Comparison ofSchizosaccharomyces pombeexpression systems

Abstract

(2). The other regulated system was constructed from the tetracycline-inducible system described by Faryar and Gatz (3). The two constitutive promoters were the previously described vectors pART1 (containing the adh promoter; .(4) and pSMl (containing the SV40 promoter; 5).The expression vector REP3, containing the thiamine-inducible nmtl promoter (1), and its derivatives REP41 and REP81 (2), which have lower levels of activity due to mutation, all contain an ATG within their polylinker. This was destroyed by insertion of a Xho linker; these derivatives are called REP3X (full strength nmtl), REP41X (slightly weaker; nmtl*) and REP81X (much weaker; nmtl **). The tetracycline expression system was originally described in (3) and modified for more general use. The expression vector, pSLF101, has the constitutive CaMV promoter adjacent to the tet operator as described in (3); in addition, it contains a polylinker, and the LEU2 and arsI markers for selection in fission yeast. A ura4+ marked version, pSLF102, was also constructed but not tested. The tet represser, which blocks transcription from the otherwise constitutive CaMV promoter, was provided by integration of plasmid pSLF104 into a strain of genotype h-ade6-704 leul-32 ura4-D18. The plasmid contains the marker sup3-5, which suppresses ade6-704, and the tet represser gene under control of the S.pombe adh promoter, as described by Faryar and Gatz (3). The resulting fission yeast strain, FY191, thus produces a constitutive tetracycline repressor protein.A 3 kb BamHI cassette containing the beta-galactosidase gene (6) was cloned into the BamHI site in the polylinkers of all the vectors, and transformed into a fission yeast strain of genotype h-leul-32 ura4-D18. Media and methods were as described in (7). The tet-inducible pSLF101-lacZ was also transformed into the strain with the integrated tetracycline repressor. Betagalactosidase assays were carried out as described in (8). All assays were carried out after growth for 24 hrs at 30°C on strains grown to

Roles of a Fimbrin and an α-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis

“…Checking by PCR identified a G418-resistant transformant (strain JW46) that had sustained the desired integration event and segregated 2:2 for G418 resistance when crossed to strain 975. In addition, ain1 ϩ was placed under the control of three versions of the thiamine-regulated nmt1 promoter (the wild-type 3nmt1, the attenuated 41nmt1, and the still weaker 81nmt1) (Basi et al, 1993;Forsburg, 1993), with or without an associated NH 2 -terminal GFP tag (Bähler et al, 1998b). The ain1-specific sequences (70 bp) of the forward primer corresponded to nucleotides Ϫ163 to Ϫ94, and those (76 or 70 bp) of the reverse primers corresponded to the complement of the NH 2 -terminal codons.…”

“…To confirm the presence of these introns, the absence of other introns within the fim1 coding region, and the identification of the putative start and stop codons, cDNA sequences were amplified by PCR using the Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN), one or the other of two cDNA libraries as template, and primers corresponding to fim1 genomic sequences or vector sequences. One library (kindly provided by Dr. C. Albright, Vanderbilt University, Nashville, TN) was constructed in vector pREP3X (Basi et al, 1993;Forsburg, 1993), and the other (Becker et al, 1991) was constructed in vector pDB20. The PCR products were cloned into the pGEM-T Easy vector and sequenced.…”

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

“…In the S. cerevisiae complementation test, the Spt4 proteins are expressed from singlecopy CEN plasmids, but no such single-copy plasmid system is available for fission yeast. Instead, we used high-copy vectors that are standard in the field (Forsburg 1993). Therefore, mutations that cause functional deficits at low gene dosage in budding yeast might be missed at high gene dosage in fission yeast, e.g., because increased production of the mutant Spt4 results by mass action in sufficient Spt5-Spt4 complex to support cell growth at 37°C.…”

“…Therefore, mutations that cause functional deficits at low gene dosage in budding yeast might be missed at high gene dosage in fission yeast, e.g., because increased production of the mutant Spt4 results by mass action in sufficient Spt5-Spt4 complex to support cell growth at 37°C. We attempted to down-regulate transcription of the plasmid-borne nmt1-spt4 + cassette by inclusion of thiamine in the growth medium (Forsburg 1993) and found that the plasmid still complemented growth of the spt4D strain at 37°C (data not shown), implying that Spt4 is indeed produced in functional excess from the high copy plasmid.…”

“…For expression of Spt4 in fission yeast, DNA fragments encoding wild-type or mutant Spt4 proteins were excised from the respective pYN132-spt4 plasmids and inserted into pREP81x, wherein Spt4 is expressed under the transcriptional control of a weakened nmt1 promoter (Forsburg 1993). Leu + transformants were selected and the growth of the various pREP81x-spt4 strains was compared to spt4D cells that had been transformed with the empty pREP81x plasmid.…”