Double muscling in cattle due to mutations in the myostatin gene

Abstract

Myostatin (GDF-8) is a member of the transforming growth factor beta superfamily of secreted growth and differentiation factors that is essential for proper regulation of skeletal muscle mass in mice. Here we report the myostatin sequences of nine other vertebrate species and the identification of mutations in the coding sequence of bovine myostatin in two breeds of double-muscled cattle, Belgian Blue and Piedmontese, which are known to have an increase in muscle mass relative to conventional cattle. The Belgian Blue myostatin sequence contains an 11-nucleotide deletion in the third exon which causes a frameshift that eliminates virtually all of the mature, active region of the molecule. The Piedmontese myostatin sequence contains a missense mutation in exon 3, resulting in a substitution of tyrosine for an invariant cysteine in the mature region of the protein. The similarity in phenotypes of double-muscled cattle and myostatin null mice suggests that myostatin performs the same biological function in these two species and is a potentially useful target for genetic manipulation in other farm animals.

Figure 1

Amino acid sequence alignment of murine, rat, human, baboon, bovine, porcine, ovine, chicken, turkey, and zebrafish myostatin. Shaded residues indicate amino acids matching the consensus. Amino acids are numbered relative to the human sequence. Dashed lines indicate gaps.

Figure 2

A fullblood Belgian Blue bull showing the double muscling phenotype.

Figure 3

Myostatin mutations in Belgian Blue (Left) and Piedmontese (Right) cattle compared with wild-type Holstein cattle. The nucleotides immediately preceding (A936) and following (C948) the Belgian Blue 11-nucleotide deletion are marked. Nucleotide and amino acid sequences are given below and numbered relative to wild type. The Belgian Blue 11-nucleotide deletion (Δ937–947) is boxed, and the Piedmontese G1056A transition is marked. Bold letters indicate nucleotide and amino acid changes. Arrows identify the locations of the mutations in the myostatin coding sequence. Shading indicates the signal sequence (gray), pro region (white) and mature C-terminal region (black).

Figure 4

Representative Southern blot hybridization showing the presence of the Belgian Blue and Piedmontese mutant sequences only in double-muscled breeds of cattle. Exon 3 PCR products were hybridized to oligonucleotide probes spanning the wild-type sequence of the region of the Belgian Blue mutation (top row), the Belgian Blue mutation Δ937–947 (second row), the wild-type sequence at nucleotide 1,056 (third row), and the Piedmontese mutant sequence at nucleotide 1,056 (bottom row). Differences in band intensity reflect differences in amounts of PCR products loaded, as judged by ethidium bromide staining (data not shown). Homozygosity for the mutations was seen only in double-muscled cattle and not in any conventional cattle as described in the text (P < 0.001 by χ²).