Construction of a UMP synthase expression cassette from Dictyostelium discoideum.
We have prepared a DNA cassette containing the UMP synthase (UMPS)-encoding gene (PYR5-6) from Dictyostelium discoideum. This gene contains no introns and can be used for expression of the UMPS protein. Due to the high percentage of AT in the flanking regions, useful restriction sites were absent, therefore the PYR5-6 was subcloned as three separate parts, manipulated, and religated to make a full-length clone. After reconstructing the coding region, we examined its functionality by introducing this gene under the control of the yeast GAL1 promoter into several uracil-requiring mutants of Saccharomyces cerevisiae. These studies demonstrated that the reconstructed PYR5-6 gene was functional and could complement independent ura3 and ura5 mutations in yeast.
Department of Plant Pathology, Iowa State University, Ames 50011.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics*; D005800:Genes, Fungal; D005809:Genes, Regulator; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae
D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; C027157:uridine 5'-monophosphate synthase; D009962:Orotate Phosphoribosyltransferase; D009964:Orotidine-5'-Phosphate Decarboxylase