Recipient DNA Research Articles

DNA gyrase is a host factor required for transposition of Tn5

We show that DNA gyrase is required for transposition of Tn5. Coumermycin, a potent inhibitor of DNA gyrase subunit B, inhibits transposition in a wild-type strain, but has no effect on strains carry ing a coumermycin-resistant allele in gyrB. In addition, strains containing a thermolabile subunit A of gyrase ( gyrA43) are defective for transposition at a nonpermissive temperature. The requirement for gyrase is due to a requirement for supercoiled DNA. We showed this by introducing into the gyrA43 strain a deletion of the gene encoding topoisomerase I. The introduction of the second mutation caused an increase in the superhelical density of DNA as well as an increase in the transposition frequency. This also implies that if the DNA is supercoiled there is no further requirement for gyrase. Experiments with coumermycin support this, because the drug does not inhibit transposition if the recipient DNA remains supercoiled. This indicates that if the DNA acting as recipient of the transposon is deficient in supercoils, it will be a poor substrate for transposition. We also describe a system in which a gene on a multicopy plasmid can be efficiently introduced into the Escherichia coli chromosome.

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis. I. Processing of B. pumilus and B. licheniformis DNA in B. subtilis.

Competent Bacillus subtilis cells were exposed to radioactive and density labeled donor DNA extracted from B. pumilus and B. licheniformis. The DNA from these strains hybridized with B. subtilis DNA in vitro at a rate of 24% and 11%, respectively. After entry the vast majority of heterologous DNA was found at the single-strand DNA position in CsCl gradients, and was gradually degraded during incubation. Much less donor DNA than expected from the hybridization values participated in the formation of the donor-recipient complex (DRC). By subjecting the heterologous DRC to sonication and alkaline CsCl gradient centrifugation, it was established that the DRC consisted of three components: (1) recipient DNA in which breakdown products of donor DNA were incorporated through DNA synthesis, (2) recipient DNA in which donor DNA was covalently integrated and (3) recipient DNA in which the donor moiety was not covalently integrated.

Specialized transduction with lambda plac5: dependence on recB.

Genetically disabled lambda plac5 transducing phage derivatives were used to study the recB dependence of recombination during specialized transduction. The frequency of transduction was normalized to colony-forming units, and the end product of recombination was monitored by scoring for addition and substitution transductants. When a chromosomal lac gene was the recipient DNA substrate molecule, both the normalized transduction frequency and the proportion of addition and substitution transductants showed essentially no recB dependence. There was a pronounced recB dependence for both normalized transduction frequency and recombination end product formation when F42 lac was the recipient DNA substrate. recB appears to have no significant role in the recombination that occurs between the two lac regions in an addition transductant. UV irradiation of the transducing phages increased the absolute level of both addition and substitution transductants obtained with a chromosomal lac gene but resulted in a considerable change in the relative frequency of addition versus substitution transductants.

Insertion sites and the terminal nucleotide sequences of the Tn4 transposon.

The nucleotide sequences at the ends of the Tn4 transposon (mercury spectinomycin and sulfonamide resistance) have been determined. They are inverted repeated sequences of 38 nucleotides with three mismatched base pairs. These sequences are strongly homologous with the terminal sequences of Tn501 (mercury resistance) but less so with those of Tn3 (ampicillin resistance). The Tn4 transposon generates pentanucleotide members (Tn3, Tn1000, Tn501, Tn551, IS2) with the exception of Tn1721 and bacteriophage Mu. Among the three Tn4 insertion sites examined here, two of them occurred near a nonanucleotide sequence in perfect homology with part of the terminal inverted-repeat sequence of Tn4 and the third insertion occurred near a sequence of partial homology to one end of Tn4. All three insertions were in the same orientation such that IRb is proximal to its homologous sequence on the recipient DNA.

Uptake and fate of bacteriophage phi W-14 DNA in competent Bacillus subtilis.

Phage phi W-14 DNA (in which one-half of the thymine residues are replaced by alpha-putrescinyl thymine) was taken up by competent Bacillus subtilis cells at a rate threefold higher than the rate of homologous DNA uptake. In contrast to other types of heterologous DNA, the amount of phi W-14 DNA taken up in 15 min exceeded the amount of homologous DNA taken up by a factor of two to three, as measured in terms of acid-precipitable material. The amount of phi W-14 DNA taken up was even greater than this analysis indicated if allowance was made for the fact that phi W-14 DNA was degraded more rapidly after uptake than homologous DNA. Competition experiments showed that the affinity of phi W-14 DNA for homologous DNA receptors was lower than the affinity of homologous DNA and was similar to the affinities of other types of heterologous DNA. The more rapid and more extensive uptake of phi W-14 DNA appeared to occur via receptors other than the receptors for homologous DNA, and these receptors (like those for homologous DNA) were an intrinsic property of competent cells. Uptake of phi W-14 DNA was affected by temperature, azide, EDTA, and chloramphenicol, as was uptake of homologous DNA. This was consistent with entry of both DNAs by means of active transport. After uptake, undegraded phi W-14 [3H]DNA was found in the cells in a single-stranded form, whereas a portion of the label was associated with recipient DNA, presumably as a result of incorporation of monomers resulting from degradation. Acetylation of the amino groups of the putrescine side chains in phi W-14 DNA decreased the affinity of this DNA for its receptors without affecting its ability to compete with homologous DNA.

Integration of Rous sarcoma virus DNA during transfection

We have investigated the organization and integration sites of Rous sarcoma virus (RSV) DNA in NIH 3T3 mouse cells transformed by transfection with unintegrated and integrated donor RSV DNAs. RSV DNAs of different cell lines transformed by unintegrated donor DNA were flanked by different cellular DNA sequences, indicating that RSV DNA integrates at multiple sites during transfection. The RSV genomes of cells transformed by transfection were colinear with unintegrated RSV DNA, except that deletions within the terminal repeat units of RSV DNA were detected in some cell lines. These results suggested that the terminal repeat sequences of RSV DNA did not necessarily provide a specific integration site for viral DNA during transfection. In addition, cell lines transformed by integrated RSV DNAs contained both the RSV genomes and flanking cellular sequences of the parental cell lines, indicating that integration of integrated viral DNA during transfection occurred by recombinational events within flanking cellular DNA sequences rather than at the terminal of viral DNA. Integration of RSV DNA during transfection thus appears to differ from integration of RSV DNA in virus-infected cells, where the terminal repeat units of viral DNA provide a highly specific integration site. Integration of donor DNA during transfection of NIH 3T3 cells instead appears to proceed by a pathway which is nonspecific for both donor and recipient DNA sequences.

Bacteriophage P1-mediated generalized transduction in Escherichia coli: Fate of transduced DNA in Rec + and RecA − recipients

P1 transducing particles labeled with 32P and 5-bromouracil were used to transduce [ 3H]thymidine-labeled Escherichia coli. Cesium chloride gradient analysis of DNA extracted from the cultures showed that about 7–15% of the 32P counts were associated with the recipient DNA in native and alkali-denatured samples from both Rec + and RecA − bacteria. At most, 9–15% of the associated 32P label could be released as DNA fragments larger than 2 × 10 5 daltons but only when Rec + recipient DNA and not when RecA − DNA was sheared or sonicated. The released 32P-labeled DNA equilibrated primarily in the heavy regions of CsCl gradients when samples were extracted during the first hour after transduction indicating that P1 transducing DNA can be integrated into bacterial DNA as double-stranded fragments. The size of the inserted DNA was estimated to be at least 5 × 10 6 daltons but substantially less than 6 × 10 7 daltons, the size of P1 transducing DNA. Such recombinant structures were not formed or were formed at reduced frequency in RecA − bacteria.

Restriction and modification in B. subtilis: the role of homology between donor and recipient DNA in transformation and transfection.

Non-modified DNAs from phages SPO2 and phi 105, and prophage DNAs extracted from lysogens carrying these phages, were used to transfect isogenic r+m+ B. subtilis recipients which were either non-lysogenic, or had been lysogenized with a homologous or a non-homologous phage. Restriction of transfecting phage and prophage DNA occurred in non-lysogenic recipients and in recipients lysogenic for a non-homologous phage. No effect of restriction was observed when phage or prophage DNA was used to transfect recipients carrying a homologous prophage. This is analogous to the absence of restriction in transformation and indicates that in B. subtilis the distinction between transforming and transforming and transfecting DNA is not made at the initial stages of DNA uptake and processing, but rather at later stages, where recognition of homologous regions in donor and recipient DNA plays an important role.

Transformation of Streptococcus pneumoniae with S. pneumoniae-lambda phage hybrid DNA: induction of deletions.

The genetic fate of a fragment of Streptococcus pneumoniae DNA cloned into a derivative of the Escherichia coli bacteriphage lambda has been studied in pneumococcal transformation. Transforming activity of this hybrid DNA is 8 times higher than standard S. pneumoniae DNA. Hybrid DNA is mutagenic for the recipient bacteria. Mutations are induced at a rate of 2 per 1000 transformation events. Most of these mutations are deletions adjacent to the cloned pneumococcal fragment, starting at or near its extremities and extending outside. The length of these deletions, estimated by genetic analysis or by gel electrophoresis of DNA fragments generated by restriction endonucleases, is quite variable, ranging from 150 base pairs to more than 1800 base pairs. Insertion of lambda DNA bas been detected in two large deletions by using DNAxDNA hybridization as a probe. This suggests that nonhomologous regions adjacent to the cloned fragment may be illegitimately integrated by the tranformation process. During the genetic analysis of these induced mutations we have observed that not only these deletions but also spontaneous deletions drastically increase recombination rates when present on donor DNA in transformation of neighboring markers. Such an effect is interpreted as partial exclusion of deletions from synapsis between donor and recipient DNA.

DNA sequence analysis of the transposon Tn3: Three genes and three sites involved in transposition of Tn3

The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid β-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.

Uptake of Homologous DNA into Nuclei of Seedlings and by Isolated Nuclei of a Higher Plant

Summary Uptake of homologous radioactively and density labelled donor DNA by plant seedlings (Matthiola incana) into the nuclei and by isolated nuclei in vitro has been followed. The recipient DNA has been characterised by caesium chloride density gradient centrifugation. Depending on the incubation time the incorporated DNA is recovered from the nuclear fraction in unaltered and macromolecular form (after 5 hours of incubation) or is partly integrated into the recipient genome resulting in a fraction with intermediate density (after 20 hours of incubation). No qualitative difference has been observed between DNA extracted from intact recipient plants and DNA extracted only from the nuclear fraction suggesting that processes altering the density of the donor DNA occur more or less inside the nucleus. Isolated nuclei of Matthiola incana which are shown to be morphologically stable and physiologically active incorporate exogenous DNA through their nuclear membrane. The incorporated DNA is not significantly degraded but is retained with unaltered molecular weight and buoyant density for at least 2 hours. The amount of foreign DNA per nucleus corresponds to the DNA content of a diploid Matthiola cell (3 pg).

Mechanisms of recombination by the RecBC and the RecF pathways following conjugation in Escherichia coli K12

The recombinational processes directed by the RecBC and the RecF pathways following conjugation in E. coli have been compared. The viable recombinant products of the RecF pathway show a higher incidence of mismatch correction, higher percentage of heterogeneous clones produced by single ex-conjugants and a much slower rate of integration and segregation compared to the RecBC pathway. There are reasons to suspect that the product of recB and recC genes may be necessary for conversion of the single stranded donor DNA in the zygote to double stranded DNA. Theoretical considerations suggest that an exchange involving only one strand of DNA may be a much slower process, with more stringent homology requirement for the entire exchanged segment, than a double strand exchange of a comparable length; the latter should be much faster, with stringent homology requirements for only the terminal regions of the exchanged segments. It is suggested that the RecF pathway mainly mediates replacement of relatively long stretches of single strands of recipient DNA by the corresponding strands of donor DNA while the RecBC pathway mediates exchange of mostly double stranded DNA between the donor and the recipient; in addition, the RecBC pathway may also catalyze the integration of very small segments of single strands of the donor DNA. A model based on the above basic hypothesis is described. It is further suggested that the enzymes exonucleaseV and exonucleaseI Control the relative yields of the recombinants produced by the two pathways by regulating the supply of the donor substrates required by these pathways; the former diverts the potential substrate of the RecF pathway (single stranded DNA) to the duplex substrates of the RecBC pathway while the latter destroys the substrates of the RecF pathway, especially in absence of exonucleaseV.

Nucleotide sequence at the insertion sites of a kanamycin transposon.

THE TRANSPOSON Tn903 carrying a gene for kanamycin resistance, is 3,100 base pairs in size and contains an inverted repeat sequence of 1,050 base pairs at both ends1–4. We report here the generation of a 9-base pair repeated sequence at the insertion site of Tn903. Tn903 can be transposed to at least nine different sites on the coliphage fd DNA1,2. We have also transposed Tn903 to three sites on small colicin E1 plasmid (ColE1) derivatives of about 1,600 base pairs. The presence of these many insertion sites on small recipient DNA molecules implies that no specific sequence is involved in the target sites on the recipient. To investigate the mechanism of transposition of Tn903 at the molecular level, we have now analysed three independent insertions of Tn903 on the small ColE1 DNA molecules. The nucleotide sequences of the three target sites and of the corresponding junctions between Tn903 and the small ColE1 have been determined.

Hyper-recombination in Escherichia coli K-12 mutants constitutive for protein X synthesis

Genetic recombination was studied in Escherichia coli F- strains in which synthesis of the recA gene product protein X is increased due to mutation in either recA (tif-1) or lexA (spr). When a single donor marker was selected, the recombination proficiency of these strains was not significantly altered in Hfr crosses. However, linkage of unselected, proximal Hfr markers was found to be much reduced among the progeny tested, and more of the progeny showed evidence of multiple exchanges between donor and recipient DNA. These effects were much more apparent when the recipient carried both tif-1 and spr mutations, but in this case recombination proficiency was reduced compared with those strains carrying either mutation alone, particularly in crosses with Hfr Cavalli. A lexA mutation was found to suppress the effect of tif-1 on the recombinant genotype.

Fate of heterospecific transforming DNA bound to Streptococcus sanguis.

The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.

Genetic fate of DNA in a strain of Bacillus subtilis which is impaired in genetic transformation

A mutation in Bacillus subtilis call recC4 which results in an impairment of genetic transformation was transferred to a new strain using the closely linked marker mit-2 (mitomycin C-resistance) for selection. This derived strain was in turn impaired in transformation but showed normal levels of sensitivity to ultraviolet irradiation and methyl methane sulfonate. The genetic and molecular fate of transforming DNA in the recC4 strain was studied. Normal amounts of DNA were taken up by the cells and this DNA or parts of it became associated with recipient DNA. Linkage between genes on donor and recipient molecules was, however, not established and transformants were not generated. The recC4 mutation therefore affects a step in the recombination pathway during transformation. Either the association between donor and recipient DNA molecules is abnormal or the cells are deficient in the further processing of the associated complex.

Fate of homospecific transforming DNA bound to Streptococcus sanguis.

The fate of [3H]DNA from Streptococcus sanguis str-r43 fus-s donors in [14C]S. sanguis str-s fus-r1 recipients was studied by examining the lysates prepared from such recipients at various times after 1 min of exposure to DNA. The lysates were analyzed in CsCl and 10 to 30% sucrose gradients; fractions from the gradients were tested for biological activity and sensitivity to nucleases, subjected to various treatments and retested for nuclease sensitivity, and run on 5 to 20% neutral and alkaline sucrose gradients. The results demonstrate that donor DNA bound to S. sanguis cells in a form resistant to exogenous deoxyribonuclease is initially single stranded and complexed to recipient material. Donor DNA can be removed from the complex upon treatment of the complex with Pronase, phenol, or isoamyl alcohol-chloroform. Within the complex, donor DNA is relatively insensitive to S1 endonuclease but can regain its sensitivity by treatment with phenol. With time the complex moves as a whole to associate physically with the recipient chromosome. After a noncovalent stage of synapsis, donor material is covalently bonded to and acquires the nuclease sensitivity of recipient DNA, while donor markers regain transforming activity and become linked to resident markers.

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis.

In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5(1), 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that basepairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation.

Uptake and early fate of metaphase chromosomes ingested by the Wi-L2 human lymphoid cell line.

Aspects of the ingestion and early intracellular fate of homologous. [3H]-thymidine-labeled chromosomes (donor) were studied in recipient Wi-L2 cells in the absence of reutilized radioactivity. As much as 67% of the cell-associated radioactivity was resistant to hydrolysis by DNase I after 4 h of incubation. Cell fractionation and electron microscope autoradiography indicated that chromosome uptake was rapid, into both cytoplasmic and nuclear fractions and was facilitator and dose dependent. Sedimentation analysis demonstrated that at 4 h donor DNA of approximate single-strand mol wt of 1--6 X 10(6), as compared to 6--12 X 10(6) for chromosomal DNA, was recoverable in cell fractions. By 6 h, a significant portion of the nucleus-associated donor DNA was converted into material of higher mol wt, although no evidence was found for integration into recipient DNA. Cytoplasmic donor DNA continued to be degraded. An average number of chromosome equivalents of nucleus-associated donor DNA to recipient cell nuclei of 1--4 was obtained and its relationship to the lower frequency of chromosome-mediated gene transfer is discussed.

The shape of the ultraviolet inactivation curve for transforming DNA

THE very unusual inverse square kinetics of ultraviolet inactivation of transforming DNA were first reported by Rupert and Goodgal1, and subsequently there was speculation that these kinetics result from blocks to recombination formed by ultraviolet lesions in the DNA2 (ref. 2). We show here that when the recipient cell lacks both excision arid postreplication repair, the ultraviolet inactivation of transforming DNA becomes a simple exponential rather than an inverse square relation. The data suggest that the square root kinetics observed with repair-proficient recipient cells result from repair processes themselves rather than from the recombination between irradiated transforming DNA and recipient DNA.