Two-step method for constructing unmarked insertions, deletions and allele substitutions in the yeast genome

We describe three extensions of the method of site-specific genomic (SSG) mutagenesis. These three extensions of SSG mutagenesis were used to generate precise insertion, deletion, and allele substitution mutations in the genome of the budding yeast, Saccharomyces cerevisiae. These mutations are termed precise because no attached sequences (e.g., marker genes or recombination sites) are retained once the method is complete. Because the method is PCR-based, neither DNA cloning nor synthesis of long oligonucleotides is required. We demonstrated the efficacy of these methods by deleting an ORF, inserting the tandem affinity purification (TAP) tag, and replacing a wild-type allele with a mutant allele.     

Scarless and site-directed mutagenesis in <Emphasis Type="Italic">Salmonella enteritidis</Emphasis>chromosome

Background  

A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome.     

A method for clone sequence confirmation using a mismatch-specific DNA endonuclease

Site-directed mutagenesis and polymerase chain reaction (PCR)-based cloning are well-established methods carried out routinely in most modern molecular biology laboratories. Application of these methods requires confirmation of the DNA sequence of the target gene by sequencing of DNA purified from multiple colonies, a laborious process. We have developed an alternative approach to screen DNA amplified directly from colony DNA for both desired and undesired mutations. This approach is based on the use of a plant mismatch DNA endonuclease, Surveyor Nuclease, to directly screen clones derived by site-directed mutagenesis. We have also used this approach to identify error-free clones of three genes from celery cDNA produced by PCR and TOPO cloning. Sequence confirmation using Surveyor Nuclease provides a fast and simple approach to obtain desired clones from site-directed mutagenesis and PCR-based cloning methods without the necessity of sequencing DNAs purified from multiple clones.     

Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis

The polymerase chain reaction catalyzed by Taq DNA polymerase has been used for site-specific mutagenesis. The amplification was primed by two oligodeoxyribonucleotides complementary to insulin receptor cDNA. To direct the synthesis of mutant DNA, mismatches were introduced into one of the primers. Six different mutations were constructed by this technique. Of twelve clones whose sequences were determined, ten (83%) had the correct sequence. This technique, which does not require the use of single-stranded DNA templates, provides a simple and efficient approach to site-specific mutagenesis.     

Alteration of ultraviolet-induced mutagenesis in yeast through molecular modulation of the REV3 and REV7 gene expression

DNA damage can lead to mutations during replication. The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations. DNA polymerase zeta (Pol zeta), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway. Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy. In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system. Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Pol zeta inhibition. Lower levels of Pol zeta significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis. In contrast, elevating the Pol zeta level by enhanced expression of both REV3 and REV7 genes led to a approximately 3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays. In vivo, UV lesion bypass by Pol zeta requires the Rev1 protein. Even overexpression of Pol zeta could not alleviate the defective UV mutagenesis in the rev1 mutant cells. These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.     

Use of synthetic DNA in expression of foreign proteins

Synthetic DNAs and oligonucleotides, which can be prepared conveniently by combining chemical synthesis and enzymatic methods, have been used extensively in recombinant DNA research. Examples include total gene synthesis, probes for the isolation of specific genes from cDNA or genomic libraries, linkers containing specific restriction sites for cloning, primers for DNA and RNA sequencing, and primers for the construction of specific mutations (either deletion, insertion or point mutations) by oligonucleotide-directed site-specific mutagenesis.This article reviews recent advances in the chemical and enzymatic synthesis of oligo- and polynucleotides and the application of synthetic DNA to the expression of foreign proteins. The synthesis of genes, including structural genes and regulatory genes are reviewed. Oligonucleotide-directed site-specific mutagenesis and use of synthetic DNA to optimize foreign protein expression are also discussed.     

Searching for Sequence Directed Mutagenesis in Eukaryotes

Sequence directed mutagenesis is a mechanism by which imperfect repeats “repair” each other to become perfect, generating mutations. This process is known to be prevalent in prokaryotes and it has been implicated in several human genetic diseases. Here we test whether sequence directed mutagenesis occurs in the protein coding sequences of eukaryotes using extensive DNA sequence data from humans, mice, Drosophila, nematodes, yeast, and Arabidopsis. Using two tests we find little evidence of sequence directed mutagenesis. We conclude that sequence directed mutagenesis is not prevalent in eukaryotes and that the examples of human diseases, apparently caused by sequence directed mutagenesis, are probably coincidental. [Reviewing Editor: Dr. Richard Kliman]     

The role of recombination and RAD52 in mutation of chromosomal DNA transformed into yeast.

While transformation is a prominent tool for genetic analysis and genome manipulation in many organisms, transforming DNA has often been found to be unstable relative to established molecules. We determined the potential for transformation-associated mutations in a 360 kb yeast chromosome III composed primarily of unique DNA. Wild-type and rad52 Saccharomyces cerevisiae strains were transformed with either a homologous chromosome III or a diverged chromosome III from S. carlsbergensis. The host strain chromosome III had a conditional centromere allowing it to be lost on galactose medium so that recessive mutations in the transformed chromosome could be identified. Following transformation of a RAD+ strain with the homologous chromosome, there were frequent changes in the incoming chromosome, including large deletions and mutations that do not lead to detectable changes in chromosome size. Based on results with the diverged chromosome, interchromosomal recombinational interactions were the source of many of the changes. Even though rad52 exhibits elevated mitotic mutation rates, the percentage of transformed diverged chromosomes incapable of substituting for the resident chromosome was not increased in rad52 compared to the wild-type strain, indicating that the mutator phenotype does not extend to transforming chromosomal DNA. Based on these results and our previous observation that the incidence of large mutations is reduced during the cloning of mammalian DNA into a rad52 as compared to a RAD+ strain, a rad52 host is well-suited for cloning DNA segments in which gene function must be maintained.     

Oligodeoxynucleotide-directed mutagenesis using the yeast transformation system

In this report we describe a highly efficient method for site-specific mutagenesis using the yeast transformation system. The method is based on the observation that Saccharomyces cerevisiae can be transformed at high frequency with single-stranded circular DNA vectors [Singh et al., Gene 20 (1982) 441-449]. The model system studied was the TRP1 gene of S. cerevisiae cloned into a derivative of the phage M13mp9 vector containing the yeast URA3 gene. ARS1, located adjacent to the TRP1 gene, allows the plasmid to replicate autonomously in yeast. Synthetic 5'P-oligodeoxynucleotides, 19 and 35 nucleotides (nt) in length, designed to produce an A----T transversion mutation within the TRP1 gene, were annealed to ss DNA of the M13 vector at a molar ratio of 30:1 and directly transformed into yeast. The intended single nt mutation was obtained at frequencies of 24 and 43%, respectively. The latter approaches the theoretical limit of 50%. In the absence of the 5'-terminal phosphate, both the transformation frequency and the efficiency of mutagenesis by the synthetic oligodeoxynucleotide (oligo) were decreased by 2-4 fold. This procedure completely obviates the need for any enzymatic manipulations in vitro after forming the heteroduplex with the oligo primer containing the desired mutation. For yeast genes, direct phenotypic selection is possible in the recipient strain.     

A simple vector modification to facilitate oligonucleotide-directed mutagenesis.

We describe a simple modification of commonly used single-stranded cloning vectors that permits the efficient recovery of mutant DNA molecules in oligonucleotide-directed mutagenesis experiments, even when the absolute efficiency of mutagenesis is very low. The modification consists of the insertion of a short synthetic DNA fragment into the vector's polylinker and permits the identification of mutant clones based on a standard chromogenic plate assay for bacterial colonies or phage plaques producing functional beta-galactosidase. Other useful properties of the original vector are retained in the modified version. In vitro mutagenesis reactions are carried out with two oligonucleotides, one to introduce the mutation of interest, and the second to correct a frameshift mutation introduced into the beta-galactosidase gene of the modified vector. We have found that these two sequence changes are closely linked following transformation of an appropriate E. coli strain with the products of the in vitro mutagenesis reaction, and have thereby recovered desired mutations at a frequency of about 50% even when the overall mutagenesis efficiency is less than 1%. By alternately correcting and re-introducing the beta-galactosidase frameshift mutation, we have shown that multiple rounds of mutagenesis can be carried out on the same template with a high efficiency of mutant recovery in each step. Modifications similar or identical to those we describe here should be feasible for most commonly used single-stranded cloning vectors and should increase the usefulness of these vectors by providing an additional option for oligonucleotide-directed mutagenesis to be used in conjunction with or in lieu of other commonly used approaches.     

Cloning of industrial Saccharomyces 2-microns plasmid variants by in vivo site-specific recombination

Southern analyses defined several industrial Saccharomyces yeast strains with extensive 2-microns DNA polymorphism. Variants included insertions and deletions up to several hundred base pairs. To facilitate the investigation of yeast plasmid evolution we developed a novel method of cloning 2-microns plasmids by taking advantage of 2-microns circle in vivo site-specific recombination and an SMRI gene as a dominant selectable marker. This method can be applied to other organisms for the isolation of plasmid variants and provides a new approach to in vivo plasmid construction.     

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast. Identification of point mutations that "silence" a constitutively active mutant M3 receptor and greatly impair receptor/G protein coupling

The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V-VII to random mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V-VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function.     

YAC cloning of telomeres

Human telomeres have been succesfully cloned in Saccharomyces cerevisiae by complementing deficient yeast artificial chromosomes (YACs). This technique allows cloning of DNA sequences that can recognize particular chromosomal ends, and therefore facilitates the mapping of eukaryotic genomes. Although the biology of adopting foreign telomeres in yeast is not fully understood, the cloning system itself seems to be a useful tool for constructing telomeric DNA libraries from higher eukaryotes. Here we describe the techniques that are currently being used in cloning of telomeric DNA.     

S-(2-chloroethyl)glutathione-generated p53 mutation spectra are influenced by differential repair rates more than sites of initial dna damage

Several steps occur between the reaction of a chemical with DNA and a mutation, and each may influence the resulting mutation spectrum, i.e. nucleotides at which the mutations occur. The half-mustard S-(2-bro-moethyl)glutathione is the reactive conjugate implicated in ethylene dibromide-induced mutagenesis attributed to the glutathione-dependent pathway. A human p53-driven Ade reporter system in yeast was used to study the factors involved in producing mutations. The synthetic analog S-(2-chloroethyl)glutathione was used to produce DNA damage; the damage to the p53 exons was analyzed using a new fluorescence-based modification of ligation-mediated polymerase chain reaction and an automated sequencer. The mutation spectrum was strongly dominated by the G to A transition mutations seen in other organisms with S-(2-chloroethyl)glutathione or ethylene dibromide. The mutation spectrum clearly differed from the spontaneous spectrum or that derived from N-ethyl,N-nitrosourea. Distinct differences were seen between patterns of modification of p53 DNA exposed to the mutagen in vitro versus in vivo. In the four p53 exons in which mutants were analyzed, the major sites of mutation matched the sites with long half-lives of repair much better than the sites of initial damage. However, not all slowly repaired sites yielded mutations in part because of the lack of effect of mutations on phenotype. We conclude that the rate of DNA repair at individual nucleotides is a major factor in influencing the mutation spectra in this system. The results are consistent with a role of N(7)-guanyl adducts in mutagenesis.     

Nonselective enrichment for yeast adenine mutants by flow cytometry

The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.     

Positional cloning and characterization of mouse mei8, a disrupted allelle of the meiotic cohesin Rec8

A novel mutation, mei8, was isolated in a forward genetic screen for infertility mutations induced by chemical mutagenesis of ES cells. Homozygous mutant mice are sterile. Mutant females exhibit ovarian dysgenesis and lack ovarian follicles at reproductive maturity. Affected males have small testes due to arrest of spermatogenesis during meiotic prophase I. Genetic mapping and positional cloning of mei8 led to the identification of a mutation in Rec8, a homolog of the yeast meiosis-specific cohesin gene REC8. Analysis of meiosis in Rec8(mei8)/Rec8(mei8) spermatocytes showed that, while initiation of recombination and synapsis occurs, REC8 is required for the completion and/or maintenance of synapsis, cohesion of sister chromatids, and the formation of chiasmata, as it is in other organisms. However, unlike yeast and Caenorhabditis elegans, localization of REC8 on meiotic chromosomes is not required for the assembly of axial elements.     

Inhibition of DNA repair by neighbouring mismatched bases in Streptococcus pneumoniae

A set of pneumococcal strains containing immediately adjacent or nearby double mutations at the amiA locus, conferring resistance to amethopterin, has been isolated by oligonucleotide site-specific mutagenesis. Repair of these double mutations has been measured by transformation of wild-type strains with DNA extracted from these strains. In several transformations we have observed an inhibition of repair by neighbouring mismatches. This inhibition ranges from mild to severe depending upon the interfering mismatch. Unrepaired mismatches can strongly inhibit repair of an adjacent repairable mutation. This suggests that the repair-complex proteins attach not only to repairable mismatches but also to some mismatches known to escape the repair system.     

Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis.

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.     

One-step random mutagenesis by error-prone rolling circle amplification

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3–4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique.