Kelly W G, Xu S, Montgomery M K, Fire A
Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210, USA.
Genetics. 1997 May;146(1):227-38. doi: 10.1093/genetics/146.1.227.
In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences.
在筛选秀丽隐杆线虫胚胎致死突变的过程中,我们鉴定出一个必需基因(let-858),它编码一种富含酸性和碱性残基的核蛋白。我们将该产物命名为核胺磷蛋白。酵母、植物和哺乳动物中的同源序列显示出在真核生物中具有很强的进化保守性。核胺磷蛋白存在于秀丽隐杆线虫的所有细胞核中,在早期发育和组织分化过程中是必需的。在早期胚胎中,通过反义介导降低LET-858的活性会导致一种致死表型,类似于阻止胚胎基因表达的特征性处理。利用转基因拯救技术,我们证明了let-858在幼虫生殖系中的额外需求。这种广泛的需求使得能够研究基因表达中的体细胞与生殖系差异。当导入标准转基因阵列时,let-858(与许多其他秀丽隐杆线虫基因一样)在体细胞中功能良好,但在生殖系中功能较差。我们观察到在转化后的最初几代中,简单的let-858阵列逐渐沉默,并推测这种沉默可能反映了阵列被识别为重复序列或异染色质样序列。为了使转基因具有更生理的背景,我们在注射的DNA中加入了过量的随机基因组片段。所得转基因在生殖系和体细胞中均表现出强劲的表达。我们的结果提示了沉默重复序列在生殖系中不必要表达的协同机制的可能性。