Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.

Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.     

Expression of GFP using Pichia pastoris vectors with zeocin or G‐418 sulphate as the primary selectable marker

Pichia pastoris is a popular host organism for expressing heterologous proteins, and various expression vectors for this yeast are currently available. Recently, vectors containing novel dominant antibiotic resistance markers have become a strong and developing field of research for this methylotropic yeast strain. We have developed new P. pastoris expression vectors, the pPICKanMX6 and pPICKanMX6α series. These vectors were constructed by replacing the zeocin resistance gene of the pPICZA, B, C and pPICZαA, B and C vectors with the Tn903 kan^R marker from pFA6a KanMX6, which confers G‐418 sulphate resistance in P. pastoris. The limits of antibiotic resistance in two transformant yeast strains were investigated, and the selection marker was shown to be stably retained. To demonstrate their usefulness, a gene encoding hexa‐histidine‐tagged green fluorescent protein (GFPH6) was cloned into one of the new vectors and GFP expression examined in P. pastoris cells. The protein expression levels using the pPICKanMX6B vector were comparable with that using the original plasmid, based on zeocin resistance as seen by yeast cell fluorescence. Moreover, GFPH6 was able to be isolated by immobilized metal ion affinity chromatography (IMAC) from lysates of both yeast strains. A model reporter construct has been used to demonstrate successful recombinant protein expression and its subsequent purification using these new vectors. Corresponding vectors can now also be engineered with foreign gene expression under the control of various different promoters, to increase the flexibility of P. pastoris as a cellular factory for heterologous protein production. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct selection of Pichia pastoris expression strains using new G418 resistance vectors

The methylotrophic yeast, Pichia pastoris, is widely used as a host organism for the expression of heterologous proteins. Currently, the Zeocin and blasticidin resistance genes are the only dominant selectable markers that can be used for primary selection of transformants. In this report we describe new expression vectors that can be used to select directly for P. pastoris transformants using G418 resistance conferred by a modified Tn903kan(r) gene. Compared to other dominant markers, this system is more economical and offers a higher transformation efficiency, due to the small sizes of the cloning vectors, pKAN B and pKANalpha B (GenBank Accession Nos EU285585 and EU285586, respectively). Additionally, multicopy transformants can be generated using these new vectors.     

Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae

Heterologous gene replacement cassettes are powerful tools for dissecting gene function in Saccharomyces cerevisiae. Their primary advantages over homologous gene replacement cassettes include reduced gene conversion (leading to efficient site-specific integration of the cassette) and greater independence of strain background. Perhaps the most widely used cassettes are the MX cassettes containing the dominant selectable kanamycin resistance gene (kanr), which confers resistance to G418 (Wach et al., 1994). One limitation of the kanMX cassettes is that they are not counterselectable and therefore not readily recyclable, which is important when constructing strains with more than one gene deletion. To address this limitation, and to expand the choices of heterologous markers, we have created two new MX cassettes by replacing the kanr ORF from plasmids pFA6-kanMX3 and pFA6-kanMX4 with the Candida albicans URA3 ORF. These plasmids, pAG60 (CaURA3MX4) and pAG61 (CaURA3MX3) are identical to the kanMX cassettes in all other respects but have the added advantage of being counterselectable and therefore readily recyclable in S. cerevisiae.     

Development of a genetic transformation system using new selectable markers for fission yeast Schizosaccharomyces pombe

We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12(+), tyr1(+) and ade7(+)) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed.     

紫杉烯合酶基因遗传转化金针菇的研究

利用紫杉烯合酶基因（ts）和潮霉素抗性基因（hph）,采用PEG转化法共转化金针菇的原生质体。研究结果表明,18个拟转化子中有8个转化子同时整合了ts基因和hph基因,两个基因的共转化率为44.44%;RT-PCR鉴定结果表明,7个金针菇转化子实现了外源ts基因的转录。金针菇转化子在不含潮霉素（HmB）的PDA平板上经5次继代培养后仍检测到有HmB抗性,说明外源基因在金针菇转化子的无性繁殖（即有丝分裂）过程中是稳定的。本研究为通过基因工程手段定向、快速改良金针菇品种以及利用金针菇作为生物反应器生产一些具有重大经济价值的外源基因产物奠定了基础。      

A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift

Completion of the fission yeast genome sequence has opened up possibilities for post-genomic approaches. We have constructed a DNA microarray for genome-wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR-amplified from a genomic DNA template, that represent > 99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3'-end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25 degrees C to 36 degrees C, conditions widely used when studying temperature-sensitive mutants. Obligingly, the vast majority of genes do not change more than two-fold, supporting the widely held view that temperature-shift experiments specifically reveal phenotypes associated with temperature-sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat-shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On-line Supplementary Information and are also available commercially.     

Mutational analysis of the gene for Schizosaccharomyces pombe RNase MRP RNA,mrp1, using plasmid shuffle by counterselection on canavanine

Reverse genetics in fission yeast is hindered by the lack of a versatile established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene for the RNA component of RNase MRP, mrp1, we have developed a system for plasmid shuffling in fission yeast using counterselection on canavanine. The system takes advantage of the ability of the Saccharomyces cerevisiae CAN1 gene to complement a Schizosaccharomyces pombe can1-1 mutation. Two general use plasmids were constructed that allow directional cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries auxotrophic markers for ade6, leu1, ura4 and his3 along with the can1-1 mutation. Using this system we examined several partial deletions and point mutations in conserved nucleotides of Schizosaccharomyces pombe RNase MRP RNA for their ability to complement a chromosomal deletion of the mrp1 gene. The degree of background canavanine resistance as well as plasmid–plasmid recombination encountered in these experiments was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmids will be widely useful.     

Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe

The ease of construction of multiple mutant strains in Schizosaccharomyces pombe is limited by the number of available genetic markers. We describe here three new cassettes for PCR-mediated gene disruption that can be used in combination with commonly used fission yeast markers to make multiple gene deletions. The natMX6, hphMX6 and bleMX6 markers give rise to resistance towards the antibiotics nourseothricin (NAT), hygromycin B and phleomycin, respectively. The cassettes are composed of exogenous sequences to increase the frequency of integration at targeted loci, and have a structure similar to the commonly used pFA6a-kanMX6 modular plasmid system. This allows a simple exchange of the kanMX6 marker in existing strains with any of the three new cassettes. Alternatively, oligonucleotide primers designed for the modular kanMX6 cassettes can be used to make the transforming PCR fragments for gene disruption. We illustrate the construction of a mutant strain with six independent gene disruptions, using the novel antibiotic cassettes in combination with existing genetic markers.     

Using promoter replacement and selection for loss of heterozygosity to generate an industrially applicable sake yeast strain that homozygously overproduces isoamyl acetate

By application of the high-efficiency loss of heterozygosity (HELOH) method for disrupting genes in diploid sake yeast (Kotaka et al., Appl. Microbiol. Biotechnol., 82, 387–395 (2009)), we constructed, from a heterozygous integrant, a homozygous diploid that overexpresses the alcohol acetyltransferase gene ATF2 from the SED1 promoter, without the need for sporulation and mating. Under the conditions of sake brewing, the homozygous integrant produced 1.4 times more isoamyl acetate than the parental, heterozygous strain. Furthermore, the homozygous integrant was more genetically stable than the heterozygous recombinant. Thus, the HELOH method can produce homozygous, recombinant sake yeast that is ready to be grown on an industrial scale using the well-established procedures of sake brewing. The HELOH method, therefore, facilitates genetic modification of this rarely sporulating diploid yeast strain while maintaining those characteristics required for industrial applications.     

In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus

An in vivo-directed evolutionary strategy was used to obtain a thermostabilized Escherichia coli hygromycin B phosphotransferase, using a host-vector system of Thermus thermophilus. Introduction of the mutant gene containing two amino acid substitutions, S52T and W238C, which was previously reported by Cannio et al. [J. Bacteriol., 180, 3237-3240 (1998)], did not confer hygromycin resistance on T. thermophilus cells at 55 degrees C; however, five spontaneously-generated independent mutants were obtained by selection of the transformants at this temperature. Each mutant gene contained one amino acid substitution of either A118V or T246A. Further selection with increasing temperature, at 58 degrees C and then 61 degrees C, led to acquisition of three more substitutions: D20G, S225P and Q226L. These mutations cumulatively influenced the maximum growth temperature of the T. thermophilus transformants in the presence of hygromycin; T. thermophilus carrying a mutant gene containing all the five substitutions was able to grow at up to 67 degrees C. This mutant gene, hph5, proved useful as a selection marker in the T. thermophilus host-vector system, either on the plasmid or by genome integration, at temperatures up to 65 degrees C.     

Construction of novel vectors for transformation of Lentinula edodes using a chitin synthase gene promoter

Novel vectors (pLCHS-hph and pChG-bar) containing expression unit driving hph as a selectable marker gene by chitin synthase gene promoter were constructed for Lentinula edodes transformation. Expression of the hph gene in random selected transformants was confirmed by RT-PCR method. Thus, both vectors are useful for L. edodes transformation.     

The complete sequence of a 6146 bp fragment of Saccharomyces cerevisiae chromosome III contains two new open reading frames.

As part of the EEC project to sequence the entire chromosome III of Saccharomyces cerevisiae we have sequenced a total of 11,040 bp from near the right end of the chromosome. A new protein kinase gene was found at one extremity of the sequenced region (Wilson et al., 1992), while the previously sequenced actin binding protein gene, ABP1, (Drubin et al., 1990) was found at the other extremity. We present here the sequence of the region between these two genes which has the potential to code for two new open reading frames (ORFs).     

An improved tetO promoter replacement system for regulating the expression of yeast genes

Regulatable promoters are commonly used to control the expression of, especially, essential genes in a conditional manner. Integration of such promoters upstream of an ORF using one-step PCR-mediated homologous recombination should be particularly efficient. However, integration of the original KanMX4-tetO promoter cassette (Belli et al., 1998a) into the relatively short upstream regions of many yeast genes is often problematic, presumably due to the size (3.9 kb) of the replacement cassette. We have created a new, shorter, KanMX4-tetO cassette by removing the transactivator (tTA) sequence from the original cassette. The transactivator (tTA) has been integrated into the yeast genome to create a new strain for use with the new system, which has a greatly increased efficiency of promoter substitution. With it, we have been able to create strains that could not be made with the original cassette. To increase the throughput of promoter substitutions, we have developed a new assay for testing doxycycline sensitivity, based on liquid culture using microtitre trays. Altogether, the components of this new 'tool kit' greatly increase the efficiency of systematic promoter substitutions.     

The end1 gene of Schizosaccharomyces pombe coding for a DNase is identical with the pnu1 gene coding for an RNase

The DNA nuclease activity encoded by the end1 gene, and its inactivation by mutation, was described in connection with the characterization of DNA topoisomerases in the fission yeast Schizosaccharomyces pombe (Uemura and Yanagida, 1984). Subsequently, end1 mutant strains were used for the preparation of cell extracts for the study of enzymes and intermediates involved in DNA metabolism. The molecular identification of the end1 gene and its identity with the pnu1 gene is presented. The end1-458 mutation alters glycine to glutamate in the conserved motif TGPYLP. The pnu1 gene codes for an RNase that is induced by nitrogen starvation (Nakashima et al., 2002b). Thus, the End1/Pnu1 protein, like related mitochondrial proteins in other organisms, is an example of a sugar-non-specific nuclease. The analysis of strains carrying a pnu1 deletion revealed no defects in meiotic recombination and spore viability.     

Characterization of a disulphide-bound Pir-cell wall protein (Pir-CWP) of Yarrowia lipolytica

In this work we have studied the disulphide-bound group of cell wall mannoproteins of Yarrowia lipolytica and Candida albicans. In the case of Y. lipolytica, SDS-PAGE analysis of the beta-mercaptoethanol-extracted material from the purified cell walls of the yeast form, showed the presence of a main polypeptide of 45 kDa and some minor bands in the 100-200 kDa range. This pattern of bands is similar to that obtained in identical extracts in Saccharomyces cerevisiae (Moukadiri et al., 1999), and besides, all these bands cross-react with an antibody raised against beta-mercaptoethanol-extracted material from the purified cell walls of S. cerevisiae, suggesting that the 45 kDa band could be the homologue of Pir4 of S. cerevisiae in Y. lipolytica. To confirm this possibility, the amino-terminal sequences of two internal regions of the 45 kDa protein were determined, and degenerate oligonucleotides were used to clone the gene. The gene isolated in this way codes a 286 amino acid polypeptide that shows homology with the Pir family of proteins of S. cerevisiae (Russo et al., 1992; Toh-e et al., 1993), accordingly we have named this gene YlPIR1. Disruption of YlPIR1 led to a slight increase in the resistance of the cells to calcofluor white, Congo red and zymolyase, but did not cause changes in cell morphology, growth rate or morphological transition.