Genetic properties of chromosomally integrated 2 mu plasmid DNA in yeast

We obtained strains of yeast with large segments of 2 mu plasmid DNA integrated at several chromosomal locations by selecting genetically for recombination between a chromosomal sequence carried on a 2 mu-circle-containing hybrid plasmid and a homologous sequence on the chromosome. In all diploids examined, the presence of 2 mu circle sequences causes a marked instability of the chromosome into which the 2 mu DNA is inserted. Although in some cases the loss of genetic markers is due to physical loss of the entire chromosome, in most cases the loss of markers appears to be due to a mitotic homozygotization of markers: the allelic information from the homologous chromosome replaces the information distal to the integrated 2 mu DNA. The instability caused by integrated 2 mu DNA sequences requires the activity of the specialized site-specific recombination system encoded by the 2 mu plasmid. We propose that the presence of integrated 2 mu DNA allows efficient integration of additional copies of the intact 2 mu plasmid by the action of the plasmid-coded special recombination system. Unequal sister-strand exchanges within the inverted repetition would result in the formation of dicentric chromosomes whose breakage during mitosis might begin a cycle analogous to the breakage-fusion-bridge cycle described many years ago in maize.     

Two autonomously replicating sequences near oli-1 gene of yeast mitochondrial DNA

We cloned two autonomously replicating sequences from a short segment of mtDNA of an oligomycin-resistant petite yeast, O-111, into a vector pYleu 12 constructed from yeast LEU 2 gene and pBR 322. These plasmids, pYmit 4 and pYmit 1, had frequencies of transformation of yeast as high as that of YEp 13, having a replicator of 2 mu DNA. They were maintained as plasmids in yeast under selective conditions and shuttled from yeast to E. coli. No evidence was obtained that these plasmids were incompatible with the wild-type mitochondrial genome. These sequences were located in intergenic regions.     

Copy number control by a yeast centromere

Plasmids containing a cloned yeast (Saccharomyces cerevisiae) centromere (CEN3) in combination with a suitable DNA replication system are maintained in yeast at the low copy number typical of a chromosome. In composite plasmids containing CEN3 plus the yeast 2 mu plasmid, the CEN3 copy number control is dominant over the amplification system that normally drives the 2 mu plasmids to high copy number. The CEN3-2 mu composite plasmids are relatively stably maintained in yeast at a copy number of about one per haploid genome, and segregate through meiosis in a typical Mendelian pattern. Some of the CEN3-2 mu composite plasmids isolated from yeast contain deletions of variable size that remove the functional centromere, resulting in loss of the CEN3 control and reversion to high copy number. Formation of the CEN3 deletions requires the specialized recombination system (inverted repeat sequences and FLP gene) of the yeast 2 mu plasmid.     

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.     

Overlap hybridization screening: isolation and characterization of overlapping DNA fragments surrounding the leu2 gene on yeast chromosome III.

A set of four plasmids containing overlapping segments comprising a total of about 30 kbp of cloned DNA from chromosome III of yeast (Saccharomyces cerevisiae) has been isolated and characterized by restriction endonuclease analyses and DNA:DNA hybridizations. Colony hybridization was carried out with labeled pYe(leu2)10, a plasmid carrying the yeast leu2 gene, to a bank of bacterial colonies containing recombinant plasmids constructed from the vector ColE1 and random fragments of yeast DNA. This resulted in the detection of two plasmids, pYe11G4 and pYe40C3, with DNA inserts which partially overlap the original cloned segment and contain additional DNA extending in opposite directions on the chromosome. By carrying out a second round of colony hybridization with pYe40C3, the cloned region was further extended in one direction. A region of DNA that is repeated at least ten times in the yeast genome was identified by hybridization of pYe11G4 to an EcoRI digest of total yeast DNA. The procedure described in this paper should allow the isolation of large sections of chromosomes, including non-transcribed regions, surrounding cloned genes.     

``break Copy'''' Duplication: A Model for Chromosome Fragment Formation in Saccharomyces Cerevisiae

Introduction of a chromosome fragmentation vector (CFV) into the budding yeast Saccharomyces cerevisiae results in a targeted homologous recombination event that yields an independently segregating chromosome fragment (CF) and an alteration in the strain's karyotype. Fragmentation with an acentric CFV directed in a centromere-proximal orientation generates a CF that contains all sequences proximal to the targeting segment and results in loss of the endogenous targeted chromosome to yield a 2N-1+CF karyotype. In contrast, fragmentation with a centric CFV directed in a centromere-distal orientation generates a CF that contains all sequences distal to the targeting segment and retention of the endogenous targeted chromosome to yield a 2N+CF karyotype. We have termed this phenomenon ``break copy' duplication. Using yeast strains in which the centromere had been transposed to a new location, it was demonstrated that the centromere inhibited break copy duplication. These data suggest that CF formation is the product of an unscheduled DNA replication event initiated by the free end of the CFV and is analogous to a ``half' double-strand break gap-repair reaction. We suggest that break copy duplication may have evolved as a mechanism for maintenance of ploidy following DNA breakage.     

Molecular cloning and genetic mapping of the DNA topoisomerase II gene of Saccharomyces cerevisiae

The structural gene for DNA topoisomerase II from the yeast Saccharomyces cerevisiae has been cloned. The clones were selected from a YEp13 plasmid bank of yeast DNA by complementing a temperature-sensitive mutation (top2-1) in the topoisomerase II gene, TOP2. Chromosomal integrants of the clone were derived by homologous recombination in strains lacking the 2 mu circle plasmid. Genetic analysis of these integrants indicates that we have cloned the TOP2 gene and not an extragenic suppressor. A YEp13-TOP2 hybrid plasmid integrant was used to localize the TOP2 gene to the left arm of chromosome XIV by the 2 mu circle-directed marker loss method. Results from standard meiotic mapping experiments indicate that TOP2 is about 16 centi-Morgans to the centromere proximal side of MET4. Northern blot analysis of TOP2 RNA isolated from a wild-type strain and from an rna2 mutant shows the RNA to be 4.5 kb long in both cases, thus indicating that the TOP2 gene has no large introns.     

Sequential cloning by a vector walking along the chromosome.

A procedure was devised for sequential cloning of chromosomal DNA by cyclical integration and excision of a plasmid vector so that slightly overlapping chromosomal segments are successively cloned. The method depends on circular integration of the vector into the chromosome of a host nonpermissive for its replication, and on excision and reduction of a recombinant plasmid by use of an appropriately designed set of restriction enzyme sites in the vector. A vector suitable for cloning in Escherichia coli was constructed by combining a segment of pBR322 with a gene encoding chloramphenicol resistance expressible in many species. Sequential cloning was demonstrated in Streptococcus pneumoniae by extending a previously cloned segment of the region of the chromosome encoding maltosaccharide utilization by 8 kb in three cycles of cloning. Accuracy of the method was confirmed by hybridization of cloned DNA with chromosomal restriction fragments. It is pointed out that the similarity of the requisite genetic processes in bacteria and yeasts should allow use of the method for sequential cloning of yeast chromosomal DNA and of human or other mammalian DNA in artificial chromosomes of yeast.     

A complex translocation at the murine kappa light-chain locus.

We have previously reported that a segment of DNA from a murine plasmacytoma comprises DNA from three chromosomes, the immunoglobulin kappa light-chain locus on chromosome 6, the S mu locus on chromosome 12, and a region on chromosome 15. We now report that the reciprocal product contains DNA from only the kappa locus and chromosome 15 and not from S mu. We conclude that a complex series of events, including both a transposition of DNA and a translocation between chromosomes, generated these imperfect reciprocal products.     

Construction of hybrid plasmids containing the yeast replicator

In Saccharomyces cerevisiae strain 6-1G-P188 about 10 per cent of rRNA genes exist as extrachromosomal copies of rDNA repeating units. These extrachromosomal copies can be isolated as covalently closed molecules with lengths around 3mu. We have constructed a set of hybrid plasmids containing the bacterial vector pBR325, the LEU2 gene of yeast encoding beta-isopropylmalatedehydrogenase and various EcoRI restriction fragments of the 3mu DNA. We have tested the ability of our hybrid plasmids to transform LEU2 strain DC5 to leucine prototrophy. One of the plasmids Rcp21/11 transforms DC5 at the frequency comparable with that obtained with YEp13, containing the 2mu DNA replication origin. The 2400 bp EcoRI-B fragment of the 3mu DNA in Rcp21/11 carries a gene for 5S rRNA and two spacers. Our results on transformation experiments allow un to suggest that this EcoRI fragment also carries the 3mu DNA replication origin. Yeast transformants containing this plasmid are highly unstable but during the prolonged growth in selective conditions the stabilization of the LEU+ phenotype is observed being most likely a result of integration of Rcp21/11 into the yeast chromosome.     

Molecular cloning of the SUF2 frameshift suppressor gene from Saccharomyces cerevisiae

A genetic approach to the molecular cloning of frameshift suppressor genes from yeast is described. These suppressors act by suppressing +1 G:C base-pair insertion mutations in glycine or proline codons. The cloning regimen involves an indirect screen for yeast transformants which harbor a functional suppressor gene inserted into the autonomously replicating “shuttle” vector YEp13, followed by transfer of the hybrid plasmid from yeast into Escherichia coli. Using this procedure a 10.7-kb DNA fragment carrying the SUF2 frameshift suppressor gene has been isolated. This suppressor acts specifically on +1 G:C insertions in proline codons. When inserted into an integrative vehicle and reintroduced into yeast by transformation, this fragment integrates by homologous recombination in the region of the SUF2 locus on chromosome III. A large proportion of the fragment overlaps with another cloned DNA segment which carries the closely linked CDC10 gene. The SUF2 fragment carries at least two tRNA genes. The SUF2 gene and one of the tRNA genes are located on a 0.85-kb restriction fragment within the 10.7-kb segment. A method is also described for the isolation of DNA fragments carrying alternative alleles of the SUF2 locus. Using this procedure, the wild-type suf2⁺ allele has been cloned.     

Analysis of a 1.6-μm circular plasmid from the yeast Kluyveromyces drosophilarum: Structure and molecular dimorphism

A new plasmid has been found in the yeast Kluyveromyces drosophilarum. It is a double-stranded circular DNA, 1.6 micron in length (4.8 kilobase pairs). As in the case of Saccharomyces 2 mu circles, this plasmid occurs in two isomeric forms corresponding to the inversion of a segment between two 346-bp-long inverted repeats within the molecule. Each form has been separately cloned into bacterial plasmids. The new yeast plasmid, called pKD1, contains sequences that allow its replication in Saccharomyces cerevisiae.     

Sensitivity to the Yeast Plasmid 2mu DNA is conferred by the nuclear allele nibl.

Two strains of Saccharomyces carlsbergensis that lacked the plasmid 2mu DNA responded differently when the plasmid was introduced into them. In one strain, cells lacking 2mu DNA ("cir0") produced the normal "smooth" colony morphology, but cells bearing 2mu DNA ("cir+") produced heterogeneous "nibbled" colonies. In the second strain, both cir+ and cir0 strains exhibited a smooth colony morphology. Crosses between these strains revealed that a single recessive nuclear gene, called nibl, conferred the nibbled colony morphology in the presence of 2mu DNA. By a series of backcrosses, nibl was introduced into a Saccharomyces cerevisiae background. nibl caused a nibbled colony morphology in this background just as it did in S. carlsbergensis. nibl was mapped to the left arm of chromosome XVI. Twelve independent smooth revertants were isolated from two nibl [cir+] strains. Seven were analyzed, and all were found to be chromosome VII disomes. Chromosome VII disomy and suppression of the nibbled phenotype cosegregated in crosses. Thus, chromosome VII disomy can suppress the nibbled phenotype. The results of other experiments (C. Holm, Cell 29:585-594, 1982) indicate that the nibbled colony morphology is the result of lethal sectoring and that the lethality is caused by a high copy number of 2mu DNA. I suggest, therefore, that the product of the nibl gene may play a role in controlling the copy number of 2mu DNA. Possible models for the suppression of the nibbled phenotype by chromosome VII disomy are discussed.     

Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid.

We have developed an effective method to delete or invert a chromosomal segment and to create reciprocal recombination between two nonhomologous chromosomes in Saccharomyces cerevisiae, using the site-specific recombination system of pSR1, a circular cryptic DNA plasmid resembling 2 microns DNA of S. cerevisiae but originating from another yeast, Zygosaccharomyces rouxii. A 2.1-kilobase-pair DNA fragment bearing the specific recombination site on the inverted repeats of pSR1 was inserted at target sites on a single or two different chromosomes of S. cerevisiae by using integrative vectors. The cells were then transformed with a plasmid bearing the R gene of pSR1, which encodes the site-specific recombination enzyme and is placed downstream of the GAL1 promoter. When the transformants were cultivated in galactose medium, the recombination enzyme produced by expression of the R gene created the modified chromosome(s) by recombination between two specific recombination sites inserted on the chromosome(s).     

pPAC-ResQ: A yeast-bacterial shuttle vector for capturing inserts from P1 and PAC clones by recombinogenic targeted cloning

We have developed a method to capture inserts from P1 and P1 artificial chromosome (PAC) clones into a yeast-bacteria shuttle vector by using recombinogenic targeting. We have engineered a vector, pPAC-ResQ, a derivative of pClasper, which was previously used to capture inserts from yeast artificial chromosome clones. pPAC-ResQ contains DNA fragments flanking the inserts in P1 and PAC vectors as recombinogenic ends. When linearized pPAC-ResQ vector and P1 or PAC DNA are cotransformed into yeast, recombination between the two leads to the transfer of inserts into pPAC-ResQ. pPAC-ResQ clones thus obtained can be further modified in yeast for functional analysis and shuttled to Escherichia coli to produce large quantities of cloned DNA. This approach provides a rapid method to modify P1/PAC clones for functional analysis.     

Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene.

We have constructed a plasmid, YEp13, which when used in conjunction with transformation in yeast is a suitable vector for isolating specific yeast genes. The plasmid consists of pBR322, the LEU2 gene of yeast, and a DNA fragment containing a yeast origin of replication from 2 mu circule. We have demonstrated the utility of this cloning system by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.     

A bacterial model system for chromosomal targeting.

A system that permits efficient site-specific chromosomal targeting of foreign DNA on the Escherichia coli chromosome has been developed, using the FLP site-specific recombination system derived from the yeast 2 mu plasmid. The system demonstrates the feasibility of using site-specific recombination for this purpose, and provides a means to gather information on parameters that may affect chromosomal targeting to guide efforts to establish similar systems in higher eukaryotes. In this model system, the efficiency of integration of foreign DNA is affected by the location of the target site in the chromosome, and the structure of the recombination sites.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.     

Transformation of protoplasted yeast cells is directly associated with cell fusion.

The frequency of cell fusion during transformation of yeast protoplasts with various yeast plasmids with a chromosome replicon (YRp or YCp) or 2 mu DNA (YEp) was estimated by two methods. In one method, a mixture of protoplasts of two haploid strains with identical mating type and complementary auxotrophic nuclear markers with or without cytoplasmic markers was transformed. When the number of various phenotypic classes of transformants for the nuclear markers was analyzed by equations derived from binominal distribution theory, the frequency of nuclear fusion among the transformants was 42 to 100% in transformations with the YRp or YCp plasmids and 28 to 39% with the YEp plasmids. In another method, a haploid bearing the sir mutation, which allows a diploid (or polyploid) homozygous for the MAT (mating type) locus to sporulate by the expression of the silent mating-type loci HML and HMR, was transformed with the plasmids. Sporulation ability was found in 43 to 95% of the transformants with the YRp or YCp plasmids, and 26 to 31% of the YEp transformants. When cytoplasmic mixing was included with the nuclear fusion, 96 to 100% of the transformants were found to be cell fusants. Based upon these observations, we concluded that transformation of yeast protoplasts is directly associated with cell fusion.