. Overlapping deletions were produced on the inserts using the Exonuclease III-Mung Bean Nuclease system (kit from Stratagene). Additional deletions were obtained through the use of restriction sites within the vector polylinker and the cDNA inserts. The cDNA insert of cBE30 contains two internal EcoRI sites but no HindIII site: it was cloned into M13mp18 and M13mp19 vectors as three separate EcoRI pieces, and into Bluescript KS- vector as a HindIII fragment containing sequences from .lambda.gt10 on both sides. Single-stranded cDNAs were produced according to the manufacturer's instructions. Both strands of the cDNAs were sequenced using the dideoxynucleotide chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467) and the Sequenase kit (U.S. Biochemical Corp.), with the M13 universal and reverse primers. cDNA sequences that were not accessible by these methods were obtained using synthetic primers complementary to part of a previously determined sequence. Genomic sequences of interest from phages A25 or I2 were subcloned into convenient restriction sites of Bluescript KS- and sequenced using synthetic primers.
DNA sequence manipulations were performed using Intelligenetic's PC-GENE software. Open reading frame prediction and plotting were performed using 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 amino acid sequence using the FASTA program (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444-2448) available by the GenBank FASTA server through BITNET.
6.3.4. EXPRESSION CONSTRUCTS AND IMMUNOLOGICAL PROCEDURES
cDNA representing the entire deltex coding region was inserted into the pGex1 expression vector and expressed in E. coli XL1-Blue (see Smith and Johnson, 1988, Gene 67:31-40). The bacterial pellet was washed first in ice-cold 50 mM Tris pH 8.0, 50 mM EDTA, 50 mM NaCl before being resuspended in ice-cold 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mg/ml lysozyme, 1 mM phenylmethylsulfonylfluoride (PMSF). After .about.1 hour on ice, the cells were lysed by the addition of Triton X-100 to 1% final concentration. The lysate was sonicated briefly to shear DNA and then cleared by centrifugation. After overnight incubation at 4.degree. C. with glutathione-agarose beads (Sigma), the fusion protein was eluted from the PBS-washed beads with 5 mM reduced glutathione, 50 mM Tris pH 8.0 (see Smith and Johnson, 1988, Gene 67:31-40). Standard procedures were used to immunize Sprague-Dawley rats with soluble fusion protein (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, N.Y.). Immune sera was tested for positive reactivity by immunofluorescent staining of transiently-transfected S2 tissue culture cells (see Fehon et al., 1990, Cell 61:523-534) and by immunostaining of protein blots (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, N.Y.). The expression vector used in the transfections was pRmHa-3 (Bunch et al., 1988, Nucl. Acids Res. 16:1043-1061), into which deltex cDNA sequences were inserted. Hybridoma fusions were performed by the Howard Hughes Medical Institute's Hybridoma Facility at Yale University. The antibody supernatant used in this study derived from the cell line C645.17A. Immunofluorescent staining and confocal imaging of embryos was essentially as described by Fehon et al. (1991, J. Cell Biol. 113:657-669).
6.3.5. STRAINS AND CROSSES
Stocks were maintained and crosses were performed using standard procedures described previously (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). Genetic markers and strains not specifically mentioned here can be found in Lindsley and Grell (1968, Genetic Variations of Drosophila melanogaster, Publication number 627, Carnegie Institute of Washington, Washington D.C.) or Lindsley and Zimm (1985, Dros. Inf. Serv. 62), Lindsley and Zimm (1986, Dros. Inf. Serv. 64), Lindsley and Zimm (1987, Dros. Inf. Serv. 67), and Lindsley and Zimm (1990, Dros. Inf. Serv. 68).
6.3.6. PHENOTYPIC REVERSION OF DX.sup.P
Homozygous dx.sup.P sn.sup.3 females were mated to cn; P(ry.sup.+ ; D2-3) ry.sup.506 Sb/TM6 males, which provide a stable source of P transposase (Robertson et al., 1988, Genetics 118:461-470). dx.sup.P sn.sup.3 /Y; cn/+; P(ry.sup.+ ;D2-3)ry.sup.506 Sb/+F1 males were then mated to C(1)A, y females. From .about.3000 males scored in the F2 generation, we recovered seven phenotypically wild-type males. These were mated to C(1)A, y females to establish single lines and their genomic DNA was analyzed further by standard Southern blot procedures (Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Laboratory, N.Y.)
5 6.3.7. COMPLEMENTATION OF DELTEX ALLELES BY THE DELTEX TRANSPOSONS TXO5B AND TXO12G
y dx.sup.ENU sn.sup.3, dx.sup.SM t.sup.2 v, ec dx and dx.sup.P sn.sup.3 females were mated to cn TXO5B/cn+; ry.sup.506 males and to cn TXO12G/cn+; ry.sup.506 males to assess effects on adult phenotypes. To assess the effects on embryonic lethality, y dx.sup.ENU sn.sup.3 females were crossed to y dx.sup.ENU sn.sup.3 /Y, cn TXO5B/+ males and to dx.sup.ENU sn.sup.3 /Y; cn TXO12G/+ males; dx.sup.SM t.sup.2 v females were crossed to dx.sup.SM t.sup.2 v/Y; cn TXO12G/+ males. From this cross both sibling progeny containing or lacking the transposon were mated to males of identical X chromosome genotype and the percentage of unhatched embryos was calculated.
6.3.8. ORIGIN OF SU(DX).sup.SP
The mutation (Su(dx).sup.sp) was found in a homozygous Ax.sup.E2 dx.sup.ENU stock maintained in our laboratory and was shown to segregate with the second chromosome. The following crosses were performed: An Ax.sup.E2 dx.sup.ENU ; Su(dx).sup.sp /+female was mated to FM6/Y; Adv/SM1 males. Individual F1 Ax.sup.E2 dx.sup.ENU /FM6; Su(dx).sup.sp /SM1 or Ax.sup.E2 dx.sup.ENU /FM6; +/SM1 females were mated to individual F1 Ax.sup.E2 dx.sup.ENU /Y; Su(dx).sup.sp /SM1 males. The individual F2 Ax.sup.E2 dx.sup.ENU /Ax.sup.E2 dx.sup.ENU ; Su(dx).sup.sp /SM1 females were then mated to individual Ax.sup.E2 dx.sup.ENU /Y; Su(dx).sup.sp /SM1 males to establish stock lines. No homozygous Su(dx).sup.sp animals were found in the F3 offsprings from these crosses, indicating that the triple homozygous mutants were lethal. Su(dx).sup.sp homozygotes and Su(dx).sup.sp /Su(dx) heterozygotes were viable and displayed no obvious mutant phenotypes.
6.3.9. CROSSES BETWEEN SU(DX) MUTATIONS AND DELTEX ALLELES
y dx.sup.ENU sn.sup.3, dx.sup.SM t.sup.2 v, ec dx and dx.sup.P sn.sup.3 females were mated to Su(dx).sup.sp, Su(dx) and ed Su(dx).sup.2 males to observe double mutant effects of dx and Su(dx). y dx sn.sup.ENU sn.sup.3 ; +/SM1 females were mated to y dx.sup.ENU sn.sup.3 /Y, Su(dx)/+ males. F1: y dx.sup.ENU sn.sup.3 ; Su(dx)/SM1 females x y dx.sup.ENU sn.sup.3 /Y; Su(dx)/SM1 male. F2: y dx.sup.ENU sn.sup.3, Su(dx) females x y dx.sup.ENU sn.sup.3 /Y; Su(dx) males.
6.3.10. CROSSES BETWEEN SU(DX) AND MUTATIONS OF THE NOTCH LOCUS
y w Ax.sup.9B2 /FM7C.times.Ax.sup.E2 dx.sup.ENU /Y; Su(dx)SM1. F1 female progeny: y w Ax.sup.9B2 /Ax.sup.E2 dx.sup.ENU ; Su(dx)/+, y w Ax.sup.9B2 /Ax.sup.E2 dx.sup.ENU ; SM1/+, FM7C/Ax.sup.E2 dx.sup.ENU ; Su(dx)/+, FM7C/Ax.sup.E2 dx.sup.ENU ; SM1/+.
y Ax.sup.9B2 sn3.times.Ax.sup.E2 dx.sup.ENU /Y; Su(dx).sup.sp /Adv. F1 female progeny: y Ax.sup.9B2 sn.sup.3 /Ax.sup.E2 dx.sup.ENU ; Su(dx)/+ and y Ax.sup.9B2 sn.sup.3 /Ax.sup.E2 dx.sup.ENU ; SM1/+.
y w Ax.sup.9B2 /FM7C.times.Ax.sup.E2 dx.sup.ENU /Y. F1 female progeny: y w Ax.sup.9B2 /Ax.sup.E2 dx.sup.ENU and FM7C/Ax.sup.E2 dx.sup.ENU.
Ax.sup.E2 dx.sup.ENU females.times.Su(dx).sup.sp, Su(dx) or ed Su(dx).sup.2 males.
w.sup.a nd dx.sup.ENU sn.sup.3 /FM7C.times.Su(dx). F1 male progeny: w.sup.a nd dx.sup.ENU sn.sup.3 /Y; Su(dx)/+ and FM7C/Y; Su(dx)/+.
w.sup.a nd dx.sup.ENU sn.sup.3 /FM7C.times.Su(dx).sup.sp /Y. F1 male progeny: w.sup.a nd dx.sup.ENU sn.sup.3 Y; Su(dx).sup.sp /+ and FM7C/Y; Su(dx).sup.sp /+.
nd.sup.2 dx.sup.ENU sn.sup.3 /FM7C.times.Su(dx)/Y. F1 male progeny: nd.sup.2 dx.sup.ENU sn.sup.3 /Y; Su(dx)/+ and FM7C/Y; Su(dx)/+.
nd.sup.2 dx.sup.ENU sn.sup.3 /FM7C.times.Su(dx).sup.sp /Y. F1 male progeny: nd.sup.2 dx.sup.ENU sn.sup.3 /Y; Su(dx).sup.sp /+ and FM7C/Y; Su(dx).sup.sp /+.
nd.sup.2 dx.sup.ENU sn.sup.3 /FM7C.times.ed Su(dx).sup.2 /Y. F1 male progeny: nd.sup.2 dx.sup.ENU sn.sup.3 Y; ed Su(dx).sup.2 /+ and FM7C/Y; ed Su(dx).sup.2 /+.
y w.sup.a N.sup.5419 dx.sup.ENU sn.sup.3 /FM7C.times.y dx.sup.ENU sn.sup.3 /Y; Su(dx). F1 female progeny: y w.sup.a N.sup.5419 dx.sup.ENU sn.sup.3 /y++dx.sup.ENU sn.sup.3 ; Su(dx)/+, FM7C/y dx.sup.ENU sn.sup.3 ; Su(dx)/+ and y w.sup.a N.sup.5419 dx.sup.ENU sn.sup.3 /FM7C; Su(dx)/+.
Ax.sup.E2 sn.sup.3 ; Su(dx).sup.sp /SM1 females.times.ed Su(dx).sup.2, Su(dx) and Ax.sup.E2 sn.sup.3 /Y; Su(dx).sup.sp /SM1 males.
6.3.11. CROSSES INVOLVING SU(DX), DELTEX, DELTA AND MASTERMIND
y dx.sup.ENU sn.sup.3 ; Su(dx) females.times.D1.sup.9P39 /TM1 males. F1 male progeny: y dx.sup.ENU sn.sup.3 /Y; Su(dx)/+; D1.sup.9P39 /+ and y dx.sup.ENU sn.sup.3 /Y; Su(dx)/+; TM1/+.
y dx.sup.ENU sn.sup.3 ; Su(dx) females.times.cn bw sp mam.sup.IL115 /Cyo males. F1 male progeny: y dx.sup.ENU sn.sup.3 /Y; Su(dx)++++/+ cn bw sp mam.sup.IL115 and y dx.sup.ENU sn.sup.3 /Y; Su(dx)/Cyo.
6.3.12. CROSSES COMPARING SU(DX) MUTATIONS
Ax.sup.E2 dx.sup.ENU ; Su(dx).sup.sp /Cyo females.times.Ax.sup.E2 dx.sup.ENU /Y; Su(dx).sup.sp /Cyo.
Ax.sup.E2 dx.sup.ENU /FM7C; Su(dx)/Sm1 females.times.Ax.sup.E2 dx.sup.ENU /Y; Su(dx)SM1.
Ax.sup.E2 dx.sup.ENU ; Su(dx).sup.sp /Cyo females.times.Ax.sup.E2 dx.sup.ENU /Y; Su(dx)/SM1. F1 progeny: Ax.sup.E2 dx.sup.ENU /Y; Cyo or SM1/Su(dx).sup.sp or Su(dx). Ax.sup.E2 dx.sup.ENU /Ax.sup.E2 dx.sup.ENU ; Cyo or SM1/Su(dx).sup.sp or Su(dx).
Ax.sup.E2 dx.sup.ENU ; Su(dx).sup.sp /Cyo females.times.ed Su(dx).sup.2 males. F1 progeny: Ax.sup.E2 dx.sup.ENU /++; Su(dx).sup.sp /ed Su(dx).sup.2, Ax.sup.E2 dx.sup.ENU /Y; Cyoled Su(dx).sup.2, Ax.sup.E2 dx.sup.ENU /Ax.sup.E2 dx.sup.ENU ; Cyoled Su(dx).sup.2.
6.3.13. CROSSES INVOLVING DUPLICATIONS OF THE DELTEX LOCUS
y w.sup.a N.sup.5419 /FM6 females.times.Tp(1:Y) J104 y B.sup.5 ;Yy.sup.+ (6E; Y.sup.6) males. F1: y w.sup.a N.sup.5419 /Tp(1:Y) J104 y B.sup.5 ; Yy.sup.+ (6E; Y.sup.6) females.times.FM7C/Y males. F2: Tp(1:Y) J104 y w.sup.a N.sup.5419 B.sup.5 ; Yy.sup.+ (6E; Y.sup.6)/FM7C individual females.times.Ax.sup.E2 dx.sup.ENU /Y males. F3: Tp(1:Y) J104 y w.sup.a N.sup.5419 B.sup.5 ; Yy.sup.+ (6E; Y.sup.6)/Ax.sup.E2 dx.sup.ENU /Y virgins.times.Ax.sup.E2 dx.sup.ENU /Y males. F4: Ax.sup.E2 dx.sup.ENU /Ax.sup.E2 dx.sup.ENU /DpJ104 y w.sup.a N.sup.5419 B.sup.5 (1A; 6E) animals.
7. DELTEX IMPLICATED IN NOTCH-MEDIATED SIGNAL TRANSDUCTION: EVIDENCE FOR CYTOSOLIC INTERACTION WITH NOTCH CDC10/SW16/ANKYRIN REPEATS
As described herein, we have demonstrated a direct interaction between the novel cytoplasmic protein encoded by the deltex locus and the transmembrane receptor encoded by Notch, by expression studies conducted in cultured cells, in yeast, and in the imaginal wing disc. deltex binds specifically to the Notch cdc10/SW16/ankyrin ("ANK") repeats, a region that is crucial for Notch signaling and that constitutes the most conserved domain among Notch family members. In addition, we describe a new class of viable Notch allele that is associated with a missense mutation within the ANK repeat region and that, like mutations of deltex, behaves as a dominant intragenic suppressor of the Abruptex `negative complementation`.
7.1. DELTEX INTERACTS WITH NOTCH INTRACELLULAR DOMAIN
We explored the possibility of protein-protein interactions between deltex and Notch by examining the relative subcellular localization of the two proteins after co-expressing them in Drosophila Schneider 2 (S2) cultured cells (Schneider, 1972, J. Embryol. Exp. Morph. 27:353-365). S2 cells were co-transfected with plasmid expression constructs that placed Notch and deltex under the inducible control of the Drosophila metallothionein and Hsp70 promoters, respectively. S2 cells do not express endogenous Notch (Fehon et al., 1990, Cell 61:523-534) and express deltex at levels too low for reliable immunofluorescent detection (see Section 6 hereinabove and data not shown). Notch expression was induced prior to heat-shock induction of deltex to ensure proper cell surface localization of Notch. By aggregating cells expressing both Notch and deltex with cells expressing Delta, a presumptive membrane-bound ligand of Notch (Fehon et al., 1990, Cell 61:523-534; Heitzler and Simpson, 1991, Cell 64:1083-1092), a dramatic co-capping of these proteins was induced at the point of cellular contact. FIG. 8a shows co-localization between deltex and Notch, indicating molecular interaction between the two proteins. Moreover, this co-localization was evident even in the absence of capping with Delta, although in this case Notch and deltex were mis-localized within peri-nuclear regions of the cell (FIG. 8b). This latter result suggests that there is no ligand dependency of deltex association with Notch.
When the same aggregation experiment was performed using plasmids expressing deltex in combination with others expressing either Delta or Serrate (another presumptive ligand of Notch; Rebay et al., 1991, Cell 67:687-699), no co-localization was observed between deltex and these transmembrane proteins (FIG. 8c, d). This indicates that overexpression itself is not causing deltex to concentrate in the regions of cellular contact where Notch and Delta accumulate (Fehon et al., 1990, Cell 61:523-534). Moreover, the results indicate that the genetic interaction previously noted between deltex and Delta (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677) is likely an indirect reflection of a direct Notch/deltex interaction.
FIG. 8 Methods: Cell culture conditions and media were as described (Fehon et al., 1990, Cell 61:523-534). For transfections, cells were transferred in fresh media to 6-well plates (Falcon) and allowed to attach to plastic for 1-3 h (cell density .about.3/4 confluent). Ten micrograms total plasmid DNA in 0.5 ml serum-free media (SFM) were mixed with 0.5 ml SFM containing 50 ml LipofectACE.TM. (Life Technologies, Inc.). After 15-30 min, mixture was added to cells previously rinsed with SFM. After .about.6 h incubation, transfection mixture was replaced with media plus serum. Expression from the metallothionein promoter was induced .about.24 h later by CuSO.sub.4 addition as described (Fehon et al., 1990, Cell 61:523-534), followed 12-18 h later with induction of the Hsp 70 promoter that consisted of two 30 min incubations at 37.degree. C. with an intervening 30 min at 25.degree. C. Three to five hours later, cells were incubated with anti-Notch antibody for 30 min and fixed in PLP fixative (Tomlinson and Ready, 1987, Dev. Biol. 120:336-376). Antibody incubations and cell mounting were as described (Fehon et al., 1990, Cell 61:523-534) using anti-deltex antibodies followed by FITC- and Texas Red-conjugated goat anti-mouse and anti-rat antibodies (Jackson ImmunoResearch Laboratories, Inc.). Third instar larvae from transgenic line 124A.sup.D (see Section 6) were heat shocked as above to induce deltex expression and dissected wing discs were fixed and incubated with antibodies as outlined above. Confocal images were obtained as described (Xu et al., 1992, Development 115:913-922); cropping and pseudo-coloring were performed using Adobe Photoshop (Adobe Systems, Inc.) computer program.
7.2. NOTCH AND DELTEX CO-LOCALIZATION IN VIVO
The above demonstration of Notch/deltex interaction in transfected cells prompted us to explore whether such interaction was detectable in vivo. We examined the imaginal wing disc, a tissue that requires both proteins for its proper development (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). In addition, Notch displays a polarized distribution to the apical surface of the disc epithelium (Fehon et al., 1991, J. Cell Biol. 113:657-669). Because the endogenous level of deltex protein accumulation is below the threshold necessary for confident immunofluorescent detection (see Section 6 hereinabove), we used a transformant fly line carrying an inducible deltex construct to elevate transiently the in vivo pool of deltex protein. Over- and/or ectopic-expression from this transposon rescues deltex mutant defects and has no obvious phenotypic consequences in otherwise wild-type animals (see Section 6 hereinabove). FIG. 8e shows a coincidence of deltex and Notch proteins at the apical surface of the wing disc epithelium, confirming the co-localization data of the S2 cell culture assay and suggesting that Notch/deltex interactions normally occur in vivo.
7.3. NOTCH ANKYRIN REPEATS ARE BOTH NECESSARY AND SUFFICIENT FOR DELTEX ASSOCIATION
The 938-amino-acid intracellular domain of Notch contains several structural motifs (Stifani et al., 1992, Nature Genetics 2:119-127; Wharton et al., 1985, Cell 43:567-581; Kidd et al., 1986, Mol. Cell. Biol. 6:3094-3108; Breeden and Nasmyth, 1987, Nature 329:651-654). To identify those regions involved in deltex binding, we expressed deltex along with a set of Notch deletion constructs (FIG. 9a) in the S2 cell culture assay. The phenotypic consequences of expressing these same deletions of Notch were also recently examined in transgenic flies (Rebay et al., 1993, Cell 74:319-329). The constructs .DELTA.B-S and A S-S, which delete sequences C-terminal of the ANK repeats, did not visibly impede deltex association (FIG. 8f, g). Overexpression of these deletions within the fly produced only mild phenotypes similar to those resulting from overexpression of an intact version of Notch (Rebay et al., 1993, Cell 74:319-329). This stands in contrast to the results obtained with deletions that removed the ANK repeats (ECN or .DELTA.cdc10). In the S2 cell assay, we detected no association of deltex with these versions of Notch (FIG. 8h, i). In the fly, overexpression of both the ECN and .DELTA.cdc10 constructs resulted in severe dominant-negative phenotypes, demonstrating an essential role for the ANK repeats in Notch signal transduction (Rebay et al., 1993, Cell 74:319-329). Combined, these results not only implicate the ANK repeats in mediating Notch/deltex interaction (see below), but also suggest a role for deltex in intracellular signaling events.
To determine whether the ANK repeats alone were sufficient to promote deltex binding, we produced an expression construct, pMTD1/NANK, that affixed the Notch ANK repeats to the cytoplasmic domain of Delta (FIG. 9b). Because Delta shows no physical association with deltex (FIG. 8c), co-localization of the two proteins at the cell surface would be a consequence of the Notch ANK repeats. FIG. 8, panels j and k show this to be the case. Indeed, the deltex/Notch-ANK-repeat interaction is emphasized in FIG. 8k, which is an electronic merging of the side-by-side image displayed in FIG. 8j. Unlike the staining patterns obtained from other transfection experiments, this shows that the co-localization is offset, i.e., not coincident, reflecting the labeling of Notch in one cell and that of deltex (through hybrid Delta/Notch-ANK-repeat protein) in an adjacent cell. Thus, the Notch ANK repeats are both necessary and sufficient for deltex binding to occur.
Because ANK repeats, in general, are a conserved feature of many proteins, we sought to address the specificity of deltex binding for those repeats of Notch. We replaced the ANK repeats of pMTD1/NANK with those of the Drosophila gene, cactus, to produce the expression plasmid, pMTD1/CANK (FIG. 9b). cactus encodes an I-.kappa.B cognate that has been shown to bind, via ANK repeats, a Drosophila NF-.kappa.B cognate, dorsal (Geisler et al., 1992, Cell 71:613-621; Kidd, 1992, Cell 71:623-635). In the S2 cell expression assay, no association of deltex for the ANK repeats of cactus was observed (FIG. 8l), although immunoblot analysis indicated a hybrid Delta/cactus protein of proper size was expressed in these cells (FIG. 9c). This result indicates a specific association of deltex for the ANK repeats of Notch and serves as a control for the concern that overexpression of ANK repeats, per se, promotes deltex association.
FIG. 9 Methods: All constructs were based on the expression vector pRmHa-3 (Bunch et al., 1988, Nucl. Acids Res. 16:1043-1061), which uses the inducible Drosophila metallothionein promoter to drive expression. a, Details of plasmid construction have been described (Rebay et al., 1993, Cell 74:319-329). b, Encoded Notch ANK repeats were isolated as a PCR-derived DNA fragment containing artificial NdeI and BspEI sites. The following synthetic oligonucleotide primers (H.H.M.I. facility, Yale University) were used: 5' GCG CAT CAG GAT CAT ATG AAG CAC GAT GTG GAT GCA 3' (SEQ ID NO:20) and 5' GGC CAC ATC GTC CGG AAA TCG ATC CAT GTG ATC 3' (SEQ ID NO:21). To generate pMTD1/NANK, the following three-piece ligation was performed: a 2.6-kb EcoRI-NdeI DNA fragment from pMTDl1 (Fehon et al., 1990, Cell 61:523-534) and the NdeI/BspEI-digested PCR fragment were inserted into the EcoRI and XmaI sites of pRmHa-3. The resulting construct encoded Notch protein sequences beginning four residues before the first ANK repeat and ending nine residues after the sixth ANK repeat. For pMTD1/CANK, the same three-piece ligation was performed except a 0.7-kb AseI-BspEI DNA fragment from the cactus cDNA pcact5B (Geisler et al., 1992, Cell 71:613-621) replaced the fragment encoding the Notch ANK repeats. The resulting construct encoded eight residues of cactus before the first ANK repeat and five residues of cactus after the sixth ANK repeat. For both constructs, a TAG stop codon was provided by the XbaI site within the vector polylinker sequence. c, Standard procedures (Harlow and Lane, 1988, in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) were used for the preparation of cell lysates, PAGE-electrophoresis (3-15% gel) and immunoblot analysis. The anti-Delta monoclonal antibody C584.9B was used in conjunction with goat anti-mouse peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) and the LumiGLO.TM. (Kirkegaard & Perry Laboratories, Inc.) chemiluminescent detection kit according to manufacturer's instructions.
7.4. DIRECT NOTCH/DELTEX INTERACTION INDICATED BY YEAST EXPRESSION STUDIES
The above demonstration of a specific association between deltex and Notch ANK repeats leaves open the question of whether the interaction is mediated directly or through other cellular components. To address this question, we have conducted similar expression studies in yeast using the so-called `interaction trap` technique (Zervos et al., 1993, Cell 72:223-232). The "interaction trap" system involves three plasmids co-transfected in a yeast cell. The first plasmid is designed to express a fusion protein with a DNA binding domain of a yeast transcription factor LexA. The second plasmid is designed to produce a fusion protein with an acidic activation transcription domain (ACT). The third plasmid is a lacZ reporter gene which is activated by the binding of functional LexA protein to its upstream elements. Since the LexA fusion protein does not have an acidic transcription activation domain, it cannot activate the lacZ reporter gene. However, if there is a protein-protein interaction between a LexA fusion and an ACT fusion, a functional LexA protein is reconstructed which activates lacZ reporter gene transcription (resulting in synthesis of .beta.-galactosidase).
Two expression plasmids were constructed. One (LexA-Notch ICN1) encoded a LexA DNA-binding domain fused to a portion of the Notch intracellular domain (amino acids 1827-2258; Wharton et al., 1985, Cell 43:567-581) that included the ANK repeats. The other plasmid (pJG4-5-deltex) encoded the entire deltex protein fused to an acidic transcription activation domain (ACT-deltex). These were co-transformed into yeast carrying a reporter gene (LexAoperator-lacZ) plasmid. Notch/deltex interactions would be expected to mediate the formation of a complex between the LexA-Notch ICN1 and ACT-deltex proteins resulting in the restoration of transcriptional activity. This would be detected as a blue yeast colony due to induced .beta.-galactosidase synthesis. As presented in FIG. 10, a specific interaction was indeed detected between Notch and deltex within the yeast cell. This result suggests that the Notch/deltex interaction observed within Drosophila cells is the consequence of a direct protein-protein interaction.
FIG. 10 Methods: The `interaction trap` method was as described (Zervos et al., 1993, Cell 72:223-232). R. Finley and R. Brent generously provided yeast strain EGY40 and plasmids LEX202+PL (to make LexA fusions), pJG4-5 (to make acidic activation domain fusions), pSH18-34 (LexAop-lacZ reporter gene) and pRFHMI (LexA-bicoid fusion). The entire groucho coding region (Delidakis et al., 1991, Genetics 129:803-823) was isolated by PCR and inserted into the EcoRI restriction sites of LEX202+PL and pJG4-5 to create LexA-groucho and pJG4-5-groucho. A PCR-derived DNA fragment containing artificial EcoRI and SalI sites and encoding amino acids 1827-2258 of Notch was inserted into LEX202+PL to create LexA-Notch ICN1. The entire coding region of deltex was recovered by PCR and inserted into the XhoI site of pJG4-5 to create pJG4-5-deltex. Yeast transformations were performed as described (Gietz et al., 1992, Nucl. Acids Res. 20:1425).
7.5. INTRAGENIC SUPPRESSOR MUTATION MAPS WITHIN ANK REPEAT
In the same genetic screen (Xu et al., 1990, Genes Dev. 4:464-475; Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677) that identified deltex as a potentially interacting partner of Notch, an unusual allele of Notch was also recovered. This allele, denoted N.sup.su42c, is homozygous viable, and like mutations of deltex, Delta or mastermind, suppresses completely the pupal lethality associated with the Ax.sup.E2 /Ax.sup.9B2 `negative complementation`.
Intra-genic recombination analyses indicated that the N.sup.su42c lesion was positioned centromere-proximal, and thus 3', to the Ax.sup.9B2 mutation located within the genomic region encoding the 23rd EGF-like repeat. We have sequenced Ax.sup.9B2 su42c genomic DNA encompassing the 17th EGF-like repeat to the C-terminal end of the protein and find a missense mutation that results in an alanine to valine substitution within the fifth ANK repeat (FIG. 11). This alanine is conserved among all Notch homologs in mice, rats, humans and Xenopus (Coffman et al., 1990, Science 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; Ellisen et al., 1991, Cell 66:649-661; Stifani et al., 1992, Nature Genetics 2:119-127; Franco del Amo et al., 1993, Genomics 15:259-264), but does not fall within a conserved position in the consensus sequence compiled for ANK repeats in general (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; Robbins et al., 1992, J. Virol. 66:2594-2599).
Interestingly, the su42c mutation, which is the first mutation known to affect the ANK region of Notch, confers upon adult flies a subset of deltex-like mutant phenotypes (not shown). These include outstretched wings and variably-fused ocelli (Gorman and Girton, 1992, Genetics 131:99-112; Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677) which are not displayed by the parental Ax.sup.9B2 mutant. Thus, both the genetic behavior of this mutation as well as its position within the ANK domain implicate this region in Notch/deltex interactions. Biochemical studies should provide insights into whether this mutation alters the affinity of deltex binding and/or interferes with requisite protein conformational changes.
FIG. 11 Methods: Genomic DNA was obtained by PCR and subcloned into Bluescript KS-(Stratagene). Several clones of at least two different PCR reactions were sequenced (both strands) by the dideoxy chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467) and covered nucleotides 2850 to 8880 (numbering based on Wharton et al., 1985, Cell 43:567-581). Eleven base-pair changes were discovered, three of which predicted amino acid substitutions. Of these, one at position 3584 corresponded to the previously described Ax.sup.9B2 mutation (Kelly et al., 1987, Cell 51:539-548; Hartley et al., 1987, EMBO J. 6:3407-3417) and another at 7510 coincided with a formerly described strain polymorphism (Kidd et al., 1986, Mol. Cell. Biol. 6:3094-3108). A third base-pair change at position 6920 was taken to be the su42c mutation and was confirmed by direct sequencing of the PCR product.
7.6. DISCUSSION
We present three independent lines of evidence, the S2 cell assay, in vivo co-localization data, and the yeast `interaction trap` assay, to demonstrate a specific interaction between deltex and the ANK repeats of the Notch intracellular domain. This reveals deltex to be the first cytoplasmic component of a postulated Notch signal transducing complex that also includes Delta and Serrate.
Although we have demonstrated that the ANK repeats are both necessary and sufficient to promote deltex binding, other regions of the intracellular domain may influence the Notch/deltex protein complex. This possibility is raised by the lethal interaction that results when deltex mutants also contain the Notch mutation notchoid.sup.1 (nd.sup.1), which is associated with missense mutations near the C terminus (Xu et al., 1990, Genes Dev. 4:464-475). Such influence would be reminiscent of that which occurs in the binding of cactus and dorsal, which are Drosophila counterparts of I.kappa.B and NF-.kappa.B, respectively. Although the ANK repeats of cactus bind to the centrally located Rel homology domain of dorsal, deletion analyses indicate that a region near the carboxy terminus of dorsal also is necessary to keep dorsal sequestered in the cytoplasm when it is bound to cactus (Kidd, 1992, Cell 71:623-635; Robbins et al., 1992, J. Virol. 66:2594-2599; Rushlow et al., 1989, Cell 59:1165-1177; Isoda et al., 1992, Genes Dev. 6:619-630; Norris and Manley, 1992, Genes Dev. 6:1657-1667).
Models analogous to those which have emerged from studies of I.kappa.B and NF-.kappa.B have been proposed recently to explain the activity of Notch family members (Kidd, 1992, Cell 71:623-635; LaMarco et al., 1991, Science 253:789-792; Kodoyianni et al., 1992, Molec. Biol. Cell 3:1199-1213): Notch may represent a novel I.kappa.B-like protein that, in response to an external stimulus, causes the dissociation of an ANK repeat-bound transcription factor, which is then transported into the nucleus. However, we find no evidence to support the possibility that deltex may be such a factor. In S2 cells expressing either the ECN or .DELTA.cdc10 deletion constructs (FIG. 8h, i) or in embryos and imaginal tissues over-expressing the deltex protein (see Section 6 and data not shown), we see no nuclear accumulation of deltex at the level of resolution provided by immunofluorescent microscopy (not shown). Truncations of Notch that delete the extracellular and transmembrane regions result in the production of an intracellular domain (N.sup.nucl); that localizes within the nucleus (Fortini et al, Nature, in press; Struhl et al., 1993, Cell 74:331-345). Co-expression of deltex with N.sup.nucl in S2 cells did not prevent N.sup.nucl from translocating into the nucleus and deltex remained in the cytoplasm (not shown).
Molecular analyses have shown that the Abruptex alleles of Notch represent single amino acid substitutions clustering within six adjacent EGF-like repeats (Kelly et al., 1987, Cell 51:539-548; Hartley et al., 1987, EMBO 6:3407-3417). The phenotype produced by these gain-of-function alleles results in the differentiation of greater numbers of epidermal cells at the expense of neural cells (Heitzler and Simpson, 1993, Development 117:1113-1123) (so-called `anti-neurogenic` phenotype (Palka et al., 1990, Development 109:167-175)), and contrasts with the opposite phenotype produced by loss-of-function Notch mutations. The underlying basis for the dominant phenotype produced by the Abruptex mutations is uncertain, although the phenotype is mimicked by truncated forms of Notch (Rebay et al., 1993, Cell 74:319-329; Struhl et al., 1993, Cell 74:331-345), suggesting that this is an activated form of the receptor. On the basis of genetic mosaic studies, Heitzler and Simpson (Heitzler and Simpson, 1993, Development 117:1113-1123) have suggested that Abruptex molecules may have an increased affinity for Delta, but do not constitute constitutively active forms of the receptor, as evidenced by their dependency upon the Delta ligand.
The mechanism by which certain heteroallelic combinations of Abruptex alleles `negatively complement` to result in pupal lethality is uncertain. The conventional interpretation of this phenomenon is that Notch polypeptides interact with one another either as dimers or as part of multiprotein complexes (Foster, 1975, Genetics 81:99-120; Kelly et al., 1987, Cell 51:539-548), a notion that has precedents in a number of other receptor proteins (Ullrich and Schlessinger, 1990, Cell 61:203-212). Pupae of the negatively complementing genotype exhibit a severe loss of sensory bristles compared with Abruptex homozygotes (Foster, 1975, Genetics 81:99-120), which are fully viable. This would seem to suggest an even greater gain-of-function activity or `antineurogenic` effect on the part of the negatively complementing alleles, the consequence of which is inviability. Reducing the gene dosage of either Delta, mastermind, or deltex suppresses both this lethality (Xu et al., 1990, Genes Dev. 4:464-475; Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677) and the sensory bristle loss. In the case of Delta and mastermind, both of which are neurogenic genes, restoration of viability and sensory bristles may result from a countervailing shift in the developmental equilibrium towards neural differentiation.
In light of our demonstration of a physical Notch/deltex interaction, the finding that lowering the gene dosage of deltex also attenuates the apparent `hyperactivity` of the negatively complementing genotypes implies that deltex normally functions as a positive regulator of Notch activity. This notion is supported by other genetic evidence, namely, the lethal interactions that result from deltex and nd.sup.1 (a hypomorphic allele) double mutants and from deltex mutants bearing only one wild-type copy of Notch (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677). Also consistent with this idea is the observation that overexpression of deltex in wild-type flies reveals no apparent phenotypic consequences (see Section 6).
The fact that the deltex protein is of unique sequence (see Section 6) raises the possibility that deltex may define a new class of proteins implicated in cell signaling events. Given that the ANK repeats constitute the most conserved (.about.70% identity) portion among the various Notch homologs (Stifani et al., 1992, Nature Genetics 2:119-127) and that deltex interacts with these repeats, we expect that deltex cognates will exist in higher eukaryotes and that these may function through similar biochemical modes of action.
8. EXAMPLE: PORTIONS OF DELTEX WHICH MEDIATE HETEROTYPIC AND HOMOTYPIC BINDING
Using the interaction trap system as our assay (see Section 7.4) 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 Notch were expressed as fusion constructs (LexA or ACT fusions), and assayed.
The sequences of the fragments A-D (SEQ ID NOS:5, 6, 7, and 8, respectively) of Drosophila deltex which were expressed are shown in FIG. 14.
FIG. 13 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 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 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 Wharton et al., 1985, Cell 43:567-581; see FIG. 17); Human Notch TAN-1 amino acids: 1826-2146 (see FIG. 17); Human Notch hN amino acids: 1772-2093 (see FIG. 17). All displayed interactions with fragment D. FIG. 15 summarizes schematically this interaction.
9. EXAMPLE: DELTEX CONTAINS A PUTATIVE SH3-BINDING DOMAIN
SH2 and SH3 domains are conserved protein modules so named based on their homology to the oncogene Src (Src Homology). These motifs have been implicated in mediating protein-protein interactions in a number of signal transduction pathways (reviewed in Cell 71:359-362; Science 252:668-674; Trends Cell Biol. 3:8-13; FEBS 307:55-61). Recently, a complementary motif that binds to the SH3 domain has been identified and called simply an `SH3-binding domain` ("SH3-BD") (Science 259:1157-1161). The core binding region of SH3-BD is proline-rich and approximately ten residues in length. As shown in FIG. 16, this motif, as defined from a mouse protein that experimentally bound an SH3 domain, is shown aligned to two regions (separated by 58 residues) of deltex that may represent Drosophila versions of this motif. These regions are located centrally in the deltex protein, approximately 280 residues C-terminal to the region of deltex that has been shown to bind to the ANK repeats of Notch. For reference, regions of selected Drosophila proteins, which may also contain SH3-BD, are shown. The Son of sevenless (Sos) gene, in particular, is worth noting. The encoded protein, a putative guanine nucleotide exchange factor (GNEF), has been shown to bind to an `adaptor` protein (drk) containing only SH2 and SH3 modules, although the actual residues that mediate binding have not been accurately defined (Cell 73:169-177 and 179-191).
There are currently only six SH3-containing proteins identified in Drosophila, any one of which may be a direct binding partner of deltex, and thus an indirect partner of Notch. These include the proteins encoded by the genes drk, src1, src2, abl, spectrin, and dlg. With the exception of spectrin, all encode signalling molecules. Spectrin encodes a protein associated with the cytoskeleton. Thus deltex may `tie-in` Notch to already identified signalling components or to the cytoskeleton.
10. DEPOSIT OF MICROORGANISMS
Plasmid pCaSpeR hs-dx containing a cDNA insert encoding a full-length Drosophila melanogaster deltex was deposited on Jan. 20, 1994 with the American Type Culture Collection, 1201 Parklawn Drive, Rockville, Md. 20852, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures and was assigned accession number 75640.
The present invention is not to be limited in scope by the microorganism deposited or the specific embodiments described herein. Indeed, various modifications of the 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 by reference in their entireties.
Claims
What is claimed is:
1. A substantially purified deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4).
2. A substantially purified protein comprising a fragment of a deltex protein, said deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4), said fragment consisting of at least 20 continuous amino acids of the deltex protein.
3. A fragment of a deltex protein consisting of at least 10 continuous amino acids of a deltex protein, said deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4), which fragment displays one or more functional activities associated with a full-length deltex protein.
4. The fragment of claim 3 which consists of at least 20 continuous amino acids of the deltex protein.
5. The substantially purified protein of claim 2 which is able to be bound by an antibody to said deltex protein.
6. The fragment of claim 3 which is able to be bound by an antibody to said deltex protein.
7. A substantially purified protein comprising a fragment of at least 20 continuous amino acids of a deltex protein, said deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4), which fragment comprises a glutamine-rich cluster of the deltex protein.
8. A substantially purified protein comprising a fragment of a deltex protein, said deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4), which fragment binds to a Notch protein or to a molecule comprising the cdc10/SW16/ankyrin repeats of a Notch protein.
9. A substantially purified protein comprising a fragment of a first deltex protein, which fragment binds to a second deltex protein or to a molecule comprising a fragment of a second deltex protein, said first and second deltex proteins each having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4).
10. A substantially purified protein comprising a fragment of a deltex protein, said deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4), which fragment comprises a SH3 binding domain of the deltex protein.
11. A chimeric protein comprising a functionally active fragment of at least 20 continuous amino acids of a deltex protein joined via a peptide bond to an amino acid sequence of a protein other than the deltex protein, said deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4).
12. The protein of claim 11 in which the fragment binds to a Notch protein or to a molecule comprising the cdc10/SW16/ankyrin repeats of a Notch protein.
13. The protein of claim 11 in which the fragment comprises an SH3 binding domain.
15. A peptide having an amino acid sequence in the range of 15-25 amino acids, said sequence being a portion of a deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4).
17. A composition comprising a deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4); and a pharmaceutically acceptable carrier.
18. A composition comprising the fragment of claim 3, and a pharmaceutically acceptable carrier.
19. A composition comprising the substantially purified protein of claim 8, and a pharmaceutically acceptable carrier.
20. A composition comprising the substantially purified protein of claim 9, and a pharmaceutically acceptable carrier.
21. A composition comprising the substantially purified protein of claim 10, and a pharmaceutically acceptable carrier.
22. A composition comprising the chimeric protein of claim 11, and a pharmaceutically acceptable carrier.
23. A composition comprising a derivative or analog of a deltex protein, said deltex protein having the amino acid sequence depicted in FIG. 12 (SEQ ID NO:4), which derivative or analog is characterized by the ability to bind to a Notch protein or to a molecule comprising the cdc10/SW16/ankyrin repeats of a Notch protein.
24. The fragment of claim 3 or 4 which is substantially purified.
25. The derivative or analog of claim 14 or 16 which is substantially purified.
26. The composition of claim 18 in which the fragment is substantially purified.
27. The composition of claim 23 in which the derivative or analog is substantially purified.
Patent Citations (3)
| Patent | Date | Inventor | Cited By |
|---|---|---|---|
| WOXWO89/12690 | 1989-12-01 | ||
| WOXWO92/19734 | 1992-11-01 | ||
| WOXWO92/19737 | 1992-11-01 |
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