Cloning the Schizosaccharomyces pombe lys2 + gene and construction of new molecular genetic tools (original) (raw)
Related papers
Yeast, 1998
We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4 + gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C-or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C-or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was d50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.
Extending the Schizosaccharomyces pombe molecular genetic toolbox
2014
Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1 + , rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70 + and pku80 + genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1 + to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42 + cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4 + selection to direct disruptive integration of leu1 + and lys1 + respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system.
Use of a ura5 +–lys7 + cassette to construct unmarked gene knock-ins in Schizosaccharomyces pombe
Current Genetics, 2011
While the counterselectable Schizosaccharomyces pombe ura4 + gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4 + confers 5FOA-resistant (5FOA R) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA R mutants. Relative to the same approach using the homologous URA3 + gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower eYciency of homologous recombination and a relatively high background of spontaneous 5FOA R colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we Wrst determined that 5FOA R strains carry mutations in either of two genes; ura4 + and ura5 +. We then cloned the S. pombe ura5 + orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5 + together with the S. pombe lys7 + gene. Using this doubly marked cassette to disrupt the sck1 + kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4 ¡ and ura5 ¡ mutants by screening 5FOA R colonies for the loss of the lys7 + marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that »95% of 5FOA R colonies in our studies arose from background ura4 ¡ and ura5 ¡ mutations. Keywords Schizosaccharomyces pombe • Fission yeast • ura5 + • lys7 + • Selectable marker • Gene knock-in Communicated by P. Sunnerhagen.
A new family of yeast vectors and S288C-derived strains for the systematic analysis of gene function
Yeast, 2001
The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2D0 ade8D0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.
Choosing and using Schizosaccharomyces pombe plasmids
Methods, 2004
A wide range of plasmids has been developed for molecular studies in the fission yeast Schizosaccharomyces pombe. This includes general purpose episomes, expression vectors, epitope tagging plasmids, and integration vectors. This review describes the typical features of S. pombe vectors, including replication origins, positive and negative selection markers, and constitutive and inducible promoter systems. We will also discuss vectors with epitope tags and how these can be used to modify episomal or endogenous gene sequences. Considerations for choosing and using a plasmid are presented and specialized methods are described.
The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2D0 ade8D0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.
Efficient PCR-Based Epitope Gene Tagging in Schizosaccharomyces pombe
2016
Gene deletion and tagging are important for understanding gene function in all organisms, particularly in single-cell model microorganisms like yeast. While the classical approaches involved homologous recombination to achieve gene deletion by inserting a selectable marker within the gene of insert and transforming the strain with a DNA fragment containing at least 1 kb of homology on each side of the selectable marker, the last decade has witnessed development of PCR-mediated approaches. For example, in the budding yeast Sachharomyces cerevisiae, short stretches of homology of ~20 bp flanking of either a prototrophic or a drug marker are sufficient to achieve homologous gene targeting with extremely high efficiency. However, longer regions of at least 1 kb are required for other yeasts like Schizosaccharomyces pombe, Aspergillus, etc.; DNA fragments generated with shorter regions of homology tend to be inserted at nonhomologous sites. Attempts to circumvent this problem have involv...
An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes
PROTEOMICS, 2009
Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag. We used this technique to design strategies for TAPtagging of predicted Schizosaccharomyces pombe genes (http://mendel.imp.ac.at/ Pombe_tagging/). To validate the approach, we purified the proteins, which associated with two evolutionarily conserved proteins Swi5 and Sfr1 as well as three protein kinases Ksg1, Orb6 and Sid1.