翻译起始区二级结构对荧光素酶基因在COS-7细胞中表达效率的影响

在荧光素酶基因起始密码子ATG下游插入4种串连重复的密码子(6×ATT ,3×ATT ,6×GCC与3×GCC) ,得到具有不同二级结构的翻译起始区(translationinitiationregion ,TIR) ,以研究TIR二级结构对该基因在COS 7细胞中表达的影响.Northern印迹结果显示,4种重组子mRNA的转录水平没有显著差异,而Western印迹与酶活性检测表明,与野生型结构相比,6×ATT与3×ATT能显著提高荧光素酶的表达量与活性,而6×GCC结构的表达量明显下降.使用计算机辅助分析软件,扫描上述TIR结构发现,TIR稳定性或二级结构复杂度是导致上述表达差异的主要原因.     

Regulation of gene expression in preimplantation mouse embryos: effects of the zygotic clock and the first mitosis on promoter and enhancer activities

Previous studies have reported that promoters requiring enhancers for full activity in mammalian somatic cells also require enhancers when injected into mouse two-cell embryos, whereas the same promoters can be expressed just as efficiently in the absence of an enhancer when injected into arrested one-cell embryos. Experiments were designed to determine whether this phenomenon reflected normal developmental changes at the beginning of mammalian development, or simply differences in the physiological states of these cells under the experimental conditions employed. The activity of three different promoters that function in a wide variety of mammalian cells was measured both in embryos whose morphological development was arrested and in embryos that continued development in vitro. Expression of the injected gene was related to the onset of zygotic gene expression ("zygotic clock"), the phase of the cell proliferation cycle, the use of aphidicolin to arrest cell proliferation, and formation of two-cell embryos in vitro and in vivo. The results demonstrated that promoter activity was tightly linked to zygotic gene expression, while the need for enhancers to stimulate promoter activity depended only on formation of a two-cell embryo. These results further support the hypothesis that the first mitosis induces a general repression of promoters prior to initiation of zygotic gene expression that is relieved specifically by enhancers.     

Introduction and Expression of Recombinant β-Galactosidase Genes in Cleavage Stage Mouse Embryos

In an attempt to study gene regulation in very early stages of mouse embryogenesis, we injected genes constructed by joining the coding sequence of the bacterial β-galactosidase gene to four different animal gene enhancers/promoters and to poly (A) signals, and examined the gene expression in cleavage stage embryos.
With appropriate injection volumes for each embryonic stage, ranging from 0.2 to 1.3 pl, the majority of the injected embryos underwent at least one further cleavage. Expression of injected genes, which occurred transiently after injection, required the promoter sequences but without much distinction of the source of enhancer/promoter complexes. This result was in a sharp contrast to transfection of mouse cell lines where the recombinant genes were variably expressed reflecting differential enhancer effects.
By injection at the 1-cell stage, expression of injected genes was low while the expression by injection at the 2-cell or later stages was several fold higher, which may correlate with the fact that most zygotic gene expression begins after the 2-cell stage. The low expression at the 1-cell stage was augmented by the conditions causing clea***age arrest such as inhibition of DNA synthesis with aphidicolin.