A family of multifunctional thiamine-repressible expression vectors for fission yeast

A series of thiamine-repressible shuttle vectors has been constructed to al low a more efficient DNA manipulation in Schizosaccharomyces pombe. These h igh-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 ar e all uniform in structure, containing: (a) sequences for replication (ori) and selection (Amp(R)) in Escherichia coli; (b) the fl ori sequence of the phage fl for packaging of ssDNA, making them suitable for site-directed mu tagenesis; and (c) the ar sl sequence for replication in S, pombe. The pJR vectors differ among them in: (a) the selectable marker (Saccharomyces cere visiae LEU 2 gene, which complements S, pombe leu1(-) gene and S, pombe ura 4(+) and his3(+) genes); (b) the thiamine-repressible nmt1 promoter (3X, 41 X and 81X with extremely high, moderate or low transcription efficiency, re spectively); and (c) the multiple cloning site (two multiple cloning sites, with 12 restriction sites each), The expression level of the pJR vectors h as been analysed using the beta-galactosidase gene as reporter, Three level s of expression for each nmt1 promoter version, with any selectable marker and for either repressed or induced conditions? have been found, The expres sion is dependent on the distance to the initiation codon, varying from 0.0 01 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 AIT sequence than with a GIC sequence. Finally, the beta-galactosidase mRNA levels were found to be similar in each nmt1 series, suggesting a translat ional effect on gene expression. As a result, any of these 18 new vectors a llow performing gene expression in fission yeast, as well as a more versati le cloning, sequencing and mutagenesis, directly in the plasmid without the need for subcloning into intermediary vectors. Copyright (C) 2000 John Wil ey & Sons, Ltd.