Double-muscling in mammals

• US 6,103,466 A
• Filed: 07/14/1997
• Issued: 08/15/2000
• Est. Priority Date: 07/14/1997
• Status: Expired due to Term

• Current Assignee: University of Liege
• Original Assignee: University of Liege
• Inventors: Georges, Michel, Grobet, Luc
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/891,789
• Time in Patent Office: 1,128 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/24.3, 536/23.1
• US Class Current: 435/6.11
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2217/30 Animal model comprising expression system for selective cell killing, e.g. toxins, enzyme dependent prodrug therapy using ganciclovir
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/495 Transforming growth factor [TGF]
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/124 Animal traits, i.e. production traits, including athletic performance or the like
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A gene (cDNA) encoding a bovine myostatin protein. The nucleic acid coding sequence is identified as SEQ ID NO:1 and the protein sequence is identified as SEQ ID NO:2. A mutant gene (SEQ ID NO:3) in which the coding sequence lacks an 11-base pair consecutive sequence (SEQ ID NO:11) of the sequence encoding bovine protein having myostatin activity has been sequenced. It has been shown that cattle of the Belgian Blue breed homozygous for the mutant gene lacking myostatin activity are double-muscled. A method for determining the presence of muscular hyperplasia in a mammal is described. The method includes obtaining a sample of material containing DNA from the mammal and ascertaining whether a sequence of the DNA encoding (a) a protein having biological activity of myostatin, is present, and whether a sequence of the DNA encoding (b) an allelic protein lacking the activity of (a), is present. The absence of (a) and the presence of (b) indicates the presence of muscular hyperplasia in the mammal.

Claims

1. A method for determining the presence of muscular hyperplasia in a bovine animal, the method comprising:

obtaining a sample of material containing DNA from a said animal; and

ascertaining whether DNA having a nucleotide sequence encoding a protein having biological activity of myostatin is present by amplifying the DNA in the presence of first and second primers based on first and second nucleotide sequences encoding spaced apart regions of the protein, wherein said regions flank a naturally occurring mutation and which when present in both alleles of a said animal results in said muscular hyperplasia,

wherein the absence of DNA having said nucleotide sequence indicates the presence of muscular hyperplasia in the animal.

2. The method of claim 1 wherein ascertaining whether DNA having a nucleotide sequence encoding a protein having biological activity of myostatin includes amplifying the DNA in the presence of primers based on a nucleotide sequence encoding a protein having biological activity of myostatin.

3. The method of claim 2 wherein DNA of a said bovine animal not displaying muscular hyperplasia has a nucleotide sequence which is capable of hybridizing with a nucleic acid molecule having the sequence identified as SEQ ID NO:

1 under stringent hybridization conditions.

4. The method of claim 1, wherein ascertaining whether DNA having a nucleotide sequence encoding a protein having biological activity of myostatin is present includes amplifying the DNA in the presence of primers based on a nucleotide sequence encoding the N-terminal and the C-terminal, respectively, of the protein having biological activity of myostatin.

5. The method of claim 1 wherein a DNA of said animal not displaying muscular hyperplasia contains a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence encoding a protein having a sequence identified as SEQ ID NO:

2 and said first primer is selected to be upstream of the codon encoding glutamic acid no. 275 and the second primer is selected to be downstream of the codon encoding aspartic acid no. 274.

6. The method of claim 1 wherein a DNA of said animal not displaying muscular hyperplasia contains a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence encoding a protein having a sequence identified as SEQ ID NO:

2 and the coding sequence of DNA of a said animal displaying muscular hyperplasia is known to contain an 11-base pair deletion beginning at base pair no. 821, and said primer is selected to span the nucleotide sequence including base pair nos. 820 and 821 of the DNA sequence containing said deletion.

7. The method of claim 5 or claim 6 wherein the animal is of the Belgian Blue breed.

8. The method of claim 1 wherein ascertaining whether DNA having a nucleotide sequence encoding a protein having biological activity of myostatin is present includes amplifying the DNA in the presence of a primer containing at least a portion of a naturally occurring mutation and which when present in both alleles of a said animal results in said muscular hyperplasia.

44. The method of claim 6, wherein said primer binds under stringent conditions with a nucleic acid molecule having the sequence identified as SEQ ID NO:

3.

9. A method for determining the presence of muscular hyperplasia in a bovine animal, the method comprising:

obtaining a sample of the animal containing mRNA; and

ascertaining whether an mRNA encoding a protein having biological activity of myostatin is present in the sample by amplifying the mRNA in the presence of first and second primers substantially complementary to first and second nucleotide sequences encoding spaced apart regions of the protein, wherein said regions flank a naturally occurring mutation and which when present in both alleles of a said animal results in said muscular hyperplasia,

wherein the absence of said mRNA indicates the presence of muscular hyperplasia in the animal.

10. The method of claim 9 wherein the sample is of muscle tissue.

11. The method of claim 10 wherein the tissue is skeletal muscle tissue.

12. The method of claim 9 wherein ascertaining whether mRNA having a nucleotide sequence encoding a protein having biological activity of myostatin includes amplifying the mRNA in the presence of primers substantially complementary to the nucleotide sequence encoding the protein.

13. The method of claim 12 wherein mRNA of a said bovine animal not displaying muscular hyperplasia has a nucleotide sequence which is capable of hybridizing with a nucleic acid molecule having the sequence identified as SEQ ID NO:

1 under stringent hybridization conditions.

14. The method of claim 9, wherein ascertaining whether mRNA having a nucleotide sequence encoding a protein having biological activity of myostatin is present includes amplifying the mRNA in the presence of primers substantially complementary to a nucleotide sequence encoding the N-terminal and the C-terminal, respectively, of the protein having biological activity of myostatin.

15. The method of claim 9 wherein an mRNA of said animal not displaying muscular hyperplasia contains a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence encoding a protein having a sequence identified as SEQ ID NO:

2 and said first primer is selected to be upstream of the codon encoding glutamic acid no. 275 and the second primer is selected to be downstream of the codon encoding aspartic acid no. 274.

16. The method of claim 9 wherein ascertaining whether mRNA having a nucleotide sequence encoding a protein having biological activity of myostatin is present includes amplifying the mRNA in the presence of a primer containing a nucleotide sequence complementary to at least a portion of a naturally occurring mutation in a said animal and which when present in both alleles of a said animal results in said muscular hyperplasia.

17. The method of claim 16 wherein an mRNA of said animal not displaying muscular hyperplasia contains a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence encoding a protein having a sequence identified as SEQ ID NO:

2 and the coding sequence of DNA of a said animal displaying muscular hyperplasia is known to contain an 11-base pair deletion beginning at base pair no. 821, and said primer is selected to span the deleted portion.

18. The method of claim 15 or claim 17 wherein the animal is of the Belgian Blue breed.

19. A method for determining the presence of muscular hyperplasia in a mammal, the method comprising:

obtaining a sample of material containing DNA from the mammal; and

ascertaining whether (a) a sequence of the DNA encoding a protein having biological activity of myostatin, is present, and whether (b) a naturally occurring sequence of the DNA encoding an allelic protein lacking the activity of a protein having biological activity of myostatin, is present;

wherein the absence of (a) and the presence of (b) indicates the presence of muscular hyperplasia in the mammal.

20. The method of claim 19 wherein the mammal is a human.

21. The method of claim 20 wherein ascertaining whether a sequence of the DNA encoding (a) is present, and whether a sequence of the DNA encoding (b) is present includes amplifying the DNA in the presence of primers based on a nucleotide sequence encoding a protein having biological activity of myostatin.

22. The method of claim 21 wherein said primers are based on the sequence identified as SEQ ID NO:

7.

23. A method for determining the presence of muscular hyperplasia in a mammal, the method comprising:

obtaining a sample of material containing mRNA from the mammal; and

ascertaining whether (a) a sequence of the mRNA encoding a protein having biological activity of myostatin, is present, and whether (b) a naturally occurring sequence of the mRNA encoding a truncated protein lacking the biological activity of myostatin, is present;

wherein the absence of (a) and the presence of (b) indicates the presence of muscular hyperplasia in the mammal.

25. The method of claim 23 wherein the mammal is human.

26. The method of claim 25 wherein ascertaining whether a sequence of (a) is present, and whether a sequence of the mRNA of (b) is present includes amplifying the mRNA in the presence of a pair of primers complementary to a nucleotide sequence encoding a protein having biological activity of myostatin.

27. The method of claim 26 wherein each said primer contains a nucleotide sequence substantially complementary to the sequence identified as SEQ ID NO:

7.

28. The method of claim 27 wherein the sequence of (b) contains at least 50 consecutive nucleotides substantially corresponding to 50 consecutive nucleotides of SEQ ID NO:

7.

29. The method of claim 23 wherein the mRNA of (a) and the mRNA of (b) correspond to alleles of DNA of the mammal.

24. A method for determining the presence of muscular hyperplasia in a mammal, the method comprising:

obtaining a tissue sample of containing mRNA of the mammal; and

ascertaining whether an mRNA encoding a naturally occurring mutant type myostatin protein lacking biological activity of myostatin is present,

wherein the presence of a said mRNA encoding a mutant type myostatin protein indicates the presence of muscular hyperplasia in the mammal.

30. A method for determining the myostatin genotype of a mammal, comprising:

obtaining a sample of material containing nucleic acid of the mammal, wherein the nucleic acid is uncontaminated by heterologous nucleic acid;

ascertaining whether the sample contains a (i) nucleic acid molecule encoding a protein having biological activity of myostatin; and

ascertaining whether the sample contains a (ii) allelic nucleic acid molecule encoding a naturally occurring protein lacking biological activity of myostatin.

31. The method of claim 30 wherein the mammal is human and (i) comprises a nucleic acid sequence substantially homologous with the sequence identified as SEQ ID NO:

7.

32. A method for identifying a nucleotide sequence of a naturally occurring mutant of a gene which normally encodes a myostatin protein, of a mammal displaying muscular hyperplasia, the method comprising:

obtaining a sample of material containing DNA from the mammal; and

probing the sample using a nucleic acid probe based on a nucleotide sequence of a known gene encoding myostatin in order to identify the nucleotide sequence of the mutant gene.

33. The method of claim 32, wherein the probe is based on a said nucleotide sequence identified as SEQ ID NO:

1, SEQ ID NO;

5 or SEQ ID NO;

7.

34. The method of claim 33 wherein the probe is based on SEQ ID NO:

1 or SEQ ID NO;

7.

35. The method of claim 34 wherein the probe is at least 8 nucleic acids in length.

36. The method of claim 32, wherein the step of probing the sample includes exposing the DNA to the probe under hybridizing conditions and further comprising isolating hybridized nucleic acid molecules.

37. The method of claim 36, further comprising the step of sequencing isolated DNA.

38. The method of claim 32, wherein the mammal is a bovine mammal and the probe is based on a said nucleotide sequence identified as SEQ ID NO:

1.

39. The method of claim 37, further comprising the step of isolating and sequencing a cDNA or mRNA encoding the complete mutant myostatin protein.

40. The method of claim 39, further comprising the step of isolating and sequencing a functional wild type myostatin from a said mammal not displaying muscular hyperplasia.

41. The method of claim 39, further comprising comparing the complete coding sequence of the complete mutant myostatin protein with, if the coding sequence for a functional wild type myostatin from a said mammal is previously known, (1) the known sequence, or if the coding sequence for a functional wild type myostatin from a said mammal is previously unknown, (2) the sequence determined according to claim 37 or claim 40, to determine the location of any mutation in the mutant gene.

42. A method for determining the myostatin genotype of a mammal, wherein wild type myostatin of the mammal is substantially that of claim 41, comprising:

obtaining a sample of material containing DNA from the mammal; and

ascertaining whether the DNA contains a said naturally occurring mutation determined according to claim 41.

43. A method for determining the myostatin genotype of a mammal, wherein wild type myostatin of the mammal is substantially that of claim 41, comprising:

obtaining a sample of material containing mRNA from the mammal; and

ascertaining whether the mRNA contains a said mutation determined according to claim 41.

45. A method for determining the myostatin genotype of a bovine mammal, comprising:

obtaining a sample of material containing nucleic acid of the mammal; and

amplifying the nucleic acid in the presence of a primer having the nucleotide sequence corresponding to a sequence of consecutive nucleotides of the myostatin gene (SEQ ID NO;

1) which flank the nucleotide sequence identified as SEQ ID NO;

11, in order to determine whether the nucleic acid includes a mutant myostatin coding sequence from which is absent the nucleotide sequence identified as SEQ ID NO;

11.