Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences

Summary Lysates obtained shortly after entry of transforming DNA to Bacillus subtilis contain donor-recipient DNA complexes, in which the donor moiety is associated with the recipient DNA in an unstable way. The complexes could be artificially stabilized by crosslinking with 4,5,8-trimethylpsoralen. The unstable complexes dissociated upon helix-destabilizing treatments, such as heating at 70°C, and CsCl gradient centrifugation at pH 11.2, but remained stable during CsCl gradient centrifugation at pH 10. Donor-recipient DNA complexes were not formed after entry of heterologous pUB110 DNA. These observations suggest that base-pairing is involved in the unstable association. The donor moiety of the unstable complexes was completely, or almost completely, digestible by nuclease S₁, indicating that the donor and recipient base-sequences are only paired over very short distances.The unstable donor-recipient DNA complexes are true recombination intermediates because (i) strain 7G224 (recE4) was impaired in the formation of the unstable complexes, and (ii) the unstable complexes were rapidly converted to stable complexes in recombination proficient strains, whereas their conversion was delayed in the recombination deficient strain 7G84.Unstable complexes were also formed with Escherichia coli donor DNA, but to a lesser extent. Apparently a limited degree of base-sequence homology is sufficient to initiate recombination.     

Unstable association in vitro between donor and recipient DNA in Bacillus subtilis.

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.     

Assimilation of single-stranded donor deoxyribonucleic acid fragments by nucleoids of competent cultures of Bacillus subtilis.

Lysates containing folded chromosomes of competent Bacillus subtilis were prepared. The chromosomes were supercoiled, as indicated by the biphasic response of their sedimentation rates to increasing concentrations of ethidium bromide. Limited incubation of the lysates with increasing concentrations of ribonucleases resulted in a gradual decrease in the sedimentation velocity of the deoxyribonucleic acid (DNA) until finally a constant S value was reached. Incubation with sonicated, 4,5',8-trimethylpsoralen-monoadducted, denatured, homologous donor DNA molecules at 37 degrees C and concomitant irradiation with long-wave ultraviolet light of the nucleoid-containing lysates resulted in the formation of complexes of the donor DNA molecules and the recipient chromosomes. This complex formation was stimulated when nucleoids were previously (i) unfolded by ribonuclease incubation, (ii) (partially) relaxed by X irradiation, or (iii) subjected to both treatments. Monoadducts were not essential. On the other hand, the complex-forming capacity of recipient chromosomes previously cross-linked by 4,5',8-trimethylpsoralen diadducts was greatly reduced, suggesting that strand separation of the recipient molecule was involved in the formation of the complex. None of these effects has been observed when heterologous (Escherichia coli) donor DNA has been used. When the same kind of experiments were carried out at 70 degrees C, donor-recipient DNA complexes were also formed and required strand separation and homology similar to donor-recipient complex formation at 37 degrees C. However, in contrast to what was found at 37 degrees C, unfolding plus relaxation of the nucleoids, as well as the absence of monoadducts in the donor DNA fragments, resulted in a decrease in complex formation. On the basis of these results, we assume that superhelicity can promote the in vitro assimilation of single-stranded donor DNA fragments by nucleoids of competents B. subtilis cells at 70 degrees C, but that at 37 degrees C a different mechanism is involved.     

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis: further characterization of unstable association between donor and recipient DNA and the involvement of the cellular membrane

Summary Although heterospecific transformation is extremely inefficient and very little heterologous donor DNA integrates into the recipient chromosome in a stable way, we have previously shown that B. pumilus DNA entering competent B. subtilis efficiently associates with the recipient chromosome in an unstable way. This association can be stabilized by photocrosslinking in the presence of 4,5,8-trimethylpsoralen; it depends on the recombination proficiency of the recipient strain and on strand-separation of the recipient chromosome (te Riele and Venema 1982b). The present study provides further evidence that the heterologous donor DNA and the recipient DNA are associated by regions of base-pairing. Based on the high sensitivity of the donor moiety in the complex to nuclease S1 (90%) and the high sensitivity of the complex to moderate denaturing conditions (Tm=48°C), we presume that donor and recipient DNA are associated either by several short sequences of 15–25 fairly well matched base pairs or by a region of base-pairing of about 200 bases, which contains 25% of mismatches. During incubation, the unstable complex disappears, probably due to nucleolytic degradation.The unstable heterologous donor-recipient complex (DRC) was found to be membrane-bound. However, in contrast to homologous DRC, the unstable heterologous DRC remains membrane bound during incubation. Apparently, the predominantly single-stranded character of the heterologous DRC prevents release of the complex from the membrane.Abbreviations DRC donor-recipient complex - TMP 4,5,8-trimethyl-psoralen - DNAase I deoxyribonuclease 1 - TCA trichloroacetic acid     

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

Summary 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, 5¹, 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that base-pairing 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.     

Effect of ethylenediaminetetraacetic acid on deoxyribonucleic acid entry and recombination in transformation of a wild-type strain and a rec-1 mutant of Haemophilus influenzae

In transformation of Haemophilus influenzae, donor deoxyribonucleic acid (DNA) enters into competent cells in the presence of ethylenediaminetetraacetic acid (EDTA), which prevents the formation of single stranded regions in the donor DNA that has entered. If after entry of DNA the recipient cells were first incubated at 17 degrees C and then at 37 degrees C in the continuous presence of EDTA, almost no integration occurred. On the other hand, if after entry of DNA the cells were incubated first at 17 degrees C in the absence of EDTA, allowing the generation of single-stranded regions (integration is blocked at this temperature), and then at 37 degrees C in the presence of EDTA, donor-recipient DNA complexes were formed. These results suggest that single-stranded regions are required for integration. Integration to completion was strongly inhibited by EDTA. In a rec-1 mutant of H. influenzae no donor-recipient DNA complexes carrying recombinant-type activity were formed during incubation at 37 degrees C in the absence of EDTA. If rec-1 cells were incubated at 37 degrees C in the presence of EDTA, which strongly inhibited breakdown of DNA, donor-recipient DNA complexes were formed if previously single-stranded regions in the donor DNA that had entered were generated by incubation at 17 degrees C in the absence of EDTA. This suggests that the rec-1 protein protects the initial donor-recipient DNA complex against degradation, so that further steps in the recombination process can proceed.     

Heterospecific transformation in Bacillus subtilis: protein composition of a membrane-DNA complex containing unstable heterologous donor-recipient complex

Summary Previously it was demonstrated that, in contrast to the homologous donor-recipient complex, the unstable heterologous donor-recipient complex remains bound to the cellular membrane. To examine whether proteins known to be involved in the processing of transforming DNA in Bacillus subtilis are associated with membrane fragments which carry chromosomal DNA, a crude membrane-DNA complex was subjected to electrophoresis through a sucrose gradient. This resulted in the separation of membrane fragments associated with DNA and free membrane fragments. By means of two-dimensional gel electrophoresis several proteins, either uniquely present or considerably enriched in the purified membrane-DNA complex, were detected. Among these proteins we identified the 45 kD recE gene product, required for recombination, the 18 kD binding protein involved in the binding of transforming DNA and a 17 kD nuclease involved in the entry of transforming DNA.These results suggest that the membrane sites at which donor DNA integrates into the recipient chromosome are in the vicinity of the sites of entry of donor DNA through the membrane.Abbreviations DNAase I deoxyribonuclease I - DRC donor-recipient DNA complex - PEG polyethyleneglycol - PMSF phenylmethylsulphonylfluoride - SSC standard saline citrate - TCA trichloroacetic acid     

On the Nature of Recombinants Formed during Transformation in Hemophilus influenzae

During the process of transformation in Hemophilus influenzae integration of donor DNA, i.e. the formation of recombinant DNA, involves the incorporation of single-stranded DNA. Evidence was obtained from cesium chloride density gradient centrifugation of DNA from donor-recipient complexes that integration was accompanied by the formation of hybrid DNA with a density intermediate with respect to heavy, ²H, ¹⁵N, donor and light, ¹H, ⁴N recipient DNA. On denaturation the position of the heavy donor DNA moved closer to, but not all the way toward, the density position of the original donor DNA. In addition to supporting the idea of single-stranded incorporation, this evidence suggested that the integrated donor DNA was covalently linked to light recipient DNA. The DNA was taken up in the double-stranded form and no detectable amounts of denatured DNA could be found during the transformation process. However, during the process of integration an amount of donor atoms, equivalent to the amount of hybrid DNA formed, appeared in recipient DNA, and indicated that while one strand of DNA was integrated the other was broken down and resynthesized. The density of the hybrid DNA, as well as rebanding of denatured hybrid, indicated that the size of the integrated piece of DNA was large, approximately 6 x 10⁶ daltons.     

Effect of 4,5'',8-trimethylpsoralen interstrand cross-links present in recipient Bacillus subtilis on the integration of transforming DNA.

When recipient Bacillus subtilis carrying chromosomal trimethylpsoralen cross-links were transformed, the donor marker activity decreased with the extent of cross-linking. Additional donor marker activity was lost upon incubation of the reextracted DNA with nuclease S1, particularly at higher levels of cross-linking. Physical analysis of the reextracted DNA showed that the donor DNA was progressively excluded from heteroduplex formation as the frequency of cross-links in the recipient DNA increased. In the donor-recipient complexes still being formed, increasing amounts of donor DNA became susceptible to nuclease S1 digestion under these conditions. These results suggest that resident interstrand cross-links interfere both with initiation of recombination and with the completion of heteroduplex formation.     

Damage at two levels of DNA folding measured by fluorescent halo technique in X-irradiated L5178Y-R and L5178Y-S cells. II. Repair

In the preceding paper we described the properties of nucleoids analyzed with the fluorescent halo assay at pH 6.9 and 9, as well as in the presence of reducing and chelating agents and after X-irradiation. We found analogies between the properties of type I and II nucleoids, as examined by Lebkowski and Laemmli (1982), and nucleoids analyzed with the fluorescent halo assay. We concluded that radiation-inflicted damage at two levels of DNA folding is measured at pH 6.9 and 9. In this paper we examined repair of damage to the nucleoid structure as assayed by the fluorescent halo method in X-irradiated L5178Y (LY) sublines; R (radiation resistant,D ₀=1.4 Gy) and S (radiation sensitive,D ₀=0.5 Gy). Halo diameters were measured after cell lysis in the presence of propidium iodide (PI; 0.5 to 50 µg/ml) at pH 6.9 and 9. The ability of DNA to be rewound at 10–50 µg/ml of PI was impaired by X-irradiation and partly restored during 90-min post-irradiation incubation, indicating damage to the superhelical structure and its partial restoration. The exponential time constants for repair were 10.1 min (LY-S, 6 Gy), 11.2 min (LY-R, 12 Gy), and 20.3 min (LY-s, 12 Gy) when measured at pH 9. In X-irradiated (12 Gy) LY-S cells, slower restoration of DNA supercoiling was observed at pH 9 than at pH 6.9. The presence of labile lesions at pH 9 did not prevent restoration of the higher-order DNA structure, as estimated from DNA rewinding at pH 6.9 in LY-S cells.Work performed at SUNY-Health Science Center at Brooklyn     

Kinetic analysis of the products of donor deoxyribonucleate in transformed cells of Bacillus subtilis

This paper describes the major transmutations of donor deoxyribonucleic acid (DNA) after uptake by competent Bacillus subtilis cells. Kinetic experiments confirm that after exposure to competent cells, donor DNA is converted to double-stranded fragments (DSF) which can be isolated as early as 30 s from the beginning of the reaction. At this time, DSF represent the only identifiable product of donor origin. After 1 to 2 min, DSF are converted to deoxyribonuclease-resistant forms, identified as single-stranded DNA fragments (SSF). SSF are intermediates in the transformation process leading to the formation of donor-recipient complex. This component makes its appearance between 2 to 4 min from the beginning of the transformation process. All the donor-recipient complexes found at the end of the reaction can be accounted for quantitatively by the DSF and the SSF found in the initial stages of transformation. A quantitative discussion of the transformation process is included.     

Transformation in Bacillus subtilis: characterization of an intermediate in recombination observed during transformation.

Summary From recombination-proficient competent cells of Bacillus subtilis in which the donor DNA entered at 17°, and which were kept at the same temperature, a complex of donor DNA and the recipient chromosome can be obtained which has a relatively high buoyant density in CsCl gradients. Exposure of the isolated complex to nuclease S1 liberates donor radioactivity. The limited biological activity of DNA re-extracted from cells attempting to recombine at 17° is decreased upon incubation with nuclease S1. If recombination is allowed to proceed at 30°, the high buoyant density of the donor-recipient complex decreases to normal values and less radioactivity can be liberated from the complex by nuclease S1. Concomitantly the biological activity of re-extracted DNA becomes less vulnerable to nuclease S1 under these conditions. On the basis of these observations we assume that the intermediate complex partly consists of unpaired single-stranded donor DNA.Support for the correctness of this assumption is derived from experiments with a mutant, which is delayed in the processing of high buoyant density donor-recipient complex to normal buoyant density donor-recipient complex. This delay is reflected in the time of acquisition of resistance to nuclease S1 digestion of the isolated complex.     

Competence Mutants II. Physical and Biological Fate of Donor Transforming Deoxyribonucleic Acid

Transformation-deficient (com(-)) mutants, which are able to bind donor transforming deoxyribonucleic acid (DNA) without yielding a significant number of transformants, were studied with regard to the fate of donor DNA. In no case was there any detectable degradation into acid-soluble radioactivity after donor DNA uptake. Physical experiments showed that some of these mutants are deficient in their ability to associate donor DNA with the recipient's chromosome (dad(-) mutants, for donor association defective), whereas others are able to form what appear to be normal donor-recipient complexes. In spite of physical evidence for integration, none of the dad(-) mutants contains biologically active recombinant DNA, suggesting that they might be deficient in the recombination process (dab(-) mutants, for donor association biologically defective). Donor biological activity is not replicated in any of the mutant strains, and in some cases there is a 10-fold reduction of donor transforming DNA within 60 min after DNA uptake.     

Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA

We examined changes in zeta potential (the surface charge density, zeta) of the complexes of liposome (nmol)/DNA (microg) (L/D) formed in water at three different ratios (L/D=1, 10 and 20) by changing the ionic strength or pH to find an optimum formulation for in vivo gene delivery. At high DNA concentrations, zeta of the complexes formed in water at L/D=10 was significantly lowered by adding NaCl (zeta=+8.44+/-3.1 to -27.6+/-3.5 mV) or increasing pH from 5 (zeta=+15.3+/-1.0) to 9 (zeta=-22.5+/-2.5 mV). However, the positively charged complexes formed at L/D=20 (zeta=+6.2+/-3.5 mV) became negative as NaCl was added at alkaline pH as observed in medium (zeta=-19.7+/-9.9 mV). Thus, the complexes formed in water under the optimum condition were stable and largely negatively charged at L/D=1 (zeta=-58.1+/-3.9 mV), unstable and slightly positively charged at L/D=10 (zeta=+8.44+/-3.7 mV), and unstable and largely positively charged at L/D=20 (zeta=+24.3+/-3.6 mV). The negatively charged complexes efficiently delivered DNA into both solid and ascitic tumor cells. However, the positively charged complexes were very poor in delivering DNA into solid tumors, yet were efficient in delivering DNA into ascitic tumors grown in the peritoneum regardless of complex size. This slightly lower gene transfer efficiency of the negatively charged complexes can be as efficient as the positively charged ones when an injection is repeated (at least two injections), which is the most common case for therapy regimes. The results indicate that optimum in vivo lipofection may depend on the site of tumor growth.     

Telomeres,telomerase, and stability of the plant genome

Telomeres, the complex nucleoprotein structures at the ends of linear eukaryotic chromosomes, along with telomerase, the enzyme that synthesizes telomeric DNA, are required to maintain a stable genome. Together, the enzyme and substrate perform this essential service by protecting chromosomes from exonucleolytic degradation and end-to-end fusions and by compensating for the inability of conventional DNA replication machinery to completely duplicate the ends of linear chromosomes. Telomeres are also important for chromosome organization within the nucleus, especially during mitosis and meiosis. The contributions of telomeres and telomerases to plant genome stability have been confirmed by analysis of Arabidopsis mutants that lack telomerase activity. These mutants have unstable genomes, but manage to survive up to ten generations with increasingly shortened telomeres and cytogenetic abnormalities. Comparisons between telomerase-deficient Arabidopsis and telomerase-deficient mice reveal distinct differences in the consequences of massive genome damage, probably reflecting the greater developmental and genomic plasticity of plants.     

Comparative cytological and molecular analysis of common wheat introgression lines containing genetic material of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> Zhuk

A total of 40 introgression lines of common wheat (2 n = 42) Triticum aestivum L × T. timopheevii Zhuk., resistant to leaf rust and partly to powdery mildew, were examined. Based on cytological analysis of meiosis in pollen mother cells (PMC), hybrid lines were subdivided into two groups characterized by either stable or unstable meiosis. In cytologically stable lines, chromosome configuration at the MI stage of meiosis was mostly bivalent (21II) with small proportion of defect cells (almost 10%), which at most contained two univalents (20II + 2I). Cytologically unstable group was comprised of the lines, containing high proportions of cells with abnormal chromosome pairing in meiotic PMC, as well as the cells with multivalents, and the lines containing aneuploid plants. Localization of the T. timopheevii fragments performed with the use of SSR markers showed that the lines with unstable meiosis were characterized by higher numbers of introgressions compared to stable lines. The influence of certain chromosomes of T. timopheevii on chromosome pairing stability was also demonstrated. In cytologically unstable lines, the increased frequency of 2A substitutions along with the high frequency of introgression of T. timopheevii genetic material into chromosome 7A was observed. Multivalents were scored in all cases of introgression in chromosome 7A. It was suggested that the reason for the genome instability in hybrid forms lied in insufficient compensating ability of certain T. timopheevii chromosomes and/or their parts, involved into recombination processes.     

Single-stranded regions in Streptococcus pneumoniae chromosomal deoxyribonucleic acid and their relation to transformation.

Deoxyribonucleic acid (DNA) in lysates of both completent and noncompetent streptococcus pneumoniae cells was characterized by chromatography on benzoylated, naphthoylated diethylaminoethyl-cellulose columns, by sensitivity to Aspergillus oryzae S1 endonuclease, and by sucrose gradient analysis. The DNAs from both competent and noncompetent cells were found to contain similar extents of single-stranded regions. These single-stranded regions appeared to be intact, unpaired regions in double-stranded DNA rather than gaps, nicks, or unpaired ends in the DNA. Inhibition of cells with rifampin prior to lysis increased the amount of such single strandedness in the DNA. Lysates made at various times after [14C]thymidine-labeled cells had bound [3H]thymidine-labeled transforming DNA were also characterized by benzoylated, naphthoylated diethylaminoethyl-cellulose chromatography. Changes in the elution profiles of DNA from cells exposed to homospecific (S. pneumoniae) donor DNA were indicative of the formation of complexes between donor DNA and the single-stranded regions of recipient DNA. In contrast, profiles of DNA from cells exposed to heterospecific (S. sanguis) DNA did not show significant changes, indicating that few such donor-recipient complexes were formed during heterospecific transformation.     

Characterization of sexual progenies of male-sterile somatic cybrids between Brassica napus and Brassica tournefortii

Cytogenetic studies were performed on four male-sterile progenies derived from four different cybrids produced between Brassica napus and B. tournefortii using the donor-recipient protoplast fusion method. The objective of these studies was to characterize the nuclear constitution of the plants. Mitotic investigation revealed that three of the four male-sterile lines had 38 chromosomes, which is equal to that of B. napus. The fourth line, C6, had variable chromosome numbers, ranging from 39 to 42 in different plants. The meiotic behavior in each progeny varied distinctly. Of the plants having 38 chromosomes, fairly high chromosome pairing, on average 18.08 bivalents per cell, was detected at metaphase-I. However, univalents with an average of 1.39 per cell, and very low frequencies of trivalents and/or tetravalents, were also observed in the lines. These results revealed that male-sterile cybrid lines were obtained with 38 chromosomes and a relatively high level of chromosome-pairing ability, indicating their potential for establishing a stable male-sterile rapeseed line. Received:15 December 1998 / Accepted:30 January 1999     

S-phase dependent forms of DNA - nuclear membrane complexes in HeLa cells.

DNA - nuclear membrane complexes were isolated from HeLa cells and examined by either zone sedimentation analysis or isopycnic centrifugation in sucrose/CsCl gradients. The data suggest that the complexes formed during the first 10 min of the S-phase remain as stable structures throughout the cell cycle. Other DNA - nuclear membrane complexes are formed at later times during replication. These later complexes appear as multiple species and the association of DNA and the nuclear membrane seems to be of a transient nature. Together, these results suggest that both the replicative origins and the replication points of the DNA are associated with the nuclear membrane. Although the complexes formed at the start of the S-phase and at later times during the S-phase appear to differ, these differences may provide them with the needed properties to serve as spatial organizers for the temporal regulation of DNA replication.     

Association of the Folded Chromosome with the Cell Envelope of Escherichia coli: Nature of the Membrane-Associated DNA

Membrane-associated folded chromosomes isolated from Escherichia coli in the presence of spermidine sedimented at about 5,800S. The folded chromosome and the membrane fragment were each stable in the absence of the other; a 1,700S folded chromosome was obtained after removal of the membrane by a Sarkosyl treatment, and a 4,000S membrane fragment remained after digestion of the chromosomal DNA with deoxyribonuclease I. The interaction between the folded chromosome and the membrane fragment was stable, and, even when the DNA was unfolded, both components remained associated and cosedimented. The large frictional effect of the unfolded DNA reduced the sedimentation rate of the complex to about 2,000S. Partial removal of this unfolded DNA with restriction endonucleases caused the membrane fragments and the remaining associated DNA to sediment faster, at about 3,500S. The DNA remaining associated with the membrane fragments after restriction endonuclease treatment, about 4.5% of the total DNA when EcoRI was used, was indistinguishable from the DNA released from the membranes by three criteria: (i) DNA size distribution in agarose gels after electrophoresis, (ii) reassociation kinetics, and (iii) thermal elution from hydroxylapatite. This finding, that random DNA sequences rather than specific ones were responsible for the majority of the DNA-membrane interactions, argues against the folded chromosome's being a static structure with specific DNA sequences interacting with the cell envelope.