Plasmid construction by homologous recombination in yeast

We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.     

Identification of proteins that interact with a protein of interest: Applications of the yeast two-hybrid system

The yeast two-hybrid system is a molecular genetic test for protein interaction. Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.     

Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments

We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes. Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid. All three vectors contain a kanamycin resistance gene. Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A. Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest. In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules. Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).     

Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation.

The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp50 bearing the yeast ARS1, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product.     

A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector

A set of genomic plasmid banks was constructed using the centromere-containing yeast shuttle vector YCp50. The centromere-containing vector is useful for the isolation of genes that are toxic to yeast when present in high copy number. Fourteen independent banks were prepared each with an average representation of two to three times the yeast genome. Any individual plasmid from a given bank is guaranteed to be of independent origin from plasmids obtained from each of the other banks. The banks were constructed from three different size classes of DNA fragments that resulted from varying conditions of partial digestion with Sau3A. This avoided the bias caused by differential sensitivity of sites to cleavage with Sau3A. Insert DNA is sufficiently large that most genes will be present in the set of plasmid banks at a frequency of about 0.1%.     

Characterization of an essential Orc2p-associated factor that plays a role in DNA replication.

The Saccharomyces cerevisiae Orc2 protein is a subunit of the origin recognition complex, ORC, which binds in a sequence-specific manner to yeast origins of DNA replication. With screens for orc2-1 synthetic lethal mutations and Orc2p two-hybrid interactors, a novel Orc2p-associated factor (Oaf1p) was identified. OAF1 is essential, its gene product is localized to the nucleus, and an oaf1 temperature-sensitive mutant arrests as large budded cells with a single nucleus. The mutant oaf1-2, isolated in the synthetic lethal screen, loses plasmids containing a single origin of DNA replication at a high rate, but it maintains plasmids carrying multiple potential origins of DNA replication. In addition, the OAF1 gene product tagged with the hemagglutinin antigen epitope binds to a DNA affinity column containing covalently linked tandem repeats of an essential origin element. These results suggest a role for OAFI in the initiation of DNA replication. Mutant alleles of cdc7 and cdc14 were also isolated in the orc2-1 synthetic lethal screen. Cdc7p, like Oaf1p, also interacts with Orc2p in two-hybrid assays.     

Genetic synthetic lethality screen at the single gene level in cultured human cells

Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.     

Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control

A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability. Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-microns plasmid DNA and an intact actin gene, were constructed. Neither plasmid alone yielded transformants in the haploid Leu- strain AH22, but when cotransformed, a number of colonies were obtained. Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-microns plasmid. The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level. The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event. Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a high frequency of transformants resulting primarily from single-crossover events between the two plasmids. This was presumably because such events no longer generated an intact actin gene on a multicopy plasmid. Infrequently a transformant from a plasmid with an intact gene was recovered, but in these cases the plasmid was not present in multiple copies. These cells exhibited a slower growth rate, and Northern blot analysis revealed an elevated level of actin mRNA.     

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.     

Study of the stability of hybrid plasmids replicating in Saccharomyces cerevisiae due to DNA fragments from polyoma virus

Hybrid plasmid pSP97 carrying the entire genome of polyoma virus (PY), inserted into bacterial vector psV3, transforms yeast cells with the frequency 1 x 10(-2). Plasmid pSP97 is capable of autonomous replication in S. cerevisiae, while its structure remains unaltered, the stability of hybrid plasmid in transformants is 44%--100%. Plasmid pSP155 consisting of Ori-containing DNA segment from polyoma, pBR322 and yeast gene arg4, transforms yeast cells with the frequency 5 x 10(-3), the stability of plasmid in transformants is 23%--29%. Two types of plasmids were isolated from transformants: one was identical to SP155, while the another differed structurally and phenotypically from SP155. Plasmids pSP113 and pSP114, in addition to pBR322 and yeast gene arg4, contain a viral DNA segment that encodes genes from small and middle T-antigens. These plasmids transform yeast cells with low frequency (2 x 10(-4), 3 x 10(-5)), the stability of plasmids in yeast transformants is 100%. However, hybrid plasmids identical to pSP113 were isolated from transformants. Structural rearrangements have been observed in pSP114, which carries the arg4 gene in reversed orientation compared to pSP113.     

Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.     

Insertion of nonhomologous DNA into the yeast genome mediated by homologous recombination with a cotransforming plasmid

Bacterial plasmids containing no detectable homology with yeast DNA sequences were inserted into the yeast genome by cotransforming with a plasmid containing a yeast gene. Analysis of the yeast transformants confirmed that recombination events occurred between the prokaryotic sequences shared by the two plasmids and between the yeast sequences common to the cotransforming plasmid and to the genome. Multiple copies of the two plasmids, in both tandem and interspersed arrays, are inserted by this method. Populations of cells grown from individual transformants are heterogenous for the number of integrated sequences. The number of integrated bacterial sequences is greatly reduced after 100 generations of growth in the populations that initially contained large numbers of sequences, while it is stable in those populations that initially contained either a single or a small number of copies.     

Functional selection and analysis of yeast centromeric DNA

A direct selection procedure has been used to isolate 11 distinct yeast genomic DNA fragments that eliminate the extreme segregation bias characteristic of autonomously replicating yeast plasmids. The selection scheme takes advantage of the fact that the cloned ochre suppressing tRNA gene, SUP11, is lethal at high copy number and therefore causes cell death when present on an ARS plasmid that lacks a cis-acting partition function. Each of the cloned DNA sequences was mapped to specific yeast chromosomes by hybridization to chromosome-sized DNA molecules separated by alternating field electrophoresis. Ten of the cloned fragments correspond to chromosomal centromeres; one fragment corresponds to the cis-acting locus required for endogenous 2 mu plasmid stability. Nucleotide sequence comparison of the ten centromere DNAs gives a new picture of conserved centromere DNA elements.     

Insertion of transposon Tn9 into the spinal (Escherichia coli-Saccharomyces cerevisiae) plasmids and the expression of the prokaryotic gene of chloramphenicol resistance in yeast cells

Transposon Tn9 carrying camr gene which controls resistance to chloramphenicol has been introduced in vivo (in cells of Escherichia coli) into two chimeric shuttle plasmids pYF91 and YEp13. These plasmids consist of the different parts of the E. coli plasmid pBR322, the yeast 2mkm DNA plasmid and the yeast LEU2 structural gene. The plasmidis able to autonomously replicate in both yeast and bacterial cells. A recipient yeast strain carrying cams and leu2 markers was constructed to study the functional expression of the prokaryotic camr gene in eukaryotic yeast cells. The chimeric plasmids pYF91::Tn9 and YEp13::Tn9 were introduced into the yeast and bacterial recipient strains by transformation. The camr LEU2 yeast transformants were isolated. They were genetically unstable when grown on non-selective medium and they simultaneously lost camr and LEU2 markers with a frequency of 10 to 30%. The E. coli transformants were genetically stable under nonselective conditions and they maintain all plasmid markers. The chimeric plasmid pYF91::Tn9 was isolated from the yeast transformants and reintroduced into the cams leuB bacterial strain by transformation. The camr LEUB transformants were obtained. All these data confirm the possibility of the expression of the prokaryotic camr gene in yeast cells and present evidence for introduction of transposon Tn9 into chimeric plasmids.     

FLP-mediated recombination in the vector mosquito, Aedes aegypti.

The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti. In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos. FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids. In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used. The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid. This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.     

Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae

Targeted integration of the yeast plasmid pMIRY2 into the ribosomal DNA (rDNA) of Saccharomyces cerevisiae by homologous recombination results in transformants carrying 100-200 copies of the plasmid per cell which are stably maintained over a large number of generations [Lopes et al., Gene 79 (1989) 199-206]. These properties make pMIRY2 an attractive vector for high-level production of (heterologous) proteins by yeast cells. We have investigated the mechanism underlying high-copy-number (hcn) integration of pMIRY-type plasmids and show that either targeting to a location outside the rDNA locus or use of the wild-type LEU2, instead of the deficient LEU2d gene, as selection marker reduces the copy number to the low value characteristic of standard integrating (YIp-type) yeast plasmids. Further experiments demonstrate that the hcn of pMIRY-type plasmids is achieved by amplification of a small number of copies initially integrated into the rDNA locus. Amplification depends upon the strong selection pressure created by the extremely low expression of the deficient LEU2d gene, but not on the presence of this gene per se. The hcn integration also occurs when either the TRP1 or URA3 gene is used as the selection marker, provided expression of the marker gene is severely curtailed, e.g., by removal of most of its 5'-flanking region.