|Chase, Chadwick - Chad|
|Elsasser, Theodore - Ted|
|Hammond, Andrew - Andy|
|Welsh, Jr., T|
Submitted to: Domestic Animal Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2009
Publication Date: 6/10/2009
Publication URL: http://www.domesticanimalendo.com/article/S0739-7240(09)00034-4/abstract
Citation: Mccormack, B.L., Chase, C.C., Olson, T.A., Elsasser, T.H., Hammond, A.C., Welsh, Jr., T.H., Jiang, H., Randel, R.D., Okamura, C.A., Lucy, M.C. 2009. A Miniature Condition in Brahman Cattle is Associated with a Single Nucleotide Mutation Within the Growth Hormone Gene. Domestic Animal Endocrinology. 37(2):104-111. Interpretive Summary: Miniature Brahman cattle at the STARS in Brooksville, Florida location are described as having normal proportioned growth but are approximately 70% of normal mature height and weight. Previous studies were conducted to describe the physiology of growth and reproduction of miniature Brahman compared to normal Brahman cattle. In miniature compared to normal Brahman, a consistent finding is that circulating concentrations of growth hormone (GH) are elevated in miniatures but that circulating concentrations of insulin like growth factor-I (IGF-I) are depressed. This is similar to a condition in humans known as Laron dwarfism. Results from earlier studies indicated that GH administered to young miniature Brahman did not significantly affect IGF-I. A most recent study using older miniature Brahman cattle demonstrated an elevation in IGF-I when GH was administered. This suggested that there may be differences in the potency between the GH produced by miniature Brahman and that produced by normal Brahman or that produced synthetically (bGH). Thus, the present study was to clone the GH cDNA from miniature Brahman, compare its sequence to normal Brahman, and determine activities of the GH protein. Results indicated that at amino acid 200 there was a mutation (threonine to methionine). The site of this mutation is in a region responsible for the binding of GH to the GH receptor (an event prior to production of IGF-I). Of 21 Brahman tested (12 miniature and 9 normal), 12 miniatures were homozygous (-/-) for the mutation, 7 normals were homozygous (+/+), and 2 normals were heterozygous (+/-). When isolated from miniature Brahman cattle, GH had approximately 60% of the activity of GH isolated from normal Brahman cattle. In summary, miniature Brahman cattle were homozygous for a mutation of an amino acid involved in binding of GH to the GH receptor. In bovine GH, threonine 200 is required for normal growth in cattle.
Technical Abstract: Miniature Brahman cattle at the USDA ARS in Brooksville, FL have normal proportioned growth but are approximately 70% of normal mature height and weight when compared with Brahman cattle in the same heard. Pedigree analyses suggest that the condition is inherited as a recessive allele. The miniature Brahman cattle in the Brooksville herd have been used for studies of growth and reproduction, but the underlying causative mutation is unknown. Presumably, the miniature condition could arise from a mutation in the GH gene. The objective therefore, was to clone the GH cDNA from Brooksville miniature Brahman cattle, compare its sequence to normal Brahman cattle, and test the activity of native GH protein. Messenger RNA was isolated from pituitary and a cDNA for the protein coding region of the GH gene was amplified by reverse transcription (PCR) from each of two miniature Brahman bulls. The cDNA were cloned into plasmid vectors and top and bottom strands were sequenced by automated DNA sequencing. The sequence of both cDNA clones derived from miniature cattle differed from Bos Indicus GH (GenBank AF034386) at base number 641 because there was a cytosine (C) instead of thymine (T). The C to T change encoded a mutation (threonine to methionine) at amino acid 200 (T200M mutation). The mutation was confirmed by sequencing of an additional two miniature cattle and comparing their sequence to two normal cattle. The threonine is located in the fourth alpha helix of GH and is one of 8 amino acids that participate in binding of GH to the GH receptor. Twelve miniature Brahman and 9 normal Brahman cattle were tested by using a restriction fragment length polymorphism analysis that employed the BsmBI restriction enzyme (specific for mutated nucleotide). The 12 miniature Brahman cattle were homozygous for the mutation (-/-). Seven of the normal cattle were homozygous for the wild type allele (+/+), and two were heterozygous (+/-). When tested in a cell-based bioassay, GH isolated from the pituitary of -/- cattle (n=4) had approximately 60% activity when compared with GH isolated from the pituitary of +/+ cattle (n=5). In summary, miniature Brahman cattle were homozygous for a single nucleotide polymorphism that encodes a mutation in an amino acid involved in binding of GH to the GH receptor. Cattle of normal stature had at least one copy of the normal GH allele. Threonine 200 in bovine GH is required for normal growth in cattle.