Characteristics of RecA-independent recombination of plasmids in E. coli cells producing restriction endonuclease EcoRI

The restriction endonuclease EcoRI dependent recombination of compatible plasmids has been studied in RecA cells of Escherichia coli. Plasmid RP4 and the isogenic ColE1 type plasmids pSA14 or pSA25, differing in restriction-modification RM EcoRI genes, have been used to study this type of recombination. EcoRI dependent recombination of plasmids is demonstrated in RecA cells and, thus, is independent of general system of homologous recombination. The classes of recombinant plasmids isolated from RecA cells differ from the classes isolated from wild type cells. Levels of tetracycline resistance conferred by plasmid RP4 are shown to be dependent on the alleles of RecA+ gene, being extremely low in RecA cells. This property is demonstrated to be useful for obtaining the multicopy recombinant plasmids resulting from EcoRI dependent recombination in RecA cells of Escherichia coli.     

In vivo restriction of Escherichia coli-transforming DNA by endonuclease R.M.EcoRI

Transformation of Escherichia coli K-12 for various chromosomal markers was accomplished by using AB1157 recBC+ strain as a recipient. The yield of transformants was reduced 10-fold, as compared with that obtained in JC7623 recBC sbcB recipient. Elimination of transformation has been obtained for arg, pro, his markers in AB1157 (pSA14) harbouring the R.M.EcoRI coding plasmid. Production of restriction endonuclease in this strain did not affect the efficiency of transformation for thr, leu markers. The presence of pSA25 which is isogenic to pSA14 but devoid of R.M.EcoRI genes has been irrelevant to transformation for leu, arg, pro, his, thr markers. Correlation between the restriction of transformed markers in vivo and in vitro is discussed.     

Cleavage of single stranded oligonucleotides by EcoRI restriction endonuclease.

The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied. It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively. The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower. To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E. coli DNA polymerase. EcoRI cleaved this immobilized oligomer into specific fragments.     

Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.     

High level secretion of TaqI restriction endonuclease by recombinant Escherichia coli

The production and high level secretion of TaqI restriction endonuclease using bacterial secretion signal within the malE gene was achieved by cloning the PCR-amplified gene from Thermus aquaticus into E. coli. The maltose binding protein (MBP) part of the MBP-TaqI fusion protein expressed by this construct did not interfere with the biological activity of the TaqI restriction endonuclease. E. coli XL1 carrying pH185 produced 332 U ml^–1 TaqI endonuclease 81% of which was secreted into the medium without apparent cell lysis. Optimization of culture conditions and selection of the host strain were found to be important for the efficient extracellular production of this protein.     

Cloned truncated recA genes in E. coli II. Effects of truncated gene products on in vivo recA+ protein activity

The ability of plasmids carrying truncated recA genes to sensitize recA+ cells to UV-irradiation was dependent upon the size of the cloned recA gene fragment. Radiosensitization correlated with the inhibition of recombinational repair, and the in vivo reduction of recA protein recombinase activity, as measured by lambda bio 11 plating efficiency. W-reactivation was also abolished by the radiosensitizing plasmids, whilst DNA degradation control, naladixic acid induced filamentation and lambda induction were unaffected. UV-induced mutagenesis in excision proficient E. coli was unaffected, whilst excision deficient strains were hypermutable. It is suggested that these effects of plasmids bearing 22% or more of the recA gene are the result of the interaction of full-sized and truncated protein subunits to generate multimers unable to catalyze recombination.     

The structures and fidelity of replication of mouse mitochondrial DNA-pSC101 EcoRI recombinant plasmids grown in E. coli K12

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (LA9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs.Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments.A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165±10 nucleotide pairs.     

A DNA sequence cleaved by restriction endonuclease R.EcoRI in only one strand.

Restriction endonuclease EcoRI cuts both strands of the DNA sequence

generating two separate frayed ends (Hedgpeth et al., 1972). Here it is shown that under standard digestion conditions, the enzyme also attacks the sequence

but cuts only one strand. The resulting nick is an efficient initiation point for DNA synthesis by Escherichia coli DNA polymerase I, allowing the selective labelling of one strand of the DNA duplex.In buffers of low molarity and high pH (8.5), EcoRI cleaves sequences with the form

(Polisky et al., 1975). Thus it seems that under both sets of conditions the enzyme recognises the four-base-pair core sequence

and that its ability to cleave different adjacent phosphodiester bonds varies with pH and ionic strength.     

Specific recombination in vitro promoted by the restriction endonuclease HgaI.

We describe the use of the restriction endonuclease HgaI from Haemophilus gallinarum for the efficient construction in vitro of recombinant molecules. Using bacteriophage f 1 DNA, we show that only HgaI fragments that were originally adjacent on the genome are ligated, that upon ligation infectious molecules are reassembled with high efficiency, and that recombinant genomes can thus be easily constructed. The method relies upon the unique properties of HgaI and is applicable to any viral or plasmid DNA that contains several HgaI recognition sites.     

Activity of restriction endonuclease Sso II in Escherichia coli strains transformed by Shigella sonnei 47 plasmids

The inverse dependence of activity of restriction endonuclease SsoII preparations on the number of low molecular mass plasmids of Shigella sonnei transforming Escherichia coli recipient cells producing the enzyme has been shown. Escherichia coli strain producing efficiently one of two Shigella sonnei 47 restriction endonucleases SsoII has been isolated. The producer strain harbours two of the nine Shigella sonnei 47 plasmids. One of them P4 codes for SsoII+ phenotype while the other P9 determines the plasmids conjugation transfer. Biochemical and physiological characteristics of the producer strain XS13 are identical to the ones of the recipient Escherichia coli strain PS200. XS13 is unable to induce keratoconjunctivitis in guinea pigs in pathogenicity test.     

In situ distribution of EcoRI methylase and restriction endonuclease in cells of Escherichia coli Bs 5

Specific IgG antibodies were raised in rabbits against purified EcoRI methylase and restriction endonuclease. Post embedding labeling experiments, using the protein A-gold technique, were made with paraformaldehyde-glutaraldehyde fixed cells, embedded in Lowicryl K4M resin at low temperatures. Labeling with methylase-specific antibodies showed 60-70% of gold particles in the cytoplasm and 30-40% at the cell envelope, whereas the use of restriction enzyme-specific antibodies led to a distribution of 10-30% in the cytoplasm and 70-90% in the cell envelope. The results coincide with the proposed function of the enzymes: in the cytoplasm methylase protects the cells' own DNA from self-destruction, and the restriction endonuclease cuts foreign DNA when entering the cell.     

Characterization of SmSu plasmids by restriction endonuclease cleavage and compatibility testing.

Twelve plasmids carrying genes for streptomycin and sulfonamide resistance were studied for the number and distribution of sites on the plasmid moleucles susceptible to cleavage by the restriction endonuclease EcoRI. Ten of the twelve were found to have a single cut site, one plasmid (R678) had three such sites, and plasmid PB165, which was isolated as three supercoiled deoxyribonucleic acid species with molecular weights 7.4 x 10(6), 14.7 x 10(6), and 21.4 x 10(6) was reduced to a single (linear) species of molecular weight 7.6 x 10(6) after cutting with EcoRI. We conclude that PB165 forms oligomers in Escherichia coli and that the number of copies of these per chromosome is more consistant and that the number of copies of these per chromosome is more consistent with a negative than a positive control mechanism for plasmid replication. Compatibility testing of a positive control mechanism for plasmid replication. Compatibility testing of these plasmids showed they all belong to the same incompatibility group, which we designate IncQ, suggesting that they may have come from a common ancestor.     

Polypeptide sequences involved in the cleavage of DNA by the restriction endonuclease EcoRI

We have prepared a variety of fragments of the restriction endonuclease EcoRI by partial or total CNBr or acid cleavage of the protein. These fragments were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were analyzed in a qualitative manner for phosphodiesterase activity. Antibodies against these fragments were elicited in rats and tested for binding to native EcoRI in an enzyme-linked immunoassay. We conclude from these experiments that the DNA binding site of EcoRI is located in the COOH-terminal half of the molecule, close to and probably comprising amino acid residues 137 to 157. This conclusion is reinforced by the observation that this sequence shows homology to the sequences of the recognition helix of other gene-regulatory proteins.     

Improvement in segregational stability of recombinant plasmids by retransformation of E. coli host cells

Summary Strains of E. coli JM103 harboring recombinant pUC8 plasmids were found to exhibit severe segregational instability in the absence of antibiotic selection. This was reversed by retransformation into fresh JM103. pUC8 was considerably more stable than its recombinant derivatives.     

Plasmid recombination by the RecBCD pathway of Escherichia coli.

Previously, we demonstrated that exonuclease I-deficient strains of Escherichia coli accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids carrying the chi sequence (5'- GCTGGTGG-3') (M. M. Zaman and T. C. Boles, J. Bacteriol. 176:5093-5100, 1994). Since high-molecular weight linear DNA is believed to be the natural substrate for RecBCD-mediated recombination during conjugation (A. J. Clark and K. B. Low, p. 155-215, in K. B. Low, ed., The Recombination of Genetic Material, 1988), we analyzed the recombination frequencies of chi+ and chi0 plasmids in sbcB strains. Here, we report that chi sites stimulate plasmid recombination frequency by 16-fold in sbcB strains. Chi-stimulated plasmid recombination is dependent on RecBCD but is independent of RecF pathway genes. The distribution of recombination products suggests that high-molecular-weight linear plasmid DNA is a substrate for RecBCD-mediated recombination. Surprisingly, our data also suggest that chi+ plasmids also recombine by the RecBCD pathway in rec+ sbcB+ cells.     

Negative complementation of recA protein by recA1 polypeptide: in vivo recombination requires a multimeric form of recA protein

Summary Recombination in vivo was studied in recA ^- heterozygous lacZ merodiploids by performing -galactosidase assays after infection with precA ⁺. Recombination as measured by -galactosidase production was a linear function of pecA ⁺ multiplicity of infection (MOI) when the strain contained a deletion of the chromosomal recA gene. However, when the strain carried a recA1 missense allele, a higher precA ⁺ MOI was required to obtain levels of recombination comparable to the (recA) strain, and the slope of the dose-response curve increased to approximately two. It is proposed that negative complementation occurs in mixed tetramers of wild-type and missense recA polypeptides, and that in vivo recombination is a property of a multimeric form of recA protein.     

The 'endo-blue method' for direct cloning of restriction endonuclease genes in E. coli.

A new E. coli strain has been constructed that contains the dinD1::LacZ+ fusion and is deficient in methylation-dependent restriction systems (McrA-, McrBC-, Mrr-). This strain has been used to clone restriction endonuclease genes directly into E. coli. When E. coli cells are not fully protected by the cognate methylase, the restriction enzyme damages the DNA in vivo and induces the SOS response. The SOS-induced cells form blue colonies on indicator plates containing X-gal. Using this method the genes coding for the thermostable restriction enzymes Taql (5'TCGA3') and Tth111l (5'GACNNNGTC3') have been successfully cloned in E. coli. The new strain will be useful to clone other genes involved in DNA metabolism.     

Kinetics of recA function in conjugational recombinant formation.

Summary Genetic recombination was studied in F^- strains of E. coli carrying a mutation (recA200) that confers a thermosensitive Rec^- phenotype. Recombination during Hfr matings at 35C was monitored by raising the temperature of incubation to 42C at various intervals so that only merozygotes that had completed those functions dependent on the activity of the recA gene product could form recombinant progeny. The results indicated that no more than 1–2% of the merozygotes present while mating was in progress were able to form recombinant colonies at 42C. Separation of mating pairs reduced the yield of recombinants obtained at 35C by 50 to 200-fold if plating on agar medium was delayed for 15–30min by continuing incubation in broth medium. recA200 merozygotes that were also recB21 sbcB15 proved relatively stable when plating was delayed in this manner, which suggested that Hfr DNA is prone to exonuclease inactivation in recA200 merozygotes after mating pairs have separated. Post-mating incubation in high salt medium or on agar plates promoted the recovery of recombinants at 35C. However, the majority of recA200 merozygotes did not acquire the ability to form recombinant colonies at 42C under these more stable conditions until mating pairs had been separated and incubation continued at 35C for 40–60 min. It was concluded that recA200 strains are partially defective for recombination even at low temperature but that terminating mating promotes the recovery of recombinants. A mechanism involving the stimulation of RecA activity by mating pair separation is postulated to account for the efficient recovery of recombinants from HfrxF^- recA200 crosses at 35C.