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.     

Mutagenesis of Neisseria meningitidis by in vitro transposition of Himar1 mariner

Now that the meningococcal genome sequence has been completed, the lack of a suitable method for saturation mutagenesis remains a major obstacle to the unraveling of the pathogenic propensity of Neisseria meningitidis. Here, we demonstrate that in vitro Himar1 mariner transposition on chromosomal or PCR-amplified meningococcal DNA, which is subsequently reintroduced into N. meningitidis by natural transformation, is an extremely efficient mutagenesis method. Southern blot analysis, sequencing the Himar1 insertion point in numerous transposition mutants, and a limited screening of the mutant libraries for clones impaired in maltose catabolism confirmed that Himar1 transposed randomly in N. meningitidis. Taken together, these data demonstrate that Himar1 in vitro transposition can lead to the exhaustive mutagenesis of N. meningitidis, allowing for the first time a genomic-scale mutational analysis of this important human pathogen.     

A new approach to random mutagenesis in vitro

Random mutagenesis is a powerful tool for studying the effects of a large number of permutations of a particular DNA sequence and its encoded products. Here we describe a new strategy of conducting in vitro random mutagenesis using ethyl methane sulfonate (EMS). The Bacillus aprN18 gene, coding for a serine protease with fibrinolytic activity, was used as a target gene. To study the mutations of the coding region, rather than the whole plasmid, the 1.4 kb gene fragment was cut out from an expression plasmid and treated with 10 mM EMS at 37 degrees C for 1 h. The treated fragment was then ligated back into the original expression vector and a library of random mutants was constructed in a protease-deficient Bacillus subtilis strain. A plate assay-based screening method was used to select for mutant clones with altered enzyme activity, and the change of activity was then confirmed by a semi-quantitative enzyme assay using liquid culture supernatant. The inserts of five clones with altered enzyme activity were randomly chosen for sequencing analysis. Among the point mutations detected, GC --> AT transition accounts for 42.1%, AT --> GC transition 34.2% and GC/CG transversion 23.7%, respectively. To our knowledge this is the first application of EMS for in vitro mutagenesis of a defined DNA sequence.     

Mutagenesis of the glycosylation site of human ApoCIII. O-linked glycosylation is not required for ApoCIII secretion and lipid binding

We have used site-directed in vitro mutagenesis to alter the codon ACT of human apoCIII gene, specifying Thr-74, to GCT (Ala-74). The normal and mutant apoCIII genes were then placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus vector and were used for cell transfection and selection of stable cell lines. Blotting analysis of RNA isolated from several independent cell clones showed that both the normal and mutant genes produced apoCIII mRNA in amounts larger than that found in human fetal liver. Pulse-chase analysis of cell clones expressing the normal and mutant apoCIII genes showed that only the normal apoCIII is modified intracellularly to produce a disialated form (apoCIIIs2). Cell clones expressing the normal apoCIII gene secrete exclusively the disialated form, whereas those expressing the mutant gene secrete the unmodified form. The amount of mutant apoCIII protein produced by C127 cell clones expressing the mutant gene was reduced as compared to that produced by the control cells. Density gradient ultracentrifugation analysis of the secreted apoCIII showed that the flotation properties of the secreted normal and mutant proteins were similar. These findings suggest that the intracellular glycosylation of apoCIII is not required for its intracellular transport and secretion. Furthermore, lack of glycosylation has no effect on the relative affinities of apoCIII for plasma very low density lipoproteins and high density lipoproteins.     

Clone size distributions of mutations induced by ethyl methanesulfonate in bacteriophage T4

Size distributions of mutant clones can reveal important aspects of the mutation process. Previously published data on mutant clones induced by ethyl methanesulfonate (EMS) in bacteriophage T4 generated a distribution that was essentially flat, implying a mutagenic mechanism involving only rare mispairing by reacted bases. Here, methods for estimating the spontaneous component of such a distribution are used to generate a corrected distribution. The corrected distribution is strongly peaked, implying frequent (but not obligatory) mispairing. Frequent mispairing is in accord with current views of the fates of DNA lesions believed to mediate EMS-induced mutagenesis.     

A novel strategy for the generation of T cell lines lacking expression of endogenous alpha- and/or beta-chain T cell receptor genes

Mutant T cell lines that do not express the endogenous alpha- and/or beta-chain genes of the TCR were generated from the alpha beta TCR/CD3+ tumor cell line C6VL with a combination of classical mutagenesis methods and selection of somatic hybrid variants. This novel strategy obviated the need for repeated mutagenesis and screening of a large number of individual clones. The loss of either the alpha- or the beta-chain expression in the mutant cells was associated with the loss of surface TCR/CD3 complex, which could be rescued by the transfection of appropriate exogenous alpha- and/or beta-chain gene constructs. Because these cells express a single TCR molecule on the cell surface, they are useful for the study of the assembly and function of the alpha beta TCR. This strategy is also generally applicable for the generation of homozygous mutant cell lines lacking other gene products.     

Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.     

Development of a flow-cytometric HLA-A locus mutation assay for human peripheral blood lymphocytes.

A flow-cytometric technique was developed to measure the frequency of variant lymphocytes lacking expression of HLA-A2 or A24 allele products among donors heterozygous for HLA-A2 or A24. It was found that the variant frequency of lymphocytes in peripheral blood was of the order of 10(-4) and increased with donor age. Molecular analyses of mutant clones revealed that about one-third were derived from somatic recombinations and that the remaining two-thirds did not show any alterations after Southern blotting analysis. In contrast, mutants obtained after in vitro X-ray mutagenesis study were found to be mostly derived from large chromosomal deletions. A small-scale study on atomic bomb survivors did not show a significant dose effect.     

Generation of mutant murine cytomegalovirus strains from overlapping cosmid and plasmid clones

We have developed a cosmid and plasmid system to generate mutant strains of murine cytomegalovirus (MCMV). The system is based on a series of seven overlapping cosmid clones that regenerate MCMV when cotransfected into mouse cells. The unaltered cosmids produce MCMV that is indistinguishable from wild-type MCMV based on restriction enzyme digest patterns of virus DNA and growth rates both in vitro and in vivo. Analysis of viral DNA from plaque-purified recombinant isolates taken from in vitro and in vivo stocks indicated that regeneration did not introduce novel mutations in the recombinant viral genomes. Isolation of specific genes and subsequent generation of specific mutant MCMVs was accomplished by replacement of cosmids with overlapping plasmid subclones. A new vector, PmeSUB, featuring a multiple cloning site and a stringent origin of replication, was constructed to make large subclones for use with smaller subclones containing the gene of interest. The utility of this system was demonstrated by the generation of two different mutant MCMVs from different combinations of overlapping plasmid subclones of one cosmid. The advantages of this system are that (i) target genes are maintained as small clones making them amenable to standard in vitro mutagenesis manipulations and that (ii) no reporter or selection genes are necessary to identify mutants.     

Isolation of poly-3-hydroxybutyrate metabolism genes from complex microbial communities by phenotypic complementation of bacterial mutants

The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate D-3-hydroxybutyrate due to absence of the enzyme D-3-hydroxybutyrate dehydrogenase activity. Clones that conferred D-3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored D-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful.     

The use of site-directed mutagenesis, transient transfection, and radioligand binding

Receptor-ligand interactions have traditionally been evaluated using a number of biochemical techniques including radioligand binding, photoaffinity labeling, crosslinking, and chemical modification. In modern biochemistry, these approaches have largely been superseded by site-directed mutagenesis in the study of protein function, owing in part to a better understanding of the chemical properties of oligonucleotides and to the ease with which mutant clones can now be generated. The Altered Sites II in vitro Mutagenesis System from the Promega Corporation employs oligonucleotides containing two mismatches to introduce specific nucleotide substitutions in the nucleic acid sequence of a target DNA. One of these mismatches will alter the primary sequence of a given protein, whereas the second will give rise to a silent restriction site that is used to screen for mutants. Transient transfection of tsA201 cells with mutant cDNA constructs using calcium phosphate as a carrier for plasmid DNA permits expression of recombinant receptors that can be characterized using radioligand binding assays. In this article, we focus on site-directed mutagenesis, heterologous expression in eukaryotic cells, and radioligand binding as a methodology to enable the characterization of receptor-ligand interactions.     

Efficient site-directed in vitro mutagenesis using phagemid vectors

Several methods have been developed that enhance the efficiency of in vitro, site-directed mutagenesis. Kunkel (8,9) has developed a method which uses a strong selection for the mutated strand and, hence, is highly efficient, but yet simple and rapid. This method originally used M13 phage as the vector. In this paper, we describe a refinement of this method using phagemid vectors, which combine the advantages of plasmids (such as high copy number and stability of cloned DNA) with the single-stranded DNA generating capability of M13 phage. We demonstrate that high efficiency of mutant production can be obtained with these vectors. We also analyzed by sequencing 11 mutated clones and found no second-site mutations, suggesting that alterations other than the site-directed mutation rarely occur in our system.     

Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.     

Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis

The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme.     

Recombinant circle PCR and recombination PCR for site-specific mutagenesis without PCR product purification.

Two simple methods for site-specific mutagenesis are described and compared. In each method, the PCR is used in two separate amplifications to mutate the site of interest and to add ends to one PCR product that are homologous to the ends of the other PCR product. In the first method, the two products are combined, denatured and reannealed prior to transformation of E. coli in order to form recombinant circles in vitro, while in the second method, the two linear products are co-transfected directly into E. coli without prior manipulation, resulting in transformation of E. coli with the recombinant of interest by recombination in vivo. Each PCR amplification uses a plasmid template that has been linearized by restriction enzyme digestion outside the region to be amplified. This permits use of unpurified PCR products in these two protocols and generation of the mutant of interest with no other enzymatic manipulation in vitro apart from PCR amplification. In each protocol greater than or equal to 50% of the resulting clones contained the mutation of interest without detected errors.     

Complementation of an Erwinia carotovora subsp. carotovora protease mutant with a protease-encoding cosmid

Summary We report the complementation of a genetic lesion in the genome of Erwinia carotovora subsp. carotovora (Ecc), a pathogenic bacterium that incites soft rot of plants. A Sau3AI genomic library of Ecc was constructed using the conjugal cosmid pLAFR-3 as a vector. Sixteen cosmid clones encoding various plant tissue-degrading enzymes were identified, including a proteolytic clone, five cellulolytic clones, and ten pectolytic clones. We detected a mutant of Ecc with no proteolytic activity following transposon mutagenesis with an unstable Tn5-carrying plasmid. Conjugal transfer of the protease-encoding cosmid to this mutant restored near-wildtype extracellular protease production. Further manipulation and study of genes encoding pathogenic determinants in Ecc will be possible using this system.     

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.     

Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis

Several cosmid clones from Streptomyces ambofaciens containing the spiramycin resistance gene srmB were introduced into S. fradiae PM73, a mutant defective in tylosin synthesis, resulting in tylosin synthesis. The DNA responsible for this complementation was localized to a 10.5-kilobase EcoRI fragment. A 32-kilobase DNA segment which included the srmB spiramycin resistance gene and DNA which complemented the defect in strain PM73 were mutagenized in vivo with Tn10 carrying the gene for Nmr (which is expressed in Streptomyces spp.) or in vitro by insertional mutagenesis with a drug resistance gene (Nmr) cassette. When these mutagenized DNA segments were crossed into the S. ambofaciens chromosome, three mutant classes blocked in spiramycin synthesis were obtained. One mutant accumulated two precursors of spiramycin, platenolide I and platenolide II. Two mutants, when cofermented with the platenolide-accumulating mutant, produced spiramycin. Tylactone supplementation of these two mutants resulted in the synthesis of a group of compounds exhibiting antibiotic activity. Two other mutants failed to coferment with any of the other mutants or to respond to tylactone supplementation.     

Detection of human somatic cell structural gene mutations by two-dimensional electrophoresis

The feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two-dimensional approach for mutagenesis studies at the protein level.