Thiamine-repressible expression vectors pREP and pRIP for fission yeast

Maundrell, Kinsey

doi:10.1016/0378-1119(93)90551-d

Cited by 1,005 publications

(963 citation statements)

References 24 publications

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“…The plasmids were transformed into S. pombe leu1-32 strains and maintained by selection for leucine prototrophy on minimal medium in the presence of thiamine (2 μM). Induction of gene expression from the nmt (no message in thiamine) promoter was achieved by culturing cells in the absence of thiamine, as described (Maundrell, 1993).…”

Section: Construction and Regulation Of Prep41-ntap-rec12 Vectorsmentioning

confidence: 99%

A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination

DeWall¹,

Davidson²,

Sharif³

et al. 2005

Gene

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The Rec12 (Spo11) protein of the fission yeast Schizosaccharomyces pombe is a meiosis-specific ortholog of the catalytic subunit of type VI topoisomerases and is thought to catalyze doublestrand DNA breaks that initiate recombination. We tested the hypothesis that the rec12-117 allele affects the choice of pathways by which recombination is resolved. DNA sequence analysis revealed a single missense mutation in the coding region (rec12-G202E). The corresponding glycine-202 residue of Rec12 protein is strictly conserved in proteins of the Rec12/Spo11/Top6A family. It maps to the base of the DNA binding pocket in the crystal structure of the archaeal ortholog, Top6A. The rec12-G202E mutants lacked crossover and non-crossover recombination, demonstrating that rec12-G202E does not affect choice of resolution pathway. Like rec12-D15 null mutants, the rec12-G202E mutants suffered chromosome segregation errors in meiosis I. The Rec12-G202E protein was as stable as wild-type Rec12, demonstrating that glycine-202 is essential for a biochemical activity of Rec12 protein, rather than for its stability. These findings suggest that Rec12 facilitates binding of the meiotic recombinase to its substrate, DNA. Interestingly, the bulk of Rec12 protein persisted until the time of anaphase I, and a portion of Rec12 protein persisted until the time of anaphase II, after which it was undetectable. This suggests that Rec12 protein has additional meiotic functions after completion of recombination in prophase, as inferred previously from genetic studies.

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Section: Construction and Regulation Of Prep41-ntap-rec12 Vectorsmentioning

confidence: 99%

A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination

DeWall¹,

Davidson²,

Sharif³

et al. 2005

Gene

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“…The PCR product was digested with BamHI and inserted into a vector plasmid pREP1 (Maundrell 1993), named pREP(spo6). The expression was driven by the thiaminerepressible nmt1 promoter.…”

Section: Over-expression Of Spo6pmentioning

confidence: 99%

The Schizosaccharomyces pombe spo6⁺ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation

2000

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“…The ability to induce and regulate the expression of recombinant proteins at will, is a valuable tool to correlate protein levels with biological functions, to localize proteins in the cell and to visualize protein dynamics in vivo at various stages of development. This approach is exemplified by the wide use of conditional promoters such as lacZ in Escherichia coli (Donovan et al, 1996), GAL4 in budding yeast (Johnston, 1987) and nmt1 in Published in Plant Molecular Biology 59, issue 5, 697-711, 2005 which should be used for any reference to this work fission yeast (Maundrell, 1993). Efficient high protein expression is also central to the production of recombinant proteins for biotechnology and pharmaceutics.…”

Section: Introductionmentioning

confidence: 99%

Controlled Expression of Recombinant Proteins in Physcomitrella patens by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

et al. 2005

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The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using b-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25°C. In contrast, a short non-damaging heat-treatment at 38°C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25°C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.Abbreviations: ASA, acetyl salicylic acid; BA, benzyl alcohol; GFP, green fluorescent protein; GT, GFPtalin; GUS, b-glucuronidase; Fv/Fm, the maximal photochemical efficiency of PSII; MU, 4-methylumbelliferone; MUG, 4-methylumbelliferyl-b-D-glucuronide; PSII, photosystem II; SA, specific activity; sHSP, small heat-shock protein; TSP, total soluble proteins; Ubi, ubiquitin; X-Gluc, 5-bromo-4-chloro-3-indolyl-b-D-glucuronide

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Thiamine-repressible expression vectors pREP and pRIP for fission yeast

Cited by 1,005 publications

References 24 publications

A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination

A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination

The Schizosaccharomyces pombe spo6⁺ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation

Controlled Expression of Recombinant Proteins in Physcomitrella patens by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

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Thiamine-repressible expression vectors pREP and pRIP for fission yeast

Cited by 1,005 publications

References 24 publications

A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination

A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination

The Schizosaccharomyces pombe spo6+ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation

Controlled Expression of Recombinant Proteins in Physcomitrella patens by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

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The Schizosaccharomyces pombe spo6⁺ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation