Zhu H, Green C B
NSF Center for Biological Timing, Department of Biology, University of Virginia, Charlottesville, VA 22904, USA.
Mol Vis. 2001 Aug 29;7:210-5.
To clone Xenopus laevis cryptochromes (crys) and to understand their role in the Xenopus retinal clock.
We designed degenerate PCR primers based on homology between mouse and human crys. DNA fragments generated from these PCR reactions were used to screen a Xenopus retinal cDNA library. Three independent clones were identified and sequenced. The temporal and spatial expression of these genes in retina were studied by Northern blot analysis and in situ hybridization.
We cloned three cry homologs from Xenopus laevis retina. We named them xcry1, xcry2a, and xcry2b based on their high homology to the mouse crys. Sequence analysis shows that these Xenopus CRYs have more than 85% identity to mouse CRYs at the amino acid level. Northern blot analysis demonstrated that all three xcrys are rhythmically expressed in the retina with peaks at different times of the day. The xcrys are expressed in a variety of tissues. In retina, they are expressed predominantly in photoreceptor cells.
Our finding of cry expression in Xenopus photoreceptor cells further supports the idea of independent circadian oscillators being present in these cells. The sequence similarities to mouse crys suggest similar functions in the circadian clock. However, their distinct temporal expression patterns suggest some unique role for xCRY in the Xenopus retina.
克隆非洲爪蟾隐花色素（crys）并了解其在非洲爪蟾视网膜生物钟中的作用。
我们根据小鼠和人类隐花色素之间的同源性设计了简并PCR引物。这些PCR反应产生的DNA片段用于筛选非洲爪蟾视网膜cDNA文库。鉴定并测序了三个独立的克隆。通过Northern印迹分析和原位杂交研究了这些基因在视网膜中的时空表达。
我们从非洲爪蟾视网膜中克隆了三个隐花色素同源物。根据它们与小鼠隐花色素的高度同源性，我们将它们命名为xcry1、xcry2a和xcry2b。序列分析表明，这些非洲爪蟾隐花色素在氨基酸水平上与小鼠隐花色素的同一性超过85%。Northern印迹分析表明，所有三个xcrys在视网膜中均有节律性表达，在一天中的不同时间达到峰值。xcrys在多种组织中表达。在视网膜中，它们主要在光感受器细胞中表达。
我们在非洲爪蟾光感受器细胞中发现隐花色素表达，进一步支持了这些细胞中存在独立昼夜节律振荡器的观点。与小鼠隐花色素的序列相似性表明它们在生物钟中具有相似的功能。然而，它们不同的时间表达模式表明xcRY在非洲爪蟾视网膜中具有一些独特的作用。
