The Impact of Mutations Biology 138 the Belgian Blue Mound of Beef
Proc Natl Acad Sci U S A. 1997 November 11; 94(23): 12457–12461.
Genetics
Double muscling in cattle due to mutations in the myostatin factor
Received 1997 Aug 12; Accepted 1997 Aug 26.
Abstruse
Myostatin (GDF-viii) is a member of the transforming growth gene β superfamily of secreted growth and differentiation factors that is essential for proper regulation of skeletal musculus 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 Bluish and Piedmontese, which are known to have an increment in muscle mass relative to conventional cattle. The Belgian Blue myostatin sequence contains an xi-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 three, 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 nil mice suggests that myostatin performs the same biological function in these 2 species and is a potentially useful target for genetic manipulation in other farm animals.
The transforming growth gene β superfamily encompasses a large group of secreted growth and differentiation factors that play important roles in regulating development and tissue homeostasis (1). We have recently described a member of this family, myostatin, that is expressed specifically in developing and adult skeletal musculus and functions as a negative regulator of skeletal musculus mass in mice (two). Myostatin nil mice generated by gene targeting show a dramatic and widespread increase in skeletal muscle mass. Individual muscles in myostatin aught mice counterbalance two- to 3-fold more than those of wild-type mice, primarily due to an increased number of musculus fibers without a corresponding increment in the amount of fatty. To pursue potential therapeutic and agricultural applications of increasing muscle mass past inhibition of myostatin activeness, we accept been characterizing myostatin in animals other than mice. Here we report that the myostatin cistron is highly conserved among vertebrate species and that two breeds of cattle that are characterized by increased muscle mass (double muscling), Belgian Blue (3) and Piedmontese (4), have mutations in the myostatin coding sequence. These results demonstrate that the part of myostatin has been highly conserved amid vertebrates.
METHODS
Cloning of Myostatin.
Poly(A)-containing RNA was isolated from homo (obtained from the International Institute for the Advancement of Medicine, Exton, PA), Holstein moo-cow, sheep (Ruppersberger and Sons, Baltimore), pig (Bullock'due south Country Meats, Westminster, Md), White Leghorn craven (Truslow Farms, Chestertown, Doc), turkey (kindly provided by D. Boyer and D. Miller, Wampler Foods, Oxford, PA) and zebrafish (kindly provided by Due south. Fisher and M. Halpern, Carnegie Establishment of Washington) skeletal muscle tissue every bit described (5). cDNA libraries were synthetic in the λZAP Two vector (Stratagene) according to the instructions provided past the manufacturer and screened without amplification. Rat and baboon skeletal muscle cDNA libraries and a bovine (Holstein) genomic library were purchased from Stratagene. Library screening and analysis of clones were carried out as described (5), except that the last washes were carried out in 25 mM sodium phosphate (pH 8.5), 0.5 Yard NaCl, ii mM EDTA, and 0.5% SDS at 65°C.
Mapping.
Fluorescence in situ hybridization was performed on human metaphase spreads (Bios, New Haven, CT) using a digoxigenin-labeled homo genomic myostatin probe.
Sequencing of Bovine Genomic DNA.
Claret from cattle was spun at 3,400 × g for 15 min, resuspended in 150 mM NaCl and 100 mM EDTA, and digested with 200 μg⋅ml−1 proteinase K and ane% SDS at 44°C. Semen (Select Sires, Rocky Mount, VA) was digested in fifty mM Tris (pH 8.0), 20 mM EDTA, 1% sarcosyl, 0.ii M 2-mercaptoethanol, and 200 μg⋅ml−ane proteinase K. DNAs were purified on a CsCl gradient. Exons were amplified by PCR from 1 μg genomic Deoxyribonucleic acid using primer pairs 133ACM v′-CGCGGATCCTTTGGCTTGGCGTTGCTCAAAAGC-3′ and 134ACM v′-CGCGGATCCTTCTCATGAACACTAGAACAGCAG-3′ (exon ane), 135ACM 5′-CGCGGATCCGATTGATATGGAGGTGTTCGTTCG-3′ and 136ACM v′-CGCGGATCCGGAAACTGGTAGTTATTTTTCACT-3′ (exon 2), and 137ACM 5′-CGCGGATCCGAGGTAGGAGAGTGTTTTGGGATC-3′ and 138ACM v′-CGCGGATCCCACAGTTTCAAAATTGTTGAGGGG-3′ (exon 3) at 94°C for 1 min, 52°C for two min, and 72°C for ii min for 40 cycles. PCR products were digested with BamHI, subcloned into pBluescript, and sequenced.
Southern Blot Analysis of Mutant Sequences.
I-5th of exon iii amplification products were electrophoresed on two% agarose gels, blotted to nylon membranes, hybridized with 32P-labeled thirteen-mers as described (6), and done in 30 mM sodium citrate, 300 mM NaCl, and 0.1% SDS. Primers used were 146 ACM five′-ATGAACACTCCAC-3′ (Holstein wild-type sequence, nucleotides 936–948), 145ACM 5′-TTGTGACAGAATC-3′ (Belgian Blue mutation, nucleotides 931–936 with 948–954), 673SJL 5′-GAGAATGTGAATT-3′ (Holstein wild-type sequence, nucleotides 1050–1062), and 674SJL 5′-GAGAATATGAATT-3′ (Piedmontese mutation, G1056A).
RESULTS AND Give-and-take
To clone the myostatin factor from other species, cDNA libraries were constructed from RNA isolated from skeletal muscle tissue and screened with a mouse myostatin probe corresponding to the conserved C-last region, which is mature, active portion of the molecule. An alignment of the predicted amino acid sequences of murine, rat, human, baboon, bovine, porcine, ovine, chicken, turkey, and zebrafish myostatin, deduced from nucleotide sequence analysis of total-length cDNA clones, is shown in Fig. 1. All of these sequences contain a putative signal sequence for secretion and a putative RXXR proteolytic processing site (amino acids 263–266) followed by a region containing the conserved C-final cysteine residues found in all transforming growth factor β family members (1). As seen from this alignment, myostatin is highly conserved beyond species. In fact, the sequences of murine, rat, human, porcine, chicken, and turkey myostatin are 100% identical in the C-terminal region following the putative proteolytic processing site, and baboon, bovine, and ovine myostatin contain just one to three amino acrid differences in the mature protein. Zebrafish myostatin is considerably more diverged and is only 88% identical to the others in this region.
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.
The high degree of sequence conservation of myostatin across species suggests that the role of myostatin has as well been conserved. To determine whether myostatin plays a role in regulating muscle mass in animals other than mice, we investigated the possibility that mutations in the myostatin gene might account for the increased muscle mass observed in double-muscled livestock breeds. Double muscling, which has been observed in many breeds of cattle for the past 190 years, appears to be inherited equally a single major autosomal locus with several modifiers of phenotypic expression, resulting in incomplete penetrance (7). In the about extensively studied double-muscled breed of cattle, Belgian Blue, the double muscling phenotype (Fig. 2) segregates as a single genetic locus designated muscular hypertrophy (mh) (8). The mh mutation, which is partially recessive, causes an average increase in muscle mass of 20–25%, a subtract in mass of about other organs (9, x), and a decrease in intramuscular fatty and connective tissue (11). The mh locus is tightly linked to markers on a region of bovine chromosome two (12) that is syntenic to a region of human chromosome two (2q32) (13) to which nosotros had mapped the human myostatin factor by fluorescence in situ hybridization (information not shown).
A fullblood Belgian Blue bull showing the double muscling phenotype.
The similarities in phenotype between the myostatin null mice and the Belgian Blue cattle brood and the like map positions of the myostatin gene and the mh locus suggested the bovine homolog of myostatin as a candidate gene for the mh locus. To decide whether the bovine myostatin gene is mutated in the Belgian Bluish breed, all 3 exons of the factor from the full-blood Belgian Blue balderdash shown in Fig. two were amplified past PCR, subcloned, and sequenced. The Belgian Blue myostatin coding sequence was identical to the Holstein sequence except for a deletion of nucleotides 937–947 in the third exon (Fig. 3). This xi-nucleotide deletion causes a frame-shift which is predicted to result in a truncated protein that terminates 14 codons downstream of the site of the mutation. The deletion is expected to be a null mutation because it occurs afterward only the beginning 7 amino acids of the C-final region, resulting in a loss of 102 amino acids (amino acids 274–375). This mutation is similar to the targeted mutation in myostatin null mice in which the unabridged region encoding the mature protein was deleted (2). By Southern blot analysis, using oligonucleotides corresponding to the wild-type or mutant sequence, this mutation was plant in both alleles in xiv/xiv fullblood Belgian Blue cattle examined (data not shown).
Myostatin mutations in Belgian Blue (Left) and Piedmontese (Right) cattle compared with wild-type Holstein cattle. The nucleotides immediately preceding (A936) and post-obit (C948) the Belgian Blue 11-nucleotide deletion are marked. Nucleotide and amino acrid sequences are given below and numbered relative to wild blazon. The Belgian Blueish xi-nucleotide deletion (Δ937–947) is boxed, and the Piemontese 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 (grey), pro region (white) and mature C-last region (black).
We also sequenced the myostatin gene in another cattle breed, Piedmontese, in which double muscling occurs at an extremely high frequency (four). The Piedmontese sequence contained 2 nucleotide changes relative to the Holstein sequence. Ane was a C to A transversion in exon 1, resulting in a conservative commutation of leucine for phenylalanine (amino acid 94). The second was a G to A transition in exon iii, resulting in a cysteine to tyrosine commutation in the mature region of the poly peptide (amino acid 313) (Fig. iii). By Southern blot assay, this mutation was found in both alleles in ten/10 double-muscled Piemontese cattle examined. This mutation is likely to issue in a consummate or almost complete loss of part, as this cysteine residue is invariant not only among all myostatin sequences but likewise among all known members of the transforming growth factor β superfamily (ane). This cysteine residuum is known to be one of the amino acids involved in forming the intramolecular cystine knot structure in members of this superfamily for which the three-dimensional structure is known (14–17). Furthermore, when the respective cysteine in activin A (cysteine-44) was mutated to alanine, the mutant poly peptide had only ii% of wild-type receptor binding and biological activity (18).
The similar map positions of the myostatin gene and the mh locus and the identification of relatively severe mutations in the myostatin cistron of two different double-muscled cattle breeds propose that these mutations are responsible for the double muscling phenotype. To farther back up this hypothesis, we analyzed DNA isolated from 120 individual fullblood or purebred cattle in 16 other breeds that are not classified as double-muscled (11 Angus, 11 Charolais, x Holstein, x Brown Swiss, 10 Polled Hereford, ten Gelbvieh, 9 Simmental, ix Jersey, ix Guernsey, ix Ayrshire, vii Limousin, 4 Brahman, 4 Polled Shorthorn, 4 Ruddy Angus, 2 Chianina, and i Texas Longhorn) for the presence of each of these mutations (Fig. iv). By Southern absorb analysis, the cysteine to tyrosine substitution present in the Piedmontese breed was not detected in any of the 120 individuals. The eleven-nucleotide deletion present in the Belgian Blue breed was detected in one allele of a single Red Angus not-double-muscled total-blood bull. In this regard, it has been suggested that the double muscling phenotype that is occasionally seen in many breeds may be due to a single mutation or very few mutations that migrated into many of the European breeds of cattle during the development of the mod breeds (seven). Our results demonstrate that myostatin mutations which cause double muscling have occurred at least twice in cattle.
Representative Southern blot hybridization showing the presence of the Belgian Blue and Piedmontese mutant sequences but in double-muscled breeds of cattle. Exon three 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 one,056 (3rd row), and the Piedmontese mutant sequence at nucleotide ane,056 (lesser 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 non in any conventional cattle as described in the text (P < 0.001 by χtwo).
Finally, to rule out the presence of other myostatin mutations in non-double-muscled breeds, we determined the complete sequence of the myostatin coding region of 11 of these breeds (Angus, Charolais, Brown Swiss, Polled Hereford, Gelbvieh, Guernsey, Ayrshire, Limousin, Brahman, Polled Shorthorn, and Texas Longhorn). This assay revealed but polymorphisms that were either silent changes in the coding sequences or were present in the introns and untranslated regions.
Unlike in mice, a myostatin null mutation in cattle causes a reduction in sizes of internal organs and only a pocket-sized increase in musculus mass (20–25% in the Belgian Blue breed as compared with 200–300% in myostatin-deficient mice). It is possible that cattle may be nearer to a maximal limit of muscle size after generations of selective breeding for large muscle mass, unlike mice, which take not been similarly selected. In this regard, even in cattle breeds that are non heavily muscled, the myostatin sequence contains two side by side nonconservative amino acrid differences (EG vs. KE) in the C-terminal region, compared with all other species examined. Although the functional significance of these differences is unknown, it is possible that these two changes represent a partial loss-of-part allele that became fixed in the population during many years of cattle breeding.
For agricultural applications, there are some disadvantages to double-muscled cattle, namely the reduction in female person fertility, lower viability of offspring, and filibuster in sexual maturation (19). Nevertheless, in the Belgian Blue breed, the increased musculus mass and increased feed efficiency largely offset these drawbacks (20). The fact that a cipher mutation in the myostatin gene in cattle results in animals that are yet viable and fertile and produce loftier-quality meat demonstrates the potential value of producing an increase in muscle mass in other meat animals such equally sheep, hog, chicken, turkey, and fish past disrupting myostatin office. Indeed, the high degree of sequence conservation in animals ranging from mammals to birds to fish suggests that the biological function of myostatin has been conserved widely throughout the brute kingdom.
Acknowledgments
We thank Dee Garrels and Chet Pennington (Lakeview Belgian Blue Ranch, Stockton, MO) for providing claret and photographs of Belgian Blue cattle. This work was supported by enquiry grants from the Edward Mallinckrodt, Jr., Foundation and MetaMorphix, Inc. (to S.-J.50). Under an agreement between MetaMorphix, Inc. and the Johns Hopkins Academy, the authors are entitled to a share of sales royalty received past the University from MetaMorphix, Inc. The Academy, A.C.M., and South.-J.L. also own MetaMorphix stock, which is bailiwick to sure restrictions under University policy. S.-J.50. is a consultant to MetaMorphix, Inc. The terms of this arrangement are existence managed by the University in accordance with its conflict of interest policies.
Footnotes
Information deposition: The sequences reported in this newspaper have been deposited in the GenBank database [baboon (accretion no. {"blazon":"entrez-nucleotide","attrs":{"text":"AF019619","term_id":"2623565"}}AF019619), bovine (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF019620","term_id":"2623567"}}AF019620), chicken (accretion no. {"type":"entrez-nucleotide","attrs":{"text":"AF019621","term_id":"2623569"}}AF019621), ovine (accretion no. {"blazon":"entrez-nucleotide","attrs":{"text":"AF019622","term_id":"2623571"}}AF019622), porcine (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF019623","term_id":"2623573"}}AF019623), rat (accretion no. {"type":"entrez-nucleotide","attrs":{"text":"AF019624","term_id":"2623575"}}AF019624), turkey (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF019625","term_id":"2623577"}}AF019625), zebrafish (accretion no. {"blazon":"entrez-nucleotide","attrs":{"text":"AF019626","term_id":"2623579"}}AF019626), and human (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF019627","term_id":"2623581"}}AF019627)].
A commentary on this article begins on page 12249.
References
ane. McPherron A C, Lee S-J. In: Growth Factors and Cytokines in Health and Disease. LeRoith D, Bondy C, editors. 1B. Greenwich, CT: JAI; 1996. pp. 357–393. [Google Scholar]
ii. McPherron A C, Lawler A M, Lee Due south-J. Nature (London) 1997;387:83–90. [PubMed] [Google Scholar]
3. Hanset R. In: Muscle Hypertrophy of Genetic Origin and Its Employ to Improve Beef Product. King J Westward B, Ménissier F, editors. The Hague, The Netherlands: Nijhoff; 1982. pp. 437–449. [Google Scholar]
iv. Masoero Grand, Poujardieu B. In: Musculus Hypertrophy of Genetic Origin and Its Use to Improve Beef Product. King J West B, Ménissier F, editors. The Hague, The Netherlands: Nijhoff; 1982. pp. 450–459. [Google Scholar]
half-dozen. Gärtner J, Moser H, Valle D. Nat Genet. 1992;ane:16–23. [PubMed] [Google Scholar]
7. Ménissier F. In: Muscle Hypertrophy of Genetic Origin and Its Use to Meliorate Beef Production. King J Due west B, Ménissier F, editors. The Hague, Kingdom of the netherlands: Nijhoff; 1982. pp. 387–428. [Google Scholar]
9. Ansay M, Hanset R. Livest Prod Sci. 1979;half-dozen:v–13. [Google Scholar]
10. Hanset R. In: Breeding for Affliction Resistance in Farm Animals. Owen J B, editor. Wallingford, U.K.: CAB International; 1991. pp. 467–478. [Google Scholar]
xi. Hanset R, Michaux C, Dessy-Doize C, Burtonboy M. In: Muscle Hypertrophy of Genetic Origin and Its Utilize to Ameliorate Beefiness Product. King J W B, Ménissier F, editors. The Hague, Holland: Nijhoff; 1982. pp. 341–349. [Google Scholar]
12. Charlier C, Coppieters W, Farnir F, Grobet L, Leroy P L, Michaux C, Mni M, Schwers A, Vanmanshoven P, Hanset R, Georges M. Mamm Genome. 1995;6:788–792. [PubMed] [Google Scholar]
13. Solinas-Toldo Southward, Lengauer C, Chips R. Genomics. 1995;27:489–496. [PubMed] [Google Scholar]
14. Daopin South, Piez One thousand A, Ogawa Y, Davies D R. Science. 1992;257:369–373. [PubMed] [Google Scholar]
fifteen. Schlunegger M P, Grütter M Yard. Nature (London) 1992;358:430–434. [PubMed] [Google Scholar]
sixteen. Griffith D L, Keck P C, Sampath T K, Rueger D C, Carlson Due west D. Proc Natl Acad Sci USA. 1996;93:878–883. [PMC costless commodity] [PubMed] [Google Scholar]
17. Mittl P R, Priestle J P, Cox D A, McMaster Chiliad, Cerletti N, Grütter M G. Protein Sci. 1996;5:1261–1271. [PMC costless commodity] [PubMed] [Google Scholar]
nineteen. Ménissier F. In: Muscle Hypertrophy of Genetic Origin and Its Utilise to Improve Beefiness Product. Male monarch J Westward B, Ménissier F, editors. The Hague, The Netherlands: Nijhoff; 1982. pp. 23–53. [Google Scholar]
Manufactures from Proceedings of the National Academy of Sciences of the The states of America are provided here courtesy of National Academy of Sciences
geisslerilthaddly.blogspot.com
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC24998/
0 Response to "The Impact of Mutations Biology 138 the Belgian Blue Mound of Beef"
Post a Comment