A new type of fusion analysis applicable to many organisms: protein fusions to the URA3 gene of yeast.
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.
Alani
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E
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Kleckner
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N
D002262:Carboxy-Lyases / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005800:Genes, Fungal; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D003062:Codon; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase