A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae

A simple assay called the sectoring shuffle was developed to monitor the mutational state of essential genes cloned into yeast centromeric plasmids. The essence of this assay is the creation of a conditional phenotype, colony color sectoring, for an essential gene in the absence of conditional thermosensitive or cold-sensitive alleles of that gene. This allows the quick determination of the mutational state of a cloned essential gene by observing its effect on the sectoring phenotype of the tester strain. During the course of this work we developed a family of 20 Escherichia coli-yeast shuttle vectors, pUN plasmids, containing ARS1 CEN4 and a variety of selectable markers as well as the SUP11 gene which can act as a color marker in the proper background. These vectors are compact and have been very useful for the sectoring-shuffle assay and for gene analysis in general. This paper decribes these vectors, the sectoring shuffle and several applications of sectoring phenotypes.     

Set of vectors for the expression of histidine-tagged proteins in vaccinia virus recombinants

Vaccinia virus expression vectors are widely used to direct the expression of proteins in eukaryotic cells. Here, we describe a new set of plasmid vectors designed for the expression of histidine-tagged proteins in the vaccinia system. To facilitate the rapid isolation of virus recombinants, the plasmids contain a viral gene (F13L) that serves as an efficient selection marker based on virus plaque phenotype. Histidine codons and restriction sites derived from pET-16b bacterial expression plasmid were included, thus facilitating the transfer of genes between E. coli and vaccinia expression plasmids. Plasmids in which the gene is placed downstream of either a strong vaccinia virus or a T7 promoter were constructed, allowing for constitutive or conditional expression, respectively, of the foreign protein.     

High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.     

Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1.

Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype. The wild-type copB gene has been cloned on various plasmid vectors. The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras. However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100). Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids. Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.     

Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains.

A model system for one-step gene disruption for an asporogenous methylotrophic yeast, Candida boidinii, is described. In this system, the 3-isopropylmalate dehydrogenase gene (C. boidinii LEU2) was selected as the target gene for disruption to derive new host strains for transformation. First, the C. boidinii LEU2 gene was cloned, and its complete nucleotide sequence was determined. Next, the LEU2 disruption vectors, which had the C. boidinii URA3 gene as the selectable marker, were constructed. Of the Ura+ transformants obtained with these plasmids, more than half showed a Leu- phenotype. Finally, the double-marker strains of C. boidinii were derived. When vectors with repeated flanking sequences of the C. boidinii URA3 gene were used for gene disruption, Leu- Ura+ transformants changed spontaneously to a Leu- Ura- phenotype ca. 100 times more frequently than they did when plasmids without the repeated sequences were used. Southern analysis showed that these events included a one-step gene disruption and a subsequent popping out of the C. boidinii URA3 sequence from the transformant chromosome.     

Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction

A small 2.1-kb plasmid called pLP1 was extracted from Lactobacillus plantarum CCM 1904 (ATCC 8014) and cloned into the Escherichia coli pUC19 plasmid. As determined by DNA-DNA Southern hybridization with a pLP1-radioactively labeled probe, other lactic acid bacteria such as L. curvatus, L. sake, Carnobacterium, and Leuconostoc mesenteroides harbor pLP1-related plasmids. Shuttle vectors based on the pLP1 replicon were constructed by inserting the erythromycin-resistance gene from pVA891 into the various pUC19-pLP1 constructions. pLP1-based shuttle vector transformation efficiencies (TE) by electroporation were compared to TE of a broad-host-range plasmid pGK12 in different lactobacilli strains. Expression of the pUC19-pLP1 plasmids in Escherichia coli maxicells showed that pLP1 encodes for a 37,000 MW protein which can act in trans allowing the replication of plasmids in which this protein is truncated. The pLP1-based shuttle vectors producing this protein replicate in lactobacilli and also in Bacillus subtilis. A pLP1-free strain was obtained by incompatibility with a pLP1-based shuttle vector introduced in L. plantarum CCM 1904 by electroporation. The absence of pLP1 has no incidence on the strain phenotype suggesting that pLP1 is not essential for the strain in our laboratory conditions.     

Plasmid vectors for selecting IS1-promoted deletions in cloned DNA: sequence analysis of the omega interposon.

We have constructed two plasmid vectors which allow selection for in vivo deletions within cloned DNA fragments. The plasmids are derivatives of pBR322 which carry the Escherichia coli rpsL (strA) gene, known to confer a dominant streptomycin (Sm)-sensitivity phenotype to the host cell, and a copy of the IS1 transposable element. Sm-resistant strains that harbor these plasmids display sensitivity to Sm. Spontaneous IS1-promoted deletions across the rpsL gene can be isolated simply by selection for Sm resistance. Hence, nested sets of deletions of a cloned DNA can be obtained and sequenced with an IS1-specific primer. Using this approach, we have determined the complete nucleotide sequence of the omega interposon [Prentki and Krisch, Gene 29 (1984) 303-313].     

Genetic method to analyze essential genes of Escherichia coli

The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-beta-d-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.     

Vectors for regulated gene expression in the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans possesses an exceptional capacity to withstand the lethal and mutagenic effects of most form of DNA damage and has received considerable interest for use in both fundamental and applied research. Here we describe vectors that allow regulated expression of Deinococcal genes for functional analysis. The vectors contain the IPTG-regulated Spac system (Pspac promoter and lacI repressor gene), originally designed for Bacillus subtilis, that we have adapted to be functional in D. radiodurans. We show that the Spac system can control the expression of a lacZ reporter gene over two orders of magnitude depending on the inducer concentration and the copy number of the lacI regulatory gene. Furthermore, we demonstrate that the Spac system can be used to regulate the synthesis of a critical repair protein, such as RecA, resulting in a conditional mitomycin-resistant cell phenotype. We have also developed tools for the construction of conditional mutants where the expression of the target gene is regulated by an inducible promoter. The utility of these conditional gene inactivation systems is exemplified by the conditional lethal phenotype of a mutant expressing gyrA from the Pspac promoter.     

Ac-Induced Instability at the Xanthophyllic Locus of Tomato

To detect genomic instability caused by Ac elements in transgenic tomatoes, we used the incompletely dominant mutation Xanthophyllic-1 (Xa-1) as a whole plant marker gene. Xa-1 is located on chromosome 10 and in the heterozygote state causes leaves to be yellow. Transgenic Ac-containing tomato plants which differed in the location and number of their Ac elements were crossed to Xa-1 tester lines and F(1) progeny were scored for aberrant somatic sectoring. Of 800 test and control F(1) progeny screened, only four plants had aberrantly high levels of somatic sectors. Three of the plants had twin sectors consisting of green tissue adjacent to white tissue, and the other had twin sectors comprised of green tissue adjacent to tissue more yellow than the heterozygote background. Sectoring was inherited and the two sectoring phenotypes mapped to opposite homologs of chromosome 10; the green/yellow sectoring phenotype mapped in coupling to Xa-1 while the green/white sectoring phenotype mapped in repulsion. The two sectoring phenotypes cosegregated with different single, non-rearranged Acs, and loss of these Acs from the genome corresponded to the loss of sectoring. Sectoring was still observed after transposition of the Ac to a new site which indicated that sectoring was not limited to a single locus. In both sectored lines, meiotic recombination of the sectoring Ac to the opposite homolog caused the phenotype to switch between the green/yellow and the green/white phenotypes. Thus the two different sectoring phenotypes arose from the same Ac-induced mechanism; the phenotype depended on which chromosome 10 homolog the Ac was on. We believe that the twin sectors resulted from chromosome breakage mediated by a single intact, transposition-competent Ac element.     

Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae

The Saccharomyces cerevisiae gene deletion project revealed that approximately 20% of yeast genes are required for viability. The analysis of essential genes traditionally relies on conditional mutants, typically temperature-sensitive (ts) alleles. We developed a systematic approach (termed "diploid shuffle") useful for generating a ts allele for each essential gene in S. cerevisiae and for improved genetic manipulation of mutant alleles and gene constructs in general. Importantly, each ts allele resides at its normal genomic locus, flanked by specific cognate UPTAG and DNTAG bar codes. A subset of 250 ts mutants, including ts alleles for all uncharacterized essential genes and prioritized for genes with human counterparts, is now ready for distribution. The importance of this collection is demonstrated by biochemical and genetic screens that reveal essential genes involved in RNA processing and maintenance of chromosomal stability.     

Potassium permanganate as an in situ probe for B-Z and Z-Z junctions.

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.     

Vectors for a ‘double-tagging’ assay for protein-protein interactions: localization of the CDK2-binding domain of human p21

We report the construction of three new vectors which can be used for the ‘double-tagging’ assay previously reported [Germino et al., Proc. Natl. Acad. Sci. USA 90 (1993) 933–937]. The vectors include two plasmids (pTrc. BCCP and pTrc. EZZ::BCCP) which encode different ‘tag’ for the capture of a target protein of interest on a filter coated with either avidin or IgG, respectively. The first plasmid (pTrc. BCCP) encodes the C terminus of the biotin carboxylase carrier protein (BCCP) under the control of the P_tac, promoter, while the second produces fusions to an IgG-binding domain (EZZ). The gene encoding a protein of interest can be inserted into these plasmids and thereby direct the production of a fusion protein which is biotinylated in vivo and can bind to avidin, or a fusion protein which can bind to IgG. The third is a positive-selection, phage λ, expression vector (λFJG2) which permits the construction of lacZ::cDNA fusion proteins which retain ß-galactosidase activity. The insertion of an active ecoRVR gene between the cloning sites (EcoRI and HindIII or NotI) permits the positive selection of inserts. The C-terminal two-thirds of the mouse retinoblastoma-encoding gene (containing the EIA-binding pocket) was cloned into pTrc. BCCP and pTrc.EZZ::BCCP, while the 13S Ela gene was cloned into λFJG2. We show that the interaction between these two proteins can be detected using the ‘double-taggin’ filter assay, and that this assay has high sensitivity and specificity for detecting this interaction. Finally, we have used these vectors to localize the CDK2-binding domain of the cyclin-dependent kinase inhibitor, p21. These results closely correspond to those obtained using the yeast two-hybrid assay, as well as in vitro binding assays.     

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.     

Acetylglutamate synthase from Neurospora crassa: structure and regulation of expression

A novel gene shuffle approach has been developed for investigating the functions of genes on the cytoplasmic linear DNA killer plasmids of Kluyveromyces lactis . By transplacing k2ORF5 from the larger plasmid pGKL2(k2) onto pGKL1(k1) we have shown this gene to be essential and functionally interchangeable between plasmids. Once transferred onto k1, k2ORF5 is fully able to complement a k2ORF5⁰ deletion on k2 in trans , giving rise to yeast strains containing only the two recombinant plasmid forms. Additionally, the in vivo product of k2ORF5 has been identified as a 19.5 kDa protein by transplacing an epitope-tagged k2ORF5 allele from k2 to k1. The ease of detection of the tagged ORF5 product in comparison to TRF1, the gene product of k2ORF10, indicates that Orf5p is one of the most abundant k2 products, implying structural rather than regulatory function.