Chemical synthesis and expression of a cassette adapted ubiquitin gene.
A gene encoding the yeast ubiquitin was chemically synthesized and expressed in yeast under regulatory control of the copper metallothionein (CUP1) promoter. The gene was assembled in a one-step ligation reaction from eight oligonucleotide fragments ranging in length from 50 to 64 nucleotides. To facilitate mutagenesis and gene fusion studies, eight unique 6-base-cutting restriction enzyme sites were placed in the reading frame which did not alter the encoded protein sequence or force the utilization of rare codons. In a copper-resistant yeast strain (CUP1r), expression of the gene was induced by copper to approximately 5% of the total yeast proteins, as determined by Coomassie-stained polyacrylamide gels. The protein, purified from yeast, reacted with ubiquitin-specific antibodies and was found to be biologically active in supporting ubiquitin-dependent protein degradation in vitro.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D005800:Genes, Fungal; D008668:Metallothionein / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic; D014452:Ubiquitins / Q000235:genetics*
D014452:Ubiquitins; D008668:Metallothionein