Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan.
PLoS One. 2010 Feb 17;5(2):e9273. doi: 10.1371/journal.pone.0009273.
Cryptochromes (CRYs) are flavoproteins sharing high homology with photolyases. Some of them have function(s) including transcription regulation in the circadian clock oscillation, blue-light photoreception for resetting the clock phase, and light-dependent magnetoreception. Vertebrates retain multiple sets of CRY or CRY-related genes, but their functions are yet unclear especially in the lower vertebrates. Although CRYs and the other circadian clock components have been extensively studied in the higher vertebrates such as mice, only a few model species have been studied in the lower vertebrates. In this study, we identified two CRYs, XtCRY1 and XtCRY2 in Xenopus tropicalis, an excellent experimental model species. Examination of tissue specificity of their mRNA expression by real-time PCR analysis revealed that both the XtCRYs showed extremely high mRNA expression levels in the ovary. The mRNA levels in the ovary were about 28-fold (XtCry1) and 48-fold (XtCry2) higher than levels in the next abundant tissues, the retina and kidney, respectively. For the functional analysis of the XtCRYs, we cloned circadian positive regulator XtCLOCK and XtBMAL1, and found circadian enhancer E-box in the upstream of XtPer1 gene. XtCLOCK and XtBMAL1 exhibited strong transactivation from the XtPer1 E-box element, and both the XtCRYs inhibited the XtCLOCK:XtBMAL1-mediated transactivation, thereby suggesting this element to drive the circadian transcription. These results revealed a conserved main feedback loop in the X. tropicalis circadian clockwork and imply a possible physiological importance of CRYs in the ovarian functions such as synthesis of steroid hormones and/or control of estrus cycles via the transcription regulation.
隐花色素（CRYs）是与光解酶具有高度同源性的黄素蛋白。它们中的一些具有包括调节生物钟振荡的转录、重置生物钟相位的蓝光光感受和光依赖性磁感受等功能。脊椎动物保留了多套 CRY 或与 CRY 相关的基因，但它们的功能尚不清楚，尤其是在低等脊椎动物中。尽管 CRY 及其它生物钟成分在高等脊椎动物（如小鼠）中得到了广泛研究，但在低等脊椎动物中仅对少数模式物种进行了研究。在这项研究中，我们在非洲爪蟾（Xenopus tropicalis）中鉴定出两种 CRY，即 XtCRY1 和 XtCRY2，这是一种优秀的实验模型物种。通过实时 PCR 分析检查其 mRNA 表达的组织特异性，结果显示两种 XtCRY 在卵巢中表现出极高的 mRNA 表达水平。卵巢中的 mRNA 水平分别比下一个丰富组织（视网膜和肾脏）高约 28 倍（XtCry1）和 48 倍（XtCry2）。为了对 XtCRYs 的功能进行分析，我们克隆了生物钟正调控因子 XtCLOCK 和 XtBMAL1，并在 XtPer1 基因的上游发现了 circadian enhancer E-box。XtCLOCK 和 XtBMAL1 从 XtPer1 E-box 元件表现出强烈的转录激活作用，并且两种 XtCRY 都抑制了 XtCLOCK:XtBMAL1 介导的转录激活，这表明该元件驱动了生物钟的转录。这些结果揭示了非洲爪蟾生物钟工作机制中的一个保守的主要反馈回路，并暗示 CRY 可能在卵巢功能（如类固醇激素的合成和/或通过转录调控控制发情周期）中具有重要的生理意义。
