Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element.

We characterized a number of widely used yeast-Escherichia coli shuttle vectors in the fission yeast Schizosaccharomyces pombe. The 2 micron vectors pDB248 and YEp13 showed high frequency of transformation, intermediate mitotic and low meiotic stability, and a low copy number in S. pombe, analogous to their behavior in [cir0] strains of Saccharomyces cerevisiae. The S. cerevisiae integration vectors pLEU2 and pURA3 transformed S. pombe at very low frequencies but, surprisingly, in a nonintegrative fashion. Instead, they replicated autonomously, and they showed very high copy numbers (up to 150 copies per plasmid-containing cell). This could reflect a lack of sequence specificity for replication of plasmid DNA in S. pombe. pFL20, an S. pombe ars vector, and a series of plasmids derived from it were studied to analyze the unusually high stability of this plasmid. Mitotic stability and partitioning of the plasmids was measured by pedigree analysis of transformed S. pombe cells. An S. pombe DNA fragment (stb) was identified that stabilizes pFL20 by improvement of plasmid partitioning in mitosis and meiosis.     

Versatile shuttle vectors and genomic libraries for use with Schizosaccharomyces pombe.

We have constructed a variety of pUC-based vectors designed for maintenance in Schizosaccharomyces pombe. These can be used for both gene bank construction and subcloning. Plasmids pUR18 and pUR19 are modifications of pUC vectors containing the Sc. pombe ars1 and ura4 sequences and retaining the lacZ XGal blue-white selection system for screening for DNA inserts. These vectors have been used to construct representative Sc. pombe and Saccharomyces cerevisiae genomic libraries. To assist in the creation of gene deletions, we have constructed another two plasmids. Combined with the technique of partially filling-in 5' overhangs created with restriction enzymes, these plasmids simplify the replacement of all or part of an open reading frame by a functional ura4 gene. Furthermore, such constructs can be excised with SfiI as a linear fragment for use in Sc. pombe transformations. When integrated into the Sc. pombe genome, the site of integration can be easily mapped by pulsed-field gel electrophoresis using the presence of a novel NotI site.     

High-frequency cotransformation by copolymerization of plasmids in the fission yeast Schizosaccharomyces pombe.

We have developed a high-frequency cotransformation system which is useful in introducing nonreplicating circular DNA plasmids into the fission yeast Schizosaccharomyces pombe. This system depends on two factors: the ability of the ural-complementing helper plasmids pFYM2 and pFYM225 to propagate autonomously in S. pombe, and the intensive recombination activity intrinsic to this yeast. If cotransformed with a helper plasmid, plasmids such as YIp5 or YIp32, Escherichia coli-Saccharomyces cerevisiae shuttle vectors incapable of replication in S. pombe, can enter S. pombe and express the gene carried on them at a frequency comparable to that of autonomously replicating plasmids (10(3) to 10(4) transformants per microgram of DNA). Even if characters of the nonreplicating DNA are not selected directly, 50 to 70% of Ura+ cells transformed with the helper have also incorporated the nonreplicating plasmid. It is shown that these two plasmids have physically recombined at a site of common DNA sequence to form a heteropolymer in the fission yeast. Since any foreign DNA cloned in pBR322 or ColE1 derivatives can be incorporated into S. pombe by using pFYM2 or pFYM225 as a helper, this cotransformation system will serve as a convenient method to examine functional expression of such cloned DNA in S. pombe. This work also demonstrates that the kanamycin resistance gene carried by the bacterial transposon Tn903 can be expressed in S. pombe, as shown by its ability to inactivate the antibiotic G418.     

Versatile use of Schizosaccharomyces pombe plasmids in Saccharomyces cerevisiae

The two model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe appear to have diverged 1000 million years ago. Here, we describe that S.?pombe vectors can be propagated efficiently in S.?cerevisiae as pUR19 derivatives, and the pREP and pJR vector series carrying the S.?cerevisiae LEU2 or the S.?pombe ura4(+) selection marker are maintained in S.?cerevisiae cells. In addition, genes transcribed from the S.?pombe nmt1(+) promoter and derivatives are expressed in budding yeast. Thus, S.?pombe vectors can be used as shuttle vectors in S.?cerevisiae and S.?pombe. Our finding greatly facilitates the testing for functional orthologs of protein families and simplifies the cloning of new S.?pombe plasmids by using the highly efficient in vivo homologous recombination activity of S.?cerevisiae.     

General purpose tagging vectors for fission yeast

We have designed a series of vectors for use in the fission yeast Schizosaccharomyces pombe that allow fusion of any protein of interest to a triple HA epitope or a GST domain. The HA epitope may be placed at the N terminus or the C terminus under three different versions of the nmt1 promoter, to allow varying levels of gene expression. The GST tag may be placed at the N terminus or C terminus under control of a fully active nmt1 promoter. This family of vectors has compatible restriction sites and modular design, so that the protein under study may be exchanged easily between different plasmids. Using the Cdc19p protein as a test case, we have demonstrated that these plasmids can express functional tagged proteins in the fission yeast cell.     

A novel copper-regulated promoter system for expression of heterologous proteins in Schizosaccharomyces pombe

The increasing use of the fission yeast Schizosaccharomyces pombe as a model organism for elucidating the mechanisms of critical biological processes such as cell-cycle control, DNA replication, and stress-mediated signal transduction has fostered the development and utilization of expression systems for gene function analysis. Using the promoter of the ctr4(+) copper transporter gene from S. pombe, we created a series of vectors, named pctr4(+)-X, which regulate the expression of heterologous genes as a function of copper availability. In this system, the addition of copper ions at levels that are non-toxic to yeast cells represses gene expression, while copper deprivation strongly induces gene expression. Conveniently, changes of growth medium or carbon sources are not required to shut down or induce gene expression. The Cu-starvation-mediated inducible expression system is rapid, producing heterologous proteins within 3 h, with sustained expression of proteins that persists for several hours. The pctr4(+)-X expression vectors harbor unique restriction sites constructed in-frame to DNA sequences encoding for epitope tags, which facilitate the detection or purification of the heterologous proteins using commercially available antibodies and affinity columns. Furthermore, the pctr4(+)-X copper-regulatable protein expression vectors have been constructed with three different selectable markers, offering more versatility for studying gene function in fission yeast.     

Improvement of bacterial two-hybrid vectors for detection of fusion proteins and transfer to pBAD-tandem affinity purification, calmodulin binding peptide, or 6-histidine tag vectors

The original vectors of the bacterial two-hybrid technique developed by Karimova et al. in 1998 did not enable detection of the recombinant proteins. Here, we propose two methods resolving this problem, either using new plasmids containing the Flag epitope, or using a trick to detect the T18 domain of adenylate cyclase. Furthermore, we describe a set of vectors for TAP, CBP or 6-histidine tagging that possess the same cloning site as our two-hybrid vectors.     

A vector system for genomic FLAG epitope-tagging in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a popular model organism to study various cellular processes, although research tools available for S. pombe are relatively inadequate. To facilitate genetic and biochemical investigation in S. pombe, we report here a system of vectors for genomic FLAG epitope-tagging. These vectors enable us to amplify gene-targeting fragments for integration into specific loci of the S. pombe genome. All vectors in this report were designed to express FLAG epitope-tagged proteins from their endogenous genomic loci. Vectors for N-terminal FLAG epitope-tagging allow us to control protein expression levels using the wild-type nmt1 promoter, its weaker derivatives, and the urg1 promoter. These vectors are available with various antibiotic markers including kanMX6, hphMX6, natMX6 and bleMX6, and the his3(+) marker. Vectors for C-terminal FLAG epitope-tagging were designed to express FLAG-fusion proteins under the control of their native promoters at their own genomic loci, allowing us to characterize protein functions under physiological conditions. These vectors are available with kanMX6, hphMX6, nat-MX6 and bleMX6 markers. The series of vectors described in this report should prove useful for protein studies in fission yeast.     

Transformation systems of non-Saccharomyces yeasts

This review describes the transformation systems including vectors, replicons, genetic markers, transformation methods, vector stability, and copy numbers of 13 genera and 31 species of non-Saccharomyces yeasts. Schizosaccharomyces pombe was the first non-Saccharomyces yeast studied for transformation and genetics. The replicons of non-Saccharomyces yeast vectors are from native plasmids, chromosomal DNA, and mitochondrial DNA of Saccharomyces cerevisiae, non-Saccharomyces yeasts, protozoan, plant, and animal. Vectors such as YAC, YCp, YEp, YIp, and YRp were developed for non-Saccharomyces yeasts. Forty-two types of genes from bacteria, yeasts, fungi, and plant were used as genetic markers that could be classified into biosynthetic, dominant, and colored groups to construct non-Saccharomyces yeasts vectors. The LEU2 gene and G418 resistance gene are the two most popular markers used in the yeast transformation. All known transformation methods such as spheroplast-mediating method, alkaline ion treatment method, electroporation, trans-kingdom conjugation, and biolistics have been developed successfully for non-Saccharomyces yeasts, among which the first three are most widely used. The highest copy number detected from non-Saccharomyces yeasts is 60 copies in Kluyveromyces lactis. No general rule is known to illustrate the transformation efficiency, vector stability, and copy number, although factors such as vector composition, host strain, transformation method, and selective pressure might influence them.     

Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids.     

酵母作为外源基因表达系统的研究进展

甲醇营养型酵母和裂殖酵母作为外源基因表达的有效系统,正引起人们广泛研究。甲醇营养型酵母具有易诱导调控、适用于高密生长、能高效表达外源蛋白的特点。裂殖酵母有许多与高等真核细胞相似的特点,是研究真核分子生物学和真核基因表达有用的工具。本文综述了这两个酵母表达系统的特点。     

Establishment of the nuclear location of Dictyostelium discoideum plasmids

The nuclear location of the Dictyostelium discoideum plasmids was studied using a biochemical approach based on the presence of plasmid sequences in nucleosomes. This analysis revealed that all four of the known plasmids (Ddp1, Ddp2, Ddp3, Ddp5) are present in chromatin. This evidence establishes that the D. discoideum plasmids are not cytoplasmic but located in the nucleus. D. discoideum is unique among eukaryotes in possessing a group of nonhomologous endogenous plasmids in its nucleus. These plasmids are excellent starting material for construction of nuclear transformation and expression vectors. Such vectors upon transformation into D. discoideum are also present in chromatin as expected for DNA located in the nucleus.     

Yeast telomere repeat sequence (TRS) improves circular plasmid segregation, and TRS plasmid segregation involves the RAP1 gene product.

Telomere repeat sequences (TRSs) can dramatically improve the segregation of unstable circular autonomously replicating sequence (ARS) plasmids in Saccharomyces cerevisiae. Deletion analysis demonstrated that yeast TRSs, which conform to the general sequence (C(1-3)A)n, are able to stabilize circular ARS plasmids. A number of TRS clones of different primary sequence and C(1-3)A tract length confer the plasmid stabilization phenotype. TRS sequences do not appear to improve plasmid replication efficiency, as determined by plasmid copy number analysis and functional assays for ARS activity. Pedigree analysis confirms that TRS-containing plasmids are missegregated at low frequency and that missegregated TRS-containing plasmids, like ARS plasmids, are preferentially retained by the mother cell. Plasmids stabilized by TRSs have properties that distinguish them from centromere-containing plasmids and 2 microns-based recombinant plasmids. Linear ARS plasmids, which include two TRS tracts at their termini, segregate inefficiently, while circular plasmids with one or two TRS tracts segregate efficiently, suggesting that plasmid topology or TRS accessibility interferes with TRS segregation function on linear plasmids. In strains carrying the temperature-sensitive mutant alleles rap1grc4 and rap1-5, TRS plasmids are not stable at the semipermissive temperature, suggesting that RAP1 protein is involved in TRS plasmid stability. In Schizosaccharomyces pombe, an ARS plasmid was stabilized by the addition of S. pombe telomere sequence, suggesting that the ability to improve the segregation of ARS plasmids is a general property of telomere repeats.     

Specific initiation at an origin of replication from Schizosaccharomyces pombe.

Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S. pombe. A 2.8-kb subclone of the fragment has the same replication properties. Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2- or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments. This region is also a site of replication initiation in the S. pombe chromosome where the fragments normally reside. These results provide strong evidence that initiation of replication in S. pombe is localized and mediated by specific DNA sequence signals.     

Rapid tagging and integration of genes in Giardia intestinalis

We developed a series of plasmids that allow C-terminal tagging of any gene in its endogenous locus in Giardia intestinalis, with different epitope tags (triple hemagglutinin [3HA] and triple Myc [3Myc]) and selection markers (puromycin, neomycin, and a newly developed marker, blasticidin). Using these vectors, cyclin B and aurora kinase were tagged, expressed, and localized.     

System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae

Integrative, centromeric, and episomal plasmids are essential for easy, fast, and reliable genetic manipulation of yeast. We constructed a system of shuttle vectors based on the widely used plasmids of the pRS series. We used genes conferring resistance to Geneticin (kanMX4), nourseothricin (natNT2), and hygromycin B (hphNT1) as markers. The centromeric and episomal plasmids that we constructed can be used the same way as the traditional auxotrophic marker-based shuttle vectors (pRS41x and pRS42x series). Additionally, we created a set of nine yeast integrative vectors with the three dominant markers. These plasmids allow for direct integration in the LEU2, URA3, and HIS3 locus of any yeast strain and the concomitant partial deletion of the gene. This prevents multiple integrations and allows for the rapid identification of correct integrants. The set of new vectors considerably enhances the flexibility of genetic manipulations and gene expression in yeast. Most notably, the new vectors allow one to work with natural yeast isolates, which do not contain auxotrophic markers.     

Vectors--derivatives of phage lambda for construction and analysis of genome libraries

Basic features of lambda phage derived, cosmid and plasmid vectors are described. Plasmid vectors combine the most useful features of phage and plasmid vectors. Plasmids can exist in vivo as a plasmid or as a phage. Plasmid vectors are similar to large capacity phage vectors, but can be maintained in vivo without a stuffer fragment. That is why plasmids are easier in preparation for cloning than phage vectors. The yield of recombinants is higher with plasmid vectors (up to 3.10(6)) and the background of non-recombinants in the library is lower. Analysis of recombinant plasmids is more simple and effective than analysis of recombinant phages and plasmids. Probably plasmid vectors will soon be widely used instead of phage or cosmid vectors for genomic libraries construction and analysis.