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.     

Stable multicopy integration of vector sequences inHansenula polymorpha

Plasmids without an origin of replication, but bearing theURA3 gene ofSaccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously inHansenula polymorpha, indicating that parts of theS. cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function inH. polymorpha. Such plasmids, replicated in low copy number in monomeric conformation, could be rescued inE. coli, and showed a low mitotic stability under selective and non-selective conditions. Selective propagation of such transformants, however, led to the integration of plasmid sequences into theH. polymorpha genome. The integration event usually occurred in high copy number (approx. 30–50) at a single non-homologous site of the genome. The plasmid sequences were found to be present in tandem array and stable under non-selective conditions. It contrast, the use of homologousURA3 gene under similar conditions led to low-copy-number transformants.     

Species specific protein--DNA interactions may determine the chromatin units of genes in S.cerevisiae and in S.pombe.

Yeast genes, such as URA3, are chromatin units characterized by positioned nucleosomes and flanking nuclease sensitive regions (NSRs). To investigate the structural determinants at the chromatin level in vivo, the URA3 gene was dissected into three parts (U5', Umid and U3'), and the chromatin structures of the individual parts were analysed after insertion into minichromosomes and after chromatin assembly in vivo in Saccharomyces cerevisiae. While nucleosome positions were altered on Umid, the 5'-end and the 3'-end of URA3 maintained their native structures (a positioned nucleosome and a NSR each) independent of the site or orientation of insertion. This suggests that the chromatin unit of the native URA3 gene is dominated by strong protein boundaries at the 5'- and 3'-ends. In an alternative approach, we investigated whether nucleosome positions or NSRs were maintained when the whole URA3 gene was placed on a shuttle vector and assembled into chromatin by Schizosaccharomyces pombe providing different proteins, but the same nucleosomal spacing. In a complementary exchange experiment, the ade6 gene of S.pombe was shuttled to S.cerevisiae. In spite of a general conservation of histone proteins and nucleosome core structures, neither nucleosome positions nor NSRs were maintained in the heterologous background. The results demonstrate that chromatin structures are species specific and that the structural boundaries of yeast genes may be dominated by strong species specific protein-DNA interactions.     

Comparison of different signal peptides for secretion of heterologous proteins in fission yeast

In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae alpha-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The alpha-factor signal did not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.     

Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombination

The genetic manipulation of the human fungal pathogen Candida albicans is difficult because of its diploid genome, the lack of a known sexual phase and its unusual codon usage. We devised a new method for sequential gene disruption in C. albicans that is based on the repeated use of the URA3 marker for selection of transformants and its subsequent deletion by FLP-mediated, site-specific recombination. A cassette was constructed that, in addition to the URA3 selection marker, contained an inducible SAP2P-FLP fusion and was flanked by direct repeats of the minimal FLP recognition site (FRT). This URA3 flipper cassette was used to generate homozygous C. albicans mutants disrupted for both alleles of either the CDR4 gene, encoding an ABC transporter, or the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily. After insertion of the URA3 flipper into the first copy of the target gene, the whole cassette could be efficiently excised by induced FLP-mediated recombination, leaving one FRT site in the disrupted allele of the target gene. The URA3 flipper was then used for another round of mutagenesis to disrupt the second allele. Deletion of the cassette from primary and secondary transformants occurred exclusively by intrachromosomal recombination of the FRT sites flanking the URA3 flipper, whereas interchromosomal recombination between FRT sites on the homologous chromosomes was never observed. This new gene disruption strategy facilitates the generation of specific, homozygous C. albicans mutants as it eliminates the need for a negative selection scheme for marker deletion and minimizes the risk of mitotic recombination in sequential disruption experiments.     

Genetic selection for reciprocal translocation at chosen chromosomal sites in Saccharomyces cerevisiae.

We have constructed viable Saccharomyces cerevisiae strains containing a reciprocal translocation between the URA2 site of chromosome X and the HIS3 site of chromosome XV. Our methodology is an extension of the method originally developed to introduce an altered cloned sequence at the chromosomal location from which the parent sequence was derived (S. Scherer and R.W. Davis, Proc. Natl. Acad. Sci. U.S.A. 76:4951-4955, 1979). It comprises three essential steps. First, a nonreverting ura2- strain was constructed by deleting a 3.7-kilobase fragment from the coding sequence of the wild-type URA2 gene. Second, part of the coding sequence of the wild-type URA2 gene (without promotor) was inserted at the HIS3 locus of the ura2- strain. Third, after several generations of growth on uracil-supplemented medium, ura2+ colonies were selected which resulted from mitotic recombination between the nonoverlapping deletions of URA2 located on chromosomes X and XV.     

Improved shuttle vectors for cloning and high-level Cu(2+)-mediated expression of foreign genes in yeast

New yeast episomal vectors having a high degree of utility for cloning and expression in Saccharomyces cerevisiae are described. One vector, pYEULlacZ, is based on pUC19 and employs the pUC19 multiple cloning site for the selection of recombinants in Escherichia coli by lacZ inactivation. In addition, the vector contains two genes, URA3 and leu2-d, for selection of the plasmid in ura3 or leu2 yeast strains. The presence of the leu2-d gene appears to promote replication at high copy numbers. The introduction of CUP1 cassettes allows these plasmids to direct Cu(2+)-regulated production of foreign proteins in yeast. We show the production of a helminth antigen as an example of the vector application.     

Impact of plasmid architecture on stability and yEGFP3 reporter gene expression in a set of isomeric multicopy vectors in yeast

Multicopy episomal plasmids in yeast, used whenever elevated levels of foreign or homologous gene expression are necessary, are known to be less stable compared to the endogenous 2-μm plasmid they are based on, at least without selective pressure. Considering that rich medium favors growth rate and, simultaneously, is less expensive than selective medium, enhancing stability in non-selective medium is extremely desirable. In this study, we changed the architecture of a multicopy model expression plasmid, creating six isoforms (same size, same DNA content but different positions and orientations of the expression block) and studied mitotic stability, copy number, as well as reporter yEGFP3 expression between isoforms. With one isoform being significantly more stable than the others and another one exhibiting elevated plasmid copy numbers in rich medium, we show that consideration of the arrangement of the plasmid elements might be crucial for productivity employing Saccharomyces cerevisiae as a host. We strongly believe that the ideal architecture has to be assessed for each case and assembly strategy has to begin by evaluating the stability of the vector backbone before insertion of the desired gene. For the plasmid set studied, yEGFP3 reporter production depends more on mitotic stability than on elevated plasmid copy numbers in a small number of cells retaining the plasmid under non-selective conditions.

     

Autocloning and amplification of LIP2 in Yarrowia lipolytica

We synthesized a Yarrowia lipolytica strain overproducing lipase for industrial applications by using long terminal repeat (zeta) of the Y. lipolytica retrotransposon Ylt1 and an allele of URA3 with a promoter deletion to construct JMP3. JMP3 is a derivative of plasmid pHSS6 carrying a NotI-NotI cassette which contains a defective URA3 allele, a polylinker sequence, and the zeta region for targeting to multiple sites in the genome of the recipient. We inserted the LIP2 gene (encoding extracellular lipase) under the control of the strong POX2 promoter into JMP3 to generate JMP6. The pHSS6 region was removed by NotI digestion prior to transformation. Two Y. lipolytica strains transformed with the JMP6 LIP2 cassette had a mean of 10 integrated copies devoid of the Escherichia coli region, corresponding to an autocloning event. The copy number in the transformants was stable even after 120 generations in nonselective and lipase-inducing conditions. The resulting strains could produce 0.5 g of active lipase per liter in the supernatant, 40 times more than the single-copy strain with the LIP2 promoter. This work provides a new expression system in Y. lipolytica that results in strains devoid of bacterial DNA and in strains producing a high level of lipase for industrial uses, waste treatment, and pancreatic insufficiency therapy.     

The fission yeast ubiquitin-conjugating enzymes UbcP3, Ubc15, and Rhp6 affect transcriptional silencing of the mating-type region

Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.     

Initiation of meiotic recombination by double-strand DNA breaks in S. pombe

Mitotic gene conversion and reciprocal recombination have recently been shown to be efficiently initiated by double-strand DNA breaks (DSBs) in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. We tested whether DSBs could also initiate meiotic recombination at the mat1 locus in S. pombe. The mat1 switching-mechanism-generated DSB found in mitotically growing cells can be repaired without mat1 switching, since strains deleted for both donor loci (mat2-P and mat3-M) have the break but do not produce inviable cells. A (mat1-P X mat1-M) cross produced a high frequency (20%) of 3:1 gene conversions of mat1 in meiotic tetrads. Gene conversion events were associated with the recombination of flanking markers. Strains lacking the DSB failed to convert. Thus, the DSB at mat1 promotes efficient meiotic recombination in fission yeast.     

Construction of an effective host-vector system for the yeast Saccharomyces exiguus Yp74L-3.

An effective host-vector system specific to the yeast Saccharomyces exiguus Yp74L-3 was constructed to promote the molecular genetic analyses for the yeast. To obtain a stable reversionless host strain, we constructed an S. exiguus strain carrying leu2::ScURA3 by disrupting the S. exiguus LEU2 gene with the S. cerevisiae URA3 gene. A vector plasmid unique to S. exiguus was subsequently developed by inserting both the LEU2 gene and an ARS cloned from S. exiguus into an Escherichia coli phagemid, pUC119. The vector constructed, pTH119 was able to transform the S. exiguus leu2::ScURA3 strain to Leu+ efficiently. The stability of the vector in the S. exiguus host cells resembled that of a YRp-type vector in S. cerevisiae.