WO1994008037A1 - Homologues humains de l'activateur du type transducine de genes fractionnes et procedes s'y rapportant - Google Patents

Homologues humains de l'activateur du type transducine de genes fractionnes et procedes s'y rapportant Download PDF

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WO1994008037A1
WO1994008037A1 PCT/US1993/009333 US9309333W WO9408037A1 WO 1994008037 A1 WO1994008037 A1 WO 1994008037A1 US 9309333 W US9309333 W US 9309333W WO 9408037 A1 WO9408037 A1 WO 9408037A1
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protein
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pro
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ala
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WO1994008037A9 (fr
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Spyridon Artavanis-Tsakonas
Stefani Stifani
Nicola J. Redhead
Robert E. Hill
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Yale University
Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to human transducin-like Enhancer of split (TLE) genes and their encoded protein products.
  • the invention also relates to derivatives and analogs of the human TLE proteins. Production of human TLE proteins, derivatives and antibodies is also provided.
  • the central player of the Notch group is the Notch (N) locus which encodes a transmembrane protein containing EGF-like repeats in its extracellular domain (Wharton et al., 1985, Cell 43:567-581 ; Kidd et al., 1986, Mol. Cell. Biol. 3:194-3108).
  • This protein has been shown to interact molecularly and genetically with two other transmembrane, EGF-containing proteins of the Notch group: Serrate and Delta (Vaessin et al., 1985, J. Neurogenetics 2:291-308; Fehon et al., 1990, Cell 61:523-534; Fleming et al.. 1990, Genes Dev.
  • 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).
  • a human Notch (TAN-1) malfunction has been associated with a lymphatic cancer (Ellisen et al, 1991, Cell 66:649-661).
  • E(spl) is a complex locus comprised of at least ten genetically related transcription units which have been separated into two distinct groups, both of which display genetic interactions with specific Notch mutations (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; Delidakis et al., 1991, Genetics 129:803-823).
  • the first group codes for proteins containing the helix-loop-helix motif (Klambt et al., 1989, EMBO J. 8:203-210) while the second displays homology to the ⁇ subunit of transducin (Hartley et al., 1988, Cell 55:785-795).
  • Knust et al. (1987, EMBO J. 6:4113-4123) have numbered the transcripts in the E(spl) region and, according to their numbering system, the transcripts coding for the transducin-homologous protein are termed m9/10.
  • m9/10 Several embryonic lethal alleles affecting this gene were isolated.
  • the mutation groucho which affects bristle development in Drosophila, is allelic to the Enhancer of split m9/10 gene (Hartley et al., 1988, Cell 55:785-795; Preiss et al., 1988, EMBO J. 7:3917-3927).
  • the 719 amino acid long product of the E(spl) m.9110 gene contains four tandemly arranged repeats spanning the carboxyl-terminal ⁇ 300 amino acid residues of the protein (Hartley et al., 1988, Cell 55:785-795). Each repeat is approximately 40 residues in length and is characterized by the presence of the conserved motif WDL.
  • Such repeats are found similarly arranged in the ⁇ subunits of G proteins and have been referred to as the "WD-40 repeat" (for review, see Simon et al., 1991, Science 252:802-808).
  • WD-40 repeat Several other proteins containing this structural motif include the products of the yeast cell cycle gene CDC 4 (Yochem and Byers, 1987, J. Mol. Biol. 195:233-245) and of the TUP1 gene, a mediator of glucose repression (Williams and Trumbly, 1990, Mol. Cell. Biol. 10:6500-6511.).
  • the present invention relates to nucleotide sequences of the human transducin-like Enhancer of split ("TLE") genes, and amino acid sequences of their encoded TLE proteins.
  • the invention further relates to fragments and other derivatives, and analogs, of human TLE 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. Binding partners of TLE proteins, and multiprotein complexes containing TLE proteins are also provided.
  • the invention provides sequences of four distinct human homologs of the Drosophila TLE gene, and sequences of their unique encoded TLE proteins.
  • the TLE proteins and their Drosophila homolog contain a motif implicated in nuclear/cytoplasmic protein transport, called the casein kinase II site/cdc2 kinase site/nuclear localization sequence motif (CcN motif).
  • CcN motif a motif implicated in nuclear/cytoplasmic protein transport
  • the invention relates to human TLE protein derivatives and analogs of the invention which are functionally active, or which comprise one or more domains of a human TLE protein, including but not limited to the "Q domain,” “GP domain,” “CcN domain,” “SP domain,” “WD-40 domain,” or a WD-40 repeat, casein kinase II (CK II) site, cdc2 kinase (cdc2) site, or nuclear localization sequence motif, or consensus sequences for any of the foregoing, or any combination of the foregoing.
  • CK II casein kinase II
  • cdc2 cdc2 kinase
  • FIG. 5 Nucleotide sequence (SEQ ID NO: 5) and deduced amino acid sequence (SEQ ID NO:6) of TLE 3.
  • Figure 4. Partial nucleotide sequence (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO: 8) of TLE 4.
  • FIG. 1 Comparison of the amino acid sequence of Drosophila E(spl) m9/10 (SEQ ID NO: 10) and human TLE proteins. Amino acids are numbered on the left side. Identical residues in all compared sequences are boxed, while residues identical in either three out of four or four out of five sequences are indicated in boldface type. Alignments maximize continuity between all sequences. Underlined amino acid residues correspond to the CcN motif.
  • FIG. 6 Comparison of the WD-40 domains of Drosophila E(spl) m9/10 and TLE proteins. Amino acids are numbered on the left side. Those residues that are identical in each of the five sequences are boxed, while residues identical in four out of five sequences are indicated in boldface type. Those amino acids that are present at a given position in at least 10 out of 20 repeats define the consensus residues (SEQ ID NO:9) indicated at the bottom of the figure.
  • MTN Blot "MTN Blot", catalog #7760-1; 2 ⁇ g/lane) was obtained from Clontech. Northern blotting experiments were performed at 42° C in a buffer containing 50% formamide, 5X SSPE, 5X Denhardt's solution, 0.5% SDS, and 100 ⁇ g/ml of salmon sperm DNA. After hybridization for 16 hr in the presence of [ 32 P]-labeled probes, blots were washed in IX SCC, 0.1 % SDS once at room temperature and 3 times at 68°C, followed by three washes at 68°C in 0.2X SSC, 0.1 % SDS.
  • TLE 1 (a), residues 260 through 435; TLE 2 (b), 32 through 342; TLE 3 (c), 350 through 440; TLE 4 (d), the region corresponding to that covered by the TLE 3 probe.
  • RNA size markers (in kb) are indicated at the left of each autoradiogram. The arrows on the right of each panel indicate the sizes of the major TLE-specific transcripts.
  • FIG. 8 Immunocytochemical characterization of TLE proteins, (a) Western blotting analysis of TLE proteins. Protein extracts from human thymus (lane 1; 250 ⁇ g of protein/lane), spleen (lane 2; 250 ⁇ g of protein/lane), lung (lane 3; 200 ⁇ g of protein/lane), heart (lane 4; 180 ⁇ g of protein/lane), kidney (lane 5; 200 ⁇ g of protein/lane), SUP-T1 cells (Ellisen et al., 1991, Cell 66:649-661) (lane 6; 180 ⁇ g of protein/lane), and HeLa cells (lane 7; 150 ⁇ g of protein/lane) were prepared and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on a 6% gel as described in Section 6.3.
  • PAGE SDS-polyacrylamide gel electrophoresis
  • FIG. 9 Western blot visualization of multiprotein complexes containing TLE proteins after non-denaturing polyacrylamide gel electrophoresis (PAGE).
  • a high speed supernatant fraction from human HeLa cell lysates was subjected to non-denaturing PAGE, proteins were transferred to nitrocellulose filters and probed in a Western blotting procedure with monoclonal antibody C597.4A, which binds to all TLE proteins.
  • Two major immunoreactive species were detected, with apparent molecular weights of greater than 670,000 daltons.
  • FIG. 10 Gel filtration chromatography of multiprotein complexes containing TLE proteins.
  • High speed supernatant fractions from HeLa cells were subjected to gel filtration chromatography using a Sephacryl S-300 matrix.
  • the fractions collected from the column were analyzed for the presence of TLE proteins in Western blotting experiments with monoclonal antibody C597.4A.
  • Panel A shows the results after SDS-PAGE under reducing conditions
  • Panel B shows the results after SDS-PAGE under nonreducing conditions.
  • Positions of elution of Dextran Blue (D.B.) and of molecular weight standards of 116 kD and of 80 kD are shown at the top of Panel A.
  • FIG. 11 Western blots of cross-linked protein complexes containing TLE proteins.
  • Protein extracts from Drosophila embryos (lanes 1-3), HeLa ceils (lanes 4-6) or SUP-T1 cells (lanes 7-9) were incubated in the presence of increasing concentrations of the chemical cross-linker, DTSSP. Concentrations used of DTSSP were as follows (in mM): Lanes 1, 4, and 7: 0; Lane 2: 0.2; Lane 3: 0.5; Lane 5: 0.06; Lane 6: 0.18; Lane 8: 0.06; Lane 9: 0.18.
  • the present invention relates to nucleotide sequences of the human transducin-like Enhancer of split [E(spl)] ("TLE”) genes, and amino acid sequences of their encoded TLE proteins.
  • TLE transducin-like Enhancer of split [E(spl)]
  • the invention further relates to fragments and other derivatives, and analogs, of human TLE 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. Binding partners of TLE proteins, and multiprotein complexes containing TLE proteins are also provided.
  • the invention provides sequences of four distinct human homologs of the Drosophila TLE gene, and sequences of their unique encoded TLE proteins.
  • the TLE proteins and their Drosophila homolog contain a motif implicated in nuclear/cytoplasmic protein transport, called the casein kinase II site/cdc2 kinase site/nuclear localization sequence motif (CcN motif).
  • CcN motif a motif implicated in nuclear/cytoplasmic protein transport
  • the invention also relates to human TLE 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) TLE protein.
  • Such functional activities include but are not limited to antigenicity [ability to bind (or compete with a TLE protein for binding) to an anti-TLE protein antibody], immunogenicity (ability to generate antibody which binds to a TLE protein), ability to bind (or compete with a TLE protein for binding) possibly to Notch or other toporythmic proteins or fragments thereof, ability to bind (or compete with a TLE protein for binding) to a receptor or ligand for a TLE protein.
  • Topicalthmic proteins refers to the protein products of Notch, Delta, Serrate, Enhancer of split, and deltex, as well as other members of this interacting gene family which may be identified, e.g. , by virtue of the ability of their gene sequences to hybridize, or their homology to Delta, E(spl), Serrate, or Notch, or the ability of their genes to display phenotypic interactions.
  • the invention further relates to fragments (and derivatives and analogs thereof) of a human TLE protein which comprise one or more domains of a human TLE protein (see Section 6), including but not limited to the "Q domain,” “GP domain,” “CcN domain,” “SP domain,” “WD-40 domain,” or a WD-40 repeat, casein kinase II (CK II) site, cdc2 kinase (cdc2) site, or nuclear localization sequence motif, or consensus sequences for any of the foregoing, or any combination of the foregoing.
  • CK II casein kinase II
  • cdc2 cdc2 kinase
  • TLE proteins are additionally provided.
  • E(spl) plays a critical role in development and other physiological processes.
  • the nucleic acid and amino acid sequences and antibodies thereto of the invention can be used for the detection and quantitation of human TLE mRNA, to study expression thereof, to produce human TLE proteins, fragments and other derivatives, and analogs thereof, in the study and manipulation of differentiation and other physiological processes, and may be of therapeutic or diagnostic use, e.g. , for neoplastic and pre-neoplastic conditions such as the detection of cervical squamous metaplasias, dysplasias, and malignancies.
  • the invention is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of four human homologs of D. melanogaster E(spl); the construction and recombinant expression of human TLE chimeric/fusion derivatives and production of antibodies thereto, and multiprotein complexes containing TLE proteins, and an about 17 kD component of such complexes.
  • TLE Genes; (ii) Expression of the Human TLE Genes; (iii) Identification and Purification of the Expressed Gene Products; (iv) Structure of the Human TLE Genes and Proteins;
  • human TLE nucleic acids comprise the
  • TLE 1, TLE 2, TLE 3, or TLE 4 cDNAs The invention provides nucleic acids consisting of at least 8 nucleotides (i.e. , a hybridizable portion) of a human TLE sequence; in other embodiments, the nucleic acids consist of at least 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a human TLE sequence. In a preferred, but not limiting, aspect of the invention, a human TLE
  • DNA can be cloned and sequenced by the method described in Section 6, infra.
  • nucleic acids hybridizable to or complementary to the foregoing sequences.
  • 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 human TLE gene.
  • a human expression library is obtained or is constructed by methods known in the art. For example, human mRNA 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 human TLE product. In one embodiment, anti-TLE protein antibodies can be used for selection. In another preferred aspect, PCR is used to amplify the desired sequence in the, library, prior to selection. Oligonucleotide primers representing known TLE protein sequences can be used as primers in PCR.
  • an expression vector e.g. , a bacteriophage derivative
  • Various screening assays can then be used to select for the expressed human TLE product.
  • anti-TLE protein antibodies can be used for selection.
  • PCR is used to amplify the desired sequence in the, library, prior to selection. Oligonucleotide primers representing known TLE
  • the oligonucleotide primers encode at least part of the conserved segments of strong homology between Drosophila and human TLE proteins (e.g. , in the Q domain, CcN domain, or WD-40 domain).
  • the synthetic oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA), preferably a cDNA library, of potential interest. PCR can be carried out, e.g. , by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp").
  • the DNA being amplified can include human mRNA or cDNA or genomic DNA.
  • 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 cell.
  • cloned DNA e.g. , a DNA "library”
  • cDNA cloning or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell.
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will lack introns and will contain only exon sequences. Whatever the source, the gene should be moleculariy 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 poly acrylamide gel electrophoresis and column chromatography.
  • 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 TLE (of any species) gene or its specific RNA, or a fragment thereof, e.g. , a Q or WD-40 domain (see Section 5.6), is available and can be purified, or synthesized, and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196: 180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize.
  • the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map, either available or deduced from a known nucleotide sequence. 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, binding activity, or antigenic properties as known for a TLE protein. By use of an antibody to a TLE protein, the TLE protein may be identified by binding of labeled antibody to the putatively TLE protein synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay)-type procedure.
  • ELISA enzyme-linked immunosorbent assay
  • the TLE 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 TLE DNA of human or of another species (e.g. , Drosophila). Immunoprecipitation analysis or functional assays (e.g. , binding to a receptor or ligand; see 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 a TLE protein.
  • a radiolabelled TLE 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 TLE DNA fragments from among other genomic DNA fragments.
  • RNA for cDNA cloning of the human TLE gene can be isolated from cells which express a TLE protein (see Section 6.1.3). 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.
  • 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.
  • the ends of the DNA molecules may be enzy atically modified.
  • 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.
  • the cleaved vector and TLE 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.
  • 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 fractionation, can be done before insertion into the cloning vector.
  • transformation of host cells with recombinant DNA molecules that incorporate the isolated TLE gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene.
  • 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. 5.2.
  • the nucleotide sequence coding for a human TLE 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 TLE gene and/or its flanking regions.
  • 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 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.
  • a chimeric protein comprising the nuclear localization signal or other motif or domain of a human TLE protein is expressed.
  • a full-length human TLE cDNA is expressed, or a sequence encoding a functionally active portion of a human TLE protein.
  • a fragment of a human TLE protein comprising a domain of the protein, or other derivative, or analog of a human TLE 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 human TLE protein or peptide fragment may be regulated by a second nucleic acid sequence so that the TLE protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a TLE protein may be controlled by any promoter/enhancer element known in the art.
  • Promoters which may be used to control TLE 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.
  • ⁇ P L ⁇ P L
  • 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.
  • 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.
  • 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.
  • 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 human TLE 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.
  • 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 TLE gene.
  • 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.
  • recombinants containing the E(spl) insert can be identified by the absence of the marker gene function.
  • 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 TLE gene product in in vitro assay systems, e.g. , binding to a ligand or receptor, binding with antibody, possible aggregation (binding) with Notch.
  • 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.
  • 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 TLE protein may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post- translational processing and modification (e.g. , phosphorylation) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • cDNA and genomic sequences can be cloned and expressed.
  • 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. (see Section 6, infra).
  • a human TLE protein Once a human TLE 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.
  • chromatography e.g. , ion exchange, affinity, and sizing column chromatography
  • centrifugation e.g., 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
  • the amino acid sequence of a human TLE 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).
  • human TLE proteins include but are not limited to those containing, as a primary amino acid sequence, all or part of the amino acid sequences substantially as depicted in Figures 1-4 (SEQ ID NOS:2, 4, 6, and 8), as well as fragments and other derivatives, and analogs thereof.
  • the cloned DNA or cDNA corresponding to the TLE 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, New York), and DNA sequence analysis (see Section 6.3.1 and Figs. 1-4). Polymerase chain reaction (PCR; U.S. Patent Nos.
  • Southern hybridization can be used to determine the genetic linkage of TLE.
  • Northern hybridization analysis can be used to determine the expression of the TLE genes.
  • Various cell types, at various states of development or activity can be tested for TLE gene expression.
  • Restriction endonuclease mapping can be used to roughly determine the genetic structure of the human TLE 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. Patent No. 4,795,699; Sequenase, U.S. Biochemical Corp.), or Taq polymerase, or use of an automated DNA sequenator (e.g. , Applied Biosystems, Foster City, CA).
  • the cDNA sequence of three human TLE genes comprises the sequence substantially as depicted in Figures 1-3 (SEQ ID NOS:l, 3, and 5), and described in Section 6, infra.
  • the cDNA sequence of a portion of a fourth human TLE gene is shown in Figure 4 (SEQ ID NO:7) and is described in Section 6, infra.
  • the amino acid sequence of a human TLE 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 human TLE protein comprises one of the sequences substantially as depicted in Figures 1-4, and detailed in Section 6, infra.
  • the TLE 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 TLE protein and the corresponding regions of the gene sequence which encode such regions.
  • 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.
  • a TLE protein 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.
  • antibodies which specifically bind to human TLE proteins are produced.
  • such an antibody recognizes the human TLE proteins TLE 1, TLE 2, TLE 3, and TLE 4, or a portion thereof.
  • such an antibody specifically binds to one human TLE protein selected from among TLE 1, TLE 2, TLE 3, and TLE 4, but does not bind to a different human TLE protein.
  • antibodies to a particular domain of a TLE protein are produced.
  • Various procedures known in the art may be used for the production of polyclonal antibodies to a human TLE protein or derivative or analog.
  • rabbit polyclonal antibodies to an epitope of one of the TLE proteins encoded by a sequence depicted in Figure 1, 2, 3 or 4, or a subsequence thereof can be obtained.
  • various host animals can be immunized by injection with a native TLE protein, or a synthetic version, or derivative (e.g. , fragment) thereof, including but not limited to rabbits, mice, rats, etc.
  • 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.
  • BCG Bacille Calmette-Guerin
  • polyclonal or monoclonal antibodies are produced by use of a hydrophilic portion of a TLE peptide (e.g. , identified by the procedure of Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824)).
  • a hydrophilic portion of a TLE peptide e.g. , identified by the procedure of Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824.
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • 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 can be used.
  • monoclonal antibodies can be produced in germ-free animals (PCT Publication No. WO 89/12690 dated December 28, 1989).
  • 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.
  • techniques developed for the production of "chimeric antibodies” (Morrison et al.. 1984, Proc. Natl. Acad. Sci. U.S.A.
  • Antibody fragments which contain the idiotype (binding domain) of the molecule can be generated by known techniques.
  • fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay).
  • antibodies which recognize a specific domain of a TLE protein one may assay generated hybridomas for a product which binds to a TLE fragment containing such domain.
  • an antibody specific to human TLE protein(s) one can select on the basis of positive binding to a human TLE protein and a lack of binding to Drosophila TLE protein.
  • Antibodies which bind to only one TLE protein e.g. , TLE 1 or TLE 2 selected from among TLE 1, 2, 3 and 4 can be selected by appropriate binding assays.
  • antibodies to a non-TLE protein component of multiprotein complexes containing a TLE protein are provided. Such antibodies can be obtained by a method comprising immunizing an animal with such multiprotein complexes.
  • 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. , see Section 5.7, infra), e.g. , for imaging these proteins, measuring levels thereof in appropriate physiological samples, etc.
  • the invention further relates to derivatives (including but not limited to fragments) and analogs of human TLE proteins.
  • the derivative or analog is functionally active, i.e. , capable of exhibiting one or more functional activities associated with a full-length, wild-type human TLE protein.
  • such derivatives or analogs which have the desired immunogenicity or antigenicity can be used, for example, in immunoassays, for immunization, for promotion or inhibition of TLE protein activity, etc.
  • Such molecules which retain, or alternatively inhibit, a desired human TLE protein property e.g. , binding to a receptor or ligand, such as possibly Notch protein, can be used as inducers, or inhibitors, respectively, of such property and its physiological correlates.
  • Derivatives or analogs of TLE proteins can be tested for the desired activity by procedures known in the art, including but not limited to the assays described in Section 5.7.
  • TLE derivatives can be made by altering TLE sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • TLE sequences Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a human TLE 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 human TLE 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.
  • the TLE 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 human TLE protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • 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.
  • 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.
  • proteins consisting of or comprising a fragment of a human TLE protein consisting of at least fifty amino acids of the TLE protein is provided.
  • the fragment consists of at least 75 or 100 amino acids of the TLE protein.
  • the human TLE 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.
  • the cloned TLE gene sequence can be modified by any of numerous strategies known in the art (Maniatis, 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • the sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • restriction endonuclease(s) e.g., EcoRI
  • enzymatic modification e.g., acetylation, acetylation, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids,
  • the TLE-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 ® linkers (Pharmacia), etc.
  • Manipulations of the human TLE sequence may also be made at the protein level. Included within the scope of the invention are human TLE protein fragments or other derivatives or analogs which are differentially modified during or after translation, e.g. , by acetylation, phosphorylation, 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 4 , acetylation, formylation, oxidation, reduction, etc.
  • phosphorylation or, alternatively, dephosphorylation is carried out, which can be to various extents, on the purified human TLE protein or derivative thereof.
  • the phosphorylation state of the molecule may determine the distribution of the TLE protein between the cellular compartments of the nucleus and the cytoplasm (see Section 6, infra). Thus, controlling the phosphorylation state may allow control of intracellular localization.
  • 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.
  • analogs and derivatives of human TLE proteins can be chemically synthesized.
  • a peptide corresponding to a portion of a TLE protein which comprises the desired domain can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the human TLE protein sequence.
  • Non-classical amino acids include but are not limited to the D- isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, /3-alanine, designer amino acids such as /3-methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids.
  • the human TLE derivative is a chimeric, or fusion, protein comprising a human TLE protein or fragment thereof (preferably consisting of at least a domain or motif of the TLE protein, or at least 50 amino acids of the TLE protein) joined at its amino or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein.
  • a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a human TLE-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.
  • 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 TLE protein which comprises a domain or motif of the TLE protein, e.g.
  • a chimeric nucleic acid can be constructed, encoding a fusion protein consisting of a human TLE nuclear localization sequence (NLS) or CcN motif (see Table I, Section 6, infra) joined to a non-TLE protein.
  • NLS human TLE nuclear localization sequence
  • CcN motif see Table I, Section 6, infra
  • the invention thus provides a method for delivering any protein of interest to the nucleus of a cell, by linkage of such protein to a human TLE protein NLS or CcN motif (the CcN motif contains an NLS).
  • a recombinant molecule can be constructed according to the invention, comprising coding portions of both a human TLE gene and another toporythmic gene.
  • Another specific embodiment relates to a chimeric protein comprising a fragment of a human TLE protein of at least six amino acids.
  • a particular example of a 5 human TLE fusion protein consisting of a human TLE fragment capable of generating anti-TLE antibody fused to the carboxyl-terminus of glutathione-S-transferase, is described in Section 7 hereof.
  • the invention relates to human TLE protein derivatives and analogs, in particular human TLE fragments and
  • - - derivatives of such fragments that comprise one or more domains of a human TLE protein, including but not limited to a Q domain [amino acids (approximately) 1-131, 1-127, and 1-130 for TLE 1, TLE 2, and TLE 3, respectively], GP domain [amino acids (approximately) 132-199, 128-191, and 131-197 for TLE 1 , TLE 2, and TLE 3, respectively], CcN domain [amino acids 0 (approximately) 200-268, 192-254, and 198-267 for TLE 1, TLE 2, and TLE 3, respectively], SP domain [amino acids (approximately) 269-449, 255-422, and 268-450 for TLE 1, TLE 2, and TLE 3, respectively], WD-40 domain [amino acids (approximately) 450-770, 423-743, and 451-774, for TLE 1, TLE 2, and TLE 3, respectively, and the last —321 amino acids of T
  • a consensus WD-40 repeat is shown in Figure 6, and consists of the following sequence (SEQ ID NO:9): PXXXX(D or E)XTXXXXXXXX(I or L)X(I or L)SPDG(T or S)XLX(T or S)GGXDGXVXXWDLX, where X is any amino acid.
  • the CcN domains comprise the CcN motifs, which latter span approximately amino acids 225-269, 214-255, and 224-268, for TLE 1, TLE 2, and TLE 3, respectively.
  • the invention also provides for human TLE fragments, and analogs or derivatives of such fragments, which mediate binding to other proteins, and nucleic acid sequences encoding the foregoing. As shown in Section 7, infra,
  • TLE proteins associate in multiprotein complexes, and thus bind to other proteins.
  • a non-TLE protein component of such multiprotein complexes is an — 17 kD protein.
  • TLE proteins The functional activity of TLE proteins, derivatives and analogs can be assayed by various methods.
  • 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.
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • 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.
  • receptor or ligand binding can be assayed, e.g. , by means well known in the art.
  • physiological correlates of TLE introduction into cells can be assayed.
  • the invention further provides proteins which associate in a multiprotein complex containing TLE proteins (see Section 7, infra).
  • a protein binds to a TLE protein or a binding partner thereof.
  • Such protein components of complexes containing TLE proteins may act as effector molecules in TLE protein signal transduction events and thus have potential uses in modulation of TLE protein activity.
  • a substantially purified multiprotein complex of about 670,000 daltons that contains a TLE protein or epitope thereof (as detected e.g.
  • such a multiprotein complex has a molecular weight greater than about 670,000 daltons; in yet other aspects, such a complex has a molecular weight of about 110, 170, 190, or 230 kilodaltons. These complexes of smaller molecular weight may be components of the larger complexes.
  • the invention provides a substantially purified protein component of such a multiprotein complex, with a molecular weight in the range of about 15,000- 18,000 daltons, in particular, about 17,000 daltons, as detected by SDS- polyacrylamide gel electrophoresis.
  • the invention also provides antibodies, in particular, monoclonal antibodies, which specifically bind to the non-TLE protein components of such multiprotein complexes.
  • antibodies are obtained by using as immunogen the multiprotein TLE complexes and selecting for negative TLE protein reactivity and positive TLE complex reactivity. 6.
  • the Drosophila m.9110 gene (groucho) of the Enhancer of split [E(spl)] complex is part of a genetic circuitry, the so-called Notch group of genes, which is required for a variety of cell fate choices in Drosophila including the segregation of neural and epidermal cell lineages.
  • Notch group of genes which is required for a variety of cell fate choices in Drosophila including the segregation of neural and epidermal cell lineages.
  • TLE human cDNA clones encoding a family of proteins, designated TLE, that are homologous to the E(spl) m9/10 gene product.
  • the TLE and E(spl) m9/10 proteins share two amino acid sequence motifs.
  • the first is a tandem array of four so-called “WD-40” repeats at the carboxyl end of the molecule and the second, referred to as the "CcN motif", consists of a closely- spaced combination of a nuclear localization sequence and potential phosphorylation sites for both casein kinase II and cdc2 kinase.
  • the TLE proteins were shown to be predominantly nuclear in HeLa cells, and the Drosophila E(spl) m9/10 protein was shown to be phosphorylated.
  • This clone contains a partial open reading frame (ORF) encoding a 282 residue long polypeptide chain exhibiting homology to the portion of the E(spi) m9/10 protein that includes the four WD-40 repeats.
  • ORF partial open reading frame
  • cDNA TL ⁇ 1 , TL ⁇ 2, and TL ⁇ 3 contain entire ORFs for three distinct proteins of 770 (M r 83,000), 743 (M r 80,000), and 774 (M r 83,000) amino acids, respectively, while TLE 4 is a partial clone. All encoded proteins are homologous to E(spl) m.9110.
  • the complete nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequence ((SEQ ID NO:2) for TLE 1 are shown in Figure 1.
  • the complete nucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4) for TLE 2 are shown in Figure 2.
  • the complete nucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO: 6) for TLE 3 are shown in Figure 3.
  • the partial nucleotide sequence (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO: 8) for TLE 4 are shown in Figure 4.
  • TLE 1 , TLE 2, and TLE 3 As is the case with the E(spl) m9/10 protein (Hartley et al., 1988, Cell 55:785-795), analysis of hydropathy plots for TLE 1 , TLE 2, and TLE 3 indicated that the TLE proteins are quite hydrophilic and appear not to have a signal sequence (not shown).
  • TLE 1 is 72%
  • TLE 2 is 68%
  • TLE 3 is 71 % identical to E(spl) m9/10.
  • Adams et al. (1991, Science 252:1651-1656) have recently described partial DNA sequences of more than 600 randomly selected cDNA clones from human brain. Sequencing of ⁇ 250 nucleotides of Adams et al.
  • clone EST00256 identified a reading frame coding for a protein related to E(spl) m9/10; this short sequence maps within the first 100 residues of the amino terminus of m9/10. Comparing the corresponding region of the TLE 1, TLE 2, and 7L£ 3 cDNAs with the nucleotide and predicted amino acid sequence of cDNA EST00256, we failed to show identity among these cDNAs. This suggests that cDNA EST00256 is either pan of the sequence coding for TLE 4, the sequence of which remains to be fully determined, or part of yet another member of this family. A poorly conserved region of approximately 80 amino acid residues follows the Q domain.
  • GP domain This portion of the molecules as the "GP domain” to indicate the presence of numerous glycine and proline residues.
  • the lack of significant structural conservation in the GP domain ends approximately 200 residues from the amino terminus, in the "CcN domain” (Jans et al., 1991, J. Cell Biol. 115:1203-1212).
  • the CcN domain consists of a stretch of — 60 residues that harbors a sequence motif conforming to the definition of a casein kinase II (CK II) site/cdc2 kinase (cdc2) site/nuclear localization sequence (NLS) motif first reported for the SV40 T antigen (Jans et al., 1991, J. Cell Biol. 115: 1203-1212; Rihs et al., 1991, EMBO J. 10:633-639).
  • CK II casein kinase II
  • cdc2 cdc2 kinase
  • NLS nuclear localization sequence
  • NLS a cluster of four positively charged amino acids preceded, at a distance of ten residues, by a block of two or three basic amino acids (Kalderon et al., 1984, Nature 311:499-509; Dingwall and Laskey, 1991, Trends Biochem. Sci. 16:478-481), is in proximity to possible phosphorylation sites for both casein kinase II (defined by the consensus sequence S / T XX D / E ) and cdc2 kinase (defined by the consensus sequence S / T PXZ, with X being dispensable and Z being generally a basic residue).
  • Table I shows a comparison of the CcN motif found in E(spl) m9/10, TLE 1, TLE 2, and TLE 3.
  • E(spl) m9/10, TLE 1, and TLE 3 have conventional NLSs, while TLE 2 deviates from the general consensus. It is worth noting, however, that a certain degree of flexibility in the selection of the amino acids that form a NLS has been observed previously (Dingwall and Laskey, 1991 , Trends Biochem. Sci. 16:478-481).
  • TLE 1 212 DKRRNGP-EFSNDIKKRKVDDKDSSH-YD-SDGDKSDDNLWDVSNED-PS-S PRASPAHSPR
  • NLS nuclear localization sequence
  • CK II casein kinase II
  • cdc2 phosphorylation sites for casein kinase II
  • Table II shows the relationship between the NLS and putative phosphorylation sites in E(spl) m9/10, TLE 1, TLE 2, TLE 3, as well as other proteins bearing the CcN motif; these proteins were selected on the basis of demonstrated nuclear localization and susceptibility to phosphorylation. Most, if not all, of them play important roles in regulating nuclear functions such as transcription and mitosis, as well as other aspects of the cell cycle (for review, see Meisner and Czech, 1991, Curr. Op. Cell Biol. 3:474-483; Moreno and Nurse, 1990, Cell 61:549-551).
  • the Drosophila protein dorsal was included as one example of several other proteins bearing a putative CcN motif for which only translocation to the nucleus has been demonstrated.
  • TLE 1 mRNA migrates as a major species of 4.5 kb detectable in all adult tissues examined, with the highest level of expression in brain, liver, and muscle (Fig. 7a). Minor species of 5.8 and 3.2 kb were also detectable. Two distinct TLE 2 mRNAs were detected (Fig. 7b). One transcript, of 2.8 kb, was expressed at different levels in all tissues examined and was noticeably abundant in heart, brain, and muscle; the second transcript, of 3.5 kb, appeared to be expressed only in brain. Three distinct TLE 3 transcripts were present, having sizes of 5.8, 4.8, and 3.7 kb.
  • Placenta and lung are the only tissues where all these mRNAs were detected, while the remaining tissues only expressed either one or two of them (Fig. 7c). Finally, two major TLE 4 transcripts of 5.1 and 2.8 kb were observed. They were predominantly expressed in brain and muscle, but were also present in all other tissues investigated (Fig. 7d).
  • TLE mRNAs are expressed in all tissues examined with individual transcripts showing specific patterns of expression.
  • Figure 8a illustrates the results of Western blotting experiments performed with monoclonal antibody
  • Drosophila E(spl) m9/10 protein is phosphorylated.
  • Drosophila S2 cells were pulse-labeled with 32 P and lysed as described in Materials and Methods.
  • a single phosphorylated protein was detected in the immunoprecipitates obtained with monoclonal antibody 3C, which is directed against Drosophila E(spl) m9/10 (Delidakis et al., 1991, Genetics 129:803-823).
  • the electrophoretic mobility of this molecule corresponded to that expected for the m9/10 protein.
  • the expression profile of the Drosophila m9/10 protein during embryogenesis was revealed by Western blotting analysis. Two closely-spaced bands were detected with monoclonal antibody 3C. The lower band was predominant very early in development and became progressively less abundant at later stages, while the higher band showed exactly an opposite profile.
  • the E(spl) m9/10 protein was shown to be the product of a single gene (Hartley et al., 1988, Cell 55:785-795; Preiss et al., 1988, EMBO J. 7:3917-3927), it is possible that this electrophoretic profile reflects a developmental ly regulated post- translational modification such as phosphorylation.
  • a second noteworthy structural feature shared by E(spl) m9/10 and TLE proteins is the presence of the CcN motif (Fig. 5 and Table I).
  • the CcN motif has been found in several nuclear proteins involved in regulating cell differentiation or proliferation (Jans et al., 1991 , J. Cell Biol. 115: 1203-1212).
  • Studies with SV40 T antigen have demonstrated that absence of the Ser residue of the CK II site of the CcN motif, which can be phosphorylated, causes a reduction of the rate of nuclear transport of the protein (Rihs et al., 1991, EMBO J. 10:633-639).
  • RNA from bom fetal and adult brain were obtained from Clontech. Incubations were at 65 °C for 48 hr using double stranded [ 32 P]- labeled probes prepared by random oligonucleotide priming. 6.3.2. cDNA CLONING A human testis cDNA library (Clontech) was used for the isolation of the 1.7-kb TLE 3a cDNA.
  • This cDNA was used to screen a human fetal brain cDNA library (Stratagene) resulting in isolation of the 7LE 1, TLE 2, TLE 3, and TLE 4 cDNAs. Recombinant phage were propagated in E. coli "XLl-Blue" cells. Plaques were screened using me TLE 3a cDNA labeled with [ 32 P]-dCTP by random oligonucleotide priming as a probe. Replicate filters were hybridized at 65 °C in buffer A [500 mM sodium phosphate (pH 7.2), 5% SDS, 1 mM ⁇ DTA., and 1 % bovine serum albumin (BSA)].
  • buffer A 500 mM sodium phosphate (pH 7.2), 5% SDS, 1 mM ⁇ DTA., and 1 % bovine serum albumin (BSA)].
  • Dounce homogenizer (10 strokes; type- A pestle), and protein extracts were obtained in the presence of 1 % Triton X-100. Lysates were centrifuged at 12,000 x g for 15 min and the resulting supernatants were collected, calibrated for their protein content, and subjected to SDS-PAGE. Human tissue samples were processed essentially in the same way.
  • IMMUNOFLUORESCENCE MICROSCOPY HeLa cell monolayers were grown in tissue culture chamber slides (Nunc). Indirect immunofluorescence microscopy using the rat monoclonal antibody C597.4A (see infra) was performed essentially as described by Fehon et al. (1990, Cell 61:523-534). Cells were fixed with freshly made 2% (w/v) paraformaldehyde in 100 mM PIPES (pH 6.8), 2 mM EGTA, 1 mM MgSO 4 , and incubated for 30 min in PBS containing 0.08% Triton X-100 and 3% normal goat serum (buffer C).
  • Drosophila S2 cells were cultured as described previously (Fehon et al., 1990, Cell 61:523-534). In a typical metabolic labeling reaction with [ 3 P]- orthophosphate, 10-15 ml of cell suspension ( — 2 x IO 7 cells/ml) was used. Cells were washed twice with BSS, resuspended in 1 ml of phosphate-free M3 medium, and incubated at 24 °C for 45 min. After this time, cells were incubated for 3 hr at 24 °C in the presence of 750 ⁇ Ci/ml of [ 32 P]-orthophosphate (Amersham; 370 MBq/ml).
  • Incubations were performed in 6-well tissue culture plates with gentle rocking motion. At the end of the incubation time, cells were collected, washed twice with ice-cold buffer D (10 mM HEPES, pH 7.8, 60 mM KC1, 15 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 5 mM MgCl,, 300 mM sucrose, 1 mM PMSF, 2 ⁇ M leupeptin, 2.5 ⁇ g/ml aprotinin, 2.5 ⁇ g/ml pepstatin A), resuspended, and lysed in buffer D containing 0.5% Triton X-100.
  • buffer D 10 mM HEPES, pH 7.8, 60 mM KC1, 15 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 5 mM MgCl, 300 mM sucrose, 1 mM PMSF, 2 ⁇ M leupeptin, 2.5
  • cell lysates were mixed with the appropriate antibodies (150 ⁇ g/ml) and incubated for 2 hr at 4°C in the presence of 3.5 mg/ml of BSA.
  • Immunoprecipitates were collected by incubation with protein G- agarose beads, washed extensively with buffer E (25 mM Tris-HCl, pH 7.8, 200 mM NaCl, 2 mM EDTA, 2 mM EGTA, 20 mM NaF, 10 mM sodium pyrophosphate, and 0.5% Triton X-100), and subjected to SDS-polyacrylamide gel electrophoresis.
  • buffer E 25 mM Tris-HCl, pH 7.8, 200 mM NaCl, 2 mM EDTA, 2 mM EGTA, 20 mM NaF, 10 mM sodium pyrophosphate, and 0.5% Triton X-100
  • Triton X-100 0.002% Triton X-100.
  • Dechorionated embryos were washed twice in PBS and then homogenized by 10 strokes of a Dounce homogenizer (type-A pestle) in buffer F (10 M HEPES, pH 7.8, 150 mM NaCl, 2 mM MgCl 2 , 1 mM PMSF, 2 ⁇ M leupeptin, 2.5 ⁇ g/ml aprotinin, and 2.5 ⁇ g/ml pepstatin A). All operations were carried out at 4 °C. After incubation in the presence of 1 % Triton X-100, homogenates were centrifuged at 12,000 x g for 15 min and the supernatants were collected. Determination of the protein concentration of embryonic and cell extracts was obtained using the Biorad protein assay kit using BSA as standard.
  • Fusion proteins were obtained using the pGEX-3X expression vector system in E. coli BL21DE3 cells.
  • the 1.7-kb testis cDNA subcloned in Bluescript SK II was excised with BamHI and EcoRV and ligated in frame into pGEX-3X. This fragment encodes the carboxyl-terminal 282 amino acid residues of the TLE 3 protein.
  • This fusion construct produced an ⁇ 55-kDa chimeric protein containing the carboxyl terminus of glutathione S-transferase. Fusion proteins were produced and purified according to standard procedures (Smith and Johnson, 1988, Gene 67:31-40) and utilized for immunization of Long Evans rats according to the schedule described in Stifani et al. (1988, Biochem. J. 250:467- 475).
  • the hybridoma cell line C597.4A was obtained from a rat immunized with the TLE 3 fusion protein as described above.
  • TLE both in Drosophila and man the TLE proteins (the terminology "TLE” applies in general to both the fly and human homologs) are present as part of high-molecular-weight (high M r ) multiprotein complexes. This was demonstrated by using a combination of non-denaturing polyacrylamide gel electrophoresis (PAGE), gel filtration, and cross-linking experiments. The results of such investigations are described below, together with the details of the experimental procedures.
  • PAGE polyacrylamide gel electrophoresis
  • HeLA cells were grown at 37°C in an atmosphere of 5% CO 2 , 95% air in the presence of MEM (Eagle) supplemented with non-essential amino acids, 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, and 0.25 ⁇ g/ml fungizone.
  • MEM Eagle
  • Cells were collected by scraping, washed once with ice-cold PBS, resuspended in ice-cold buffer G (50 mM HEPES, pH 7.6, 10 mM iodoacetic acid, 10 mM KC1, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM PMSF, 2 ⁇ M leupeptin, 2.5 ⁇ g/ml aprotinin, 2.5 ⁇ g/ml pepstatin A, and 2.5 ⁇ g/ml antipain), and homogenized by 10 strokes of a Dounce homogenizer (teflon pestle).
  • a Dounce homogenizer teflon pestle
  • the homogenate was centrifuged at 100,000 x g for 1 hr and the supernatant was recovered and immediately loaded onto 3-18% non- denaturing polyacrylamide gels. (This lysis procedure results in the recovery of more than 90% of the total cellular content of TLE proteins in the high-speed supernatant fraction.) Electrophoresis was performed at 150 V for 24 hr using a running buffer containing 90 mM Tris Base, 80 mM boric acid, and 3 mM EDTA (pH 8.4). After electrophoretic transfer of proteins to nitrocellulose, Western blotting experiments were performed as described in Section 6, supra, in the presence of a 1:20 dilution of monoclonal antibody C597.4A.
  • cross-linking reactions facilitated detection of - 110-kD species (see lanes 6 and 9), likely reflecting the association of TLE proteins with low M r components.
  • the apparent molecular weights of some of them indicate that they do not simply represent oligomeric forms of the TLE proteins, but must involve other unrelated proteins.
  • the homogenate was centrifuged at 13,000 x g for 15 min and the supernatant was recovered and used in the cross-linking experiments.
  • HeLa and SUP-Tl cell lysates were prepared as described in Section 7.1 except that the homogenates were centrifuged at 8,000 x g for 5 min.
  • the resulting supernatants were collected and immediately incubated with the cross-linking agent, DTSSP.
  • Cross-linking reactions were carried out for 30 min at room temperature in the presence of the amounts of DTSSP indicated in Figure 11.
  • TLE proteins can interact with other proteins.
  • One such component of these large complexes appears to be a — 17-kD protein(s) that can be detected both in Drosophila and man.
  • the appropriate fractions containing the complex are then subjected to SDS-PAGE analysis (both under reducing and non-reducing conditions), followed by Western blotting with (a) one of the unique monoclonal antibodies described above; and (b) a monoclonal antibody against one or more of the TLE proteins.
  • An individual protein that is a member of the multiprotein TLE complex is identified by its ability (a) after SDS-PAGE under reducing conditions, to be bound by the unique monoclonal antibody; and (b) after PAGE under nondenaturing conditions, to be bound by the unique monoclonal antibody while in a multiprotein complex, which complex is also able to be bound by the anti-TLE antibody.
  • Cross-linking reagents and immunoprecipitation experiments can also be employed.
  • TLE genes Knowledge of the chromosomal location of the TLE genes allows them to find use as markers in the genetic mapping of diseases and disorders.
  • a rat monoclonal antibody against TLE 2 designated as C637.2, was obtained using standard procedures, against the synthetic peptide EEERPSGPGGGG (part of SEQ ID NO:4) at position 202-213 of the TLE 2 amino acid sequence.
  • Monoclonal antibody C637.2 specifically recognizes
  • TLE 2 does not cross-react with TLE 1 , 3, 4 or Drosophila TLE proteins.
  • a rat polyclonal antibody against TLE 1 was obtained using standard procedures, against the synthetic peptide GTDKRRNGPEFS (part of SEQ ID NO:2) at position 210-221 of the TLE 1 amino acid sequence.
  • Antibody ⁇ -TLE 1 specifically recognizes TLE 1 , and does not cross-react with TLE 2, 3, 4 or Drosophila TLE proteins.
  • Monoclonal antibody C597.4A recognizes both human and Drosophila TLE proteins.
  • CAG GGC CAC ACG GAC GGC GCC AGC TGC ATT GAT ATT TCC GAT TAC GGC 1828 Gin Gly His Thr Asp Gly Ala Ser Cys He Asp He Ser Asp Tyr Gly 590 595 600
  • ATC GGG CAG CAG CAG CTC CAG GCG CAG CAC CTC TCC CAT GCC ACA CAC 435 He Gly Gin Gin Gin Leu Gin Ala Gin His Leu Ser His Ala Thr His 125 130 135

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Abstract

L'invention se rapporte à des séquences nucléotidiques de l'activateur de type transducine humaine de gènes TLE fractionnés ainsi qu'à des séquences d'acides aminés de leurs protéines TLE codées. L'invention concerne également des fragments, d'autres dérivés et analogues des protéines TLE humaines ainsi que les anticorps dirigés contre ces dernières. Les acides nucléiques codant ces fragments ou dérivés, la production desdites protéines et dérivés à l'aide, par exemple, de procédés de recombinaision, ainsi que les partenaires de liaison des protéines TLE sont également décrits. L'invention concerne également les séquences de quatre homologues humains distincts du gène TLE drosophila, et les séquences de leurs protéines codées uniques. Dans certains modes de réalisation, l'invention porte sur des dérivés et des analogues des protéines TLE humaines qui sont fonctionnellement actives ou qui comprennent un ou plusieurs domaines d'une protéine TLE humaine dont les domaines Q, GP, CcN, SP, WD-40 ou une séquence WD-40, un site de la caséine kinase II (CKII), un site de la cdc2 kinase (cdc 2), ou un motif de séquence nucléaire de localisation, des séquences consensus pour ces derniers ou toute combinaison de ces derniers.
PCT/US1993/009333 1992-09-30 1993-09-30 Homologues humains de l'activateur du type transducine de genes fractionnes et procedes s'y rapportant WO1994008037A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780300A (en) * 1995-09-29 1998-07-14 Yale University Manipulation of non-terminally differentiated cells using the notch pathway
WO1998036069A1 (fr) * 1997-02-14 1998-08-20 Incyte Pharmaceuticals, Inc. Nouvelle proteine humaine mago nashi
US6004924A (en) * 1991-12-11 1999-12-21 Imperial Cancer Research Technology, Ltd. Protein sequences of serrate gene products
US6436650B1 (en) 1997-07-23 2002-08-20 Yale University Activated forms of notch and methods based thereon
US6692919B1 (en) 1997-07-23 2004-02-17 Yale University Activated forms of notch and methods based thereon
US7754431B2 (en) 2007-11-30 2010-07-13 Applied Genomics, Inc. TLE3 as a marker for chemotherapy

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMBO J., Vol. 7, No. 12, issued 1988, PREISS et al., "The Molecular Genetics of Enhancer of Split, a Gene Required for Embryonic Neural Development in Drosophila", pp. 3917-3927. *
TRENDS GENET., Vol. 7, No. 11/12, issued November/December 1991, ARTAVANIS-TSAKONAS et al., "Choosing a Cell Fate: a View from the Notch Locus", pp. 403-408. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6004924A (en) * 1991-12-11 1999-12-21 Imperial Cancer Research Technology, Ltd. Protein sequences of serrate gene products
US6703489B1 (en) 1995-03-07 2004-03-09 Yale University Antibodies to vertebrate serrate proteins and fragments
US6149902A (en) * 1995-09-29 2000-11-21 Yale University Manipulation of non-terminally differentiated cells using the notch pathway
US5780300A (en) * 1995-09-29 1998-07-14 Yale University Manipulation of non-terminally differentiated cells using the notch pathway
WO1998036069A1 (fr) * 1997-02-14 1998-08-20 Incyte Pharmaceuticals, Inc. Nouvelle proteine humaine mago nashi
US6436650B1 (en) 1997-07-23 2002-08-20 Yale University Activated forms of notch and methods based thereon
US6692919B1 (en) 1997-07-23 2004-02-17 Yale University Activated forms of notch and methods based thereon
US7727732B2 (en) 1997-07-23 2010-06-01 Yale University Methods for identifying modulators of Notch activation
US8222213B2 (en) 1997-07-23 2012-07-17 Yale University Activated amino- and carboxy-terminal forms of Notch
US7754431B2 (en) 2007-11-30 2010-07-13 Applied Genomics, Inc. TLE3 as a marker for chemotherapy
US7816084B2 (en) 2007-11-30 2010-10-19 Applied Genomics, Inc. TLE3 as a marker for chemotherapy
US8785156B2 (en) 2007-11-30 2014-07-22 Clarient Diagnostic Services, Inc. TLE3 as a marker for chemotherapy
US9005900B2 (en) 2007-11-30 2015-04-14 Clarient Diagnostic Services, Inc. TLE3 as a marker for chemotherapy

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