Nucleotide and protein sequences of vertebrate delta genes and methods based thereon

An isolated nucleic acid comprising a nucleotide sequence encoding the amino acid sequence depicted in SEQ ID NO:23, SEQ ID NO:65, SEQ ID NO:72 or SEQ ID NO:77.

An isolated nucleic acid comprising a fragment of a vertebrate Delta gene consisting of at least 50 nucleotides, said Delta gene comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:24 and SEQ ID NO:26.

An isolated nucleic acid comprising a nucleotide sequence encoding a fragment of a human Delta protein which is able to display a functional activity of a Delta protein, said human Delta protein comprising the amino acid sequence depicted in SEQ ID NO:23, SEQ ID NO:65, SEQ ID NO:72 or SEQ ID NO:77, which activity is selected from the group consisting of antigenicity, immunogenicity, binding to a Notch protein and binding to a second Delta protein.

An isolated nucleic acid comprising a nucleotide sequence encoding a fragment of a vertebrate Delta protein comprising a domain of the protein selected from the noun consisting of extracellular domain, DSL domain, domain amino terminal to the DSL domain, epidermal growth factor-like repeat domain, transmembrane domain and intracellular domain, said Delta protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:23, SEQ ID NO:65, SEQ ID NO:72 and SEQ ID NO:77.

An isolated nucleic acid comprising a nucleotide sequence encoding a fragment of a vertebrate Delta protein, which fragment is able to bind a Notch protein, said Delta protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:23, SEQ ID NO:65, SEQ ID NO:72 and SEQ ID NO:77.

An isolated nucleic acid comprising a nucleotide sequence encoding a protein, said protein comprising amino acid numbers 1-175 of the human Delta sequence depicted in SEQ ID NO:23, or a complement of said nucleotide sequence.

An isolated nucleic acid comprising a nucleotide sequence encoding a chimeric protein comprising a fragment of a vertebrate Delta protein consisting of at least 20 amino acids fused via a covalent bond to an amino acid sequence of a second protein, in which the second protein is not a Delta protein, said vertebrate Delta protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:23, SEQ ID NO:65, SEQ ID NO:72 and SEQ ID NO:77.

A recombinant cell transformed with a recombinant nucleic acid, which nucleic acid encodes a vertebrate Delta protein, said vertebrate Delta protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence depicted in SEQ ID NO:2, the amino acid sequence depicted in SEQ ID NO:12, the amino acid sequence depicted in SEQ ID NO:23, the amino acid sequence depicted in SEQ ID NO:65, the amino acid sequence depicted in SEQ ID NO:72 and the amino acid sequence depicted in SEQ ID NO:77.

A recombinant cell transformed with a recombinant nucleic acid encoding a fragment of a vertebrate Delta protein comprising a domain of the protein selected from the group consisting of extracellular domain, DSL domain, domain amino terminal to the DSL domain, epidermal growth factor-like repeat domain, transmembrane domain and intracellular domain, said Delta protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:23, SEQ ID NO:65, SEQ ID NO:72 and SEQ ID NO:77.

A recombinant cell transformed with a recombinant nucleic acid encoding a protein, said protein comprising amino acid numbers 1-175 of the human Delta sequence depicted in SEQ ID NO:23.

An isolated nucleic acid comprising the nucleotide sequence depicted in SEQ ID NO:26.

An isolated nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence depicted in SEQ ID NO:26.

An isolated nucleic acid, which nucleic acid (a) hybridizes under conditions of high stringency to a vertebrate Delta sequence depicted in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:26, or a sequence comprising an at least 50 nucleotide portion of said Delta sequence or its complement, said high stringency conditions comprising pretreatment for 8 to 12 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA:

• hybridization for 48 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.0 1% Ficoll, and 0.01% SDS and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC, and (b) encodes, or is the antisense strand to a nucleic acid which encodes, a protein which is able to be bound by an anti-vertebrate Delta antibody.

An isolated nucleic acid, which nucleic acid (a) hybridizes under conditions of high stringency to a second nucleic acid consisting of the nucleotide sequence depicted in SEQ ID NO:14, or comprising an at least 50 nucleotide portion of said sequence, said high stringency conditions comprising pretreatment for 8 to 12 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;

• hybridization for 48 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% SDS and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC, and (b) encodes, or is the antisense strand to a nucleic acid which encodes, a protein which is able to be bound by an anti-vertebrate Delta antibody.

An isolated nucleic acid, which nucleic acid hybridizes under conditions of high stringency to a nucleic acid consisting of the consensus nucleotide sequence depicted in SEQ ID NO:24, or comprising an at least 50 nucleotide portion of said sequence, said high stringency conditions comprising pretreatment for 8 to 12 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;

• hybridization for 48 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% SDS and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC, and (b) encodes, or is the antisense strand to a nucleic acid which encodes, a protein which is able to be bound by an anti-vertebrate Delta antibody.

An isolated nucleic acid consisting of a vertebrate Delta sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:24 and SEQ ID NO:26.

An isolated nucleic acid comprising a vertebrate Delta sequence selected from the group consisting of the mouse Delta sequence of SEQ ID NO:11, the complementary strand to the mouse Delta sequence of SEQ ID NO:11, the chick Delta sequence of SEQ ID NO:1, the complementary strand to the chick Delta sequence of SEQ ID NO:1, the chick Delta sequence of SEQ ID NO:4, the complementary strand to the chick Delta sequence of SEQ ID NO:4, the human Delta sequence of SEQ ID NO:14, the complementary strand to the human Delta sequence of SEQ ID NO:14, the human Delta sequence of SEQ ID NO:26, the complementary strand to the human Delta sequence of SEQ ID NO:26, the human-mouse Delta consensus sequence of SEQ ID NO:24, and the complementary strand to the human-mouse Delta consensus sequence of SEQ ID NO:24.

A recombinant cell transformed with a recombinant nucleic acid, which nucleic acid (a) hybridizes under conditions of high stringency to a vertebrate Delta sequence depicted in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:26, or a sequence comprising an at least 50 nucleotide portion of said Delta sequence, said high stringency conditions comprising pretreatment for 8 to 12 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;

• hybridization for 48 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% SDS and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC, and (b) encodes, or is the antisense strand to a nucleic acid which encodes, a protein which is able to be bound by an anti-vertebrate Delta antibody.

An isolated nucleic acid, which nucleic acid (a) hybridizes under conditions of low stringency to a vertebrate Delta sequence depicted in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:26, or a sequence comprising an at least 50 nucleotide portion of said Delta sequence, said low stringency conditions comprising pretreatment for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA;

• hybridization for 18-20 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml denatured salmon sperm DNA, and 10% (wt./vol.) dextran sulfate;
• washing for 1.5 hours at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS;
• and a second washing for 1.5 hours at 60° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS, and (b) encodes, or is the antisense strand to a nucleic acid which encodes, a protein which is able to be bound by an anti-vertebrate Delta antibody.

A recombinant cell transformed with a recombinant nucleic acid, which nucleic acid (a) hybridizes under conditions of low stringency to a vertebrate Delta sequence depicted in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:26, or a sequence comprising an at least 50 nucleotide portion of said Delta sequence, said low stringency conditions comprising pretreatment for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA;

• hybridization for 18-20 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCL (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml denatured salmon sperm DNA, and 10% (wt/vol.) dextran sulfate;
• washing for 1.5 hours at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS;
• and a second washing for 1.5 hours at 60° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS, and (b) encodes, or is the antisense strand to a nucleic acid which encodes, a protein which is able to be bound by an anti-vertebrate Delta antibody.

An isolated nucleic acid comprising a nucleotide sequence encoding a fragment of at least 20 amino acids of a vertebrate Delta protein, said fragment being able to display one or more functional activities of a Delta protein, said vertebrate Delta protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence depicted in SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:23, SEQ ID NO:65, SEQ ID NO:72 and SEQ ID NO:77.

An isolated nucleic acid comprising a fragment of a vertebrate Delta gene consisting of at least 25 nucleotides of a vertebrate Delta nucleotide sequence depicted in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:26, or a complement of said nucleotide sequence.

A recombinant cell transformed with a recombinant nucleic acid comprising at least 25 nucleotides of a vertebrate Delta nucleotide sequence depicted in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:26, or a complement of said nucleotide sequence.

An isolated nucleic acid comprising a nucleotide sequence encoding a fragment of at least 20 amino acids of a vertebrate Delta protein, said protein comprising a sequence selected from the group consisting of the chick Delta sequence depicted in SEQ ID NO:2, the mouse Delta sequence depicted in SEQ ID NO:12, the human Delta sequence depicted in SEQ ID NO:23, the human Delta sequence depicted in SEQ ID NO:65, the human Delta sequence depicted in SEQ ID NO:72 and the human Delta sequence depicted in SEQ ID NO:77, which fragment (a) is capable of being bound by an anti-vertebrate Delta antibody;

• and (b) lacks the transmembrane and intracellular domains of the protein.

The nucleic acid in which the vertebrate Delta protein has the amino acid sequence depicted in SEQ ID NO:23.

An isolated nucleic acid comprising a nucleotide sequence encoding a fragment of at least 10 amino acids of a vertebrate Delta protein, said protein comprising a sequence selected from the group consisting of the chick Delta sequence depicted in SEQ ID NO:2, the mouse Delta sequence depicted in SEQ ID NO:12, the human Delta sequence depicted in SEQ ID NO:23, the human Delta sequence depicted in SEQ ID NO:65, the human Delta sequence depicted in SEQ ID NO:72 and the human Delta sequence depicted in SEQ ID NO:77, which fragment (a) is capable of being bound by an anti-vertebrate Delta antibody;

• and (b) lacks the extracellular domain of the protein.

An isolated nucleic acid comprising a nucleotide sequence encoding a protein comprising a fragment of at least 20 amino acids of a vertebrate Delta protein, said vertebrate Delta protein comprising a sequence selected from the group consisting of the chick Delta sequence depicted in SEQ ID NO:2, the mouse Delta sequence depicted in SEQ ID NO:12, the human Delta sequence depicted in SEQ ID NO:23, the human Delta sequence depicted in SEQ ID NO:65, the human Delta sequence depicted in SEQ ID NO:72 and the human Delta sequence depicted in SEQ ID NO:77, which fragment is able to bind to a Notch protein.

An isolated nucleic acid comprising a nucleotide sequence encoding a protein comprising a fragment of at least 20 amino acids of the chick Delta sequence depicted in SEQ ID NO:2, said fragment comprising a domain of the protein selected from the group consisting of the extracellular domain, DSL domain, epidermal growth factor-like repeat domain, cysteine-rich domain, transmembrane domain, and intracellular domain.

An isolated nucleic acid comprising a nucleotide sequence encoding a protein comprising a fragment of at least 20 amino acids of the mouse Delta sequence depicted in SEQ ID NO:12, said fragment comprising a domain of the protein selected from the group consisting of the extracellular domain, DSL domain, epidermal growth factor-like repeat domain, cysteine-rich domain, transmembrane domain, and intracellular domain.

An isolated nucleic acid which encodes a fragment of a vertebrate Delta protein, which fragment consists of a domain of the protein selected from the group consisting of the extracellular domain, the DSL domain, epidermal growth factor-like repeat domain, transmembrane domain, and intracellular domain, which nucleic acid hybridizes under high stringency conditions to the consensus nucleotide sequence depicted in SEQ ID NO:24 or its complement, which fragment is able to be bound by an anti-vertebrate Delta antibody, said high stringency conditions comprising pretreatment for 8 to 12 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;

• hybridization for 48 hours at 65° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% SDS and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC.

An isolated nucleic acid which encodes a fragment of a vertebrate Delta protein, which fragment consists of a domain of the protein selected from the group consisting of the extracellular domain, the DSL domain, epidermal growth factor-like repeat domain, transmembrane domain, and intracellular domain, which nucleic acid hybridizes under low stringency conditions to the consensus nucleotide sequence depicted in SEQ ID NO:24 or its complement, which fragment is able to be bound by an anti-vertebrate Delta antibody, said low stringency conditions comprising pretreatment for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA;

• hybridization for 18-20 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml denatured salmon sperm DNA, and 10% (wt./vol.) dextran sulfate;
• washing for 1.5 hours at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS;
• and a second washing for 1.5 hours at 60° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS.

The nucleic acid of claim which hybridizes under low stringency conditions to a vertebrate Delta nucleotide sequence depicted in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:24 or SEQ ID NO:26, or a complement thereof, said low stringency conditions consisting of pretreatment for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA;

• hybridization for 18-20 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml denatured salmon sperm DNA, and 10% (wt./vol.) dextran sulfate;
• washing for 1.5 hours at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS;
• and a second washing for 1.5 hours at 60° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS.

An isolated nucleic acid comprising a nucleotide sequence encoding a protein, said protein comprising amino acid numbers 1-192 of the human Delta sequence depicted in SEQ ID NO:65, or the complement of said nucleotide sequence.

A recombinant cell transformed with a recombinant nucleic acid encoding a protein, said protein comprising amino acid numbers 1-192 of the human Delta sequence depicted in SEQ ID NO:65.