WO1993012141A1 - Nucleotide and protein sequences of the serrate gene and methods based thereon - Google Patents

Nucleotide and protein sequences of the serrate gene and methods based thereon Download PDF

Info

Publication number
WO1993012141A1
WO1993012141A1 PCT/US1991/009240 US9109240W WO9312141A1 WO 1993012141 A1 WO1993012141 A1 WO 1993012141A1 US 9109240 W US9109240 W US 9109240W WO 9312141 A1 WO9312141 A1 WO 9312141A1
Authority
WO
WIPO (PCT)
Prior art keywords
serrate
protein
fragment
nucleic acid
substantially purified
Prior art date
Application number
PCT/US1991/009240
Other languages
French (fr)
Inventor
Robert J. Fleming
Spyridon Artavanis-Tsakonas
Original Assignee
Yale University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yale University filed Critical Yale University
Priority to PCT/US1991/009240 priority Critical patent/WO1993012141A1/en
Priority to CA002125767A priority patent/CA2125767A1/en
Priority claimed from CA002125767A external-priority patent/CA2125767A1/en
Publication of WO1993012141A1 publication Critical patent/WO1993012141A1/en

Links

Classifications

    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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 Serrate genes and their encoded protein products.
  • invention also relates to derivatives and analogs of the Serrate protein. Production of Serrate proteins, derivatives, and antibodies is also provided.
  • zygotically-acting genes the so called neurogenic loci, which affect the choice of ectodermal cells between epidermal and neural pathways (Poulson, 1937, Proc. Natl. Acad. Sci. 23:133-137; Lehmann et al.,
  • Enhancer of Split (E(spl), neuralized (neu), and big brain (bib) -- result in hypertrophy of the nervous system at the expense of ventral and lateral epidermal structures. This effect is due to the misrouting of epidermal precursor cells into a neuronal pathway, and implies that neurogenic gene function is necessary to divert cells within the neurogenic region from a neuronal fate to an epithelial fate. Serrate has been identified as a genetic unit capable of interacting with the Notch locus (Xu et al., 1990, Genes Dev.
  • Notch and Delta appear to encode transmembrane proteins that span the membrane a single time.
  • the Notch gene encodes a ⁇ 300 kd protein (we use “Notch” to denote this protein) with a large N-terminal extracellular domain that includes 36 epidermal growth factor (EGF)-like tandem repeats followed by three other cysteine-rich repeats, designated Notch/lin-12 repeats (Wharton, et al., 1985, Cell 43:567-581; Kidd and Young, 1986, Mol. Cell. Biol. 3:194-3108; Yochem, et al., 1988, Nature 335:547-550).
  • Delta encodes a ⁇ 100 kd protein (we use "Delta” to denote DLZM, the protein product of the predominant zygotic and
  • EGF-like motif has been found in a variety of proteins, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell
  • the present invention relates to nucleotide sequences of Serrate genes, and amino acid sequences of their encoded proteins, as well as derivatives
  • the Serrate protein is a human protein.
  • the invention relates to Serrate 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) Serrate 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, 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 Serrate which comprise one or more domains of the Serrate protein, including but not limited to the intracellular domain, extracellular domain, transmembrane domain, membraneassociated region, or one or more EGF-like
  • underscoring the name of a gene shall indicate the gene, in contrast to its encoded protein product which is indicated by the name of the gene in the absence of any underscoring.
  • Serrate shall mean the Serrate gene
  • Serrate shall indicate the protein product of the Serrate gene.
  • Mutant wing shows typical "fig leaf” shape, distorted wing veins, and loss of the majority of marginal bristles and hairs, with the exception of the anterodistal wing margin.
  • the extra N + copy suppresses the heterozygous Ser D dominant
  • Figure 3 Molecular map of the Serrate- encoding region. Approximately 85 kb of cloned genomic DNA from the 97F chromosomal region are presented along with the restriction sites of three enzymes [(B) BamHI; (E) EcoRI; (H) HindII l]. The locations of individual DNA alteratiodns associated with Serrate allelic breakpoints are displayed above the genomic DNA (for descriptions of mutant alleles, see Section 6, infra; (rev 3 and rev 2-11) Ser rev3 and Ser rev2-11 , respectively; (R128) T(Y:3)R128. The shaded box from coordinates 0 to +3 represents the region of EGF homology detectable by Southern hybridization.
  • the BamHI site adjacent to the EGF homology was arbitrarily chosen as position 0. Map orientation is with the centromere to the left. At the bottom of the figure are shown the individual recombinant phage isolates.
  • FIG. 4 Serrate sequence analysis.
  • the complete 5561 bp sequence (SEQ ID NO:1) derived from cDNAs C1 and C3 is shown. Nucleotide numbering is at left, amino acid numbering of the predicted open reading frame (ORF) is at right.
  • the deduced protein product appears to be a transmembrane protein of 1404 amino acids (SEQ ID NO: 2). Hydrophobic regions are denoted inside dashed boxes; amino acids 51 to 80 represent the likely signal peptide; amino acids 542 to 564 represent the potential membrane associated region; amino acids 1221 to 1245 represent the
  • the first cysteine of each of the fourteen EGF-like repeats is denoted with a solid black box, and each repeat is underlined.
  • the partial EGF-like repeat is considered "degenerate," since the fourth cysteine residue of this repeat has been changed to lysine (shown in boldface type at amino acid position 268).
  • the initial cysteine of this repeat is denoted with an open box (amino acid 284), and the repeat is underlined. Amino acid insertions occur in the fourth, sixth, and tenth EGF- like repeats and are denoted by hatched underlines.
  • FIG. 5 The Serrate transcript and deduced protein product.
  • the composite transcript shown was constructed from the C1 and C3 cDNAs, which overlap by 109 bp. Selected restriction enzyme cleavage sites are shown. The hatched box represents the 4212 bp ORF. Open boxes represent the 442 bp 5'- untranslated leader and 900 bp 3'-trailer sequence,
  • SP Putative signal peptide;
  • SP signal peptide
  • TM transmembrane domain
  • Each lane contains five ⁇ g of poly(A) + RNA.
  • the stage of the embryonic RNAs is denoted in hours after egg laying; (L1, L2, and L3) RNA from the first, second and third larval instar periods; (EP and LP) early and late pupal stages; (M and F) adult male and female RNAs, respectively.
  • a composite cDNA subclone (constructed from C1 and C3) was used as a hybridization probe.
  • transcription is represented primarily as a 5.5 kb and 5.6 kb doublet beginning at 4-8 hours of
  • Embryos are oriented with anterior to the left and dorsal side up unless otherwise noted, (a) Dorsal view of an early stage 10 embryo (middorsal focal plane). Earliest expression occurs in the ectoderm of the foregut (FG) and presumptive clypeolabrum (CL). (b) Dorsal view of a germ bandextended embryo (late stage 10). Additional
  • VE presumptive trachea
  • T Ventrally, note different expression (VE) patterns in the thoracic versus abdominal segments.
  • VE expression
  • g Lateral view of an early stage 14 embryo. Outline of the presumptive trachea (T) is distinct from the overlying epidermal expression. Arrows denote the zigzag pattern of lateral expression.
  • h Dissected embryo (stage 14) opened along the dorsal midline and laid flat. Two areas of hindgut expression (HG1 and HG2) are two areas of hindgut expression.
  • Serrate see SEQ ID NO: 2 is shown above line, Delta (SEQ ID NO: 4) below. The sequence begins at Serrate amino acid position 59; the partial EGF-like repeat of both Serrate and Delta is boxed.
  • the Serrate amino acid sequence (amino acids 79-282 of Fig. 4) placed into the chimeric ⁇ EGF Notch construct and determined to be sufficient for Notch binding is presented in boldface type. The positions of the synthetic
  • degenerate primers (designated FLE1 through FLE4R) are shown; refer to Figure 9 for nucleotide composition.
  • FIG. 9 Nucleotide comparison of aminoterminal Serrate-Delta homology.
  • the nucleotide sequence corresponding to the amino acid sequence in Figure 8 is shown (Serrate sequence: see SEQ ID NO:1; Delta sequence: SEQ ID NO: 3).
  • the DNA encoding the partial EGF-repeat is boxed.
  • the Serrate nucleotide sequence (nucleotides 676-1287 of Fig. 4) placed into the chimeric ⁇ EGF Notch construct determined to be sufficient for Notch binding is presented in boldface type.
  • the sequences of the synthetic degenerate primers are given against black backgrounds.
  • the present invention relates to nucleotide sequences of Serrate genes, and amino acid sequences of their encoded proteins.
  • the invention further relates to fragments and other derivatives, and analogs, of Serrate proteins. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention.
  • the Serrate protein is a human protein. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is provided.
  • the invention relates to Serrate 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) Serrate 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, 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 Serrate which comprise one or more domains of the Serrate protein, including but not limited to the intracellular domain, extracellular domain, transmembrane domain, membrane- associated region, or one or more EGF-like (homologous) repeats of a Serrate protein, or any combination of the foregoing.
  • Antibodies to Serrate are additionally provided.
  • Serrate 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 Serrate mRNA of human and other species, to study expression thereof, to produce Serrate and 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.
  • the invention is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of D. melanogaster Serrate (Section 6); the construction and recombinant expression of a
  • Serrate chimeric/fusion derivative and production of antibodies thereto (Section 7); the recombinant expression of Serrate, a Serrate fragment lacking the EGF-like repeats present in Serrate, and a chimeric Notch-Serrate derivative, and assays for binding to Notch (Section 8); and the cloning of a human Serrate homolog (Section 9).
  • the invention relates to the nucleotide sequences of Serrate consisting of at least 8
  • nucleotides i.e., a hybridizable portion.
  • the invention relates to the nucleic acid sequence of the human Serrate gene. In another embodiment, the invention relates to the
  • Drosophila Serrate gene in a preferred, but not limiting, aspect of the invention, a Drosophila
  • Serrate DNA sequence (ATCC Accession Number __ ) can be cloned and sequenced by the method described in Section 6, infra.
  • the invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences.
  • a human expression library 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 Serrate product. In one embodiment, anti-Serrate antibodies can be used for selection.
  • an expression vector e.g., a bacteriophage derivative
  • PCR is used to amplify the desired sequence in the library, prior to selection.
  • Oligonucleotide primers representing known Serrate sequences can be used as primers in PCR.
  • oligonucleotides may be obtained
  • oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA),
  • an oligonucleotide primer pair used for PCR with a cDNA library is FLE1/FE3R,
  • FLE1/FLE4R, FLE2/FLE3R, or FLE2/FLE4R shown in Fig. 9. (see SEQ ID NO:1) (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 mRNA or cDNA or genomic DNA from any
  • Any eukaryotic cell potentially can serve as the nucleic acid source for the molecular cloning of the Serrate gene.
  • the nucleic acid sequences encoding Serrate can be isolated from human, porcine, bovine, feline, avian, equine, canine, as well as additional primate sources, insects, etc. For example, we have amplified fragments of the appropriate size in
  • Drosophila mouse, Xenopus, and human
  • 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. (See, for example, Sambrook et al.,
  • 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.
  • DNA fragments are generated, some of which will encode the desired gene.
  • the DNA may be cleaved at specific sites using various restriction enzymes.
  • DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by
  • linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and
  • 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
  • Serrate of any species gene or its specific RNA, or a fragment thereof, e.g., an extracellular domain (see Section 5.6), 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;
  • 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, receptor binding activity, in vitro aggregation activity ("adhesiveness") or antigenic properties as known for Serrate.
  • the Serrate protein may be identified by binding of labeled antibody to the putatively Serrate synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay) -type procedure.
  • the Serrate 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
  • DNA fragments may represent available, purified Serrate DNA of another species (e.g., Drosophila).
  • Immunoprecipitation analysis or functional assays e.g., aggregation ability in vitro; binding to receptor; 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.
  • specific mRNAs may be
  • a radiolabelled Serrate 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 Serrate DNA fragments from among other genomic DNA fragments.
  • genomic DNA include, but are not limited to,
  • RNA for cDNA cloning of the Serrate gene can be isolated from cells which express Serrate. 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. 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
  • linkers onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • the cleaved vector and Serrate gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection,
  • 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.
  • transformation of host cells with recombinant DNA molecules that
  • 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.
  • nucleotide sequence coding for a Serrate 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
  • 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, the adhesive portion of the Serrate gene is expressed. In other specific
  • the human Serrate gene is expressed, or a sequence encoding a functionally active portion of human Serrate.
  • a fragment of Serrate comprising the extracellular domain, or other derivative, or analog of Serrate is expressed.
  • transcriptional/translational control signals and the protein coding sequences may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).
  • nucleic acid sequence encoding a Serrate protein or peptide fragment may be regulated by a second nucleic acid sequence so that the Serrate protein or peptide is expressed in a host transformed with the recombinant DNA molecule.
  • expression of a Serrate protein may be controlled by any promoter/enhancer element known in the art.
  • Promoters which may be used to control toporythmic gene expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon,
  • 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 ⁇ -lactamase promoter (Villa- Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al.,
  • promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK
  • 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,
  • 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
  • Expression vectors containing Serrate 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 toporythmic gene.
  • 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.
  • certain "marker" gene functions e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
  • 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 Serrate gene product in vitro assay systems, e.g., aggregation (binding) with Notch, binding to a receptor, binding with antibody.
  • recombinant expression vectors can be propagated and prepared in quantity.
  • the expression vectors which can be used include, but are not limited to, the following vectors or their
  • human or animal viruses such as vaccinia virus or adenovirus
  • insect viruses such as
  • 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 Serrate protein may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage [e.g., of signal sequence]) of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • expression in a bacterial system can be used to produce an unglycosylated core protein product.
  • Expression in yeast will produce a glycosylated product.
  • Expression in mammalian cells can be used to ensure "native" glycosylation of a heterologous mammalian toporythmic protein.
  • different vector/host expression systems may effect processing reactions such as proteolytic cleavages to different extents.
  • cDNA and genomic sequences can be cloned and expressed.
  • Serrate 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
  • Serrate protein Once the Serrate protein is identified, it may be isolated and purified by standard methods including chromatography (e.g., ion exchange,
  • the amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant.
  • the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller, M., et al., 1984, Nature 310:105-111).
  • Serrate proteins whether produced by recombinant DNA techniques or by chemical synthetic methods, include but are not limited to those
  • the cloned DNA or cDNA corresponding to the Serrate gene can be analyzed by methods including but not limited to Southern hybridization (Southern, E.M., 1975, J. Mol. Biol. 98:503-517), Northern
  • Serrate-specific probe can allow the detection of the Serrate gene in DNA from various cell types.
  • Southern hybridization can be used to determine the genetic linkage of Serrate.
  • Northern hybridization analysis can be used to determine the expression of the Serrate gene.
  • Various cell types, at various states of development or activity can be tested for Serrate expression. Examples of such techiques and their results are described in Section 6, infra. The stringency of the hybridization
  • hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of
  • Restriction endonuclease mapping can be used to roughly determine the genetic structure of the
  • cleavage with restriction enzymes can be used to derive the restriction map shown in Figure 3, infra. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis.
  • 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, F., et al., 1977, Proc. Natl. Acad. Sci.
  • representative Serrate gene comprises the sequence substantially as depicted in Figure 4, and described in Section 6, infra.
  • the amino acid sequence of the Serrate 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.
  • representative Serrate protein comprises the sequence substantially as depicted in Figure 4, and detailed in
  • the Serrate protein sequence can be further characterized by a hydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad. Sci. U.S.A.
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the Serrate 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.
  • Serrate protein its fragments or other derivatives, or analogs
  • antibodies 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 to human Serrate are produced.
  • sequence depicted in Figure 4, or a subsequence thereof can be obtained.
  • various host animals can be immunized by injection with the native Serrate 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,
  • BCG Bacille Calmette-Guerin
  • monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545).
  • human antibodies may be used and can be obtained by using human hybridomas
  • idiotype 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). For example, to select ELISA (enzyme-linked immunosorbent assay).
  • antibodies which recognize a specific domain of a Serrate protein one may assay generated hybridomas for a product which binds to a Serrate fragment containing such domain. For selection of an antibody specific to human Serrate, one can select on the basis of positive binding to human Serrate and a lack of binding to Drosophila Serrate.
  • 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
  • the invention further relates to derivatives (including but not limited to fragments) and analogs of Serrate proteins.
  • the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional
  • Such molecules which retain, or alternatively inhibit, a desired Serrate property, e.g., binding to Notch or other toporythmic proteins, binding to a cell-surface receptor, can be used as inducers, or inhibitors, respectively, of such
  • a specific embodiment relates to a Serrate fragment that can be bound by an anti-Serrate antibody but cannot bind to a Notch protein or other toporythmic protein.
  • Derivatives or analogs of Serrate 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.
  • Serrate derivatives can be made by altering Serrate 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 Serrate gene may be used in the practice of the
  • the Serrate 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 Serrate 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
  • 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
  • the polar neutral amino acids include glycine, serine,
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Derivatives or analogs of Serrate include but are not limited to those peptides which are substantially homologous to Serrate or fragments thereof, or whose encoding nucleic acid is capable of hybridizing to a Serrate nucleic acid sequence.
  • Serrate 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 Serrate gene sequence can be modified by any of numerous strategies known in the art (Maniatis, T., 1990, Molecular Cloning, A
  • Serrate uninterrupted by translational stop signals, in the gene region where the desired Serrate activity is encoded.
  • Serrate-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
  • Serrate sequence may also be made at the protein level. Included within the scope of the invention are Serrate protein
  • analogs and derivatives of Serrate can be chemically synthesized.
  • a peptide corresponding to a portion of a Serrate protein which comprises the desired domain see
  • 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, ⁇ -alanine, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids.
  • the Serrate derivative is a chimeric, or fusion, protein
  • such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a Serrate-coding sequence joined in-frame to a non-Serrate coding sequence).
  • a nucleic acid encoding the protein comprising a Serrate-coding sequence joined in-frame to a non-Serrate coding sequence.
  • 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.
  • such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • a chimeric nucleic acid encoding a mature Serrate protein with a heterologous signal sequence is expressed such that the chimeric protein is expressed and processed by the cell to the mature Serrate protein.
  • a recombinant molecule can be constructed according to the invention, comprising coding portions of both Serrate and another toporythmic gene, e.g., Delta.
  • the encoded protein of such a recombinant molecule could exhibit properties associated with both Serrate and Delta and portray a novel profile of biological activities, including agonists as well as antagonists.
  • the primary sequence of Serrate and Delta may also be used to predict tertiary structure of the molecules using computer simulation (Hopp and Woods, 1981, Proc.
  • Serrate/Delta chimeric recombinant genes could be designed in light of correlations between tertiary structure and biological function. Likewise, chimeric genes comprising portions of Serrate fused to any heterologous protein-encoding sequences may be
  • a specific embodiment relates to a chimeric protein comprising a fragment of Serrate of at least six amino acids.
  • a particular example of the construction and expression of a Notch-Serrate chimera is presented in Section 8 hereof. A particular
  • the Serrate derivative is a fragment of Serrate comprising a region of homology with another toporythmic protein.
  • a region of a first protein shall be considered "homologous" to a second protein when the amino acid sequence of the region is at least 30% identical or at least 75% either identical or
  • such a Serrate fragment can comprise one or more regions homologous to Delta, including but not limited to Serrate amino acids 63-73, 124-134, 149- 158, 195-206, 214-219, 250-259, or 79-282 (or 79-246, excluding the partial EGF-like repeat) (see Figs. 4, 8), or portions of Serrate of other species most homologous to the foregoing sequences.
  • the invention relates to Serrate derivatives and analogs, in
  • Serrate protein including but not limited to the extracellular domain, transmembrane domain,
  • EGF-like repeats EGF-like repeats
  • Serrate protein In particular examples relating to the Drosophila Serrate protein (see example 6), such domains are identified as follows, with reference to Figure 4: extracellular domain, amino acids numbers
  • the fragments comprising specific portions of Serrate are those comprising portions in the respective Serrate protein most homologous to specific fragments of the Drosophila Serrate protein.
  • a fragment comprising a domain of a Serrate homolog can be identified by protein analysis methods as described in Section 5.3.2 or 6.
  • Serrate derivatives which are Serrate fragments and chimeric/fusion proteins are described by way of example in Sections 7 and 8 infra.
  • the invention also provides for Serrate fragments, and analogs or derivatives of such
  • toporythmic proteins and nucleic acid sequences encoding the foregoing.
  • the adhesive fragment of Serrate is that comprising the portion of Serrate most homologous to about amino acid numbers 85-283 or 79-282 of the Drosophila Serrate sequence (see Figure 4).
  • toporythmic protein preferably Notch
  • the ability of a Serrate fragment to bind to a Notch protein can be demonstrated by detecting the ability of the Serrate fragment, when expressed on the surface of a first cell, to bind to a Notch protein expressed on the surface of a second cell.
  • toporythmic proteins or adhesive domains 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 toporythmic genes can be identified.
  • Serrate 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,
  • 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.
  • one can carry out an in vitro aggregation assay such as described infra in Section 8.2.1 (see also Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699).
  • receptor binding can be any substance that Serrate is identified, receptor binding can be any substance that Serrate is identified, receptor binding can be any substance that Serrate is identified, receptor binding can be any substance that Serrate is identified, receptor binding can be any substance that Serrate is identified, receptor binding can be any substance that Serrate is identified, receptor binding can be any substance that Serrate is identified, receptor binding can be any substance that Serrate is identified, receptor binding can be any substance that Serrate is identified, receptor binding can be
  • Serrate binding to cells expressing a Serrate receptor can be assayed.
  • mutations in the third chromosome gene Serrate are shown to display genetic interactions with specific alleles of the neurogenic locus Notch, which encodes a transmembrane protein with epidermal growth factor homology.
  • the locus Serrate displays a striking phenotypic interaction with a specific Notch allele known to affect postembryonic development.
  • We present the molecular cloning of Serrate and show that it encodes two coordinately-expressed transcripts from a genomic interval greater than 30 kilobases in length.
  • the deduced protein product of 1404 amino acids contains a single transmembrane domain and 14
  • Serrate locus encodes an essential function, the loss of which results in embryonic lethality brought about by the disruption of both neuronal and epidermal tissues. Serrate is likely to represent an element in a network of interacting molecules operating at the cell surface during the differentiation of certain tissues.
  • Notch + Animals carrying an extra copy of Notch + normally exhibit a Confluens phenotype characterized by wing vein thickening. Surprisingly, animals bearing Ser D and an extra copy of Notch + have
  • Ser D phenotype insensitivity of the Ser D phenotype to the number of wildtype (Ser + ) copies present, that is Ser D /+/+ displays a phenotype similar to Ser D /+ and to
  • Ser D /Deficiency P. Lewis, pers. comm.
  • Flies with only one copy of wild-type Ser + i.e., individuals heterozygous for a wild-type allele over deficiencies
  • Ser D mutation is homozygous, viable adults are produced that display a more severe wing phenotype than heterozygous Ser D /+ animals ( Figure If).
  • the expression of the Ser D wing phenotype appears to be directly related to the expression of a mutant or novel gene product rather than to Ser + gene dosage.
  • transheterozygotes of nd with two different Ser revertants alleles exhibit an enhanced mutant wing phenotype as compared to nd
  • Bd G Goldschmidt (Bd G ), which also maps to the 97F region. Heterozygous adults bearing the Bd G mutation display a wing nicking phenotype that is more severe than that observed in Ser D heterozygotes (data not shown).
  • Bd G mutation unlike Ser D , is homozygous lethal.
  • Df(3R ⁇ Ser +82f24 exhibit embryonic lethality.
  • Cuticle preparations of unhatched embryos from heterozygous parents revealed a continuous and complex range of phenotypes: The progeny of a single allele from a single brood included individuals that were nearly wild-type in appearance as well as those lacking the majority of differentiated cuticle. Another commonly observed defect was the failure of germ band
  • phenotypes are exemplified by embryos with retracted germ bands that lack the cuticle of the entire
  • Fig. 2c cephalic regions and sometimes the first and second thoracic segments.
  • Other embryos fail to retract the germ band and lack head and thoracic structures, may exhibit twisted germ bands, and/or frequently lack large patches of dorsal or ventral cuticle.
  • Fig. 2d only a small cuticular patch remains (Fig. 2d).
  • the cuticle that remains in Ser D mutant embryos can be of dorsal or ventral origin.
  • anti-HRP anti-horseradish peroxidase
  • Notch gene (Rothberg et al., 1988, Cell 55:1047-1059), was used as an entry point to initiate a chromosomal walk. From this initial clone, eight recombinant phage spanning ⁇ 85 kb of genomic DNA were isolated (see Fig. 3). A BamHI site adjacent to the region of EGF homology was arbitrarily chosen as coordinate position zero.
  • Genomic Southern blots containing mutant and wild-type DNAs were probed with DNA from the
  • Ser revertant alleles Of the five Ser revertant alleles, three (Ser rev2-3 ,Ser rev5-5 , and Ser rev6-1 ) appeared cytologically normal and did not exhibit DNA polymorphisms detectable by our Southern analyses. The remaining two revertants,
  • Ser rev2-11 and Ser rrev3 , had polymorphic DNA restriction fragments within the cloned region.
  • Ser rev2-11 is an inversion of polytene bands 97F to 98C. The 97F breakpoint was localized between coordinates +1.5 to +4, within the region of strongest detectable EGF homology (Fig. 3).
  • Ser rev3 is a reciprocal
  • polymorphisms represent the 97F breakpoints of these chromosomal rearrangements.
  • T(Y:3)R128 is a reciprocal translocation that also breaks within the 97F region (Lindsley et al., 1972, Genetics
  • C1 and C3 Two overlapping cDNA clones, denoted C1 and C3, from an early pupal library (see Section 6.3). Sequence analysis of these cDNAs revealed a perfect overlap of 109 bp for a combined length of 5.6 kb, which is in excellent agreement with the larger of the two transcripts as determined by Northern analysis. Genomic probes unique to the 5' end of C3 only
  • the size difference between the 5.5 and 5.6 kb transcripts may represent an alteration in the potential protein coding capacity or an alteration of 5' untranslated sequence.
  • the composite 5.6 kb cDNA confirms that the Serrate transcription unit spans ⁇ 30 kb of genomic DNA, encompasses the EGF homologous region, and is interrupted by at least five of the six DNA
  • the complete nucleotide sequence compiled from the cDNAs C1 and C3 is 5561 bp (see Fig. 4) and agrees with the transcript sizes determined by Northern analysis. Within this sequence there is a single large open reading frame (ORF) of 4329 bp. There are two possible initiator AUG codons at positions 433 and 442. Of these, the second AUG is within a sequence context that agrees with the Drosophila consensus sequence determined for translation initiation
  • the Serrate mRNA contains an untranslated leader sequence of at least 441 base pairs, encodes an expected protein product of 1404 amino acids, and terminates with 908 bp of
  • Hydropathy plots revealed three major hydrophobic regions (Fig. 5b; see also Section 6.3). The first, beginning at amino acid 51, is likely to represent a signal peptide sequence; a potential signal cleavage site occurs at amino acid 80. A second hydrophobic domain runs from amino acid 540 to 560. This region does not have a requisite
  • the third hydrophobic domain (amino acids 1220 to 1245) is bounded by
  • the most striking structural feature of the predicted protein is the series of EGF-like repeats (see Fig. 5c). There are 14 copies of this motif with an additional partial or degenerate repeat occurring toward the amino terminus (see below). In addition, at least three of these repeats are interrupted by stretches of amino acids. The first interruption
  • the second interruption (labelled B in Fig. 5c), occurring in the sixth repeat, is ⁇ 44 amino acids long and has numerous hydrophobic residues. This region represents the putative membrane-associated domain noted earlier.
  • the final interruption (labelled C in Figure 5c), which occurs in us tenth repeat and is 29 amino acids in length, has an unusual run of
  • the latter region corresponds to the partial EGF-like repeat (designated PR in Fig. 5c), which lacks a cysteine residue but retains the other characteristic cysteines and conserved amino acids typically found in the remaining EGF-like repeats.
  • the homology between Serrate and Delta extends beyond these amino-terminal regions, since both of these proteins contain EGF-like repeats.
  • the predicted Serrate protein contains a small intracellular domain of ⁇ 160 amino acids.
  • the internal domain does not contain any significant known structural homologies, although there are numerous potential sites for phosphorylation (Those identified in the putative intracellular region by the SITES program were at amino acid positions 1283, 1292, 1297, 1349, 1365, 1371, 1389, and 1390).
  • transcripts of 5.5 kb and 5.6 kb which first appear 4 to 8 hours into embryogenesis (Fig. 6).
  • transcripts show peak expression between 8 and 12 hours of embryogenesis and diminish thereafter;
  • a smaller (3.4 kb) transcript is expressed transiently between 2 and 4 hours of embryogenesis (Fig. 6).
  • RNA localizations may represent a composite for both transcripts.
  • Serrate RNA in the yolk of pre-gastrulation embryos because faint staining of the yolk was observed consistently. Although this staining was never observed with control probes (see
  • the observed staining may correspond to the expression of the transient 3.4 kb RNA species observed by the Northern analysis of this same developmental stage.
  • each abdominal segment displayed a cluster of cells on either side of the ventral midline.
  • stage 12 the lateral epidermal cell patches broadened to form stripes that lie in the middle of each segment. A portion of these cells appeared to coalesce into an internal longitudinal stripe that was coincident with the developing tracheae (see Fig. 7 e, f, g, and h). The cells that remained on the surface extended dorsally and ventrally forming a zig-zag shaped pattern (Fig. 7g, arrows). This surface expression in the thoracic segments was wider, more intense, and extended further dorsally and ventrally than in the abdominal segments (Fig. 7g). Later in embryogenesis (stages 14 and 15) the surface epidermal expression, with the exception of the first thoracic segment, diminished relative to the tracheal expression.
  • pockets of cells may correspond to primordia of imaginal discs; in the first thoracic segment they appeared to be closely associated with opening of the anterior spiracle.
  • Hindgut expression though appearing later than foregut expression, occurred at an analogous position, that is, where ectoderm meets endoderm.
  • the initial expression in the hindgut was seen at the time of germ band retraction (stage 12) as a wide band of cells where the Malphigian tubules were forming, but never included the tubules themselves.
  • stage 14 an additional ring of expression appeared in the hindgut approximately mid-way between the insertion point of the Malphigian tubules and the proctodeum (Fig. 7h). Expression at the posterior-most end of the embryo, near the proctodeal opening, initiated early (stage 11) (Fig. 7b). This expression within the telson remained at high levels throughout
  • the fourteen EGF repeats of Serrate are not completely contiguous. At least three of these repeats contain sizeable
  • glycoprotein glutactin a basement membrane protein of Drosophila (Olson et al., 1990, EMBO J. 9:1219-1227).
  • Serrate transcripts are localized in complex patterns within these tissues.
  • the abundant and widespread expression of Serrate transcripts in the segments that make up the embryonic head and thorax correlates well with the lack of embryonic head and thoracic structures
  • Serrate is expressed in a small number of cells within the CNS, the gross morphological defects observed in the CNS of Ser- embryos may reflect contributions from two components. The first is the loss of Serrate CNS expression itself, and the second may be a consequence of mechanical stresses (e.g., lack of germ band retraction) imposed by an improperly differentiating epidermis.
  • EGF receptor homolog known as DER faint little ball or torpedo (Livneh et al., 1985, Cell 40:599-607;
  • Ser D , Df (3R) Ser +82f24 , and Bd G chromosomes were obtained from Peter Lewis.
  • the red Ser D chromosome was maintained in homozygous condition.
  • the mutations pll 11 , Bd 862.5 , and Bd 43.5 were generously provided by Kathryn Anderson.
  • the Notch duplication CosP479 is an ⁇ 40 kb P-element cosmid construct inserted into the third chromosome (Ramos et al., 1989, Genetics
  • peroxidase antibody staining of the embryonic nervous system was carried out using fluoresceinconjugated antibody (Cappel) as described in Preiss et al. (1988, EMBO J. 7:3917-3927). CNS preparations of torpedo 2C82 were used for comparison studies.
  • DNA probes labeled with 32 P were prepared by random oligonucleotide priming, as described in Feinberg and Vogelstein
  • guanidinium thiocyanate essentially as described in Chirgwin et al. (1979, Biochem. 18:5294-5299).
  • Pupal and adult RNAs were generously provided by A. Preiss (Preiss et al., 1988, EMBO J. 7:3917-3927).
  • Poly (A) + RNA was selected by serial passage over oligo(dT)- cellulose (Stratagene) according to Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring
  • the EcoRI cDNA inserts from ⁇ gt10 were subcloned directly into Bluescript KS+ and KS- vectors (Stratagene). Single-stranded DNAs were produced according to the manufacturer's instructions. Both strands of the cDNAs were sequenced using the
  • Proteinase K (Boehringer-Mannheim) treatment was 10 to 14 minutes; 100 ⁇ l (rather than 10 ⁇ l) of boiled probe was used; after washing the embryos with 1:4 hybridization buffer to PBT, they were washed twice in PBT for 20 minutes, and then twice in 1X PBS, 0.1% BSA (globin free, Sigma), 0.2% Triton-X100 for 20 minutes; the antibody treatment was done in the same PBS, BSA, Triton solution at 4°C overnight; the embryos were washed four times in the PBS, BSA, Triton solution at room temperature; after the alkaline phosphatase reaction, embryos were dehydrated twice in 70% and 100% ethanol and then cleared in xylenes; the embryos were mounted in Permount (Sigma). Dissected embryos were rehydrated, dissected in PBT, and mounted in 90% glycerol [10% Tris-HCl at pH 8.0, with 0.5% n-propylgalate (wt/vol;
  • the probe was made by runoff of a PCR reaction in 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 0.01% (wt/vol) gelatin, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.15 mM dTTP, and 0.07 mM digoxigenin-11-dUTP (Boehringer Mannheim) using 150 ng of custom synthesized primer and approximately 400 ng of linearized DNA. Probe was synthesized from cDNA coordinates 4826 to 3854; the opposite strand
  • control probe constituted the control probe and was synthesized from coordinates 4458 to 5015 (refer to Fig. 6).
  • the conditions for the PCR thermal cycler were 95°C for 45 seconds, 55°C for 30 seconds, and 72°C for 1 minute, which were run for 30 cycles.
  • the probe was ethanol precipitated twice and resuspended in 300 ⁇ l of hybridization solution.
  • Mouse anti-Serrate polyclonal antisera were made as follows: A BamHI fragment encoding amino acids 78-425 (Fig. 4) was subcloned into the pGEX-1 expression vector (Smith and Johnson, 1988, Gene
  • the Serrate Kpnl fragment (571 - 2981) was inserted and the proper orientation selected, to generate BTSer5 'PCR-Kpn.
  • BTSer5'PCR-Kpn (SacII sites in Bluescript polylinker and in Serrate (1199)) was isolated and used to replace the 5' SacII fragment of cDNA C1 (Fleming et al., 1990, Genes & Dev. 4:2188-2201), thus
  • a Serrate deletion expression construct was also made, in which nucleotides 672-1293 (encoding amino acids 77-284) (Figs. 4, 8, 9) were deleted.
  • This deletion construct was made as follows: The Sermtn construct was digested with EcoRV. which cuts at nucleotide 672, and with Sfil, which cuts at
  • nucleotide 4073 The linearized vector, lacking the EcoRV-Sfil (672-4073) fragment, was isolated. Plasmid SerFL was then digested with Ndel, which cuts at nucleotide 1289, and treated with mung bean nuclease resulting in the "trimming back" of four bases. The resulting SerFL fragment was then digested with Sfil which cuts at base 4073, and the resulting 1293-4073 fragment was isolated and ligated into the EcoRV-Sfil vector isolated above.
  • Notch-Serrate chimeric construct was made using a clone consisting of Drosophila Notch cDNA with a deletion of all the Notch EGF-like repeats (" ⁇ EGF") (see copending application Serial No. to be assigned, filed November 14, 1991 by Artavanis- Tsakonas et al.; Rebay et al., 1991, Cell 67:687-699 (Fig. 13, construct no. 25)).
  • ⁇ EGF Notch EGF-like repeats
  • Serrate EGF-like repeats (Serrate nucleotide numbers 676-1287, encoding amino acids 79-282; Figs. 8, 9) was placed into the ⁇ EGF deletion of Notch.
  • the above constructs were expressed in Drosophila S2 cells.
  • the S2 cell line (Schneider, 1972, J. Embryol. Exp. Morph. 27, 353-365) was grown in M3 medium (prepared by Hazleton Co.) supplemented with 2.5 mg/ml Bacto-Peptone (Difco), 1 mg/ml TC
  • Yeastolate (Difco), 11% heat-inactivated fetal calf serum (FCS) (Hyclone), and 100 U/ml penicillin-100 ⁇ g/ml streptomycin-0.25 ⁇ g/ml fungizone (Hazleton).
  • aggregation assays Two types were used.
  • a total of 3 ml of cells (5-10 x 10 6 cells/ml) was placed in a 25 ml Erlenmeyer flask and rotated at 40-50 rpm on a rotary shaker for 24-48 hr at room temperature.
  • cells were mixed 1-4 hr after induction began and induction was continued throughout the aggregation period.
  • ⁇ 0.6 ml of cells were placed in a 0.6 ml Eppendorf tube (leaving a small bubble) after an overnight induction (12-16 hr) at room temperature and rocked gently for 1-2 hr at 4°C. Ca 2+ dependence experiments were performed using the latter assay.
  • BSS-FCS balanced saline solution
  • PCS was dialyzed against 0.9% NaCl, 5 mM Tris [pH 7.5]) or in Ca 2+ free BSS-FCS containing 10 mM EGTA (Snow et al., 1989, Cell 59: 313-323) and then resuspended in the same medium at the original volume.
  • Notch-expressing cells for the assays were obtained similarly, using metallothionein promoterdriven plasmid constructions containing D.
  • Serrate did not appear to interact homotypically.
  • we detect no interactions between Serrate and Delta It is possible that such interactions do occur, but at an affinity such that they are below the level of detection in our assay system.
  • Notch EGF-like repeats 11 and 12 in addition to binding to Delta, also mediate
  • Serratebinding function of these repeats also appears to have been conserved in the corresponding two EGF repeats of Xenopus Notch (construct #33 ⁇ Cla+XEGF(10-13); see Rebay et al., supra).
  • Notch and Serrate appeared to aggregate less efficiently than Notch and Delta, perhaps because the Notch-Serrate interaction is weaker. For example, when scoring Notch-Delta aggregates, we detect ⁇ 40% of all Notch expressing cells in clusters with Delta expressing cells and ⁇ 40% of all Delta expressing cells in contact with Notch expressing cells. For Notch-Serrate, we find only -20% of all Notch
  • Notch-Delta may indicate a fundamental functional difference between Notch-Delta and Notch-Serrate interactions that may be significant in vivo.
  • PCR polymerase chain reaction
  • PCR reaction products were subjected to agarose gel electrophoresis, resulting in the detection of two amplified DNA fragments from the cDNA library.
  • the fragments are then each cloned into a plasmid for production of quantities thereof, using the TA cloning kit (Invitrogen).
  • the Serrate-homologous fragments amplified and obtained in this manner are then sequenced at least in part, by use of Sequenase® (U.S. Biochemical Corp.), to confirm the identity of the fragments as Serrate homologs. Upon such confirmation, the fragments are then used as probes with which to screen the same cDNA library for human Serrate clones.
  • the isolated phage ⁇ clones are converted to plasmids via the
  • the host E. coli strain is XL1-Blue (see Sambrook et al., 1989,
  • sequence of human Serrate contained in the cDNA clone(s) is determined (by use of Sequenase ® , U.S. Biochemical Corp.).
  • Expression constructs are made using the isolated clone(s).
  • the clone is excised from its vector as an EcoRI restriction fragment(s) and

Abstract

The present invention relates to nucleotide sequences of Serrate genes, and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. In a specific embodiment, the Serrate protein is a human protein. The invention relates to Serrate 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 the full-length (wild-type) Serrate protein. The invention further relates to fragments (and derivatives and analogs thereof) of Serrate which comprise one or more domains of the Serrate protein, including but not limited to the intracellular domain, extracellular domain, transmembrane region, membrane-associated region, or one or more EGF-like repeats of a Serrate protein, or any combination of the foregoing. Antibodies to Serrate, its derivatives and analogs, are additionally provided. Methods of production of the Serrate proteins, derivatives and analogs, e.g., by recombinant means, are also provided.

Description

NUCLEOTIDE AND PROTEIN SEQUENCES OF THE
SERRATE GENE AND METHODS BASED THEREON
________________________________________________________________
This invention was made in part with
government support under Grant numbers GM 29093 and NS 26084 awarded by the Department of Health and Human Services. The government has certain rights in the invention.
1. INTRODUCTION
The present invention relates to Serrate genes and their encoded protein products. The
invention also relates to derivatives and analogs of the Serrate protein. Production of Serrate proteins, derivatives, and antibodies is also provided.
2. BACKGROUND OF THE INVENTION Genetic analyses in Drosophila have been extremely useful in dissecting the complexity of developmental pathways and identifying interacting loci. However, understanding the precise nature of the processes that underlie genetic interactions requires a knowledge of the protein products of the genes in question.
Recent embryological, genetic and molecular evidence indicates that the early steps of ectodermal differentiation in Drosophila depend on cell
interactions (Doe and Goodman, 1985, Dev. Biol.
111:206-219; Technau and Campos-Ortega, 1986, Dev.
Biol. 195:445-454; Vassin et al., 1985, J. Neurogenet. 2:291-308; de la Concha et al., 1988, Genetics
118:499-508; Xu et al., 1990, Genes Dev. 4:464-475; Artavanis-Tsakonas, 1988, Trends Genet. 4:95-100).
Mutational analyses reveal a small group of
zygotically-acting genes, the so called neurogenic loci, which affect the choice of ectodermal cells between epidermal and neural pathways (Poulson, 1937, Proc. Natl. Acad. Sci. 23:133-137; Lehmann et al.,
1983, Wilhelm Roux's Arch. Dev. Biol. 192:62-74;
Jϋrgens et al., 1984, Wilhelm Roux's Arch. Dev. Biol. 193:283-295; Wieschaus et al., 1984, Wilhelm Roux's Arch. Dev. Biol. 193:296-307; Nϋsslein-Volhard et al.,
1984, Wilhelm Roux's Arch. Dev. Biol. 193:267-282). Null mutations in any one of the zygotic neurogenic loci -- Notch (N), Delta (Dl), mastermind (mam),
Enhancer of Split (E(spl), neuralized (neu), and big brain (bib) -- result in hypertrophy of the nervous system at the expense of ventral and lateral epidermal structures. This effect is due to the misrouting of epidermal precursor cells into a neuronal pathway, and implies that neurogenic gene function is necessary to divert cells within the neurogenic region from a neuronal fate to an epithelial fate. Serrate has been identified as a genetic unit capable of interacting with the Notch locus (Xu et al., 1990, Genes Dev.
4:464-475). These genetic and developmental
observations have led to the hypothesis that the protein products of the neurogenic loci function as components of a cellular interaction mechanism
necessary for proper epidermal development (Artavanis- Tsakonas, S., 1988, Trends Genet. 4:95-100).
Mutational analyses also reveal that the action of the neurogenic genes is pleiotropic and is not limited solely to embryogenesis. For example, ommatidial, bristle and wing formation, which are known also to depend upon cell interactions, are affected by neurogenic mutations (Morgan et al., 1925, Bibliogr. Genet. 2:1-226; Welshons, 1956, Dros. Inf. Serv. 30:157-158; Preiss et al., 1988, EMBO J. 7:3917-
3927; Shellenbarger and Mohler, 1978, Dev. Biol.
62:432-446; Technau and Campos-Ortega, 1986, Wilhelm Roux's Dev. Biol. 195:445-454; Tomlison and Ready, 1987, Dev. Biol. 120:366-376; Cagan and Ready, 1989, Genes Dev. 3:1099-1112).
Sequence analyses (Wharton et al., 1985, Cell 43:567-581; Kidd and Young, 1986, Mol. Cell.
Biol. 6:3094-3108; Vassin, et al., 1987, EMBO J.
6:3431-3440; Kopczynski, et al., 1988, Genes Dev.
2:1723-1735) have shown that two of the neurogenic loci. Notch and Delta, appear to encode transmembrane proteins that span the membrane a single time. The Notch gene encodes a ~300 kd protein (we use "Notch" to denote this protein) with a large N-terminal extracellular domain that includes 36 epidermal growth factor (EGF)-like tandem repeats followed by three other cysteine-rich repeats, designated Notch/lin-12 repeats (Wharton, et al., 1985, Cell 43:567-581; Kidd and Young, 1986, Mol. Cell. Biol. 6:3094-3108; Yochem, et al., 1988, Nature 335:547-550). Delta encodes a ~100 kd protein (we use "Delta" to denote DLZM, the protein product of the predominant zygotic and
maternal transcripts; Kopczynski, et al., 1988, Genes Dev. 2:1723-1735) that has nine EGF-like repeats within its extracellular domain (Vassin, et al., 1987, EMBO J. 6:3431-3440; Kopczynski, et al., 1988, Genes Dev. 2:1723-1735). Molecular studies have lead to the suggestion that Notch and Delta constitute
biochemically interacting elements of a cell
communication mechanism involved in early
developmental decisions (Fehon et al., 1990, Cell 61:523-534).
The EGF-like motif has been found in a variety of proteins, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell
53: 505-518). In particular, this motif has been found in extracellular proteins such as the blood clotting factors IX and X (Rees et al., 1988, EMBO J. 7:2053-2061; Furie and Furie, 1988, Cell 53: 505-518), in other Drosophila genes (Knust et al., 1987 EMBO J. 761-766; Rothberg et al., 1988, Cell 55:1047-1059), and in some cell-surface receptor proteins, such as thrombomodulin (Suzuki et al., 1987, EMBO J. 6:1891- 1897) and LDL receptor (Sudhof et al., 1985, Science 228:815-822). A protein binding site has been mapped to the EGF repeat domain in thrombomodulin and
urokinase (Kurosawa et al., 1988, J. Biol. Chem
263:5993-5996; Appella et al., 1987, J. Biol. Chem. 262:4437-4440).
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention relates to nucleotide sequences of Serrate genes, and amino acid sequences of their encoded proteins, as well as derivatives
(e.g., fragments) and analogs thereof. Nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences are also provided. In a specific embodiment, the Serrate protein is a human protein.
The invention relates to Serrate 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) Serrate protein. Such
functional activities include but are not limited to antigenicity [ability to bind (or compete with Serrate for binding) to an anti-Serrate antibody],
immunogenicity (ability to generate antibody which binds to Serrate), ability to bind (or compete with
Serrate for binding) to Notch or other toporythmic proteins or fragments thereof ("adhesiveness"), ability to bind (or compete with Serrate for binding) to a receptor for Serrate. "Toporythmic proteins" as used herein, 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, 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 Serrate which comprise one or more domains of the Serrate protein, including but not limited to the intracellular domain, extracellular domain, transmembrane domain, membraneassociated region, or one or more EGF-like
(homologous) repeats of a Serrate protein, or any combination of the foregoing.
Antibodies to Serrate, its derivatives and analogs, are additionally provided.
Methods of production of the Serrate
proteins, derivatives and analogs, e.g., by
recombinant means, are also provided. 3.1. DEFINITIONS
As used herein, underscoring the name of a gene shall indicate the gene, in contrast to its encoded protein product which is indicated by the name of the gene in the absence of any underscoring. For example, "Serrate" shall mean the Serrate gene, whereas "Serrate" shall indicate the protein product of the Serrate gene.
4. DESCRIPTION OF THE FIGURES Figure 1. Phenotypic interactions between Notch and Serrate. (a) wa spl wing blade showing characteristic wild-type symmetry, venation, and marginal wing bristles and hairs. (b) nd/Y male.
Distal wing notches and loss of posterior hairs are evident. (c) SerD/+ heterozygote. Note similarity to nd/Y wing blade in Fig. 1b. (d) nd/Y; SerD/+
transheterozygote wing blade. Mutant wing shows typical "fig leaf" shape, distorted wing veins, and loss of the majority of marginal bristles and hairs, with the exception of the anterodistal wing margin. (e) +/Y; SerD/Dp(3R)CosP479BE (N+) male. The extra N+ copy suppresses the heterozygous SerD dominant
phenotype (compare to Fig. 1c). Also note suppression of the Confluens phenotype (see text). (f) SerD/SerD homozygote. Note the increased severity of the phenotype relative to SerD/+ (compare to Fig. 1c).
Figure 2. Phenotypes of Serrate lethal mutations, (a-d) Cuticular preparations; (e-h) anti- HRP preparations. (a) Wild-type cuticular pattern of embryo just prior to hatching. (b) Typical non- retracted germ band Serrevertant homozygote. Note position of Filzkδrper (arrow). (c) Serrevertant homozygote lacking the cuticle of the cephalic regions and the first and second thoracic segments. (d) Severely affected
Serrevertant homozygote displaying limited cuticle
differentiation. (e) Wild-type embryo showing typical nervous system differentiation. (f) Homozygous Serrevertant embryo displaying a "mild" disruption of the
longitudinal and commissural axon tracts. (g) Serrevertant homozygote; note singular, twisted longitudinal
connective. (h) Severely affected Serrevertant homozygote with dispersed clumps of neural material remaining.
Figure 3. Molecular map of the Serrate- encoding region. Approximately 85 kb of cloned genomic DNA from the 97F chromosomal region are presented along with the restriction sites of three enzymes [(B) BamHI; (E) EcoRI; (H) HindII l]. The locations of individual DNA alteratiodns associated with Serrate allelic breakpoints are displayed above the genomic DNA (for descriptions of mutant alleles, see Section 6, infra; (rev 3 and rev 2-11) Serrev3 and Serrev2-11, respectively; (R128) T(Y:3)R128. The shaded box from coordinates 0 to +3 represents the region of EGF homology detectable by Southern hybridization. The BamHI site adjacent to the EGF homology was arbitrarily chosen as position 0. Map orientation is with the centromere to the left. At the bottom of the figure are shown the individual recombinant phage isolates. The C1 and C3 cDNAs together constitute the larger of the two Serrate messages (~5.6 kb). Intron positions and coding capacities have been approximated solely upon cross hybridization of the cDNAs with the genomic DNA regions.
Figure 4. Serrate sequence analysis. The complete 5561 bp sequence (SEQ ID NO:1) derived from cDNAs C1 and C3 is shown. Nucleotide numbering is at left, amino acid numbering of the predicted open reading frame (ORF) is at right. The deduced protein product appears to be a transmembrane protein of 1404 amino acids (SEQ ID NO: 2). Hydrophobic regions are denoted inside dashed boxes; amino acids 51 to 80 represent the likely signal peptide; amino acids 542 to 564 represent the potential membrane associated region; amino acids 1221 to 1245 represent the
putative transmembrane domain. The first cysteine of each of the fourteen EGF-like repeats is denoted with a solid black box, and each repeat is underlined. The partial EGF-like repeat is considered "degenerate," since the fourth cysteine residue of this repeat has been changed to lysine (shown in boldface type at amino acid position 268). The initial cysteine of this repeat is denoted with an open box (amino acid 284), and the repeat is underlined. Amino acid insertions occur in the fourth, sixth, and tenth EGF- like repeats and are denoted by hatched underlines.
Figure 5. The Serrate transcript and deduced protein product. (a) The composite transcript shown was constructed from the C1 and C3 cDNAs, which overlap by 109 bp. Selected restriction enzyme cleavage sites are shown. The hatched box represents the 4212 bp ORF. Open boxes represent the 442 bp 5'- untranslated leader and 900 bp 3'-trailer sequence, (b) Kyte-Doolittle hydropathy plot of the deduced 1404 amino acid protein. (SP) Putative signal peptide;
(MA) potential membrane associated region; (TM) likely transmembrane domain. (c) Cartoon representation of the gross structural features of the predicted Serrate protein. The darkly shaded region, including the partial EGF-like repeat (PR) is ~250 amino acids in length and homologous to the Delta protein. Bracketed EGF-like repeats labelled (A, B, and C) contain insertions of amino acids and thus differ from the canonical EGF-like structure. Other features of the protein include the signal peptide (SP), a cysteine rich region, a transmembrane domain (TM), and an intracellular region of ~160 amino acids.
Figure 6. Temporal profile of Serrate
transcript accumulation. Each lane contains five μg of poly(A)+ RNA. The stage of the embryonic RNAs is denoted in hours after egg laying; (L1, L2, and L3) RNA from the first, second and third larval instar periods; (EP and LP) early and late pupal stages; (M and F) adult male and female RNAs, respectively. A composite cDNA subclone (constructed from C1 and C3) was used as a hybridization probe. Serrate
transcription is represented primarily as a 5.5 kb and 5.6 kb doublet beginning at 4-8 hours of
embryogenesis. A transient 3.4 kb transcript is observed only during 2-4 hr of embryogenesis. The pupal and adult RNAs were fractionated on a separate gel for a longer period of time for better resolution. Equivalent loadings of RNA were noted by ethidium bromide staining of the RNA gels and confirmed by subsequent probing with an actin 5C probe shown at bottom; Fyrberg et al., 1983, Cell 33:115-123). Minor bands were not consistently observed in other blots and may reflect other EGF-homologous transcripts
Figure 7. Whole-mount in situ Serrate
transcripts. Embryos are oriented with anterior to the left and dorsal side up unless otherwise noted, (a) Dorsal view of an early stage 10 embryo (middorsal focal plane). Earliest expression occurs in the ectoderm of the foregut (FG) and presumptive clypeolabrum (CL). (b) Dorsal view of a germ bandextended embryo (late stage 10). Additional
expression occurs near the proctodeum (PR), within the eighth (A8) and ninth (A9) abdominal segments, and in the labial and maxillary primordia (arrow). (c)
Lateral view of an early stage 11 embryo. The lateral (LE) and ventral (VE) expression patterns are out of register and do not include the tracheal pits (TP). (d) Germ band-extended embryo (mid stage 11) dissected and flattened such that the dorsal surfaces are at the lateral edges. Extensive expression is observed between the labial (LB), maxillary (MX), and
mandibular (MN) lobes, and within the hypopharynx (HP) and clypeolabrum (CL). Expression is also apparent in the salivary gland placodes (SP) that have moved to the ventral midline. Note relationship between lateral and ventral patterns and elaboration of expression in the tail region [presumptive telson (TL)]. (e) Germ band-retracting embryo (stage 12; lateral view). Lateral expression (LE) is beginning to coalesce. (f) Lateral view of a germ bandretracted embryo (stage 13). The lateral expression is beginning to extend both dorsally and ventrally in each thoracic and abdominal segment and is most pronounced in the first thoracic segment (T1). A portion of the lateral expression now appears to include the presumptive trachea (T). Ventrally, note different expression (VE) patterns in the thoracic versus abdominal segments. (g) Lateral view of an early stage 14 embryo. Outline of the presumptive trachea (T) is distinct from the overlying epidermal expression. Arrows denote the zigzag pattern of lateral expression. (h) Dissected embryo (stage 14) opened along the dorsal midline and laid flat. Two areas of hindgut expression (HG1 and HG2) are
apparent; HG1 occurs near the origin of the Malpighian tubules. (i) Ventral view of a stage-16 embryo focusing on the ventral nerve cord (VNC). Earlier expression in the salivary gland placodes (SP in panel d) now constitutes the SD. Expression in the
proventriculus (PV) and the maxillary/mandibular region (MX/MN) is slightly out of focus. (j)
Dorsomedial focal plane of same embryo as in (i); head involution is nearly complete. The in-pocketings of expression in the thoracic segments (T1, T2, and T3) may represent imaginal disc primordia. Pharyngeal expression (PH) is a combination of clypeolabrum and hypopharyngeal expression noted earlier. (k) Dorsal view of the same embryo as in (i) and (j). Note individual expressing cells in the brain lobes (BC).
Expression in the fully differentiated trachea (T) and hindgut (H1) is evident. (1) Flattened preparation of early stage 16 embryo. Expression within the telson (TL) now constitutes a ring around the presumptive anal pads.
Figure 8. Amino acid comparison of aminoterminal Serrate-Delta homology. Conserved regions are indicated at the top of the figure (* = identical amino acids; ' = conservative changes in sequence). Serrate (see SEQ ID NO: 2) is shown above line, Delta (SEQ ID NO: 4) below. The sequence begins at Serrate amino acid position 59; the partial EGF-like repeat of both Serrate and Delta is boxed. The Serrate amino acid sequence (amino acids 79-282 of Fig. 4) placed into the chimeric ΔEGF Notch construct and determined to be sufficient for Notch binding is presented in boldface type. The positions of the synthetic
degenerate primers (designated FLE1 through FLE4R) are shown; refer to Figure 9 for nucleotide composition.
Figure 9. Nucleotide comparison of aminoterminal Serrate-Delta homology. The nucleotide sequence corresponding to the amino acid sequence in Figure 8 is shown (Serrate sequence: see SEQ ID NO:1; Delta sequence: SEQ ID NO: 3). The DNA encoding the partial EGF-repeat is boxed. The Serrate nucleotide sequence (nucleotides 676-1287 of Fig. 4) placed into the chimeric ΔEGF Notch construct determined to be sufficient for Notch binding is presented in boldface type. The sequences of the synthetic degenerate primers (designated FLE1 through FLE4R) are given against black backgrounds.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to nucleotide sequences of Serrate genes, and amino acid sequences of their encoded proteins. The invention further relates to fragments and other derivatives, and analogs, of Serrate proteins. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. In a preferred embodiment of the invention, the Serrate protein is a human protein. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is provided.
The invention relates to Serrate 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) Serrate protein. Such
functional activities include but are not limited to antigenicity [ability to bind (or compete with Serrate for binding) to an anti-Serrate antibody],
immunogenicity (ability to generate antibody which binds to Serrate), ability to bind (or compete with Serrate for binding) to Notch or other toporythmic proteins or fragments thereof ("adhesiveness"), ability to bind (or compete with Serrate for binding) to a receptor for Serrate. "Toporythmic proteins" as used herein, 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, 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 Serrate which comprise one or more domains of the Serrate protein, including but not limited to the intracellular domain, extracellular domain, transmembrane domain, membrane- associated region, or one or more EGF-like (homologous) repeats of a Serrate protein, or any combination of the foregoing.
Antibodies to Serrate , its derivatives and analogs, are additionally provided.
As demonstrated infra (see Section 6),
Serrate 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 Serrate mRNA of human and other species, to study expression thereof, to produce Serrate and 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.
The invention is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of D. melanogaster Serrate (Section 6); the construction and recombinant expression of a
Serrate chimeric/fusion derivative and production of antibodies thereto (Section 7); the recombinant expression of Serrate, a Serrate fragment lacking the EGF-like repeats present in Serrate, and a chimeric Notch-Serrate derivative, and assays for binding to Notch (Section 8); and the cloning of a human Serrate homolog (Section 9).
For clarity of disclosure, and not by way of limitation, the detailed description of the invention will be divided into the following sub-sections:
(i) Isolation of the Serrate Gene;
(ii) Expression of the Serrate Gene;
(iii) Identification and Purification of the Expressed Gene Product;
(iv) Structure of the Serrate Gene and
proteion; (v) Generation of Antibodies to
Serrate Proteins and Derivatives
Thereof;
(vi) Serrate Derivatives and Analogs;
(vii) Assays of Serrate Proteins,
Derivatives, and Analogs.
5.1. ISOLATION OF THE SERRATE GENE
The invention relates to the nucleotide sequences of Serrate consisting of at least 8
nucleotides (i.e., a hybridizable portion). In a specific embodiment, the invention relates to the nucleic acid sequence of the human Serrate gene. In another embodiment, the invention relates to the
Drosophila Serrate gene. In a preferred, but not limiting, aspect of the invention, a Drosophila
Serrate DNA sequence (ATCC Accession Number ________ ) can be cloned and sequenced by the method described in Section 6, infra. The invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences.
Nucleic acids encoding fragments and
derivatives of Serrate (see Section 5.6) are
additionally provided.
Fragments of Serrate nucleic acids comprising regions of homology to other toporythmic proteins are also provided. For example, the total region of homology with Delta spans nucleotides 627- 1290 (Fig. 4) (see SEQ ID NO:1) of the Serrate
sequence. Nucleic acids encoding conserved regions between Delta and Serrate, such as those represented by Serrate amino acids 63-73, 124-134, 149-158, 195- 206, 214-219, and 250-259 (see SEQ ID NO:2), are also provided. A preferred embodiment for the cloning of human Serrate, presented as a particular example but not by way of limitation, follows:
A human expression library 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 Serrate product. In one embodiment, anti-Serrate 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 Serrate sequences can be used as primers in PCR. In a preferred aspect, the oligonucleotide primers
represent at least part of the Serrate conserved segments of strong homology between Serrate and Delta. For example, oligonucleotides may be obtained
corresponding to parts of the four highly conserved regions between Delta and Serrate, i.e., that
represented by Serrate AA 124-134, 149-158, 214-219, and 250-259 (see SEQ ID NO:2). 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. In a specific embodiment an oligonucleotide primer pair used for PCR with a cDNA library is FLE1/FE3R,
FLE1/FLE4R, FLE2/FLE3R, or FLE2/FLE4R shown in Fig. 9. (see SEQ ID NO:1) (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 mRNA or cDNA or genomic DNA from any
eukaryotic species. 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 Serrate homolog and the known Serrate. After
successful amplification of a segment of a Serrate 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 this fashion, additional genes encoding Serrate proteins may be identified. Such a procedure is presented by way of example in Section 9, infra.
The above-methods are not meant to limit the following general description of methods by which clones of Serrate may be obtained.
Any eukaryotic cell potentially can serve as the nucleic acid source for the molecular cloning of the Serrate gene. The nucleic acid sequences encoding Serrate can be isolated from human, porcine, bovine, feline, avian, equine, canine, as well as additional primate sources, insects, etc. For example, we have amplified fragments of the appropriate size in
Drosophila, mouse, Xenopus, and human, by PCR using cDNA libraries with Drosophila Serrate primers. 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. (See, for example, Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York; 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
Serrate (of any species) gene or its specific RNA, or a fragment thereof, e.g., an extracellular domain (see Section 5.6), 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. 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, receptor binding activity, in vitro aggregation activity ("adhesiveness") or antigenic properties as known for Serrate. If an antibody to Serrate is available, the Serrate protein may be identified by binding of labeled antibody to the putatively Serrate synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay) -type procedure.
The Serrate 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 Serrate DNA of another species (e.g., Drosophila). Immunoprecipitation analysis or functional assays (e.g., aggregation ability in vitro; binding to receptor; 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 Serrate protein. A radiolabelled Serrate 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 Serrate DNA fragments from among other genomic DNA fragments.
Alternatives to isolating the Serrate
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 Serrate protein. For example, RNA for cDNA cloning of the Serrate gene can be isolated from cells which express Serrate. 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 Serrate 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 Serrate 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.
5.2. EXPRESSION OF THE SERRATE GENE
The nucleotide sequence coding for a Serrate protein or a functionally active fragment or other derivative thereof (see Section 5.6), 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 proteincoding sequence. The necessary transcriptional and translational signals can also be supplied by the native Serrate 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, the adhesive portion of the Serrate gene is expressed. In other specific
embodiments, the human Serrate gene is expressed, or a sequence encoding a functionally active portion of human Serrate. In yet another embodiment, a fragment of Serrate comprising the extracellular domain, or other derivative, or analog of Serrate 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 nucleic acid sequence encoding a Serrate protein or peptide fragment may be regulated by a second nucleic acid sequence so that the Serrate protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a Serrate protein may be controlled by any promoter/enhancer element known in the art.
Promoters which may be used to control toporythmic 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 β-lactamase promoter (Villa- Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al.,
1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303:209-213) 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 Serrate 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 toporythmic 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 Serrate gene is inserted within the marker gene sequence of the vector, recombinants containing the Serrate 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 Serrate gene product in vitro assay systems, e.g., aggregation (binding) with Notch, binding to a receptor, 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 Serrate protein may be controlled.
Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage [e.g., of signal sequence]) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in mammalian cells can be used to ensure "native" glycosylation of a heterologous mammalian toporythmic protein. Furthermore, different vector/host expression systems may effect processing reactions such as proteolytic cleavages to different extents.
Both cDNA and genomic sequences can be cloned and expressed.
5.3. IDENTIFICATION AND PURIFICATION OF THE EXPRESSED GENE PRODUCT
Once a recombinant which expresses the
Serrate 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.
Once the Serrate 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, once a Serrate protein produced by a recombinant is identified, the amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant. As a result, the the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller, M., et al., 1984, Nature 310:105-111).
In a specific embodiment of the present invention, such Serrate proteins, whether produced by recombinant DNA techniques or by chemical synthetic methods, include but are not limited to those
containing, as a primary amino acid sequence, all or part of the amino acid sequence substantially as depicted in Figure 4 (SEQ ID NO:2), as well as fragments and other derivatives, and analogs thereof. 5.4. STRUCTURE OF THE SERRATE GENE AND PROTEIN
The structure of the Serrate gene and protein can be analyzed by various methods known in the art. 5.4.1. GENETIC ANALYSIS
The cloned DNA or cDNA corresponding to the Serrate gene can be analyzed by methods including but not limited to Southern hybridization (Southern, E.M., 1975, J. Mol. Biol. 98:503-517), Northern
hybridization (see e.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction endonuclease mapping (Maniatis, T., 1982, Molecular Cloning, A Laboratory, Cold Spring Harbor, New York), and DNA sequence analysis. Polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,202, 4,683,195 and
4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7652-7656; Ochman et al., 1988,
Genetics 120:621-623; Loh et al., 1989, Science
243:217-220) followed by Southern hybridization with a
Serrate-specific probe can allow the detection of the Serrate gene in DNA from various cell types. In one embodiment, Southern hybridization can be used to determine the genetic linkage of Serrate. Northern hybridization analysis can be used to determine the expression of the Serrate gene. Various cell types, at various states of development or activity can be tested for Serrate expression. Examples of such techiques and their results are described in Section 6, infra. 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 Serrate probe used.
Restriction endonuclease mapping can be used to roughly determine the genetic structure of the
Serrate gene. In a particular embodiment, cleavage with restriction enzymes can be used to derive the restriction map shown in Figure 3, infra. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis.
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, F., 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), or use of an automated DNA sequenator (e.g., Applied Biosystems, Foster City, CA). The cDNA sequence of a
representative Serrate gene comprises the sequence substantially as depicted in Figure 4, and described in Section 6, infra.
5.4.2. PROTEIN ANALYSIS The amino acid sequence of the Serrate 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 Serrate protein comprises the sequence substantially as depicted in Figure 4, and detailed in
Section 6, infra, with the representative mature protein that shown by amino acid numbers 81-1404.
The Serrate protein sequence can be further characterized by a hydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad. Sci. U.S.A.
78:3824). A hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the Serrate protein and the corresponding regions of the gene sequence which encode such regions. A hydrophilicity profile of the Serrate protein
described in the examples section infra is depicted in
Figure 5.
Secondary, structural analysis (Chou, P. and
Fasman, G., 1974, Biochemistry 13:222) can also be done, to identify regions of Serrate 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, A., 1974, Biochem.
Exp. Biol. 11:7-13) and computer modeling (Fletterick,
R. and Zoller, M. (eds.), 1986, Computer Graphics and
Molecular Modeling, in Current Communications in
Molecular Biology, Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York). 5.5. GENERATION OF ANTIBODIES TO SERRATE
PROTEINS AND DERIVATIVES THEREOF
According to the invention, Serrate 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 specific embodiment, antibodies to human Serrate are produced. In another embodiment, antibodies to the extracellular domain of
Serrate are produced. In another embodiment,
antibodies to the intracellular domain of Serrate are produced.
Various procedures known in the art may be used for the production of polyclonal antibodies to a
Serrate protein or derivative or analog. In a
particular embodiment, rabbit polyclonal antibodies to an epitope of the Serrate protein encoded by a
sequence depicted in Figure 4, or a subsequence thereof, can be obtained. For the production of antibody, various host animals can be immunized by injection with the native Serrate 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. For preparation of monoclonal antibodies directed toward a Serrate 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). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545).
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). 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 Serrate 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. Patent 4,946,778) can be adapted to produce Serrate-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 Serrate proteins, derivatives, or analogs.
Antibody fragments which contain the
idiotype of the molecule can be generated by known techniques. For example, such 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.
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 Serrate protein, one may assay generated hybridomas for a product which binds to a Serrate fragment containing such domain. For selection of an antibody specific to human Serrate, one can select on the basis of positive binding to human Serrate and a lack of binding to Drosophila Serrate.
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. 5.6. SERRATE DERIVATIVES AND ANALOGS The invention further relates to derivatives (including but not limited to fragments) and analogs of Serrate proteins.
The production and use of derivatives and analogs related to Serrate 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 Serrate protein. As one example, such derivatives or analogs which have the desired immunogenicity or antigenicity can be used, for example, in
immunoassays, for immunization, for inhibition of Serrate activity, etc. Such molecules which retain, or alternatively inhibit, a desired Serrate property, e.g., binding to Notch or other toporythmic proteins, binding to a cell-surface receptor, can be used as inducers, or inhibitors, respectively, of such
property and its physiological correlates. A specific embodiment relates to a Serrate fragment that can be bound by an anti-Serrate antibody but cannot bind to a Notch protein or other toporythmic protein.
Derivatives or analogs of Serrate 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.
In particular, Serrate derivatives can be made by altering Serrate 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 Serrate 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 Serrate 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 Serrate 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 Serrate 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.
Derivatives or analogs of Serrate include but are not limited to those peptides which are substantially homologous to Serrate or fragments thereof, or whose encoding nucleic acid is capable of hybridizing to a Serrate nucleic acid sequence.
The Serrate 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 Serrate gene sequence can be modified by any of numerous strategies known in the art (Maniatis, T., 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. In the production of the gene encoding a derivative or analog of Serrate, care should be taken to ensure that the modified gene remains within the same translational reading frame as
Serrate, uninterrupted by translational stop signals, in the gene region where the desired Serrate activity is encoded.
Additionally, the Serrate-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, C., et al., 1978, J.
Biol. Chem 253:6551), use of TAB® linkers (Pharmacia), etc.
Manipulations of the Serrate sequence may also be made at the protein level. Included within the scope of the invention are Serrate protein
fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by glycosylation, 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, NaBH4;
acetylation, formylation, oxidation, reduction;
metabolic synthesis in the presence of tunicamycin; etc.
In addition, analogs and derivatives of Serrate can be chemically synthesized. For example, a peptide corresponding to a portion of a Serrate protein which comprises the desired domain (see
Section 5.6.1), or which mediates the desired
aggregation activity in vitro, or binding to a
receptor, 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 Serrate sequence. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids.
In a specific embodiment, the Serrate derivative is a chimeric, or fusion, protein
comprising a Serrate protein or fragment thereof fused to a non-Serrate amino acid sequence. In one
embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a Serrate-coding sequence joined in-frame to a non-Serrate coding sequence). 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. In a specific embodiment, a chimeric nucleic acid encoding a mature Serrate protein with a heterologous signal sequence is expressed such that the chimeric protein is expressed and processed by the cell to the mature Serrate 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 Serrate and another toporythmic gene, e.g., Delta. The encoded protein of such a recombinant molecule could exhibit properties associated with both Serrate and Delta and portray a novel profile of biological activities, including agonists as well as antagonists. The primary sequence of Serrate and Delta may also be used to predict tertiary structure of the molecules using computer simulation (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824- 3828); Serrate/Delta chimeric recombinant genes could be designed in light of correlations between tertiary structure and biological function. Likewise, chimeric genes comprising portions of Serrate fused to any heterologous protein-encoding sequences may be
constructed. A specific embodiment relates to a chimeric protein comprising a fragment of Serrate of at least six amino acids. A particular example of the construction and expression of a Notch-Serrate chimera is presented in Section 8 hereof. A particular
example of another Serrate fusion protein is presented in Section 7 hereof. In another specific embodiment, the Serrate derivative is a fragment of Serrate comprising a region of homology with another toporythmic protein. As used herein, a region of a first protein shall be considered "homologous" to a second protein when the amino acid sequence of the region is at least 30% identical or at least 75% either identical or
involving conservative changes, when compared to any sequence in the second protein of an equal number of amino acids as the number contained in the region. For example, such a Serrate fragment can comprise one or more regions homologous to Delta, including but not limited to Serrate amino acids 63-73, 124-134, 149- 158, 195-206, 214-219, 250-259, or 79-282 (or 79-246, excluding the partial EGF-like repeat) (see Figs. 4, 8), or portions of Serrate of other species most homologous to the foregoing sequences.
Other specific embodiments of derivatives and analogs are described in the subsections below and examples sections infra.
5.6.1. DERIVATIVES OF SERRATE CONTAINING
ONE OR MORE DOMAINS OF THE PROTEIN
In a specific embodiment, the invention relates to Serrate derivatives and analogs, in
particular Serrate fragments and derivatives of such fragments, that comprise one or more domains of the
Serrate protein, including but not limited to the extracellular domain, transmembrane domain,
intracellular domain, membrane-associated region, and one or more of the EGF-like repeats (ELR) of the
Serrate protein. In particular examples relating to the Drosophila Serrate protein (see example 6), such domains are identified as follows, with reference to Figure 4: extracellular domain, amino acids numbers
(AA) 81-541; transmembrane domain, AA 1221-1245; intracellular domain, AA 1246-1404; membrane- associated region, AA 542-564; ELR (see underscored sequences in Fig. 4).
In a specific embodiment, relating to a Serrate protein of a species other than D.
melanogaster, the fragments comprising specific portions of Serrate are those comprising portions in the respective Serrate protein most homologous to specific fragments of the Drosophila Serrate protein. Alternatively, a fragment comprising a domain of a Serrate homolog can be identified by protein analysis methods as described in Section 5.3.2 or 6.
Serrate derivatives which are Serrate fragments and chimeric/fusion proteins are described by way of example in Sections 7 and 8 infra.
5.6.2. DERIVATIVES OF SERRATE THAT MEDIATE
BINDING TO TOPORYTHMIC PROTEIN DOMAINS
The invention also provides for Serrate fragments, and analogs or derivatives of such
fragments, which mediate binding to toporythmic proteins (and thus are termed herein "adhesive"), and nucleic acid sequences encoding the foregoing.
In a specific embodiment, the adhesive fragment of Serrate is that comprising the portion of Serrate most homologous to about amino acid numbers 85-283 or 79-282 of the Drosophila Serrate sequence (see Figure 4).
The ability to bind to a toporythmic protein (preferably Notch) can be demonstrated by in vitro aggregation assays with cells expressing such a toporythmic protein as well as cells expressing
Serrate or a Serrate derivative (See Section 5.7).
That is, the ability of a Serrate fragment to bind to a Notch protein can be demonstrated by detecting the ability of the Serrate fragment, when expressed on the surface of a first cell, to bind to a Notch protein expressed on the surface of a second cell.
The nucleic acid sequences encoding toporythmic proteins or adhesive domains 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 toporythmic genes can be identified.
7. ASSAYS OF SERRATE PROTEINS,
DERIVATIVES AND ANALOGS
The functional activity of Serrate proteins, derivatives and analogs can be assayed by various methods.
For example, in one embodiment, where one is assaying for the ability to bind or compete with wild- type Serrate for binding to anti-Serrate 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 assyas, 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 a toporythmic protein, e.g., Notch, one can carry out an in vitro aggregation assay such as described infra in Section 8.2.1 (see also Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699).
In another embodiment, where a receptor for
Serrate is identified, receptor binding can be
assayed, e.g., by means well-known in the art. In another embodiment, physiological correlates of
Serrate binding to cells expressing a Serrate receptor (signal transduction) can be assayed.
In another embodiment, in insect or other model systems, genetic studies can be done to study the phenotypic effect of a Serrate mutant that is a derivative or analog of wild-type Serrate (see Section 6, infra).
Other methods will be known to the skilled artisan and are within the scope of the invention.
6. THE GENE SERRATE ENCODES A PUTATIVE EGF-LIKE
TRANSMEMBRANE PROTEIN ESSENTIAL FOR PROPER ECTODERMAL DEVELOPMENT IN DROSOPHILA MELANOGASTER
As described in the example herein (see
Fleming et al., 1990, Genes Dev. 4:2188-2201),
mutations in the third chromosome gene Serrate are shown to display genetic interactions with specific alleles of the neurogenic locus Notch, which encodes a transmembrane protein with epidermal growth factor homology. The locus Serrate displays a striking phenotypic interaction with a specific Notch allele known to affect postembryonic development. We present the molecular cloning of Serrate and show that it encodes two coordinately-expressed transcripts from a genomic interval greater than 30 kilobases in length. The deduced protein product of 1404 amino acids contains a single transmembrane domain and 14
epidermal growth factor-like repeats. Whole-mount in situ hybridization analysis revealed complex temporal and spatial patterns of RNA expression consistent with the epidermal and neuronal defects observed in mutant embryos.
We demonstrate that the Serrate locus encodes an essential function, the loss of which results in embryonic lethality brought about by the disruption of both neuronal and epidermal tissues. Serrate is likely to represent an element in a network of interacting molecules operating at the cell surface during the differentiation of certain tissues.
6.1. RESULTS
6.1.1. THE SERRATE AND NOTCH GENES INTERACT
PHENOTYPICALLY
In the course of genetic crosses designed to detect interactions between the Notch locus and other genes in Drosophila, a dramatic phenotypic interaction was observed between the Notch allele notchoid (nd) and the third chromosome mutation Serrate (designated SerD herein). The recessive nd mutation, which is associated with an amino acid substitution in the intracellular portion of the Notch protein (Xu et al., 1990, Genes Dev. 4:464-475), causes wing notches in the adult (see Fig. lb; compare to wildtype, Fig. 1a). The SerD mutation is dominant and in heterozygous condition produces an adult wing blade very similar to that of nd animals (compare Figures 1b and 1c). The phenotypic interaction seen in nd/Y; SerD/+ males is characterized by loss of anterior and posterior wing margins, as well as loss of distal wing blade tissue. Concomitant with this loss, thickening of the L3 and L5 wing veins is observed (see Fig. 1d).
Even though both the SerD and nd mutations affect wing blade development, the interaction appears to be synergistic because a novel phenotype is seen, that is, rather than just additive effects. To explore this question of synergy further, we
constructed flies carrying genetic duplications of
Notch+. Animals carrying an extra copy of Notch+ normally exhibit a Confluens phenotype characterized by wing vein thickening. Surprisingly, animals bearing SerD and an extra copy of Notch+ have
essentially wild-type wings (Fig. le), that is, both the SerD wing nicking and the Confluens phenotypes are suppressed in this combination. This interaction was noted using both Dp(1;2) 51b (a large genetic
duplication of 3C1-2; 3D6 including N+) and CosP479BE [ (N+) (86E5-6), a cosmid construct containing only the N+ gene (Ramos et al., 1989, Genetics 123:337:348)].
Because the SerD mutation is neomorphic, the interactions observed between SerD and Notch mutations might not be representative of interactions normally occurring between these gene products. We therefore examined the phenotypes of nd males heterozygous for Df(3R)Ser+82f24 (nd/Y; Df(3R)Ser+82f24/+). These animals exhibit a significantly increased mutant wing
phenotype as compared to nd alone (not shown). Thus, it appears that Notch and SerD mutually influence each other's phenotypic expression.
6.1.2. GENETIC CHARACTERIZATION OF SERRATE
Previous genetic characterizations have demonstrated that the SerD mutation maps to the 97F region of the polytene chromosomes and is neomorphic, producing the dominant wing nicking phenotype shown in Figure Id (Belt, 1971, Drosophila Inf. Serv. 46:116; P. Lewis, Yale University; anpubl.). The neomorphic nature is demonstrated genetically via the
insensitivity of the SerD phenotype to the number of wildtype (Ser+) copies present, that is SerD/+/+ displays a phenotype similar to SerD/+ and to
SerD/Deficiency (P. Lewis, pers. comm.). Flies with only one copy of wild-type Ser+ (i.e., individuals heterozygous for a wild-type allele over deficiencies) are phenotypically wild-type, demonstrating that reduction of gene product (i.e., haploinsufficiency) is not causing the dominant phenotype. Finally, when the SerD mutation is homozygous, viable adults are produced that display a more severe wing phenotype than heterozygous SerD/+ animals (Figure If). Thus, the expression of the SerD wing phenotype appears to be directly related to the expression of a mutant or novel gene product rather than to Ser+ gene dosage.
In an effort to obtain amorphic alleles of Serrate. we used X-ray mutagenesis to produce
phenotypic revertants of the dominant mutation (see Section 6.3 for details). All five of the revertants of the SerD mutation are lethal when homozygous and, consistent with the deficiency phenotypes, are
phenotypically wild-type when heterozygous with a wild-type chromosome. Complementation tests revealed that the Serrevertants are allelic. Moreover,
transheterozygotes of nd with two different Serrevertants alleles (nd/Y; Serrev2-3/+ and nd/Y; Serevv2-11/+) exhibit an enhanced mutant wing phenotype as compared to nd
mutants, in agreement with the SerD-Notch interactions noted previously. These complementation tests were extended to include another dominant mutation. Beaded of
Goldschmidt (BdG), which also maps to the 97F region. Heterozygous adults bearing the BdG mutation display a wing nicking phenotype that is more severe than that observed in SerD heterozygotes (data not shown).
Moreover, the BdGmutation, unlike SerD, is homozygous lethal. Finally, three alleles (Bd43.5, Bd862.5, and pll11) of a lethal complementation group isolated in K.
Anderson's laboratory were shown to be allelic to BdG (P. Hecht, unpubl.; a complete listing of the alleles used and their descriptions is provided in Section 6.3). Although transheterozygotes of SerD and BdG are viable, it is interesting to note that
Df(3R) Ser+82f24 and most of the Serrevertants fail to
complement the BdG mutation for viability. The
exception is the Ser rev2-3 allele, which although
homozygous lethal, complements BdG. Despite the exceptional Ser rev2-3 allele, these results suggest that the Serrate and Beaded mutations are alleles of the same gene (see also below). Consistent with this idea is the fact that Serrevertant and Bd alleles have similar phenotypes (see also below).
6.1.3. PHENOTYPIC CHARACTERIZATION OF SERRATE
LETHAL ALLELES
All of the Serrevertants, as well as the Bd
alleles we tested (BdG, Bd43.5, and Bd862.5), and the
Df(3R}Ser+82f24 exhibit embryonic lethality. Cuticle preparations of unhatched embryos from heterozygous parents revealed a continuous and complex range of phenotypes: The progeny of a single allele from a single brood included individuals that were nearly wild-type in appearance as well as those lacking the majority of differentiated cuticle. Another commonly observed defect was the failure of germ band
retraction. Although all of the alleles displayed the full range of mutant phenotypes (including Df (3R) Ser +82f24) , the proportions of weak or strong phenotypes observed for each individual allele differed.
Moreover, three alleles, Serrev2-3, Serrev2-11 and Serrev2-5 , in heterozygous combination with Df (3R) Ser+82f24, revealed the same range of mutant phenotypes as when
homozygous.
Embryos exhibiting weak mutant phenotypes often appear to have completely differentiated
cuticular structures yet fail to retract the germ band. These embryos appear "J"-shaped within the egg, with the Filzkorper residing at ~50% egg length on the dorsal surface of the embryo (Fig. 2b). Other weakly affected individuals undergo normal germ band
retraction, have faintly pigmented denticle bands and dorsal hairs, and may have "holes" in the cuticle along the length of the embryo. More severe
phenotypes are exemplified by embryos with retracted germ bands that lack the cuticle of the entire
cephalic regions and sometimes the first and second thoracic segments (Fig. 2c). Other embryos fail to retract the germ band and lack head and thoracic structures, may exhibit twisted germ bands, and/or frequently lack large patches of dorsal or ventral cuticle. Finally, in very severely affected mutant embryos, only a small cuticular patch remains (Fig. 2d). Unlike embryos from a neurogenic mutant, the cuticle that remains in SerD mutant embryos can be of dorsal or ventral origin.
We examined the nervous system of mutant embryos using anti-horseradish peroxidase (anti-HRP) antibody (Jan and Jan, 1982, Proc. Natl. Acad. Sci.
USA 79:2700-2704). Consistent with the cuticular abnormalities, a corresponding range of defects was observed for homozygous individuals of a given Serrevertant allele. Many individuals exhibited missing
commissures or breaks in the longitudinal connectives that run between segmental ganglia (Fig. 2f). In other embryos this disruption was more pronounced such that there appeared to be only one longitudinal
connective running most of the length of the embryo; this condition often correlated with improper germ band retraction or twisted germ bands (Fig. 2g).
Finally, in the most severely affected individuals, only small clumps of anti-HRP staining "neural" material were present throughout the embryo (Fig. 2h). In no case did we see hypertrophy of the central nervous system.
6.1.4. MOLECULAR CHARACTERIZATION OF SERRATE DNA
In an effort to elucidate the molecular nature of the Serrate gene product, DNA from the 97F region was cloned and characterized. A Drosophila genomic clone, previously isolated on the basis of cross hybridization to the EGF-like domain of the
Notch gene (Rothberg et al., 1988, Cell 55:1047-1059), was used as an entry point to initiate a chromosomal walk. From this initial clone, eight recombinant phage spanning ~85 kb of genomic DNA were isolated (see Fig. 3). A BamHI site adjacent to the region of EGF homology was arbitrarily chosen as coordinate position zero.
Genomic Southern blots containing mutant and wild-type DNAs were probed with DNA from the
individual phage isolates to detect and localize
rearrangement breakpoints that might be associated with the various Serrate alleles. Within the first phage isolate, ø10.2, restriction fragment polymorphisms were detected on the original SerD chromosome. The polymorphism detected with each of three restriction enzymes (EcoR., BamHI, and Hindlll) was consistent with an insertion of -5.5 kb of DNA between map coordinates 0 and -3 (Fig. 3). Subsequent Southern analysis using DNA cloned from SerD revealed a repeated DNA sequence, suggesting the presence of a mobile insertional element associated with the
mutation. In addition to the insertion, the Hindlll site at coordinate -2 has been eliminated in the SerD chromosome. Because the parental chromosome from which the SerD mutation arose is unavailable, we cannot be certain that the noted polymorphisms are causal to the SerDphenotype.
Of the five Serrevertant alleles, three (Serrev2-3,Serrev5-5, and Serrev6-1 ) appeared cytologically normal and did not exhibit DNA polymorphisms detectable by our Southern analyses. The remaining two revertants,
Serrev2-11, and Serrrev3, had polymorphic DNA restriction fragments within the cloned region. Serrev2-11 is an inversion of polytene bands 97F to 98C. The 97F breakpoint was localized between coordinates +1.5 to +4, within the region of strongest detectable EGF homology (Fig. 3). Serrev3 is a reciprocal
translocation of chromosomes 3R and 2R, with the 97F breakpoint localized between coordinates +15 and +17 (Fig. 3). in situ hybridization of the cloned wild- type genomic DNAs to polytene chromosomes of Serrev3 and Serrev2-11 confirmed that the observed DNA
polymorphisms represent the 97F breakpoints of these chromosomal rearrangements.
As noted earlier, Serrevertant alleles fail to complement BdG, suggesting that the Serrate and Bd mutations are alleles of the same gene. As with the
SerD mutation, the parental chromosome for the BdG mutation was not available; hence, unambiguous
assignment of mutant phenotypes to DNA polymorphisms cannot be made. Cytological observations of the BdG chromosome failed to reveal any visible abnormalities; however, two regions of DNA polymorphism were
detectable by Southern analysis. These regions lie between coordinates 0 to +1 and +14 to +17.
Investigations of the polymorphism at position 0 to +1 were pursued by cloning the mutant DNA sequences.
Preliminary results indicate that the polymorphisms do not result from a small inversion between these two regions but, rather, from a more complex event.
Of the three mutant chromosomes, Bd43.5, Bd862.5, and pll11, only pll11 was found to have a DNA
polymorphism, which was localized between coordinates +17 and +19 (Fig. 3). Genetic and cytological data for the pll11 mutation suggest the presence of a very small chromosomal aberration within the 97F region (P. Hecht, pers. comm.), and the molecular data are
consistent with this observation. Finally, T(Y:3)R128 is a reciprocal translocation that also breaks within the 97F region (Lindsley et al., 1972, Genetics
71:157-184) and fails to complement BdG (P. Hecht, pers. comm.). The DNA breakpoint for this
translocation resides at map coordinates +25 to +28 (Fig. 3). Taken together, these findings strengthen the genetic evidence that Serrate and Bd mutations are alleles of the same gene. In summary, of eleven tested chromosomes containing Serrate or Bd mutation, six were shown to have associated DNA rearrangements within a 30 kb region known to contain EGF homologous sequences.
To examine the structure of the Serrate
transcription unit, we probed Northern blots
containing 2- to 14-hour embryonic poly(A)+ RNA with the recombinant phages spanning this region (ø10.1, 01.3 and ø15K; Fig. 3). This analysis revealed the presence of two transcripts of ~5.5 kb and 5.6 kb.
We isolated two overlapping cDNA clones, denoted C1 and C3, from an early pupal library (see Section 6.3). Sequence analysis of these cDNAs revealed a perfect overlap of 109 bp for a combined length of 5.6 kb, which is in excellent agreement with the larger of the two transcripts as determined by Northern analysis. Genomic probes unique to the 5' end of C3 only
detected the larger 5.6 kb transcript. Thus, the size difference between the 5.5 and 5.6 kb transcripts may represent an alteration in the potential protein coding capacity or an alteration of 5' untranslated sequence. The composite 5.6 kb cDNA confirms that the Serrate transcription unit spans ~30 kb of genomic DNA, encompasses the EGF homologous region, and is interrupted by at least five of the six DNA
rearrangements that affect Serrate function (Fig. 3). From Southern analysis, at least two introns are apparent; additional introns are likely but not detectable at this level of resolution.
6.1.5. SERRATE ENCODES A PUTATIVE TRANSMEMBRANE
PROTEIN WITH 14 EGF-LIKE REPEATS
The complete nucleotide sequence compiled from the cDNAs C1 and C3 is 5561 bp (see Fig. 4) and agrees with the transcript sizes determined by Northern analysis. Within this sequence there is a single large open reading frame (ORF) of 4329 bp. There are two possible initiator AUG codons at positions 433 and 442. Of these, the second AUG is within a sequence context that agrees with the Drosophila consensus sequence determined for translation initiation
[CAAAAUG; (Cavener, 1987, Nucl. Acids Res. 15:1353- 1361)]. Predicted codon usage within this ORF is highly consistent with established Drosophila
melanogaster codon preferences (Beachy et al., 1985, Nature 313:545-550). Assuming that translation starts at the second AUG, the Serrate mRNA contains an untranslated leader sequence of at least 441 base pairs, encodes an expected protein product of 1404 amino acids, and terminates with 908 bp of
untranslated 3' sequence (Fig. 5a). However, if translation begins at the first AUG, the protein product is 1443 amino acids.
Hydropathy plots revealed three major hydrophobic regions (Fig. 5b; see also Section 6.3). The first, beginning at amino acid 51, is likely to represent a signal peptide sequence; a potential signal cleavage site occurs at amino acid 80. A second hydrophobic domain runs from amino acid 540 to 560. This region does not have a requisite
transmembrane structure and is more likely to be a membrane-associated domain. The third hydrophobic domain (amino acids 1220 to 1245) is bounded by
hydrophilic residues and is therefore likely to
represent a true transmembrane domain.
The most striking structural feature of the predicted protein is the series of EGF-like repeats (see Fig. 5c). There are 14 copies of this motif with an additional partial or degenerate repeat occurring toward the amino terminus (see below). In addition, at least three of these repeats are interrupted by stretches of amino acids. The first interruption
(labelled A in Fig. 5c), which occurs in the fourth complete EGF-like repeat (repeats are numbered
beginning from the amino terminus), is ~64 amino acids in length and is enriched for serine residues.
The second interruption (labelled B in Fig. 5c), occurring in the sixth repeat, is ~44 amino acids long and has numerous hydrophobic residues. This region represents the putative membrane-associated domain noted earlier. The final interruption (labelled C in Figure 5c), which occurs in us tenth repeat and is 29 amino acids in length, has an unusual run of
threonines [Thr(9) Ala Thr(3) ].
Within the amino-terminal region of the Serrate protein, considerable structural homology (darklyshaded region in Fig. 5c) is observed with the main protein product of the Delta locus (Vassin et al., 1987, EMBO J. 6:3431-3440; Kopczynski et al., 1988, Genes Dev. 2:1723-1735). Near the signal peptides for both of these molecules there lies a stretch of ~210 conserved amino acids. Within the first 165 amino acids, there is ~32% identity, which increases to greater than 50% for the remaining 45 amino acids.
The latter region corresponds to the partial EGF-like repeat (designated PR in Fig. 5c), which lacks a cysteine residue but retains the other characteristic cysteines and conserved amino acids typically found in the remaining EGF-like repeats. The homology between Serrate and Delta extends beyond these amino-terminal regions, since both of these proteins contain EGF-like repeats.
in addition to the extracellular EGF-like sequences, the predicted Serrate protein contains a small intracellular domain of ~160 amino acids. The internal domain does not contain any significant known structural homologies, although there are numerous potential sites for phosphorylation (Those identified in the putative intracellular region by the SITES program were at amino acid positions 1283, 1292, 1297, 1349, 1365, 1371, 1389, and 1390).
6.1.6. EXPRESSION OF SERRATE RNA Northern analysis of developmentally staged RNAs revealed that the majority of Serrate expression is represented by two coordinately regulated
transcripts of 5.5 kb and 5.6 kb, which first appear 4 to 8 hours into embryogenesis (Fig. 6). These
transcripts show peak expression between 8 and 12 hours of embryogenesis and diminish thereafter;
however, they continue to be readily detectable throughout development except for the adult stages (Fig. 6). In addition to these major transcripts, a smaller (3.4 kb) transcript is expressed transiently between 2 and 4 hours of embryogenesis (Fig. 6).
We undertook an analysis of the spatial distribution of RNA transcripts from the Serrate locus in order to identify regions of the embryo that may require Serrate function. Using the whole mount in situ method (Tautz and Pfeifle, 1989, Chromosoma
98:81-85) and employing nonradioactive probes that hybridize to both the 5.5 kb and 5.6 kb transcripts, we found that Serrate mRNA accumulates in a dynamic pattern beginning from mid-embryogenesis (late stage 10) and persisting until the latest stages examined (stage 16); (embryonic stages are those of Campos- Ortega and Hartenstein, 1985, The Embryonic
Development of Drosophila Melanogaster, Springer- Verlag, Berlin). Because the tissue distribution of the two transcripts may be independently regulated, we note that the observed RNA localizations may represent a composite for both transcripts. We also note the possibility of a low level of Serrate RNA in the yolk of pre-gastrulation embryos because faint staining of the yolk was observed consistently. Although this staining was never observed with control probes (see
Section 6.3), the presence of yolk staining is known to be a common artifact of the whole-mount in situ technique (Ashburner, 1989, Drosophila - A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). However, if this
observation is not artifactual, the observed staining may correspond to the expression of the transient 3.4 kb RNA species observed by the Northern analysis of this same developmental stage.
Initial cellular localization was seen in latestage 10 embryos and consisted of a ring of cells in the foregut. The foregut is formed by the
invagination of the stomodeum (the initial event of stage 10); thus, the foregut is actually derived from ectodermal tissue. Shortly thereafter, a bilateral patch of expressing cells appeared in the anteriormost portion of the head, the presumptive clypeolabrum (Fig. 7a). Additional areas of expression appeared abruptly at the end of stage 10 in a group of cells on the lateral edge of abdominal segment 8, followed by cells near the proctodeum and lateral epidermis of abdominal segment 9 (Fig. 7b). Later, during stage 11, expression was detected within cells located at the junction between the labial and maxillary lobes and within cells located near the tracheal pit of the first thoracic segment. The expression pattern
progressed to include a group of lateral epidermal cells located between the tracheal pits in each of the thoracic and abdominal segments (Fig. 7c). In
addition, each abdominal segment displayed a cluster of cells on either side of the ventral midline.
During germ band retraction (stage 12), the lateral epidermal cell patches broadened to form stripes that lie in the middle of each segment. A portion of these cells appeared to coalesce into an internal longitudinal stripe that was coincident with the developing tracheae (see Fig. 7 e, f, g, and h). The cells that remained on the surface extended dorsally and ventrally forming a zig-zag shaped pattern (Fig. 7g, arrows). This surface expression in the thoracic segments was wider, more intense, and extended further dorsally and ventrally than in the abdominal segments (Fig. 7g). Later in embryogenesis (stages 14 and 15) the surface epidermal expression, with the exception of the first thoracic segment, diminished relative to the tracheal expression.
Later, intense expression was observed in what
appeared to be ectodermal invaginations located dorsolaterally on the thoracic segments (Fig. 7j).
These pockets of cells may correspond to primordia of imaginal discs; in the first thoracic segment they appeared to be closely associated with opening of the anterior spiracle.
Coincident with the lateral expression, another segmentally reiterated pattern evolved in the ventral epidermis of the trunk. In the extended germ band embryo, this pattern, which consisted of stripes of expressing cells near the anterior border of the abdominal segments, lay out of register with the corresponding lateral expression (Fig. 7c). The pattern in the thorax contrasted with that in the abdomen and consisted of only small clusters of
expressing cells in the latero-ventral region (see Fig. 7f and h). The ventral expression was quite intense through stage 13 and dissipated thereafter (Fig. 71).
Serrate expression was also observed in the ectodermally-derived portions of the gut. The
earliest expression was evident in the foregut and persisted throughout embryonic development (Fig. 7a).
During germband retraction, a tightly defined,
intensely expressing ring of cells lay at the junction with the anterior midgut. The proventriculus develops from this area; however, expression was limited to the ectodermally-derived portion of this composite
structure (King, 1988, J. Morph. 196:253-282).
Hindgut expression, though appearing later than foregut expression, occurred at an analogous position, that is, where ectoderm meets endoderm. The initial expression in the hindgut was seen at the time of germ band retraction (stage 12) as a wide band of cells where the Malphigian tubules were forming, but never included the tubules themselves. Later still (stage 14), an additional ring of expression appeared in the hindgut approximately mid-way between the insertion point of the Malphigian tubules and the proctodeum (Fig. 7h). Expression at the posterior-most end of the embryo, near the proctodeal opening, initiated early (stage 11) (Fig. 7b). This expression within the telson remained at high levels throughout
embryonic development, eventually forming a ring of cells around the presumptive anal pads (Fig. 71).
Within the head region, Serrate expression was temporally and spatially dynamic. The earliest expression occurred in the presumptive clypeolabrum (stage 10; Fig. 7a) and became broader and more intense as development proceeded. Early expression between the labial and maxillary lobes increased along their borders, and expression was also seen in the anterior of the mandibular lobe during stage 12 (Fig. 7d and e). In addition, expression was now observed in the hypopharyngeal region, just posterior to the stomodeum, and at the base of the labial lobes in an area encompassing the salivary gland duct opening (Fig. 7d). There was also low level expression in the dorsal procephalic epidermal region (not shown). By the end of germ band retraction (stage 13), expression encompassed the entire mandibular lobe. As a
consequence of the cellular movements associated with head involution (stages 14-16), the expressing cells of the clypeolabrum, hypopharynx and labial lobes combined to form the pharynx. Prior expression in the area of the salivary gland placodes was now limited to the ducts of the developing salivary gland (Fig. 7i). The maxillary and mandibular lobes, which have moved to the anterior-most region of the embryo, expressed intensely at this time (Fig. 7j).
Serrate expression in the central nervous system (CNS) was apparent during stage 12 as a
segmentally-reiterated array of single cells along the lateral edge of the ventral nerve cord and within the supraesophogeal ganglia (brain hemispheres). By the end of germ band retraction (stage 13), there were now two cells that appeared to express in each hemisegment of the ventral nerve cord (not shown). However, by stage 15, ventral nerve cord expression was again limited to a single cell per hemisegment (Fig. 7i) while expression in the brain hemispheres remained unchanged (Fig. 7k).
In summary, there are a wide array of tissues that express Serrate mRNA, and the expression pattern is tightly regulated both temporally and spatially. In addition, it should be stressed that at the present level of resolution, Serrate expression appears to be restricted exclusively to cells of ectodermal origin.
6.2. DISCUSSION
Unlike Notch and Delta, the fourteen EGF repeats of Serrate are not completely contiguous. At least three of these repeats contain sizeable
interruptions consisting of insertions of long
stretches of amino acids. Similarly, interruptions have been noted in two of the thirty EGF-like repeats of the Drosophila gene crumbs (Tepass et al., 1990, Cell 61:787-799). In Serrate. the interruption that occurs in the sixth repeat is particularly intriguing because it consists largely of hydrophobic amino acids. Although hydropathy plots indicate that this region does not conform to known transmembrane
regions, it could represent a membrane-associated domain that serves to "tie" the protein back to the membrane. The interruption in the tenth repeat is also unusual in that it bears a stretch of threonines [Thr(9) Ala Thr(3) ]. A similar motif of thirteen
contiguous threonine residues is found in the
glycoprotein glutactin, a basement membrane protein of Drosophila (Olson et al., 1990, EMBO J. 9:1219-1227).
If the observed genetic interactions between Notch and Serrate had been only with the original SerD allele, it could have been argued that this neomorphic mutation is allowing two functionally disparate but structurally similar molecules to interact out of their normal contexts. But because we observe genetic interactions with other Serrate alleles, it is likely that we are observing a manifestation of normal
Serrate-Notch interactions.
we have shown that phenotypic revertants of SerD behave genetically in a similar fashion to known deficiencies for the locus; that is, they are
homozygous lethal during embryogenesis and completely recessive as heterozygotes. We also gathered evidence indicating that the mutation BdG. which was thought to belong to a distinct complementation group, may in fact be an allele of Serrate.
The embryonic lethal phenotypes of Serrev2-3,
Serrev2-11 , and Ser rev5-5, which are essentially
indistinguishable from one another, appear unchanged when in homozygous or hemizygous condition. This latter result genetically defines these alleles genetically as amorphic. However, since the Ser rev2-3 allele complements the BdG mutation, the Ser rev2-3 mutation is probably not a protein null allele.
Consistent with the defects observed in the cuticle and nervous system of Ser- embryos, Serrate transcripts are localized in complex patterns within these tissues. The abundant and widespread expression of Serrate transcripts in the segments that make up the embryonic head and thorax correlates well with the lack of embryonic head and thoracic structures
commonly seen in Ser- embryos. Likewise, the pattern of Serrate expression in the ventral epidermis of the abdominal segments correlates with the frequently absent or improperly formed denticles. Although
Serrate is expressed in a small number of cells within the CNS, the gross morphological defects observed in the CNS of Ser- embryos may reflect contributions from two components. The first is the loss of Serrate CNS expression itself, and the second may be a consequence of mechanical stresses (e.g., lack of germ band retraction) imposed by an improperly differentiating epidermis.
In the course of examining the embryonic phenotypes associated with Serrate lethal mutations, we noticed their similarity to those produced by several alleles of the gene coding for the Drosophila
EGF receptor homolog known as DER, faint little ball or torpedo (Livneh et al., 1985, Cell 40:599-607;
Price et al., 1989, Cell 56:1085-1092; Schejter and
Shilo, 1989, Cell 56:1093-1104). 6.3. MATERIALS AND METHODS
6.3.1. DROSOPHILA TURES AND STRAINS Cultures were maintained on standard cornmeal/ molasses/agar Drosophila medium supplemented with active dry yeast and were raised at 25°C. The red
SerD, Df (3R) Ser+82f24, and BdGchromosomes were obtained from Peter Lewis. The red SerD chromosome was maintained in homozygous condition. The mutations pll11, Bd862.5, and Bd43.5 were generously provided by Kathryn Anderson. The Notch duplication CosP479 is an ~40 kb P-element cosmid construct inserted into the third chromosome (Ramos et al., 1989, Genetics
123:337-348). Other mutations and chromosomes have been described previously (Lindsley and Grell, 1968, Genetic variations of Drosophila melanogaster,
Carnegie Inst. Wash. Publ. 627).
6.3.2. MUTAGENESIS
Males aged 3-7 days and homozygous for the red SerD chromosome were irradiated with approximately 4500 R (150 kV, 5 mA, 9.2 min exposure; Torrex 150 Source, Torr X-Ray Corp.) and mated immediately to
C(1)A;y/y2Y611 or C(1)Dx;yf/y2Y611 virgin females. The F1 males were scored for the absence of the SerD wing phenotype and mated to Glp1-3 fz red e/Tm2, red e virgin females to establish balanced Serrev/Tm2, red e stocks.
Mutations used in this study are shown in Table
1. 3 v2 1
3.5
.5 1
2f24
Figure imgf000062_0001
6.3.3. EMBRYONIC PHENOTYPE ANALYSIS
Cuticle preparations were according to the protocol of Wieschaus and Nϋsslein-Volhard (1986, in Drosophila. A Practical Approach, (ed. D.B. Roberts), IRL Press, Oxford, pp. 199-227) on embryos aged for a minimum of 24 hours at 25°C. Anti-horseradish
peroxidase antibody staining of the embryonic nervous system (Jan and Jan, 1982, Proc. Natl. Acad. Sci. USA 79:2700-2704) was carried out using fluoresceinconjugated antibody (Cappel) as described in Preiss et al. (1988, EMBO J. 7:3917-3927). CNS preparations of torpedo2C82 were used for comparison studies.
6.3.4. ISOLATION OF NUCLEIC ACIDS Genomic DNA was isolated as described in Pirrotta et al. (1983, EMBO J. 2:927-934).
Restriction enzyme cleavage, agarose gel
electrophoresis, capillary transfer to nitrocellulose and hybridization conditions were carried out
according to standard procedures. DNA probes labeled with 32P were prepared by random oligonucleotide priming, as described in Feinberg and Vogelstein
(1983, Anal. Biochem. 132:6-13). Stage-specific total RNAs from a Canton-S strain were extracted in
guanidinium thiocyanate essentially as described in Chirgwin et al. (1979, Biochem. 18:5294-5299). Pupal and adult RNAs were generously provided by A. Preiss (Preiss et al., 1988, EMBO J. 7:3917-3927). Poly (A)+ RNA was selected by serial passage over oligo(dT)- cellulose (Stratagene) according to Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York) and stored in ethanol. RNA was size fractionated in formaldehyde agarose gels and blotted onto Nytran membrane (Schleicher & Schuell) by capillary transfer. RNA was fixed to the membrane via UV crosslinking.
Two Drosophila genomic phage libraries (Preiss et al., 1985, Nature 313:27-32; R. Karess, unpubl.) were screened and recombinant clones were isolated as described in Benton and Davis (1977, Science 196:180- 182). cDNAs in λgt10 were isolated from the early pupal library of Poole et al. (1985, Cell 40:37-43). We isolated the Cl cDNA using the genomic EGF-like sequences from coordinates +1.5 to +4 (Fig. 3) as probe. Subsequently, we isolated the C3 cDNA using the 5' 700 bp terminal fragment of the C1 cDNA as probe.
6.3.5. SEQUENCING AND ANALYSIS
The EcoRI cDNA inserts from λgt10 were subcloned directly into Bluescript KS+ and KS- vectors (Stratagene). Single-stranded DNAs were produced according to the manufacturer's instructions. Both strands of the cDNAs were sequenced using the
dideoxynucleotide chain-termination procedure (Sanger, et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463- 5467) using the Sequenase kit (U. S. Biochemical).
Sequence was obtained using the M13 and reverse
primers for these vectors. Additional sequence was obtained by generating internal deletions through the use of restriction sites within the Bluescript
polylinker and the cDNA inserts. The remaining cDNA sequences that were not accessible by these methods were obtained by using synthetic primers (Research Genetics) complementary to the end of a previously determined sequence.
Sequences were entered by sonic digitizer and overlapping sequence compilation; manipulation,
translation, and secondary structure prediction were accomplished by using the Intelligenetics PC-GENE. 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 SITES program (PCGENE) was used to predict the location of the signal sequence, transmembrane domain, EGF-like repeats, and
phosphorylation sites.
6.3.6. WHOLE MOUNT IN SITU PROCEDURE A modification of the whole-mount in situ procedure of D. Tautz (Procedure 84a in Ashburner, 1989, Drosophila: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) was used. The differences were as follows:
Proteinase K (Boehringer-Mannheim) treatment was 10 to 14 minutes; 100 μl (rather than 10 μl) of boiled probe was used; after washing the embryos with 1:4 hybridization buffer to PBT, they were washed twice in PBT for 20 minutes, and then twice in 1X PBS, 0.1% BSA (globin free, Sigma), 0.2% Triton-X100 for 20 minutes; the antibody treatment was done in the same PBS, BSA, Triton solution at 4°C overnight; the embryos were washed four times in the PBS, BSA, Triton solution at room temperature; after the alkaline phosphatase reaction, embryos were dehydrated twice in 70% and 100% ethanol and then cleared in xylenes; the embryos were mounted in Permount (Sigma). Dissected embryos were rehydrated, dissected in PBT, and mounted in 90% glycerol [10% Tris-HCl at pH 8.0, with 0.5% n-propylgalate (wt/vol; Sigma)].
The probe was made by runoff of a PCR reaction in 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.01% (wt/vol) gelatin, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.15 mM dTTP, and 0.07 mM digoxigenin-11-dUTP (Boehringer Mannheim) using 150 ng of custom synthesized primer and approximately 400 ng of linearized DNA. Probe was synthesized from cDNA coordinates 4826 to 3854; the opposite strand
constituted the control probe and was synthesized from coordinates 4458 to 5015 (refer to Fig. 6). The conditions for the PCR thermal cycler were 95°C for 45 seconds, 55°C for 30 seconds, and 72°C for 1 minute, which were run for 30 cycles. The probe was ethanol precipitated twice and resuspended in 300 μl of hybridization solution.
7. EXPRESSION OF A SERRATE FRAGMENT
AS A FUSION PROTEIN AND PRODUCTION OF ANTIBODIES THERETO
Mouse anti-Serrate polyclonal antisera were made as follows: A BamHI fragment encoding amino acids 78-425 (Fig. 4) was subcloned into the pGEX-1 expression vector (Smith and Johnson, 1988, Gene
67:31-40). Fusion proteins were purified on
glutathione-agarose beads (SIGMA), and injected into mice for antibody production. Mouse antisera were precipitated with 50% (NH4)2SO4 and resuspended in PBS
(150 mM NaCl, 14 mM Na2HPO4, 6 mM NaH2PO4) with 0.02%
NaN3.
8. EXPRESSION OF SERRATE AND A FRAGMENT AND A
CHIMERIC DERIVATIVE THEREOF; IDENTIFICATION
OF A NOTCH-BINDING DOMAIN
We describe herein the recombinant expression of Serrate, of a deletion construct (fragment)
thereof, and of a chimeric Notch-Serrate fragment, and show that the full-length Serrate and the chimeric derivative are capable of binding to Notch in vitro. 8.1. EXPRESSION OF SERRATE AND OF
DERIVATIVES THEREOF
For the Serrate expression construct, a synthetic primer containing an artificial BamHI site immediately 5' to the initiator AUG at position 442
(all sequence numbers are according to Fleming et al.,
1990, Genes & Dev. 4:2188-2201) and homologous through position 464, was used in conjunction with a second primer from position 681-698 to generate a DNA
fragment of -260 base pairs. This fragment was cut with BamHI and Kpnl (position 571) and ligated into
Bluescript KS+ (Stratagene). This construct,
BTSer5'PCR, was checked by sequencing, then cut with
Kpnl. The Serrate Kpnl fragment (571 - 2981) was inserted and the proper orientation selected, to generate BTSer5 'PCR-Kpn. The 5' SacII fragment of
BTSer5'PCR-Kpn (SacII sites in Bluescript polylinker and in Serrate (1199)) was isolated and used to replace the 5' SacII fragment of cDNA C1 (Fleming et al., 1990, Genes & Dev. 4:2188-2201), thus
regenerating the full length Serrate cDNA minus the 5' untranslated regions. This insert was isolated by a
Sall and partial BamHI digestion and shuttled into the BamHI and Sall sites of the metallothionein promoter vector pRmHa-3 (Bunch et al., 1988, Nucl. Acids. Res. 16:1043-1061) to generate the final expression
construct, Ser-mtn.
A Serrate deletion expression construct was also made, in which nucleotides 672-1293 (encoding amino acids 77-284) (Figs. 4, 8, 9) were deleted.
This deletion construct was made as follows: The Sermtn construct was digested with EcoRV. which cuts at nucleotide 672, and with Sfil, which cuts at
nucleotide 4073. The linearized vector, lacking the EcoRV-Sfil (672-4073) fragment, was isolated. Plasmid SerFL was then digested with Ndel, which cuts at nucleotide 1289, and treated with mung bean nuclease resulting in the "trimming back" of four bases. The resulting SerFL fragment was then digested with Sfil which cuts at base 4073, and the resulting 1293-4073 fragment was isolated and ligated into the EcoRV-Sfil vector isolated above.
In addition, a Notch-Serrate chimeric construct was made using a clone consisting of Drosophila Notch cDNA with a deletion of all the Notch EGF-like repeats ("ΔEGF") (see copending application Serial No. to be assigned, filed November 14, 1991 by Artavanis- Tsakonas et al.; Rebay et al., 1991, Cell 67:687-699 (Fig. 13, construct no. 25)). An N-terminal region of Serrate with homology to Delta and including the
Serrate EGF-like repeats (Serrate nucleotide numbers 676-1287, encoding amino acids 79-282; Figs. 8, 9) was placed into the ΔEGF deletion of Notch.
The above constructs were expressed in Drosophila S2 cells. The S2 cell line (Schneider, 1972, J. Embryol. Exp. Morph. 27, 353-365) was grown in M3 medium (prepared by Hazleton Co.) supplemented with 2.5 mg/ml Bacto-Peptone (Difco), 1 mg/ml TC
Yeastolate (Difco), 11% heat-inactivated fetal calf serum (FCS) (Hyclone), and 100 U/ml penicillin-100 μg/ml streptomycin-0.25 μg/ml fungizone (Hazleton).
Cells growing in log phase at -2 x 106 cells/ml were transfected with 20 μg of DNA-calcium phosphate
coprecipitate in 1 ml per 5 ml of culture as
previously described (Wigler et al., 1979, Proc. Natl. Acad. Sci. USA 78, 1373-1376), with the exception that BES buffer (SIGMA) was used in place of HEPES buffer (Chen and Okayama, 1987, Mol. Cell. Biol. 7, 2745-
2752). After 16-18 hr, cells were transferred to conical centrifuge tubes, pelleted in a clinical centrifuge at full speed for 30 seconds, rinsed once with 1/4 volume of fresh complete medium, resuspended in their original volume of complete medium, and returned to the original flask. Transfected cells were then allowed to recover for 24 hr before
induction. Expression from the metallothionein constructs was induced by the addition of CuSO4 to 0.7 mM.
8.2. AGGREGATION ASSAYS FOR BINDING TO NOTCH
8.2.1. METHODS
Two types of aggregation assays were used. In the first assay, a total of 3 ml of cells (5-10 x 106 cells/ml) was placed in a 25 ml Erlenmeyer flask and rotated at 40-50 rpm on a rotary shaker for 24-48 hr at room temperature. For these experiments, cells were mixed 1-4 hr after induction began and induction was continued throughout the aggregation period. In the second assay, ~0.6 ml of cells were placed in a 0.6 ml Eppendorf tube (leaving a small bubble) after an overnight induction (12-16 hr) at room temperature and rocked gently for 1-2 hr at 4°C. Ca2+ dependence experiments were performed using the latter assay. For Ca2+ dependence experiments, cells were first collected and rinsed in balanced saline solution (BSS) with 11% FCS (BSS-FCS; PCS was dialyzed against 0.9% NaCl, 5 mM Tris [pH 7.5]) or in Ca2+ free BSS-FCS containing 10 mM EGTA (Snow et al., 1989, Cell 59: 313-323) and then resuspended in the same medium at the original volume.
For Viewing by immunofluorescence, cells were collected by centrifugation (3000 rpm for 20 seconds in an Eppendorf microcentrifuge) and fixed in 0.6 ml Eppendorf tubes with 0.5 ml of freshly made 2%
paraformaldehyde in PBS for 10 min at room temperature. After fixing, cells were collected by centrifugation, rinsed twice in PBS, and stained for 1 hr in primary antibody in PBS with 0.1% saponin
(SIGMA) and 1% normal goat serum (Pocono Rabbit Farm, Canadensis, PA). Sera were appropriately diluted
(e.g., 1:1000) for this step. Cells were then rinsed once in PBS and stained for 1 hr in specific secondary antibodies (double-labeling grade goat anti-rabbit and goat anti-mouse), in PBS-saponin-normal goat serum. After this incubation, cells were rinsed twice in PBS and mounted on slides in 90% glycerol, 10% 1 M Tris (pH 8.0), and 0.5% n-propyl gallate. Cells were viewed under epifluorescence on a Leitz Orthoplan 2 microscope.
Confocal micrographs were taken using the Bio- Rad MRC 500 system connected to a Zeiss Axiovert compound microscope. Images were collected using the BHS and GHS filter sets, aligned using the ALIGN program, and merged using MERGE. Fluorescent bleedthrough from the green into the red channel was
reduced using the BLEED program (all software provided by Bio-Rad). Photographs were obtained directly from the computer monitor using Kodak Ektar 125 film.
Notch-expressing cells for the assays were obtained similarly, using metallothionein promoterdriven plasmid constructions containing D.
melanogaster Notch (see copending application Serial No. to be assigned, filed November 14, 1991 by
Artavanis-Tsakonas et al.; Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699).
8.2.2. RESULTS
We found that Serrate expressing cells adhere to Notch expressing cells in a calcium dependent manner (see also Rebay et al., 1991,. Cell 67:687-699). However, unlike Delta, under the experimental
conditions tested, Serrate did not appear to interact homotypically. In addition, we detect no interactions between Serrate and Delta. It is possible that such interactions do occur, but at an affinity such that they are below the level of detection in our assay system.
We have tested a subset of our Notch deletion constructs to map the Serrate-binding domain and have found that Notch EGF-like repeats 11 and 12, in addition to binding to Delta, also mediate
interactions with Serrate. In addition, the Serratebinding function of these repeats also appears to have been conserved in the corresponding two EGF repeats of Xenopus Notch (construct #33ΔCla+XEGF(10-13); see Rebay et al., supra).
We were also able to define the Serrate region which is essential for the Notch/Serrate aggregation. Deleting nucleotides 672-1293 (i.e. amino acids 77- 284) eliminated the ability of the Serrate protein to aggregate with Notch. While both cells expressing Notch and cells expressing the Serrate fragments were detected by immunofluorescence with anti-Notch and anti-Serrate antibodies, respectively, these cells did not co-aggregate.
Aggregation assays with cells expressing Notch and cells expressing the chimeric ΔEGF Notch-Serrate construct showed binding between Notch and the
chimeric construct. These experiments thus
demonstrated that a fragment of Serrate consisting of amino acids 79-282 (see SEQ ID NO: 2) is capable of mediating binding to Notch. Similar experiments with Delta from the laboratory of M. Muskavitch (personal communication) have demonstrated that the homologous region of Delta (without the partial EGF-like repeat) was sufficient to mediate Notch-Delta binding.
Therefore, it is likely that the partial EGF-like repeat of Serrate is not essential for this binding to occur.
Work in our laboratory has shown that Notch and Delta proteins interact directly at the molecular level (Fehon et al., 1990, Cell 61:523-534; copending U.S. patent applications serial no. 07/695,189 filed May 3, 1991 and serial no. to be assigned, filed
November 14, 1991, by Artavanis-Tsakonas, et al.;
collectively incorporated by reference herein in their entireties), as demonstrated by the specific binding of Notch-expressing cells to Delta-expressing cells in vitro. We have also shown that EGF-like repeats repeats 11 and 12 of Notch are required and sufficient for Notch-Delta-mediated aggregation, and that Delta participates in heterotypic (Delta-Notch) and
homotypic (Delta-Delta) interactions mediated by its amino-terminus (id.). Thus, it is conceivable that the Serrate and Delta proteins compete for binding with the Notch protein. Such interplay could underlie the genetic interactions observed between Notch and
Serrate.
Notch and Serrate appeared to aggregate less efficiently than Notch and Delta, perhaps because the Notch-Serrate interaction is weaker. For example, when scoring Notch-Delta aggregates, we detect ~40% of all Notch expressing cells in clusters with Delta expressing cells and ~40% of all Delta expressing cells in contact with Notch expressing cells. For Notch-Serrate, we find only -20% of all Notch
expressing cells and ~15% of all Serrate expressing cells in aggregates. For the various Notch deletion constructs tested, we consistently detect a reduction in the amount of aggregation between Notch and Serrate as compared to the corresponding Notch-Delta levels, with the possible exception of two constructs which exhibit severely reduced levels of aggregation even with Delta. One trivial explanation for this reduced amount of aggregation could be that our Serrate construct simply does not express as much protein at the cell surface as the Delta construct, thereby diminishing the strength of the interaction.
Alternatively, the difference in strength of
interaction may indicate a fundamental functional difference between Notch-Delta and Notch-Serrate interactions that may be significant in vivo.
9. THE CLONING, SEQUENCING, AND
EXPRESSION OF HUMAN SERRATE
Clones for the human Serrate sequence are obtained as described below.
The polymerase chain reaction (PCR) was used to amplify DNA from a 17-18 week human fetal brain cDNA library in the Lambda Zap II vector (Stratagene).
Degenerate primers used in this reaction were designed based on amino-terminal regions of homology between Drosophila Serrate and Drosophila Delta (see Fig. 9). Synthetic oligonucleotide primers FLE1 and FLE4R
(shown in Fig. 9) were used in PCR to amplify Serratehomologous fragments in the cDNA library. The PCR reaction products were subjected to agarose gel electrophoresis, resulting in the detection of two amplified DNA fragments from the cDNA library. The fragments are then each cloned into a plasmid for production of quantities thereof, using the TA cloning kit (Invitrogen).
The Serrate-homologous fragments amplified and obtained in this manner are then sequenced at least in part, by use of Sequenase® (U.S. Biochemical Corp.), to confirm the identity of the fragments as Serrate homologs. Upon such confirmation, the fragments are then used as probes with which to screen the same cDNA library for human Serrate clones. The isolated phage λ clones are converted to plasmids via the
manufacturer's instructions, yielding the Serratehomologous fragments as cDNA inserts in the EcoRI site of pBluescript SK- (Stratagene). The host E. coli strain is XL1-Blue (see Sambrook et al., 1989,
Molecular Cloning Press, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York, p. A12).
The sequence of human Serrate contained in the cDNA clone(s) is determined (by use of Sequenase®, U.S. Biochemical Corp.).
Expression constructs are made using the isolated clone(s). The clone is excised from its vector as an EcoRI restriction fragment(s) and
subcloned into the EcoRI restriction site of each of the three pGEX vectors (Glutathione S-Transferase expression vectors; Smith and Johnson, 1988, Gene 7: 31-40). This allows for the expression of the human Serrate protein product from the subclone in the correct reading frame.
10. DEPOSIT OF MICROORGANISM
Bacteria strain XL1-Blue containing plasmid SerFL, containing an EcoRI fragment encoding a full- length Drosophila Serrate, was deposited on December 11, 1991 with the American Type Culture Collection, 1201 Parklawn Drive, Rockville, Maryland 20852, under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedures, and assigned Accession No. ___________________________ . 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.
Figure imgf000076_0001
le le le
le le
le le
Figure imgf000077_0001
le le
le le
Figure imgf000078_0001
le le le le
le le
le el e
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
T/US91/09240
Figure imgf000088_0001

Claims

WHAT IS CLAIMED IS:
1. A substantially purified Serrate protein.
2. The protein of claim 1 which is a human protein.
3. The protein of claim 1 which is a D.
melanogaster protein.
4. The protein of claim 3 which comprises the amino acid sequence substantially as set forth in Figure 4 from amino acid numbers 81-1404 (in SEQ ID NO:2).
5. The protein of claim 3 which is encoded by plasmid SerFL as deposited with the ATCC and assigned accession number ___________ .
6. A substantially purified human protein encoded by a nucleic acid hybridizable to plasmid SerFL or the Serrate sequence therein as deposited with the ATCC and assigned accession number _____________.
7. A substantially purified derivative or analog of the protein of claim 2, which is able to display one or more functional activities of a human or D. melanogaster Serrate protein.
8. A substantially purified derivative or analog of the protein of claim 3, which is able to display one or more functional activities of a human or D. melanogaster Serrate protein.
9. The derivative or analog of claim 8 which is able to be bound by an antibody directed against a human or D. melanogaster Serrate protein.
10. A substantially purified fragment of the protein of claim 1, which is able to be bound by an antibody directed against a human Serrate protein.
11. A substantially purified fragment of the protein of claim 2, which is able to be bound by an antibody directed against a D. melanogaster Serrate protein.
12. A substantially purified fragment of the protein of claim 3, which is able to be bound by an antibody directed against a D. melanocraster Serrate protein.
13. A molecule comprising the fragment of claim 10.
14. A substantially purified fragment of the protein of claim 2 which is able to display one or more functional activities of a human or D.
melanogaster Serrate protein.
15. A substantially purified fragment of a Serrate protein comprising the extracellular domain of the protein.
16. A substantially purified fragment of a Serrate protein comprising the intracellular domain of the protein.
17. A substantially purified fragment of a Serrate protein comprising the membrane-associated region of the protein.
18. A substantially purified fragment of a Serrate protein comprising the transmembrane domain of the protein.
19. A substantially purified fragment of a Serrate protein comprising an epidermal growth factorhomologous repeat of the protein.
20. A substantially purified fragment of a Serrate protein comprising a region homologous to a Notch protein or a Delta protein, and consisting of at least six amino acids.
21. A substantially purified fragment of a Serrate protein comprising amino acid numbers 63-73, 124-134, 149-158, 195-206, 214-219, or 250-259 as shown in Figure 4 (SEQ ID NO: 2).
22. A substantially purified fragment of a Serrate protein comprising the region of the protein with the greatest homology to amino acid numbers 79- 282 as shown in Figure 4 (SEQ ID NO:2).
23. A chimeric protein comprising a fragment of a Serrate protein consisting of at least 6 amino acids fused via a covalent bond to an amino acid sequence of a second protein, in which the second protein is not Serrate.
24. The chimeric protein of claim 23 in which the fragment of a Serrate protein is a fragment capable of being bound by an anti-Serrate antibody.
25. The chimeric protein of claim 24 in which the Serrate protein is a D. melanogaster protein.
26. The chimeric protein of claim 23 which is able to display one or more functional activities of a human or D. melanogaster Serrate protein.
27. The chimeric protein of claim 23 in which the fragment of a Serrate protein comprises an
epidermal growth factor-homologous repeat of a Serrate protein.
28. A substantially purified fragment of a Serrate protein which (a) is capable of being bound by an anti-Serrate antibody; and (b) lacks the
transmembrane and intracellular domains of the
protein.
29. A substantially purified fragment of a Serrate protein which (a) is capable of being bound by an anti-Serrate antibody; and (b) lacks the
extracellular domain of the protein.
30. An antibody which is capable of binding the Serrate protein of claim 1.
31. An antibody which is capable of binding the Serrate protein of claim 2.
32. The antibody of claim 1 which is
monoclonal.
33. A molecule comprising a fragment of the antibody of claim 30, which fragment is capable of binding a Serrate protein.
34. An isolated nucleic acid comprising a nucleotide sequence encoding a Serrate protein.
35. The nucleic acid of claim 34 which is DNA.
36. An isolated nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of claim 34.
37. An isolated nucleic acid comprising a nucleotide sequence encoding the Serrate protein of claim 2.
38. An isolated nucleic acid comprising a nucleotide sequence encoding the Serrate protein of claim 3.
39. An isolated nucleic acid comprising a nucleotide sequence encoding the Serrate protein of claim 4.
40. An isolated nucleic acid comprising the Serrate sequence contained in plasmid SerFL as
deposited with the ATCC and assigned accession number ________________.
41. An isolated human nucleic acid
hybridizable to plasmid SerFL or the Serrate sequence therein as deposited with the ATCC and assigned
accession number ________________ .
42. An isolated nucleic acid comprising a fragment of a Serrate gene consisting of at least 8 nucleotides.
43. An isolated nucleic acid comprising a nucleotide sequence encoding the fragment of claim 10.
44. An isolated nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of claim 43.
45. An isolated nucleic acid comprising a nucleotide sequence encoding the fragment of claim 11.
46. An isolated nucleic acid comprising a nucleotide sequence encoding the fragment of claim 12.
47. An isolated nucleic acid comprising a nucleotide sequence encoding the fragment of claim 14.
48. An isolated nucleic acid comprising a nucleotide sequence encoding the fragment of claim 20.
49. An isolated nucleic acid comprising a nucleotide sequence encoding the protein of claim 23.
50. An isolated nucleic acid comprising a nucleotide sequence encoding the fragment of claim 28.
51. An isolated nucleic acid comprising a nucleotide sequence encoding the fragment of claim 29.
52. A recombinant cell containing the nucleic acid of claim 34.
53. A recombinant cell containing the nucleic acid of claim 37.
54. A recombinant cell containing the nucleic acid of claim 38.
55. A recombinant cell containing the nucleic acid of claim 42.
56. A method of producing a Serrate protein comprising growing a recombinant cell containing the nucleic acid of claim 34 such that the encoded Serrate protein is expressed by the cell, and recovering the expressed Serrate protein.
57. A method of producing a Serrate protein comprising growing a recombinant cell containing the nucleic acid of claim 37 such that the encoded Serrate protein is expressed by the cell, and recovering the expressed Serrate protein.
58. A method of producing a Serrate protein comprising growing a recombinant cell containing the nucleic acid of claim 38 such that the encoded Serrate protein is expressed by the cell, and recovering the expressed Serrate protein.
59. A method of producing a protein comprising a fragment of a Serrate protein, which method
comprises growing a recombinant cell containing the nucleic acid of claim 43 such that the encoded protein is expressed by the cell, and recovering the expressed protein.
55. The product of the process of claim 56.
56. The product of the process of claim 57.
57. The product of the process of claim 58.
58. The product of the process of claim 59.
PCT/US1991/009240 1991-12-11 1991-12-11 Nucleotide and protein sequences of the serrate gene and methods based thereon WO1993012141A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1991/009240 WO1993012141A1 (en) 1991-12-11 1991-12-11 Nucleotide and protein sequences of the serrate gene and methods based thereon
CA002125767A CA2125767A1 (en) 1991-12-11 1991-12-11 Nucleotide and protein sequences of the serrate gene and methods based thereon

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US1991/009240 WO1993012141A1 (en) 1991-12-11 1991-12-11 Nucleotide and protein sequences of the serrate gene and methods based thereon
CA002125767A CA2125767A1 (en) 1991-12-11 1991-12-11 Nucleotide and protein sequences of the serrate gene and methods based thereon

Publications (1)

Publication Number Publication Date
WO1993012141A1 true WO1993012141A1 (en) 1993-06-24

Family

ID=25677313

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/009240 WO1993012141A1 (en) 1991-12-11 1991-12-11 Nucleotide and protein sequences of the serrate gene and methods based thereon

Country Status (1)

Country Link
WO (1) WO1993012141A1 (en)

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0662827A1 (en) * 1992-09-30 1995-07-19 Yale University Therapeutic and diagnostic methods and compositions based on notch proteins and nucleic acids
WO1995035323A1 (en) * 1994-06-20 1995-12-28 Borealis Polymers Oy Procatalyst for ethylene polymer production, method for its preparation and use
WO1996027610A1 (en) 1995-03-07 1996-09-12 Yale University Nucleotide and protein sequences of vertebrate serrate genes and methods based thereon
US5780300A (en) * 1995-09-29 1998-07-14 Yale University Manipulation of non-terminally differentiated cells using the notch pathway
EP0861894A1 (en) * 1995-11-17 1998-09-02 Asahi Kasei Kogyo Kabushiki Kaisha Differentiation-suppressive polypeptide
EP0861261A1 (en) * 1995-06-28 1998-09-02 Imperial Cancer Research Technology Limited Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
WO1998051799A1 (en) * 1997-05-14 1998-11-19 Asahi Kasei Kogyo Kabushiki Kaisha Novel differentiation inhibitor
WO1998057983A2 (en) * 1997-06-18 1998-12-23 Zymogenetics, Inc. Mammalian neuro-growth factor like protein
EP0930365A2 (en) * 1991-05-03 1999-07-21 Yale University Binding domains in serrate protein
US6004924A (en) * 1991-12-11 1999-12-21 Imperial Cancer Research Technology, Ltd. Protein sequences of serrate gene products
US6090922A (en) * 1991-05-03 2000-07-18 Yale University Antibodies to human notch proteins and fragments
US6156556A (en) * 1991-12-13 2000-12-05 Heska Corporation Flea protease proteins, nucleic acid molecules, and uses thereof
US6204010B1 (en) 1995-06-07 2001-03-20 Heska Corporation Flea protease proteins, nucleic acid molecules, and uses thereof
US6232096B1 (en) 1991-12-13 2001-05-15 Heska Corporation Flea serine protease nucleic acid molecules and uses thereof
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
WO2011127472A1 (en) 2010-04-09 2011-10-13 Fred Hutchinson Cancer Research Center Compositions and methods for providing hematopoietic function
WO2011127470A1 (en) 2010-04-09 2011-10-13 Fred Hutchinson Cancer Research Center Compositions and methods for providing hematopoietic function without hla matching
WO2013086436A1 (en) 2011-12-08 2013-06-13 Fred Hutchinson Cancer Research Center Compositions and methods for enhanced generation of hematopoietic stem/progenitor cells
WO2014110020A1 (en) 2013-01-08 2014-07-17 Fred Hutchinson Cancer Research Center Compositions and methods for expansion of embryonic hematopoietic stem cells
WO2016118780A1 (en) 2015-01-21 2016-07-28 Fred Hutchinson Cancer Research Center Point-of-care and/or portable platform for gene therapy
WO2016176652A2 (en) 2015-04-29 2016-11-03 Fred Hutchinson Cancer Research Center Modified stem cells and uses thereof
US10813949B2 (en) 2015-12-04 2020-10-27 Fred Hutchinson Cancer Research Center Uses of expanded populations of hematopoietic stem/progenitor cells
EP3995142A2 (en) 2015-04-29 2022-05-11 Fred Hutchinson Cancer Research Center Modified hematopoietic stem/progenitor and non-t effector cells, and uses thereof
EP4083062A1 (en) 2013-10-31 2022-11-02 Fred Hutchinson Cancer Center Modified hematopoietic stem/progenitor and non-t effector cells, and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4981784A (en) * 1987-12-02 1991-01-01 The Salk Institute For Biological Studies Retinoic acid receptor method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4981784A (en) * 1987-12-02 1991-01-01 The Salk Institute For Biological Studies Retinoic acid receptor method

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANNUAL REV. CELL. BIOL., Volume 7, Issued 1991, S. ARTAVANIS-TSAKONAS et al., "The Notch Locus and the Cell Biology of Neuroblast Segregation", pages 427-452. *
CELL, Volume 67, Issued 15 November 1991, I. REBAY et al., "Specific EGF Repeats of Notch Mediate Interactions with Delta and Serrate: Implications for Notch as a Multifunctional Receptor", pages 687-699. *
DEVELOPMENT, Volume 111, Issued 1991, U. THOMAS et al., "The Drosophila Gene Serrate Encodes an EGF-Like Transmembrane Protein with a Complex Expression Pattern in Embryos and Wing Discs", pages 749-761. *
GENES AND DEVELOPMENT, Volume 4, Issued 1990, R.J. FLEMING et al., "The Gene Serrate Encodes a Putative EGF-Like Transmembrane Protein Essential for Proper Ectodermal Development in Drosophila Melanogaster", pages 2188-2201. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 265, No. 35, Issued 15 December 1990, Y.B. XIE et al., "Extracellular Domain of Lutropin/Choriogonadotropin Receptor Expressed in Transfected Cells Binds Choriogonadotrophin with High Affinity", pages 21411-21414. *

Cited By (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0930365A2 (en) * 1991-05-03 1999-07-21 Yale University Binding domains in serrate protein
US6090922A (en) * 1991-05-03 2000-07-18 Yale University Antibodies to human notch proteins and fragments
EP0930365A3 (en) * 1991-05-03 1999-07-28 Yale University Binding domains in serrate protein
US6004924A (en) * 1991-12-11 1999-12-21 Imperial Cancer Research Technology, Ltd. Protein sequences of serrate gene products
US6232096B1 (en) 1991-12-13 2001-05-15 Heska Corporation Flea serine protease nucleic acid molecules and uses thereof
US6156556A (en) * 1991-12-13 2000-12-05 Heska Corporation Flea protease proteins, nucleic acid molecules, and uses thereof
US6083904A (en) * 1992-04-30 2000-07-04 Yale University Therapeutic and diagnostic methods and compositions based on notch proteins and nucleic acids
EP0662827A1 (en) * 1992-09-30 1995-07-19 Yale University Therapeutic and diagnostic methods and compositions based on notch proteins and nucleic acids
EP1197220A3 (en) * 1992-09-30 2009-04-15 Yale University Therapeutic methods and compositions based on delta proteins and nucleic acids
EP1175909A3 (en) * 1992-09-30 2003-11-26 Yale University Therapeutic methods and compositions based on serrate proteins and nucleic acids
EP2145897A2 (en) * 1992-09-30 2010-01-20 Yale University Therapeutic methods and compositions based on serrate proteins and nucleic acids
EP2145897A3 (en) * 1992-09-30 2010-03-03 Yale University Therapeutic methods and compositions based on serrate proteins and nucleic acids
EP0662827A4 (en) * 1992-09-30 1997-12-03 Univ Yale Therapeutic and diagnostic methods and compositions based on notch proteins and nucleic acids.
WO1995035323A1 (en) * 1994-06-20 1995-12-28 Borealis Polymers Oy Procatalyst for ethylene polymer production, method for its preparation and use
EP0813545A1 (en) * 1995-03-07 1997-12-29 Yale University Nucleotide and protein sequences of vertebrate serrate genes and methods based thereon
JP2010273685A (en) * 1995-03-07 2010-12-09 Yale Univ Nucleotide and protein sequence of vertebrate serrate gene, and method based thereon
US6703489B1 (en) 1995-03-07 2004-03-09 Yale University Antibodies to vertebrate serrate proteins and fragments
WO1996027610A1 (en) 1995-03-07 1996-09-12 Yale University Nucleotide and protein sequences of vertebrate serrate genes and methods based thereon
EP0813545A4 (en) * 1995-03-07 2002-04-24 Univ Yale Nucleotide and protein sequences of vertebrate serrate genes and methods based thereon
JP2008099687A (en) * 1995-03-07 2008-05-01 Yale Univ Nucleotide and protein sequence of serrate gene of vertebrate and method based thereon
US6204010B1 (en) 1995-06-07 2001-03-20 Heska Corporation Flea protease proteins, nucleic acid molecules, and uses thereof
US6783956B2 (en) 1995-06-28 2004-08-31 Yale University Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
US7118890B2 (en) 1995-06-28 2006-10-10 Yale University Antibodies to vertebrate delta proteins and fragments
EP0861261A1 (en) * 1995-06-28 1998-09-02 Imperial Cancer Research Technology Limited Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
US8415303B2 (en) 1995-06-28 2013-04-09 Imperial Cancer Research Technology, Ltd. Method of treatment using a soluble human delta protein fragment
US6262025B1 (en) 1995-06-28 2001-07-17 Imperial Cancer Research Technology, Ltd. Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
EP0861261A4 (en) * 1995-06-28 2001-08-29 Imp Cancer Res Tech Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
US7928071B2 (en) 1995-06-28 2011-04-19 Yale Universtiy Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
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
EP0861894A1 (en) * 1995-11-17 1998-09-02 Asahi Kasei Kogyo Kabushiki Kaisha Differentiation-suppressive polypeptide
EP0861894A4 (en) * 1995-11-17 2002-08-07 Asahi Chemical Ind Differentiation-suppressive polypeptide
US7141379B2 (en) 1995-11-17 2006-11-28 Asahi Kasei Kabushiki Kaisha Method of suppressing differentiation by administering a human serrate-1 polypeptide
US7179622B2 (en) 1995-11-17 2007-02-20 Asahi Kasei Kabushiki Kaisha Method of suppressing differentiation by administering a human serrate-1 polypeptide
US7198918B2 (en) 1995-11-17 2007-04-03 Asahi Kasei Kabushiki Kaisha Nucleic acid encoding a human serrate-1 polypeptide
US7253265B2 (en) 1995-11-17 2007-08-07 Asahi Kasei Kabushiki Kaisha Antibody and a method for producing an antibody that specifically binds to a full length amino acid sequence of human Delta-1
US6664098B1 (en) 1997-05-14 2003-12-16 Asahi Kasei Kabushiki Kaisha Differentiation inhibitory agent
WO1998051799A1 (en) * 1997-05-14 1998-11-19 Asahi Kasei Kogyo Kabushiki Kaisha Novel differentiation inhibitor
US7022499B2 (en) 1997-05-14 2006-04-04 Asahi Kasei Kabushiki Kaisha Nucleic acids encoding differentiation inhibitor delta 2
WO1998057983A2 (en) * 1997-06-18 1998-12-23 Zymogenetics, Inc. Mammalian neuro-growth factor like protein
WO1998057983A3 (en) * 1997-06-18 1999-03-18 Zymogenetics Inc Mammalian neuro-growth factor like protein
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
US6692919B1 (en) 1997-07-23 2004-02-17 Yale University Activated forms of notch and methods based thereon
US6436650B1 (en) 1997-07-23 2002-08-20 Yale University Activated forms of notch and methods based thereon
EP3590520A1 (en) 2010-04-09 2020-01-08 Fred Hutchinson Cancer Research Center Compositions for providing hematopoietic function without hla matching
WO2011127470A1 (en) 2010-04-09 2011-10-13 Fred Hutchinson Cancer Research Center Compositions and methods for providing hematopoietic function without hla matching
EP3097916A1 (en) 2010-04-09 2016-11-30 Fred Hutchinson Cancer Research Center Compositions and methods for providing hematopoietic function
EP3097917A1 (en) 2010-04-09 2016-11-30 Fred Hutchinson Cancer Research Center Compositions and methods for providing hematopoietic function without hla matching
WO2011127472A1 (en) 2010-04-09 2011-10-13 Fred Hutchinson Cancer Research Center Compositions and methods for providing hematopoietic function
WO2013086436A1 (en) 2011-12-08 2013-06-13 Fred Hutchinson Cancer Research Center Compositions and methods for enhanced generation of hematopoietic stem/progenitor cells
US9834755B2 (en) 2011-12-08 2017-12-05 Fred Hutchinson Cancer Research Center Compositions and methods for enhanced generation of hematopoietic stem/progenitor cells
WO2014110020A1 (en) 2013-01-08 2014-07-17 Fred Hutchinson Cancer Research Center Compositions and methods for expansion of embryonic hematopoietic stem cells
EP4083062A1 (en) 2013-10-31 2022-11-02 Fred Hutchinson Cancer Center Modified hematopoietic stem/progenitor and non-t effector cells, and uses thereof
WO2016118780A1 (en) 2015-01-21 2016-07-28 Fred Hutchinson Cancer Research Center Point-of-care and/or portable platform for gene therapy
WO2016176652A2 (en) 2015-04-29 2016-11-03 Fred Hutchinson Cancer Research Center Modified stem cells and uses thereof
EP3995142A2 (en) 2015-04-29 2022-05-11 Fred Hutchinson Cancer Research Center Modified hematopoietic stem/progenitor and non-t effector cells, and uses thereof
US10813949B2 (en) 2015-12-04 2020-10-27 Fred Hutchinson Cancer Research Center Uses of expanded populations of hematopoietic stem/progenitor cells

Similar Documents

Publication Publication Date Title
WO1993012141A1 (en) Nucleotide and protein sequences of the serrate gene and methods based thereon
US5869282A (en) Nucleotide and protein sequences of the serrate gene and methods based thereon
US6703489B1 (en) Antibodies to vertebrate serrate proteins and fragments
Fleming et al. The gene Serrate encodes a putative EGF-like transmembrane protein essential for proper ectodermal development in Drosophila melanogaster.
US5789195A (en) Human notch and delta, binding domains in toporythmic proteins, and methods based thereon
JP4430891B2 (en) Binding domains in Notch and Delta proteins
US5786158A (en) Therapeutic and diagnostic methods and compositions based on notch proteins and nucleic acids
US5856441A (en) Serrate fragments and derivatives
EP0861261B9 (en) Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
IE84505B1 (en) Human notch and delta, binding domains in toporythmic proteins, and methods based thereon
US20050112121A1 (en) Therapeutic and diagnostic methods and compositions based on notch proteins and nucleic acids
CA2125767A1 (en) Nucleotide and protein sequences of the serrate gene and methods based thereon
IE85870B1 (en) Human notch and delta, binding domains in toporythmic proteins, and methods based thereon
IL183831A (en) Fragments of a serrate protein which bind a notch protein

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2125767

Country of ref document: CA