Alani E, Kleckner N
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Genetics. 1987 Sep;117(1):5-12. doi: 10.1093/genetics/117.1.5.
We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5'-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.
我们构建了一些结构，将酿酒酵母HIS4和GAL1基因以及大肠杆菌lacZ基因的启动子序列和部分编码区与酿酒酵母URA3基因（编码乳清苷-5'-磷酸（OMP）脱羧酶）的第六个密码子连接起来，形成三种读框内的蛋白质融合体。在每种情况下，融合蛋白都具有通过互补试验测定的OMP脱羧酶活性，并且这种活性受到适当调节。一个由URA3片段加上HIS4C的一些紧邻近端氨基酸组成的便捷盒式结构，可用于构建URA3与其他感兴趣蛋白质的融合体。与其他用于基因融合分析的系统相比，URA3融合体具有几个优点：URA3指定的蛋白质小且位于胞质中；在酵母（酿酒酵母和粟酒裂殖酵母）和细菌（大肠杆菌和鼠伤寒沙门氏菌）中都存在用于鉴定URA3功能增加或减少的突变体的遗传筛选方法；并且有灵敏的OMP脱羧酶酶活性测定方法。此外，哺乳动物、果蝇和植物中都存在OMP脱羧酶活性，因此URA3融合体最终也可能适用于这些其他生物体。
