A miniature condition in Brahman cattle is associated with a single nucleotide mutation within the growth hormone gene
A miniature condition in Brahman cattle is associated with a single nucleotide mutation within the growth hormone gene
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DOI：
10.1016/j.domaniend.2009.04.001
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发表时间：
2009-08-01
影响因子：
2.1
通讯作者：
Lucy, M. C.
中科院分区：
文献类型：
--
作者：
McCormack, B. L.;Chase, C. C., Jr.;Lucy, M. C.
佛罗里达州布鲁克斯维尔的美国农业部ARS亚热带农业研究站的微型婆罗曼牛具有正常的比例生长，但与同一牛群中的婆罗曼牛相比，大约是成熟牛身高和体重的70%。家系分析表明，这种情况是通过隐性等位基因遗传的。布鲁克斯维尔牛群中的微型婆罗门牛已被用于生长和繁殖研究，但潜在的致病突变尚不清楚。据推测，这种微小的疾病可能是由于生长激素基因的突变引起的。因此，我们的目的是克隆布鲁克斯维尔小型婆罗曼牛的生长激素基因，将其序列与正常婆罗曼牛进行比较，并检测天然生长激素蛋白的生物学活性。从2头小型婆罗曼公牛的脑垂体中提取信使RNA，通过逆转录-聚合酶链式反应(RT-PCR)扩增出生长激素基因蛋白编码区的cDNA。将其克隆到载体上，并对其顶链和底链进行DNA自动测序。来自小型牛的两个克隆的序列与野牛GH(GenBank AF034386)的碱基641不同，这是因为它们含有胞嘧啶(C)而不是胸腺嘧啶(T)。C到T的改变编码了200位氨基酸的突变(苏氨酸到蛋氨酸)(T200M突变)。通过对另外两头小型牛进行测序，并将它们的序列与两头正常牛进行比较，证实了该突变。苏氨酸位于生长激素的第四个α螺旋上，是参与生长激素与生长激素受体结合的8个氨基酸之一。对12头小型婆罗曼牛和9头正常婆罗曼牛进行了BsmBI限制性内切酶的限制性片段长度多态性分析。这12头小型婆罗门牛都是突变(-/-)纯合子。正常婆罗门牛中有7头野生型等位基因纯合(+/+)，2头杂合(+/-)。在基于细胞的生物测定中，从-/-牛(n=4)脑垂体分离的GH与从+/+牛脑垂体(n=5)分离的GH相比，活性约为60%。总而言之，小型婆罗门牛是纯合的单核苷酸多态，编码与生长激素受体结合有关的氨基酸突变。正常体型的牛至少有一个正常生长激素等位基因的拷贝。牛生长激素中的苏氨酸200是牛正常生长所必需的。(C)2009 Elsevier Inc.保留所有权利。
Miniature Brahman cattle at the USDA ARS Subtropical Agriculture Research Station in Brooksville, FL have normal proportioned growth but are approximately 70% of mature height and weight when compared with Brahman cattle in the same herd. Pedigree analyses suggest that the condition is inherited through 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 biological activity of the native GH protein. Messenger RNA was isolated from the pituitary, and a cDNA for the protein coding region of the GH gene was amplified by reverse-transcription polymerase chain reaction (PCR) from each of 2 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 a 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 2 miniature cattle and comparing their sequence to 2 normal cattle. The threonine is located in the fourth alpha helix of GH and is 1 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 the mutated nucleotide). The 12 miniature Brahman cattle were homozygous for the mutation (-/-). Seven of the normal Brahman cattle were homozygous for the wild-type allele (+/+), and 2 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 size had at least 1 copy of the normal GH allele. Threonine 200 in bovine GH is required for normal growth in cattle. (C) 2009 Elsevier Inc. All rights reserved.