A series of thiamine-repressible shuttle vectors has been constructed to allow a more efficient DNA manipulation in Schizosaccharomyces pombe. These high-copy-number vectors with regulatable expression (pJR) are based on the backbone of the pREP-3X, pREP-41X and pREP-81X plasmids. The pJR vectors are all uniform in structure, containing: (a) sequences for replication (ori) and selection (AmpR) in Escherichia coli; (b) the f1 ori sequence of the phage f1 for packaging of ssDNA, making them suitable for site-directed mutagenesis; and (c) the ars1 sequence for replication in S. pombe. The pJR vectors differ among them in: (a) the selectable marker (Saccharomyces cerevisiae LEU 2 gene, which complements S. pombe leu1- gene and S. pombe ura4+ and his3+ genes); (b) the thiamine-repressible nmt1 promoter (3X, 41X and 81X with extremely high, moderate or low transcription efficiency, respectively); and (c) the multiple cloning site (two multiple cloning sites, with 12 restriction sites each). The expression level of the pJR vectors has been analysed using the beta-galactosidase gene as reporter. Three levels of expression for each nmt1 promoter version, with any selectable marker and for either repressed or induced conditions, have been found. The expression is dependent on the distance to the initiation codon, varying from 0.001 to 15 times the activity characterized for the pREP plasmids. Also, the gene expression has been found to be extremely sensitive to the nucleotide sequence prior to the initiation codon, being up to 50-fold higher with an A/T sequence than with a G/C sequence. Finally, the beta-galactosidase mRNA levels were found to be similar in each nmt1 series, suggesting a translational effect on gene expression. As a result, any of these 18 new vectors allow performing gene expression in fission yeast, as well as a more versatile cloning, sequencing and mutagenesis, directly in the plasmid without the need for subcloning into intermediary vectors.
Copyright 2000 John Wiley & Sons, Ltd.