A MODULAR SET OF LACZ FUSION VECTORS FOR STUDYING GENE-EXPRESSION IN CAENORHABDITIS-ELEGANS
A MODULAR SET OF LACZ FUSION VECTORS FOR STUDYING GENE-EXPRESSION IN CAENORHABDITIS-ELEGANS
复制标题
DOI：
10.1016/0378-1119(90)90224-f
复制
发表时间：
1990-09-14
期刊：
影响因子：
3.5
通讯作者：
DIXON, D
中科院分区：
文献类型：
--
作者：
FIRE, A;HARRISON, SW;DIXON, D
我们描述了一系列的质粒载体，其中包含模块化的功能，特别是用于研究在真核系统中的基因表达。载体含有大肠杆菌β-半乳糖苷酶（β Gal）编码区（lacZ基因），在5“和3”端侧接独特的多接头区段，以及多种模块的几种组合：选择标记（琥珀抑制tRNA）、翻译起始区、合成内含子区段、来自SV 40的早期多腺苷酸化信号和来自两个线虫基因的3“区。将编码来自SV 40 T抗原的核定位肽的区段掺入许多构建体中，导致β Gal在细胞核中积累，这可以促进复杂组织中生产细胞的鉴定。为了使功能性β Gal融合到分泌蛋白，我们构建了具有编码lacZ上游的合成跨膜结构域的替代模块的质粒。该结构域被设计为在分泌期间阻止分泌蛋白跨膜转移，允许融合多肽的β Gal结构域保留在细胞质中，从而在酶测定中发挥作用。我们已经使用的载体来分析线虫Caernorhabditis elegans中的几个基因的表达，并已在这些研究中表明，lacZ可以在各种不同的组织和细胞类型中表达。这些载体在研究C. elegans和其他实验系统中。
We describe a series of plasmid vectors which contain modular features particularly useful for studying gene expression in eukaryotic systems. The vectors contain the Escherichia coli .beta.-galactosidase (.beta.Gal)-encoding region (the lacZ gene) flanked by unique polylinker segments on the 5'' and 3'' ends, and several combinations of a variety of modules: a selectable marker (an amber suppressor tRNA), a translational initiation region, a synthetic intron segment, the early polyadenylation signal from SV40, and 3'' regions from two nematode genes. A segment encoding the nuclear localization peptide from the SV40 T antigen is incorporated into many of the constructs, leading to .beta.Gal accumulation in nuclei, which can facilitate identification of producing cells in complex tissues. To make functional .beta.Gal fusions to secreted proteins, we constructed plasmids with an alternate module encoding a synthetic transmembrane domain upstream from lacZ. This domain is designed to stop transfer of secreted proteins across the membrane during secretion, allowing the .beta.Gal domain of the fusion polypeptide to remain in the cytoplasm and thus function in enzymatic assays. We have used the vectors to analyze expression of several genes in the nematode Caernorhabditis elegans, and have demonstrated in these studies that lacZ can be expressed in a wide variety of different tissues and cell types. These vectors should be useful in studying gene expression both in C. elegans and in other experimental systems.