Sequence and expression characteristics of a shuttle chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli.
An efficiently transforming chloramphenicol-resistance (CmR) shuttle marker for Saccharomyces cerevisiae and Escherichia coli has been characterized in terms of its primary structure and expression characteristics. The complete nucleotide (nt) sequence of the CmR marker is given, with details on restriction sites, apparent expression signals for both organisms, and translation of the Cm acetyltransferase (CAT)-coding sequence. SDS-polyacrylamide gel electrophoresis and Western blotting have confirmed that the marker produced an identical CAT protein in yeast and E. coli. Each copy of the marker, whether present in multiple copies or as a single copy, gave rise to approx. 0.1% of the total soluble protein as CAT in haploid yeast cells. When compared with homologous expression of alcohol dehydrogenase (ADH-I) by the same ADC1 promoter, this represents a 27-fold reduction for CAT expression, which is typical of heterologous gene expression in yeast. When the marker was on a multicopy plasmid in yeast, up to 2.1% of the total soluble cell protein was produced as CAT, but this did not adversely affect the growth of host cells. Increase of the Cm concentration in the medium did not result in an increase in the number of plasmids nor the amount of CAT protein produced, showing that plasmid copy number and marker expression are regulated independently of the selection pressure. In E. coli, the ADC1 yeast-promoter DNA was found to contain both forwards and backwards promoter activity. The level of expression provided by these promoters was equivalent to that of an average E. coli gene.
D000123:Acetyltransferases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002701:Chloramphenicol / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000032:analysis; D004351:Drug Resistance; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005800:Genes, Fungal; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*
D004274:DNA, Recombinant; D002701:Chloramphenicol; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D004262:DNA Restriction Enzymes