Vertebrate smoothened proteins

• US 6,492,139 B1
• Filed: 04/27/2000
• Issued: 12/10/2002
• Est. Priority Date: 09/30/1996
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: de Sauvage, Frederic J., Rosenthal, Arnon, Stone, Donna M.
• Primary Examiner(s): Spector, Lorraine
• Assistant Examiner(s): Kaufman, Claire M.
• Application Number:
  US09/560,876
• Time in Patent Office: 957 Days
• Field of Search: 536/23.1, 536/23.5, 435/69.1, 435/320.1, 435/325, 435/252.3, 435/254.11, 530/350
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

Novel vertebrate homologues of Smoothened, including human and rat Smoothened, are provided. Compositions including vertebrate Smoothened chimeras, nucleic acid encoding vertebrate Smoothened, and antibodies to vertebrate Smoothened, are also provided.

Claims

1. Isolated nucleic acid which hybridizes to either a) SEQ ID NO:

1 or b) the complement thereof, wherein said nucleic acid encodes vertebrate Smoothened which directly binds to Patched, and wherein said hybridization occurs under the following conditions;

42°

C. in 50% formamide, 5×

SSC, 10×

Denhardt'"'"'s, 0.05M sodium phosphate, (pH 6.5), 0.1% sodium pyrophosphate, 50 μ

g/ml sonicated salmon sperm, followed by rinsing in 2×

SSC and washing with 0.5×

SSC and 0.1% SDS at 42°

C.

2. The nucleic acid of claim 1 wherein said nucleic acid encodes native sequence vertebrate Smoothened comprising the amino acid sequence of SEQ ID NO:

2.

3. A vector comprising said nucleic acid of claim 1.

4. The vector of claim 3, wherein said nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.

5. A host cell comprising the vector of claim 3.

6. The host cell of claim 5 which is prokaryotic.

7. The host cell of claim 5 which is mammalian.

8. A method of expressing vertebrate Smoothened nucleic acid comprising the steps of:

a) transforming a host cell with the vector of claim 4;

b) culturing the host cell under conditions suitable for expression; and

c) purifying the expression product.

9. Isolated nucleic acid which hybridizes to either a) SEQ ID NO:

3 or b) the complement thereof, wherein said nucleic acid encodes vertebrate Smoothened which directly binds to Patched, and wherein said hybridization occurs under the following conditions;

42°

C. in 50% formamide, 5×

SSC, 10×

Denhardt'"'"'s, 0.05M sodium phosphate, (pH 6.5), 0.1% sodium pyrophosphate, 50 μ

g/ml sonicated salmon sperm, followed by rinsing in 2×

SSC and washing with 0.5×

SSC and 0.1% SDS at 42°

C.

10. The nucleic acid of claim 9 wherein said nucleic acid encodes native sequence vertebrate Smoothened comprising the amino acid sequence of SEQ ID NO:

4.

11. A vector comprising said nucleic acid of claim 9.

12. The vector of claim 10, wherein said nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.

13. A host cell comprising the vector of claim 12.

14. The host cell of claim 12 which is prokaryotic.

15. The host cell of claim 12 which is mammalian.

16. A method of expressing vertebrate Smoothened nucleic acid comprising the steps of:

a) transforming a host cell with the vector of claim 12.

b) culturing the host cell under conditions suitable for expression; and

c) purifying the expression product.

17. Isolated nucleic acid comprising the coding sequence for human vertebrate Smoothened polypeptide deposited under ATTC Dep. No. 98162.

18. The nucleic acid of claim 17 comprising the human cDNA insert of ATTC Dep. No. 98162.

19. Isolated nucleic acid comprising the coding sequence for human vertebrate Smoothened polypeptide deposited under ATTC Dep. No. 98163.

20. The nucleic acid of claim 19, comprising the human cDNA insert of ATTC Dep. No. 98163.

21. Isolated nucleic acid comprising the coding sequence for rat nucleic acid deposited under ATCC Dep. No. 98165.

22. The nucleic acid of claim 21, comprising the rat cDNA insert of ATTC Dep. No. 98165.

23. A nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising residues 30 to 787 of FIG. 4 (SEQ ID NO:

4).

24. The nucleic acid of claim 23 wherein the polypeptide encoded comprises residues 1 to 787 of FIG. 4 (SEQ ID NO:

4).

25. The nucleic acid of claim 23 comprising nucleotide residues 13 through 2373 of FIG. 4 is (SEQ ID NO:

3).

26. A nucleic acid comprising a nucleotide sequence that encodes residues 33 to 793 of FIG. 1 (SEQ ID NO:

2).

27. The nucleic acid of claim 26 wherein the polypeptide encoded comprises residues 1 to 793 of FIG. 1 (SEQ ID NO:

2).

28. The nucleic acid of claim 26 comprising nucleotide residues 450 to 2828 of FIG. 1 (SEQ ID NO:

1).