IRISH-SETTER DOGS AFFECTED WITH ROD CONE DYSPLASIA CONTAIN A NONSENSE MUTATION IN THE ROD CGMP PHOSPHODIESTERASE BETA-SUBUNIT GENE
IRISH-SETTER DOGS AFFECTED WITH ROD CONE DYSPLASIA CONTAIN A NONSENSE MUTATION IN THE ROD CGMP PHOSPHODIESTERASE BETA-SUBUNIT GENE
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DOI：
10.1073/pnas.90.9.3968
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发表时间：
1993-05-01
影响因子：
11.1
通讯作者：
HURWITZ, RL
中科院分区：
文献类型：
--
作者：
SUBER, ML;PITTLER, SJ;HURWITZ, RL
患有视杆细胞/视锥细胞发育不良（基因座指定，rcd1）的爱尔兰塞特犬在出生后发育过程中视网膜 cGMP 水平显着升高。光感受器退化几乎在出生后 25 天开始，并在大约 1 岁时达到顶峰，此时视杆细胞和视锥细胞数量耗尽。组蛋白敏感的视网膜 cGMP 磷酸二酯酶（PDE；EC 3.1.4.35）活性是光感受器 PDE 的标记，之前已表明存在于未成熟、受影响的爱尔兰塞特犬的视网膜匀浆中。在这里，我们报告说，根据 HPLC 分离判断，这种活性完全源自视锥细胞光感受器，而视杆细胞 PDE 活性不存在。在受影响狗的视网膜提取物的免疫印迹上可以检测到 PDE α 亚基大小的免疫反应产物，但没有检测到 β 亚基大小的免疫反应产物，表明 PDE β 亚基基因中存在无效突变。使用爱尔兰塞特犬视网膜 cDNA 的 PCR 扩增，我们确定了杂合子和受影响动物中 PDE β 亚基的完整编码序列。受影响的 PDE β 亚基 mRNA 在密码子 807 处包含无义琥珀突变（将 TGG 转换为 TAG 的 G → A 转换），该突变被证实存在于受影响的 β 亚基基因的推定外显子 21 中。提前终止密码子将 β 亚基截断 49 个残基，从而去除翻译后加工和膜关联所需的 C 端结构域。这些结果表明，rcd1 基因编码视杆光感受器 PDE β 亚基，并且该基因中的无义突变导致产生无功能的视杆 PDE 和 rcd1/rcd1 爱尔兰塞特犬的光感受器退化。
Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences almost-equal-to 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G --> A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.