Primorac D, Stover M L, Clark S H, Rowe D W
Department of Pediatrics, University of Connecticut Health Center, Farmington.
Matrix Biol. 1994 Aug;14(4):297-305. doi: 10.1016/0945-053x(94)90195-3.
Nanomelia is a recessively inherited connective tissue disorder of chicken affecting cartilage development. Other investigators have demonstrated that it involves low aggrecan production and diminished aggrecan mRNA levels. Based on genetic linkage studies showing a high likelihood that the mutation responsible for the nanomelic phenotype lay within the aggrecan gene, a series of experiments was performed to define the molecular basis of the trait. Aggrecan mRNA was present in the nucleus of the nanomelic chondrocyte but greatly reduced in the cytoplasmic compartment, a finding suggestive of a premature stop codon within the aggrecan transcript. Since no defect in mRNA splicing could be demonstrated by ribonucleasease protection studies, direct DNA sequencing was initiated by polymerase chain reaction of the mRNA and of genomic DNA. A stop codon was demonstrated at codon 1513, which is located in the eighth repeat of the chondroitin sulfate 2 domain of the large tenth exon. The mutation creates a unique BasBI restriction site which readily distinguishes the mutant and wild-type alleles.
短肢畸形是鸡的一种隐性遗传结缔组织疾病，影响软骨发育。其他研究人员已证明，它涉及聚集蛋白聚糖产量低和聚集蛋白聚糖mRNA水平降低。基于遗传连锁研究表明，导致短肢畸形表型的突变很可能位于聚集蛋白聚糖基因内，因此进行了一系列实验来确定该性状的分子基础。聚集蛋白聚糖mRNA存在于短肢畸形软骨细胞的细胞核中，但在细胞质部分大大减少，这一发现提示聚集蛋白聚糖转录本内存在过早的终止密码子。由于核糖核酸酶保护研究未显示mRNA剪接存在缺陷，因此通过对mRNA和基因组DNA进行聚合酶链反应启动了直接DNA测序。在第1513密码子处发现了一个终止密码子，该密码子位于第十大外显子硫酸软骨素2结构域的第八个重复序列中。该突变产生了一个独特的BspBI限制性位点，可轻松区分突变和野生型等位基因。
