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.     

Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation.

The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length. To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S. cerevisiae. We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell. An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number. Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids. Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends.     

Recombinational instability of a chimeric plasmid in Saccharomyces cerevisiae.

Wild-type strains of Saccharomyces cerevisiae exhibit mitotic recombination between the chimeric plasmid TLC-1 and the endogenous 2mu circle that involves sequence homologies between the two plasmids that are not acted on by the 2mu circle site-specific recombination system. This generalized recombination can be detected because it separates the LEU2 and CAN1 markers of TLC-1 from each other through the formation of a plasmid containing only the S. cerevisiae LEU2 region and the 2mu circle. This derivative plasmid is maintained more stably during vegetative growth than TLC-1, and strains which carry it frequently lose the endogenous 2mu circle. Therefore, TLC-1 can provide a convenient selection for [cir0] cells. Formation of this new plasmid is greatly reduced, but not eliminated, in strains containing the rad52-1 mutation. This indicates that generalized mitotic recombination between plasmid sequences utilizes functions required for chromosomal recombination in S. cerevisiae.     

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.     

A Schizosaccharomyces pombe artificial chromosome large DNA cloning system.

The feasibility of using the fission yeast, Schizosaccharomyces pombe , as a host for the propagation of cloned large fragments of human DNA has been investigated. Two acentric vector arms were utilized; these carry autonomously replicating sequences ( ars elements), selectable markers ( ura4(+) or LEU2 ) and 250 bp of S. pombe terminal telomeric repeats. All cloning was performed between the unique sites in both vector arms for the restriction endonuclease Not I. Initially the system was tested by converting six previously characterized cosmids from human chromosome 11p13 into a form that could be propagated in S.pombe as linear episomal elements of 50-60 kb in length. In all transformants analysed these cosmids were maintained intact. To test if larger fragments of human DNA could also be propagated total human DNA was digested with Not I and size fractionated by pulsed field gel electrophoresis (PFGE). Fractions of 100-1000 kb were ligated to Not I-digested vector arms and transformed into S.pombe protoplasts in the presence of lipofectin. Prototrophic ura+leu+transformants were obtained which upon examination by PFGE were found to contain additional linear chromosomes migrating at between 100 and 500 kb with a copy number of 5-10 copies/cell. Hybridization analyses revealed that these additional bands contained human DNA. Fluorescent in situ hybridization (FISH) analyses of several independent clones indicated that the inserts were derived from single loci within the human genome. These analyses clearly demonstrate that it is possible to clone large fragments of heterologous DNA in fission yeast using this S.p ombe artificial chromosome system which we have called SPARC. This vector-host system will complement the various other systems for cloning large DNA fragments.     

The 5'-flanking AT-rich sequence of thioredoxin gene involved in high-frequency transformation of the fission yeast

During the cloning of a genomic DNA encoding mitochondrial thioredoxin (TRX) from the fission yeast Schizosaccharomyces pombe, its 5' flanking sequence was involved in the high-frequency of transformation. The recombinant plasmid pYEX that was constructed in the 2 mu plasmid-derived vector pYES2 gave rise to a significant high-frequency of transformation in S. pombe, compared to the vector alone. Plasmid pYEX contains 1,090 bp 5'-flanking sequences of the TRX gene that are ahead of the open-reading frame. Similar 5'-flanking sequences, which were inserted in the lacZ fusion vector YEp357R that contained the 2 mu origin of replication, also gave a high-frequency of transformation. Dissection of the 5'-flanking sequence of the TRX gene by the HindIII restriction site showed that the 782 bp flanking sequence (5' upstream of the HindIII site) was responsible for the high-frequency of transformation by the 2 mu plasmid-derived vector DNAs. The putative sequence that is involved in the high-frequency of transformation contains a very high ratio of A-T pairs. No known functions were assigned on the sequence, which was estimated from the GenBank database.     

Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903

Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS). To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (L-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol. Several ARS-containing plasmids were selected from a K. lactis recombinant DNA library in K. lactis and in Saccharomyces cerevisiae. Two of four ARS clones selected in K. lactis promoted transformation frequencies of 5-10 X 10(2) G418-resistant cells/micrograms of plasmid DNA. This frequency of transformation was at least twice as high as with ARS clones selected in S. cerevisiae. The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug. In the absence of selection pressure less than 5% of the cells retained the drug-resistance phenotype. Plasmids containing the ARS1 or 2 mu replicon of S. cerevisiae failed to transform K. lactis for G418 resistance. Inclusion of S. cerevisiae centromere, CEN4, in a K. lactis ARS recombinant plasmid did not increase the stability of the plasmid in K. lactis, and marker genes on the vector segregated predominantly 4-:0+ through meiosis. We conclude that neither the ARS sequences or the centromere of S. cerevisiae was functioning in K. lactis.     

Circular DNA is excised by immunoglobulin class switch recombination

We have purified extrachromosomal circular DNAs from adult mouse spleen cells, and cloned into a phage vector the BamHl fragments hybridizing with C mu and S gamma 1 probes. We obtained 52 S mu+S gamma 1+ clones by screening 1.4 million phage clones derived from spleen cells stimulated with bacterial lipopolysaccharide and interleukin 4. We have identified the breakpoints of six clones that contain S gamma 1 and S mu sequences fused in the 5' to 3' orientation. All these switch recombination sites were assigned to the central repetitive sequences of the S mu and S gamma 1 regions. Since the common S mu-S gamma 1 sequences at the recombination sites are at most 2 bases long, typical homologous recombination cannot account for their joining. These findings provide direct evidence that mu-gamma 1 class switching can occur by the looping out and excision of chromosomal DNA, with formation of a circle.     

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.     

Sequence analysis of ARS elements in fission yeast.

Chromosomal DNA of Schizosaccharomyces pombe contains sequences with properties analogous to ARS elements of Saccharomyces cerevisiae. Following Sau3A fragmentation of the S. pombe genome we have recovered a number of such fragments in an M13-based shuttle vector, suitable for subsequent sequence analysis. The complete nucleotide sequence has been obtained for eight ARS+ inserts derived from the Sau3A cloning and for the ARS present in pFL20 isolated previously by Losson and Lacroute (Cell, 32, 371-377, 1983). The Sau3A clones are single fragments between 0.8 and 1.8 kb. No ARS+ clones smaller than this were recovered even though the average size Sau3A fragment in S. pombe is approximately 200-300 bp. The sequence analysis revealed that all clones are AT-rich (69-75% A + T residues), and all contain a particularly AT-rich 11 bp core element represented by the consensus sequence 5' (A/T)PuTT-TATTTA(A/T) 3'. Deletion mapping indicates that the consensus in all cases is in the vicinity of a functional ARS domain. However precise excision of the consensus by in vitro mutagenesis has little effect on ARS activity as judged by the transformation assay. We argue that the association of the consensus with the ARS domain occurs too reproducibly to be explained by chance alone. We suggest that although it may not be essential for the extrachromosomal maintenance of plasmids in S. pombe, the consensus does have a function in situ in the chromosome and thus is always present as a cryptic sequence in the isolated ARS element.     

Tetrahymena micronuclear sequences that function as telomeres in yeast.

We explored the ability of S. cerevisiae to utilize heterologous DNA sequences as telomeres by cloning germline (micronuclear) DNA from Tetrahymena thermophila on a linear yeast plasmid that selects for telomere function. The only Tetrahymena sequences that functioned in this assay were (C4A2)n repeats. Moreover, these repeats did not have to be derived from Tetrahymena telomeres, although we show that micronuclear telomeres (like macronuclear telomeres) of Tetrahymena terminate in (C4A2)n repeats. Chromosome-internal restriction fragments carrying (C4A2)n repeats also stabilized linear plasmids and were elongated by yeast telomeric repeats. In one case, the C4A2 repeat tract was approximately 1.5 kb from the end of the genomic Tetrahymena DNA fragment that was cloned, but this 1.5 kb of DNA was missing from the linear plasmid. Thus, yeast can utilize internally located tracts of telomere-like sequences, after the distal DNA is removed. The data provide an example of broken chromo-some healing, and underscore the importance of the telomeric repeat structure for recognition of functional telomeric DNA in vivo.     

Complementation of a Schizosaccharomyces pombe mutant lacking the beta subunit of the mitochondrial ATPase by the ATP2 gene of Saccharomyces cerevisiae

A chimeric plasmid carrying the structural gene (ATP2) for the mitochondrial ATPase beta subunit of Saccharomyces cerevisiae has been used to complement a mutant of Schizosaccharomyces pombe lacking the beta subunit (Boutry, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477). Transformation with ATP2 restored the growth rate of S. pombe mutant on glycerol as well as the mitochondrial ATPase and 32Pi-ATP exchange activities to approximately 20% of the parental strain. Mitochondria prepared from the transformant contained a normal amount of a hybrid F1-ATPase consisting of the S. cerevisiae beta subunit assembled with the remaining subunits of the S. pombe ATPase complex. The presence of the S. cerevisiae beta subunit in the S. pombe ATPase complex conferred a sensitivity to the energy transfer inhibitors citreoviridin and oligomycin which was like that of the intact S. cerevisiae enzyme. The S. cerevisiae beta subunit assembled into the hybrid ATPase complex was the same size as the mature subunit in S. cerevisiae. These data indicate that the mechanism of mitochondrial import and the assembly of the cytoplasmically synthesized subunits is similar or identical in these evolutionary divergent yeasts. In addition, this study provides a new approach for the construction of hybrid mitochondrial ATPase complexes which can be used to examine the function of selected subunits in energy transduction.     

Identification of healed terminal DNA fragments in linear minichromosomes of Schizosaccharomyces pombe.

The minichromosome Ch16 of the fission yeast Schizosaccharomyces pombe is derived from the centromeric region of chromosome III. We show that Ch16 and a shorter derivative, Ch12, made by gamma-ray cleavage, are linear molecules of 530 and 280 kilobases, respectively. Each minichromosome has two novel telomeres, as shown by genomic Southern hybridization with an S. pombe telomere probe. Comparison by hybridization of the minichromosomes and their chromosomal counterparts showed no signs of gross rearrangement. Cosmid clones covering the ends of the long arms of Ch16 and Ch12 were isolated, and subcloned fragments that contained the breakage sites were identified. They are apparently unique in the genome. By hybridization and Bal 31 digestion, the ends appear to consist of the broken-end sequences directly associated with short stretches (about 300 base pairs) of new DNA that hybridizes to a cloned S. pombe telomere. They do not contain the telomere-adjacent repeated sequences that are present in the normal chromosomes. The sizes of the short telomeric stretches are roughly the same as those of the normal chromosomes. Our results show that broken chromosomal ends in S. pombe can be healed by the de novo addition of the short telomeric repeats. The formation of Ch16 must have required two breakage-healing events, whereas a single cleavage-healing event in the long arm of Ch16 yielded Ch12.     

Cloning ofCandida boidinii DNA fragments promoting autonomous replication of plasmids inSaccharomyces cerevisiae

Fragments of Candida boidinii chromosomal DNA were inserted into the integrative vector YIp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids in Saccharomyces cerevisiae. Restriction maps of two plasmids, designated S6/4 and S6/5, originating from the same S. cerevisiae transformant, were constructed. Southern hybridization data confirmed that the plasmids carry sequences from the C. boidinii chromosome. Both plasmids transform S. cerevisiae strains at 4-5-fold higher frequency than cloning vectors based on the replication origin of the 2 microns plasmid. Mitotic stability of the constructed plasmids is similar to that of the 2 mu-based vector pNF2 in S. cerevisiae.     

Isolation of a yeast vector marked by the mutation of multiple resistance to antibiotics

The plasmid mutation AntR determining multiple resistance to antibiotics--tetracycline and cycloheximide in Saccharomyces cerevisiae was earlier obtained and genetically characterized. In this work we describe experiments on cytoduction and transformation, proving the localization of this mutation in the yeast 2 mu DNA. As a result of cotransformation of the sensitive cells carrying a double mutation in the gene LEU2 with the yeast vector marked by LEU2 and 2 mu DNA obtained from the yeast AntR mutant, the Leu+ AntR clones were selected. Though the primary co-transformans contain both plasmids in an unlinked state, we managed to get clones in which the markers AntR and LEU2 were linked. The putative recombinant molecules were cloned in Escherichia coli and then introduced into the yeast recipient cells, differing by the presence of the endogenous 2 mu DNA. Retransformation of cir0 cells results in the appearance of the clones in which LEU2 and AntR markers segregate together. Thus, the result of cotransformation and selection in vivo is that the mutation of multiple resistance was included into the yeast vector plasmid, presumably, in its 2 mu part.     

Cloning of the ATP sulphurylase gene of Schizosaccharomyces pombe by functional complementation

The ATP sulphurylase gene of Schizosaccharomyces pombe has been cloned by complementation of cysteine auxotrophy of a selenate-resistant mutant, which supposedly had a defect in ATP sulphurylase. A sulphate nonutilizing (cysteine auxotrophic) and selenate-resistant mutant of S. pombe was transformed with a wild-type S. pombe genomic library and sulphate-utilizing clones were isolated. The open reading frame encoding the ATP sulphurylase enzyme was found to be responsible for the restoration of sulphate assimilation. Transformants became as sensitive for selenate as the wild-type strain and produced a comparable amount of ATP sulphurylase as the prototrophic strains. The cloned ATP sulphurylase gene (sua1) proved to be an efficient selection marker in an ARS vector, when different isogenic or nonisogenic S. pombe selenate-resistant mutants were used as cloning hosts. Complementation of sua1- mutations by sua1-bearing multicopy vectors functions as a useful dual positive and negative selection marker. The cloned sua1 gene also complemented the met3 (ATP sulphurylase deficient) mutation in Saccharomyces cerevisiae.     

Identification of plant cytoskeletal, cell cycle-related and polarity-related proteins using Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe has been used to identify Arabidopsis thaliana proteins that may play a role in cell shape maintenance or cell cycle regulation. An Arabidopsis thaliana cDNA library was constructed in pREP5N vector under the control of the inducible nmt 1 promoter and transformed into S. pombe . Expression of the A. thaliana sequences was induced and clones showing severe morphological changes were identified and analysed. Comparison of the sequences of the inserts with the sequence data bases revealed that several cDNAs encode proteins known to play a role in function of the cytoskeleton, the cell cycle and establishment of cell polarity. These include α-1, α-2, α-6 and β-6 tubulins, myosin heavy chain-like protein, ubiquitin conjugating enzymes UBC9 (E2), 26S protease subunits, Ran-binding protein, myb protein, PRL1 gene product and rho protein. Approximately 30% of the clones encode novel sequences. The results suggest that S. pombe phenotypic screening can be used to identify plant proteins involved in cell shape maintenance and regulation during cell cycle and development.     

A linear shuttle vector for yeast and the hypotrichous ciliate Stylonychia

A linear plasmid was constructed in vitro using the telomeres of the rDNA of Tetrahymena pyriformis. These telomeres were added to a yeast circular vector containing an ARS sequence from Dictyostelium, the LEU2 gene of yeast and the neo gene from Escherichia coli Tn5 fused with a eukaryotic promoter. The resulting plasmid was used to transform yeast. During the replication of the linear plasmid in yeast it was spontaneously modified at the extremity by the addition of 300 bp of yeast telomeric sequence for each end. Total DNA prepared from yeast transformants was used to transform the hypotrichous ciliate Stylonychia lemnae. The same plasmid isolated from Stylonychia can again be replicated in yeast.