Site-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion.

DpnI can cleave fully methylated parental DNA while leaving hemi-methylated DNA intact. Based on this observation, we developed a rapid site-directed mutagenesis method using uracil-containing, double-stranded (ds)DNA templates and DpnI digestion. A 38% mutation efficiency was achieved by DpnI treatment of the mutagenic strand-extension reaction, and it increased to 70%-91% when uracil-containing dsDNA templates were used. This method compares favorably to the most efficient current methods, but is simpler and does not require the use of single-stranded templates or phage vectors.     

Site-directed mutagenesis in the Escherichia coli recA gene

Escherichia coli RecA protein plays a fundamental role in genetic recombination and in regulation and expression of the SOS response. We have constructed 6 mutants in the recA gene by site-directed mutagenesis, 5 of which were located in the vicinity of the recA430 mutation responsible for a coprotease deficient phenotype and one which was at the Tyr 264 site. We have analysed the capacity of these mutants to accomplish recombination and to express SOS functions. Our results suggest that the region including amino acid 204 and at least 7 amino acids downstream interacts not only with LexA protein but also with ATP. In addition, the mutation at Tyr 264 shows that this amino acid is essential for RecA activities in vivo, probably because of its involvement in an ATP binding site, as previously shown in vitro.     

Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells

Plasmid DNA was used to study gamma-radiation-induced recombination and mutagenesis in Escherichia coli host cells. Plasmid pBRP1, a derivative of pBR322 containing the lac operon of E. coli, was irradiated with 60Co gamma rays prior to transformation into E. coli strains of different recA and lac genotypes. Plasmid-chromosome recombination was assayed in lacY1 host cells, whereas plasmid mutagenesis was assayed in delta lac host cells lacking chromosomal sequences homologous to the plasmid. Both recombinant and mutant plasmids were identified by the phenotypic changes in lactose utilization, and confirmed by restriction analysis of isolated plasmids. Plasmid-chromosome recombination was induced to high levels (about 20% of survivors at 700 Gy) and was dependent on the host recA gene. Plasmid mutagenesis occurred at lower levels (about 1.5% of survivors at 600 Gy) and was relatively independent of the recA gene. Plasmid survival was unaffected by the presence or absence of host recA mutations or the potential for plasmid-chromosome recombination.     

DNA cloning by homologous recombination in Escherichia coli

The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination, for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.     

A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation

Summary A technique is presented by which mutations can be introduced into the Escherichia coli chromosome by gene replacement between the chromosome and a plasmid carrying the mutant gene. The segregational instability of plasmids in E. coli is used with high efficiency to isolate E. coli mutants. The method should be applicable to construction of mutants for any E. coli chromosomal gene provided it is dispensable, and for any E. coli strain provided it is capable of homologous recombination. The use of the method was demonstrated by constructing E. coli mutants for the glycogen branching enzyme gene (glgB) and the -galactosidase gene (lacZ). The results show that recombination occurs via a reciprocal mechanism indicating that the method should, in a slightly modified form, also be useful in transferring chromosomal mutations onto multicopy plasmids in vivo.     

Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase

A new vector for the expression of phosphofructokinase (pfk-1) was constructed with pEMBL, which allows reliable, inducible, high-expression, and facile mutagenesis of the gene. Two mutants in the effector site of the enzyme were produced by site-specific mutagenesis of residue Tyr-55 to assess the role of its side chain in binding an allosteric inhibitor, phosphoenolpyruvate (PEP), and an activator, guanosine 5'-diphosphate (GDP): Tyr-55----Phe-55 and Try-55----Gly-55. The dissociation constant of PEP from the T state is unaffected by the mutations. Mutation of Tyr-55----Phe-55 only slightly increases the dissociation constant of GDP from the R state, indicating a minimal involvement of the hydroxyl group in binding. A 5.5-fold increase in the dissociation constant of GDP on the mutation of Tyr-55----Gly-55 suggests a small hydrophobic interaction of the aromatic ring of the tyrosine residue with guanine of GDP.     

Site-directed mutagenesis of the lipoate acetyltransferase of Escherichia coli.

Remote but significant similarities between the primary and predicted secondary structures of the chloramphenicol acetyltransferases (CAT) and lipoate acyltransferase subunits (LAT, E2) of the 2-oxo acid dehydrogenase complexes, have suggested that both types of enzyme may use similar catalytic mechanisms. Multiple sequence alignments for CAT and LAT have highlighted two conserved motifs that contain the active-site histidine and serine residues of CAT. Site-directed replacement of Ser550 in the E2p subunit (LAT) of the pyruvate dehydrogenase complex of Escherichia coli, deemed to be equivalent to the active-site Ser148 of CAT, supported the CAT-based model of LAT catalysis. The effects of other substitutions were also consistent with the predicted similarity in catalytic mechanism although specific details of active-site geometry may not be conserved.     

Biochemistry of homologous recombination in Escherichia coli.

Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.     

Reciprocal and non-reciprocal homologous recombination between Escherichia coli chromosomal DNA and ultraviolet light-irradiated plasmid DNA

Plasmid DNA substrates were used to study ultraviolet (UV)-induced recombination events in Escherichia coli host cells. Plasmids derived from pBR322, containing all or part of the lac operon of E. coli, were irradiated with ultraviolet light before transformation into E. coli strains of different recA and lacY genotypes. Recombinational exchanges were identified by phenotypic changes in lactose utilization and were confirmed by restriction analysis of isolated plasmids. Ultraviolet-induced reciprocal plasmid-chromosome recombination occurred at a slightly higher frequency then non-reciprocal chromosome-to-plasmid recombination, and at a much higher frequency than non-reciprocal plasmid-to-chromosome recombination. These frequencies did not depend on segregative mechanisms. The asymmetry of non-reciprocal exchange was not due to the particular arrangement of wild-type and lacY1 alleles because the same results were observed when these were interchanged. The host recA gene was required for plasmid-chromosome recombination, and slightly enhanced plasmid survival. Evidence for plasmid replication prior to recombination was found in reciprocal recombinants, but rarely in the non-reciprocal recombinants analyzed. Irradiation of competent bacterial host cells prior to transformation did not effectively induce plasmid-chromosome recombination.     

Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme

Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.     

Site-directed mutagenesis of Pro327 in the lac permease of Escherichia coli

By use of oligonucleotide-directed, site-specific mutagenesis, Pro327 in the lac permease of Escherichia coli has been replaced with Ala, Gly, or Leu. Permease with Ala at position 327 catalyzes lactose/H+ symport in a manner indistinguishable from wild-type permease. Permease with Gly at position 327, on the other hand, exhibits about one-tenth the activity of wild-type permease but catalyzes lactose accumulation to essentially the same steady-state level as wild-type permease. Finally, permease with Leu at position 327 is completely inactive. The results demonstrate that there is no relationship between permease activity and the helix-breaking (Pro and Gly) or helix-making (Ala and Leu) properties of the residue at position 327. It is suggested that it is primarily a chemical property of the side chain at position 327 (i.e., bulk, hydropathy, and/or ability to hydrogen bond) that is critical for activity and that neither cis/trans isomerization of Pro327 nor the presence of a kink at this position is important.     

rec genes and homologous recombination proteins in Escherichia coli

The twenty-five years since the first published report of recA mutants in Escherichia coli has seen the identification of more than 12 other recombination genes. The genes are usually grouped into three pathways named RecBCD, RecE and RecF for prominent genes which function in each. A proposal is made here that there are two RecF pathways, one sensitive and one resistant to exonuclease I, the SbcB enzyme. Five methods of grouping the genes functionally are discussed: 1) by enzyme activity, 2) by common indirect suppressor, 3) by common phenotype, 4) by common regulation and 5) by epistasis. Five classes of enzyme activities implicated in recombination are discussed according to their involvement in presynapsis, synapsis or postsynapsis: 1) nucleases 2) helicases 3) DNA-binding proteins 4) topoisomerases and 5) ligases. Plausible presynaptic steps for the RecBCD, RecF (SbcBS) and RecE pathways show the common feature of generating 3'-terminated single-stranded DNA (ssDNA). On this ssDNA it is proposed that a RecA protein filament is generated discontinuously. This implies the existence of nucleation and possibly measurement and 3' end protection proteins. Specific proposals are made for which recombination genes might encode such products. Finally the generality of the RecA-ssDNA-filament mechanism of synapsis in the cellular biological world is discussed.     

Site-directed mutagenesis of tyrosine residues in the lac permease of Escherichia coli

By using oligonucleotide-directed, site-specific mutagenesis, each of the 14 Tyr residues in the lac permease of Escherichia coli was replaced with Phe, and the activity of each mutant was studied with respect to active transport, equilibrium exchange, and efflux. Ten of the mutations have no significant effect on permease activity. Of the four mutations that alter activity, replacement of Tyr26 or Tyr336 with Phe severely decreases all modes of translocation, and the binding affinity of the mutant permease for p-nitrophenyl alpha-D-galactopyranoside is markedly decreased (i.e., KD is increased). In addition, the Phe336 mutant permease is inserted into the membrane to a lesser extent than wild-type permease, as judged by immunoblot experiments. Permease containing Phe in place of Tyr236 catalyzes lactose exchange approximately 40% as well as wild-type permease but does not catalyze active transport or efflux. Finally, permease with Phe in place of Tyr382 catalyzes equilibrium exchange normally, but exhibits low rates of active transport and efflux without being uncoupled, thereby suggesting that replacement of Tyr382 with Phe alters a kinetic step involving translocation of the unloaded permease across the membrane.     

Site-directed mutagenesis of the ferric uptake regulation gene of Escherichia coli

The 12 histidine and four cysteine residues of the Fur repressor of Escherichia coli were changed, respectively, to leucine and serine by site-directed mutagenesis of the fur gene. The affects of these mutations were measured in vivo by ligation of the mutated genes to a wild-type fur promoter followed by measurement of the ability of these plasmids to regulate expression of a lacZ fusion in the aerobactin operon. In vitro affects were assayed by insertion of the mutated genes in the expression vector pMON2064 attended by isolation of the altered Fur proteins and appraisal of their capacity to bind to operator DNA. The results suggest that cysteine residues at positions 92 and 95 are important for the activity of the Fur protein.     

Site-directed mutagenesis and generation of chimeric viruses by homologous recombination in yeast to facilitate analysis of plant-virus interactions

A yeast homologous recombination system was used to generate mutants and chimeras in the genome of Potato leafroll virus (PLRV). A yeast-bacteria shuttle vector was developed that allows mutants and chimeras generated in yeast to be transformed into Escherichia coli for confirmation of the mutations and transformed into Agrobacterium tumefaciens to facilitate agroinfection of plants by the mutant PLRV genomes. The advantages of the system include the high frequency of recovered mutants generated by yeast homologous recombination, the ability to generate over 20 mutants and chimeras using only two restriction endonuclease sites, the ability to introduce multiple additional sequences using three and four DNA fragments, and the mobilization of the same plasmid from yeast to E. coli, A. tumefaciens, and plants. The wild-type PLRV genome showed no loss of virulence after sequential propagation in yeast, E. coli, and A. tumefaciens. Moreover, many PLRV clones with mutations generated in the capsid protein and readthrough domain of the capsid protein replicated and moved throughout plants. This approach will facilitate the analysis of plant-virus interactions of in vivo-generated mutants for many plant viruses, especially those not transmissible mechanically to plants.     

大肠杆菌glgC基因的定点突变及功能分析

摘要: 利用PCR从Escherichia coli JM109基因组中扩增到全长为1 296 bp的glgC基因编码区, 通过PCR重组方法进行点突变, 获得氨基酸突变的3个突变体, 分别是Pro295Ser(Val121Ala, Met151Ile和Val334Asp)、Gly336 Asp单点突变和Pro295Ser/Gly336Asp(Lys109Arg), 其基因分别命名为295⁺³、336和295/ 336⁺¹。将突变和未突变的基因分别克隆到pET32-a, 构建重组质粒pET-glgC、pET-295⁺³、pET-336和pET-295/ 336+1, 在文中分别简称为a、b、c和d。转化大肠杆菌BL21(DE3), 在1 mmol/L IPTG 诱导下表达。SDS- PAGE 电泳分析显示, 在约67 kDa 处有1条明显与预期大小一致的蛋白质, 表明目的基因已得到融合表达。上述转化子的碘染和糖原含量测定结果, 第336位的Gly变成Asp后, 宿主菌的糖原含量提高; Pro295Ser/Gly336Asp(Lys109Arg)的突变导致宿主菌的糖原含量与Gly336Asp突变体相近, 表明在336突变基因的基础上增加Pro295Ser的突变没有进一步加大宿主菌中AGPase酶的反馈抑制效应的降低。已有的结果显示, Pro295Ser可以降低AGPase酶的反馈抑制效应活性, 而实验中295⁺³突变基因转入宿主菌后细胞糖原含量明显降低, 推测这个结果可能是295⁺³中的Val334Asp的突变造成, 而334位的氨基酸可能是AGPase功能域中的一个重要位点。     

Site-directed mutagenesis in Escherichia coli of a stable R772::Ti cointegrate plasmid from Agrobacterium tumefaciens

The host range of an octopine Ti plasmid is limited to Rhizobiaceae. This has been extended also to Escherichia coli in the form of a stable cointegrate with the wide-host-range plasmid R772. Its structure was studied by constructing a physical map of R772 and of the R772::pTiB6 cointegrate. An insertion sequence present in R772, called IS70, turned out to be involved in cointegrate formation. We found one intact copy of IS70 and a small segment of IS70, respectively, at the junctions of R772 and Ti DNA. The absence of a complete second copy of IS70 is a likely explanation for the stability of the cointegrate plasmid. A procedure for site-directed mutagenesis of this cointegrate plasmid in E. coli is described. The effect of mutations in the Ti plasmid part can be studied subsequently by transferring the cointegrate into Agrobacterium tumefaciens. The advantage of this procedure for Ti plasmids over other methods used at present is discussed.     

RecBCD- RecFOR-independent pathway of homologous recombination in Escherichia coli

The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3′-5′ exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3′ overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.     

Enzymatic control of homologous recombination and hyperrecombination in Escherichia coli

The RecA protein is a central homologous recombination enzyme in the bacterial cell. Forming a right-handed filament on ssDNA, RecA provides for a homology search between two DNA molecules and homologous strand exchange. RecA protects the cell from ionizing radiation and UV light and is capable of completing recombination during normal cell growth. Several mutant and natural RecA forms have a higher recombination potential in vitro and in vivo as compared with the wild-type Escherichia coli RecA, causing hyperrecombination. Recombinational hyperactivity of RecA depends to a great extent on the filamentation dynamics and DNA transferase properties, which may depend not only on specific amino acid substitutions in RecA, but also by defects in cell enzymatic machinery, including RecO, RecR, RecF, RecX, DinI, SSB, and PsiB. The functions of these proteins are currently known at the molecular level, while their roles in hyperrecombination are still incompletely understood. An increase in recombination in vivo is not always advantageous for the cell and is therefore limited by various mechanisms. In addition to the limitations imposed by cell enzymatic machinery, genomic rearrangements aimed at inhibiting the expression of hyperactive RecA are fixed through cell generations via selection against hyperrecombination. The mechanisms regulating hyperactive RecA forms in several model systems are considered.