Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, The University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK.
Mech Ageing Dev. 2009 Nov-Dec;130(11-12):762-70. doi: 10.1016/j.mad.2009.10.001.
As in yeast, flies and mammals, over-expression of the Caenorhabditis elegans sirtuin gene sir-2.1 leads to extension of lifespan and deletion of the gene shortens lifespan. The sir-2.1 gene, however, is located in an operon, an organization not taken into account in previous studies of this gene's expression.
Recombineering allowed insertion of both a mCherry and a gfp reporter gene precisely at the end of the two protein-coding regions of the 4.5kb sir-2.1 operon within a 29.3kb genomic DNA fosmid clone.
In C. elegans transgenic for this recombineered fosmid, with abundant food, the sir-2.1::mCherry distribution indicated that sir-2.1 is indeed expressed in the hypodermis and many nerve cells, as previously described, but also in the intestine and in muscles. This broader expression of sir-2.1, which would fit with an expectation that SIR2.1 function in influencing lifespan might be required in most cell types, arises from transcription starting with the gene upstream of sir-2.1 in the operon. Importantly, the expression of both genes in the operon increases upon starvation, this induction also depending on the operon promoter. Furthermore, SIR-2.1::mCherry undergoes a dynamic subcellular relocalization through starvation.
与酵母、苍蝇和哺乳动物一样,秀丽隐杆线虫中的 sir-2.1 长寿基因的过表达会延长寿命,而该基因的缺失则会缩短寿命。然而,sir-2.1 基因位于操纵子中,这在以前对该基因表达的研究中并未考虑到。
通过重组酶技术,我们可以将 mCherry 和 gfp 报告基因精确地插入到 4.5kb 的 sir-2.1 操纵子的两个蛋白质编码区的末端,该操纵子位于 29.3kb 的基因组 DNA fosmid 克隆中。
在这种重组 fosmid 的转基因秀丽隐杆线虫中,在丰富的食物条件下,sir-2.1::mCherry 的分布表明 sir-2.1 确实在表皮和许多神经细胞中表达,这与之前的描述一致,但也在肠道和肌肉中表达。这种 sir-2.1 的更广泛表达,与 SIR2.1 影响寿命的功能可能需要在大多数细胞类型中发挥作用的预期相符,这是由于操纵子上游基因的转录起始引起的。重要的是,操纵子中两个基因的表达在饥饿时增加,这种诱导也依赖于操纵子启动子。此外,SIR-2.1::mCherry 会经历一个通过饥饿引起的动态亚细胞重定位。