Construction and characterization of a series of vectors for Schizosaccharomyces pombe

A set of vectors was created to allow cloning and expression studies in Schizosaccharomyces pombe. These vectors had a uniform backbone with an efficient Sz. pombe ARS, ARS3002, but different selectable markers--his3+, leu1+, ade6+ and ura4+. The vectors functioned efficiently as autonomously replicating plasmids that could also be converted into integrating vectors. The ura4+-containing vector was used to construct a Sz. pombe genomic library.     

Construction of fission yeast vectors with a novel selection strategy that allows their use in wild-type fission yeasts

Novel vectors that use the Pichia pastoris INO1 gene as a selectable marker and exploit the natural inositol auxotrophy of the fission yeast are described. These plasmids also contained other features desirable in a plasmid cloning vector. These plasmids were evaluated in other species of Schizosaccharomyces and found to replicate autonomously in another variety of S. pombe, S. pombe var. malidevorans. These plasmids can be used for transformation of any wild-type S. pombe strain without the need for selection by induced auxotrophic mutations, or by selection by drug resistance markers, and should greatly assist genetic and molecular manipulations in these yeasts.     

Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd.     

Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance

The one-step PCR-mediated technique used for modification of chromosomal loci is a powerful tool for functional analysis in yeast. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe are amenable to this technique. However, the scarce availability of selectable markers for Sz. pombe hampers the easy use of this technique in this species. Here, we describe the construction of new vectors deriving from the pFA6a family, which are suitable for tagging in both yeasts owing to the presence of a nourseothricin-resistance cassette. These plasmids allow various gene manipulations at chromosomal loci, viz. N- and C-terminal tagging with 3HA (haemagglutinin) or 13Myc epitopes, GST (glutathione S-transferase), 4TAP (tandem affinity purification) and several GFP (green fluorescent protein) isoforms. For N-terminal modifications, the use of different promoters allows constitutive (PADH1) or regulatable (PGAL1) promoters for S. cerevisiae and derivatives of Pnmt1 for Sz. pombe expression.     

Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins

One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host-specific proteases. As an initial solution to this problem, we constructed a protease-deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine- and/or cysteine-type proteases in the culture medium. These findings also demonstrated that construction of a protease-deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required.     

Cloning and disruption of the Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers

Screening of a partial genomic database of Pichia pastoris allowed us to identify the ARG1, ARG2, ARG3, HIS1, HIS2, HIS5 and HIS6 genes, based on homology to their Saccharomyces cerevisiae counterparts. Based on the cloned sequences, a set of disruption vectors was constructed, using the previously described PpURA5-blaster as a selectable marker, and the cloned genes were individually disrupted. All disruptants exhibited the expected auxotrophic phenotypes, with only the his2 knockouts displaying a bradytroph phenotype. To allow their use as auxotrophic markers, we amplified the open reading frames and respective promoters and terminator regions of PpARG1, PpARG2, PpARG3, PpHIS1, PpHIS2 and PpHIS5. We then designed a set of integration vectors harbouring cassettes of the ARG pathway as selectable markers, to disrupt the genes of the HIS pathway and vice versa. Employing this strategy, we devised a scheme allowing for the rapid and stable introduction of several heterologous genes into the genome of P. pastoris without the need for recyclable markers or strains with multiple auxotrophies. Furthermore, simple replica-plating, instead of cost-consuming and labour-intensive colony PCR or Southern analysis, can be used to identify positive transformants, making this approach amendable for initial high-throughput applications, which can then be followed up by a more careful analysis of the selected transformants.     

New generation of loxP‐mutated deletion cassettes for the genetic manipulation of yeast natural isolates

In this work, we developed molecular tools used in standard laboratory yeast strains, such as the cre–loxP system, so that they can be used with natural and industrial prototrophic yeast species. We constructed a new generation of dominant cassettes, with mutated loxP sites (loxLE and lox2272) and selectable drug markers, to create heterothallic strains and auxotrophic mutants without incurring in the risk of generating chromosomal rearrangements. We have shown that our newly developed loxLE–hphNT1–loxRE and lox2272–natNT2–lox2272 gene‐disruption cassettes can be present in the yeast genome together with the widely used loxP–marker gene–loxP cassettes without any recombination between the lox sequences. Moreover, we also developed a new phleomycin‐resistant Cre‐expressing vector (to excise multiple markers simultaneously) and two new standard loxP deletion cassettes containing hygromicin B and cloNAT as selecatable markers. To validate these cassettes, we created heterothallic auxotrophic S. cerevisiae strains, without the risk of incurring gross chromosomal rearrangements, and we showed an example of a fitness study of intraspecific hybrids deriving from parents with different adaptations to carbon‐limited resources. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of a Schizosaccharomyces pombe mutant deficient in UDP-galactose transport activity.

In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist of galactomannan, unlike in Saccharomyces cerevisiae. We previously found that the disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, led to the complete defect of cell surface galactosylation in Sz. pombe. The Deltagms1 strain is therefore useful for the analysis of physiological properties of galactose residues in Sz. pombe. The deletion strain of gms1+ was viable; however, itshowed an aberrant cell morphology and increased sensitivities to digestion with beta-glucanase and to various drugs, such as hygromycin B, sodium orthovanadate and Calcofluor white. A reduction of galactomannan layers of the cell wall in the Deltagms1 strain was observed by scanning and transmission electron microscopic analyses. The addition of osmotic stabilizer suppressed the morphologic defect of the Deltagms1 cells, while other phenotypes were weakly suppressed. The Deltagms1 (h90) strain was incapable of sexual conjugation during nutritional starvation. These results suggest that the cell surface galactosylation is required not only for non-sexual flocculation but also for sexual conjugation in Sz. pombe.     

米曲霉3.042尿苷/尿嘧啶营养缺陷型遗传转化体系的构建

米曲霉对潮霉素等多种抗生素不敏感,对其基因改造有一定困难.该研究以米曲霉3.042为出发菌株,建立pyrG筛选标记遗传转化体系.首先,分别运用紫外诱变法和左右臂同源重组法破坏米曲霉自身的pyrG基因,在含有5-氟乳清酸和尿苷/尿嘧啶的培养基平板进行筛选,最终两种方法分别获得性状稳定的尿苷/尿嘧啶营养缺陷型突变株米曲霉O...     

Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes. © 1998 John Wiley & Sons, Ltd.     

New selectable host–marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha

Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1), MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using an HpURA3 pop‐out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5‐fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter‐selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co‐transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively. The nucleotide sequences presented here were submitted to GenBank under Accession Nos AY795576 (HpTRP1), FJ226453 (HpMET2) and FJ493241 (HpADE2), respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Isolation and characterization of the plasma membrane biotin transporter from Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is auxotrophic for biotin (vitamin H) and growth depends on biotin uptake over the plasma membrane. Here a biotin transport mutant of Saccharomyces cerevisiae is used to identify the vht1(+) gene encoding the Schizosaccharomyces pombe plasma membrane transport protein for biotin. SpVht1p belongs to the family of allantoate transporters and has only little sequence homology to the S. cerevisiae biotin transporter. Although having dissimilar primary structures, the biotin transporters in Sz. pombe and S. cerevisiae share similar biochemical properties and regulation. Like in S. cerevisiae, biotin uptake in Sz. pombe is a high-affinity process, is optimal at acidic pH values and inhibited by protonophores, indicating that SpVht1p acts as a proton-biotin symporter. Desthiobiotin, the metabolic precursor of biotin, is also imported by SpVht1p. Deletion of vht1(+) abolishes growth on low external concentrations of the vitamin, showing that vht1(+) encodes the only protein that mediates biotin uptake in Sz. pombe. Expression of vht1(+) is maximal at low external biotin concentrations, indicating that Sz. pombe can adjust the rate of biotin uptake to meet the requirement for the vitamin.     

DNA transformations of Candida tropicalis with replicating and integrative vectors.

The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.     

A new family of yeast vectors and S288C-derived strains for the systematic analysis of gene function

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 ade2 Delta 0 ade8 Delta 0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.     

Effects of the S288c genetic background and common auxotrophic markers on mitochondrial DNA function in Saccharomyces cerevisiae

Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Throughout its development as a research tool, several strain backgrounds have been utilized and different combinations of auxotrophic marker genes have been introduced into them, creating a useful but non‐homogeneous set of strains. The ade2 allele was used as an auxotrophic marker, and for ‘red–white’ screening for respiratory competence. his3 alleles that influence the expression of MRM1 have been used as selectable markers, and the MIP1[S] allele, found in the commonly used S228c strain, is associated with mitochondrial DNA defects. The focus of the current work was to examine the effects of these alleles, singly and in combination, on the maintenance of mitochondrial function. The combination of the ade2 and MIP1[S] alleles is associated with a slight increase in point mutations in mitochondrial DNA. The deletion in the his3Δ200 allele, which removes the promoter for MRM1, is associated with loss of respiratory competence at 37 °C in the presence of either MIP1 allele. Thus, multiple factors can contribute to the maintenance of mitochondrial function, reinforcing the concept that strain background is an important consideration in both designing experiments and comparing results obtained by different research groups. Copyright © 2009 John Wiley & Sons, Ltd.     

Overproduction of pentose phosphate pathway enzymes using a new CRE-loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxP-zeocin-loxP cassette using pCRE3. The 2mu-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counter-selection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers.     

Construction of diploid zygotes by interallelic complementation of ade6 in Schizosaccharomyces japonicus

The construction of diploid cells eases genetic analysis in haploid genetic systems because diploid cells allow for the characterization of essential genes. Here, we report the construction of diploid cells using ade6 point mutants that suppress each other via interallelic complementation in the fission yeast Schizosaccharomyces japonicus var japonicus (Sz. japonicus). We constructed an ade6-domK mutant in addition to the previously described ade6-domE. Phenotypes of both mutants exhibited adenine auxotrophy and red colonies. The mutations complemented the phenotypes in a mutually dependent manner. Diploid zygotes, in which the two mutations were introduced simultaneously into the same cells, were isolated by selecting for adenine independence. Such diploid cells are apparently larger in size than haploid cells, yet have a similar nuclear/cytoplasmic ratio, and thus the nuclear size control that has been reported in Sz. pombe is also present in Sz. japonicus.