Leish Z, Byrne C R, Hunt C L, Ward K A
Division of Animal Production, Commonwealth Scientific and Industrial Research Organisation, Blacktown, New South Wales, Australia.
Appl Environ Microbiol. 1993 Mar;59(3):892-8. doi: 10.1128/aem.59.3.892-898.1993.
The coding sequences of the cysE and cysK genes from Escherichia coli, which encode the enzymes of the cysteine biosynthetic pathway, namely, serine acetyltransferase (EC 2.3.1.30) and O-acetylserine sulfhydrylase (or cysteine synthase [EC 4.2.99.8]), were modified for expression in eukaryotic cells and introduced into murine L cells. A number of fusion genes comprising the cysE or cysK coding sequences joined to the promoter of the ovine metallothionein-Ia (MT-Ia) gene and various portions of the ovine growth hormone (GH) gene were prepared. Significant differences in the level of transcription were observed, depending on the amount and arrangement of the GH gene sequences used, the highest levels being obtained with the constructs MTCE10 and MTCK7, which contained only the GH 3' untranslated gene sequences. These two constructs were fused to produce the gene MTCEK1. In this single DNA sequence, each bacterial gene is under independent MT-Ia promoter control. Expression of the cysK sequence in this construct (MT-Ia promoter-cysE-3' GH sequence-MT-Ia promoter-cysK-3' GH sequence) was elevated compared with expression of the cysK gene in MTCK7. However, expression of the cysE sequence in MTCEK1 was only 40% of that of the cysE gene cloned into MTCE10. The double-promoter configuration, which enhances the expression of the second gene in MTCEK1, is proposed as a model for the modification of bacterial genes in general.
编码大肠杆菌半胱氨酸生物合成途径中酶的cysE和cysK基因的编码序列，即丝氨酸乙酰转移酶（EC 2.3.1.30）和O-乙酰丝氨酸巯基酶（或半胱氨酸合酶[EC 4.2.99.8]），被修饰用于在真核细胞中表达，并导入小鼠L细胞。制备了许多融合基因，这些融合基因包含与绵羊金属硫蛋白-Ia（MT-Ia）基因启动子和绵羊生长激素（GH）基因不同部分相连的cysE或cysK编码序列。观察到转录水平存在显著差异，这取决于所使用的GH基因序列的数量和排列，其中构建体MTCE10和MTCK7获得了最高水平，它们仅包含GH 3'非翻译基因序列。将这两个构建体融合以产生基因MTCEK1。在这个单一DNA序列中，每个细菌基因都处于独立的MT-Ia启动子控制之下。与MTCK7中cysK基因的表达相比，该构建体（MT-Ia启动子-cysE-3' GH序列-MT-Ia启动子-cysK-3' GH序列）中cysK序列的表达有所提高。然而，MTCEK1中cysE序列的表达仅为克隆到MTCE10中的cysE基因表达的40%。双启动子构型增强了MTCEK1中第二个基因的表达，被提议作为一般细菌基因修饰的模型。
