Department of Microbiology, CICESE, Ensenada, B.C., Mexico.
Fungal Genet Biol. 2010 Apr;47(4):332-41. doi: 10.1016/j.fgb.2009.12.013. Epub 2010 Jan 4.
The frequency (frq) gene of Neurospora crassa has long been considered essential to the function of this organism's circadian rhythm. Increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. To address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins such as FRQ. However, due to the difficulty of studying the expression of circadian rhythm genes in aerobic N. crassa cultures, little is known about the dynamics of this gene under physiologically realistic conditions. To address these issues we report a fluorescent fusion to the frq gene using a codon optimized version of the mCherry gene. To trace the expression and accumulation of FRQ-mCherryNC (FRQ-mCh) during the circadian rhythm, growing vegetative hyphae were scanned every hour under confocal microscopy (100x). Fluorescence of FRQ-mCh was detected only at the growing edge of the colony, and located in the cytoplasm and nuclei of vegetative hyphae for a distance of approximately 150-200microm from the apices of leading hyphae. When driven by the frq promoter, apparently there was also a second FRQ entrance into the nucleus during the circadian cycle; however the second entrance had a lower accumulation level than the first entrance. Thus this fluorescent fusion protein has proven useful in tracking the spatial dynamics of the frq protein and has indicated that the dynamics of the FRQ protein's nuclear trafficking may be more complex than previously realized.
粗糙脉孢菌（Neurospora crassa）的频率（frq）基因长期以来被认为是该生物生物钟功能所必需的。越来越多的，解析核心振荡器基因（如 frq）与生物钟输出途径的耦合已成为生物钟研究的主要焦点。为了解决这种耦合问题，关键是要有一个生物钟活性的报告器，它可以提供有关核心振荡蛋白（如 FRQ）动力学的高分辨率时空信息。然而，由于在需氧的粗糙脉孢菌培养物中研究生物钟节律基因表达的困难，因此，对于该基因在生理现实条件下的动力学知之甚少。为了解决这些问题，我们使用优化后的 mCherry 基因的密码子报告 frq 基因的荧光融合。为了在生物钟节律过程中追踪 FRQ-mCherryNC（FRQ-mCh）的表达和积累，在共聚焦显微镜下（100x）每隔一小时扫描生长的营养菌丝。仅在菌落的生长边缘检测到 FRQ-mCh 的荧光，并且位于营养菌丝的细胞质和核中，距离前沿菌丝的顶端约 150-200μm。当由 frq 启动子驱动时，显然在生物钟周期中也有第二个 FRQ 进入细胞核；然而，第二个入口的积累水平低于第一个入口。因此，这种荧光融合蛋白已被证明在跟踪 frq 蛋白的空间动力学方面非常有用，并表明 FRQ 蛋白核运输的动力学可能比以前认识的更为复杂。
