Linear pBR322 Research Articles

Determination of electroosmotic and electrophoretic mobility of DNA and dyes in low ionic strength solutions.

Nanocoding, a genome analysis platform, relies on very low ionic strength conditions to elongate DNA molecules up to 1.06 (fully stretched DNA=1). Understanding how electroosmotic and electrophoretic forces vary, as ionic strength decreases, will enable better Nanocoding devices, or other genome analysis platforms, to be developed. Using gel electrophoresis to determine overall mobility (includes contributions from electrophoretic and electroosmotic forces) in different ionic strength conditions, linear DNA molecules (pUC19 (2.7 kb), pBR322 (4.4 kb), ΦX174 (5.4 kb), and PSNAPf-H2B (6.2 kb)) were analyzed in varying gel concentrations (1.50, 1.25, 1.00, 0.75, and 0.50%). Additionally, buffer concentration (Tris-EDTA, TE) was varied to determine free solution mobility at different ionic strength solutions. As ionic strength decreased from 13.8 to 7.3mM, overall mobility increased. As TE buffer decreased (< 7.3mM), overall mobility drastically decreased as ionic strength decreased. Rhodamine B dye was utilized to determine the electroosmotic mobility. As the ionic strength decreased, electroosmotic mobility increased. The experimental electrophoretic mobility was compared to theoretical considerations for electrophoretic mobility (Pitts and Debye-Hückel-Onsager). Electroosmotic forces decreased the overall mobility of DNA molecules and bromophenol blue migration in a gel matrix as ionic strength decreased.

Synthesis of TiO2 nanorods in the presence of linear DNA plasmid pBR322 by a sol–gel process

In this work, we have synthesized titanium dioxide nanorods ranging in size from 20 to40 nm by means of the linear plasmid pBR322 and using titanium isopropoxide as aprecursor through the sol–gel process. The resulting gels were calcined and the resultingpowders were characterized by means of scanning electron microscopy, energy dispersivespectroscopy, transmission electron microscopy, x-ray diffraction, thermogravimetricanalysis and differential thermal analysis. The results show that the synthesis in vitro ofnanorods in the presence of DNA can be achieved. Thus, we report the synthesisof hybrids made of nucleic acids in inorganic materials that may have severalapplications as catalytic systems, biomaterials and nanostructured materials.

Mutational analysis of archaeal histone-DNA interactions

Site-specific mutagenesis of the hmfB gene cloned from the archaeon Methanothermus fervidus, followed by expression in Escherichia coli, has been used to generate ∼90 recombinant (r) variants of the archaeal histone HMfB. The abilities of these variants to form stable archaeal nucleosome-containing complexes with linear pBR322 DNA, and with an 89 bp restriction fragment of this DNA have been determined. Variants that failed to form such complexes, based on negative gel-shift assays, had substitutions at the N terminus or within the α1, L1 and L2 regions of the rHMfB histone fold, at sites predicted to be homologous to eucaryal histone fold residues that contact the DNA in the eucaryal nucleosome. Variants that failed to give gel shifts were further assayed for their abilities to facilitate ligase-catalyzed circularization of a linear 88 bp DNA molecule, and to reduce the ellipticity of a DNA solution at 275 nm (θ 275). Consistent with cooperative but independent sites of DNA binding, a combination of three residue substitutions, one each in α1, L1 and L2, was required to generate a rHMfB variant with no detectable DNA binding based on gel shift, circularization and θ 275 reduction assays.

DNA- and chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec; H1t is a poor condenser of chromatin.

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using both acid and salt extraction procedures. Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA. Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein

The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.

Photosensitized cleavage and cross-linking of pBR322 DNA with khellin and visnagin

The naturally occuring furanochromones khellin and visnagin have received considerable attention, largely because of their vasodilatory properties and of their ability (particularly that of khellin) to induce skin pigmentation upon ultraviolet light treatment of patients suffering from vitiligo. There are conflicting statements in the literature on whether or not they are capable of cross-linking DNA photochemically. Supercoiled and linear pBR322 DNA was used to probe this reaction. The results showed that both khellin and, to a greater extent, visnagin photosensitized DNA cross-linking. In addition, both photosensitizers induced extensive DNA cleavage.

The antileukemic alkaloid fagaronine is an inhibitor of DNA topoisomerases I and II

The antileukemic alkaloid, fagaronine, is a potent differentiation inducer of various hematopoietic cell lines. We show here that fagaronine is a DNA base-pair intercalator with a K app of 2.1 × 10 5 M −1 for calf thymus DNA. Fagaronine inhibits the catalytic activity of purified calf thymus topoisomerase I as shown by relaxation of supercoiled plasmid DNA followed by electrophoresis in neutral as well as in chloroquine-containing gels. The catalytic activity of topoisomerase I is inhibited at concentrations above 30 μM. Fagaronine also inhibits the catalytic activity of purified calf thymus topoisomerase II at concentrations above 25 μM as shown by decatenation of kinetoplast DNA. Fagaronine stabilizes the covalent DNA-enzyme reaction intermediate (the cleavable complex) between topoisomerase I and linear pBR322 DNA at concentrations up to 1 μM. Further increase of the fagaronine concentration leads to a progressive decrease in the cleavable complex formation, which is totally inhibited at 100 μM. In contrast, up to 1 μM fagaronine has no effect on cleavable complex formation between purified calf thymus topoisomerase II and linear pBR322 DNA, whereas cleavable complex formation is inhibited at higher concentrations. Exposure to fagaronine results in an increase in DNA-protein complex formation in intact P388 murine leukemia cells. P388CPT5 cells, which have an altered topoisomerase I activity, are 4-fold resistant to the growth inhibitory effects of fagaronine compared to the parental cell line. Similarly, DC-3F/9-OH-E Chinese hamster fibrosarcoma cells, which have an altered topoisomerase II activity, are about 5-fold resistant to the growth inhibitory effects of fagaronine. We conclude that fagaronine is an inhibitor of both DNA topoisomerase I and II and propose that this might play a role in the cytotoxic activity.

Early stages in RecA protein-catalyzed pairing: Analysis of coaggregate formation and non-homologous DNA contacts

RecA protein will catalyze the in vitro pairing of homologous DNA molecules. To further explore the events involved in the search for homology, we have applied a nitrocellulose filter binding assay to follow pairing, and a sedimentation assay to follow the generation of aggregates (termed coaggregates) formed between RecA-complexed single-stranded (ss) DNA and double stranded (ds) DNA. Electron microscopy (EM) was used to visualize the structures involved. RecA protein promoted the pairing of circular M13 ssDNA and linear M13mp7 dsDNA efficiently in the absence of coaggregates. Indeed, pairing of homologous ss- and dsDNAs involved coaggregate formation only if the dsDNA was circular. For DNAs containing only a few hundred base-pairs of homology, for example pUC7 dsDNA and M13mp7 ssDNA, pairing and joint formation was observed if the dsDNA was superhelical but not if it was topologically relaxed or linear with the homology internal to an end of the dsDNA. The effect of non-covalently attached heterologous dsDNA on the RecA-promoted joining of M13 ssDNA and linear M13mp7 dsDNA (with non-M13 sequences at both ends) was found to depend on the topology and concentration of the heterologous DNA. A tenfold excess of superhelical pBR322 DNA strongly inhibited pairing. However, addition of relaxed or linear pBR322 DNA to the pairing reaction had little effect. As seen by EM, superhelical pBR322 DNA inhibited joint formation by excluding the homologous dsDNA from the coaggregates. EM also revealed heterologous DNA interactions presumably involved in the search for homology. Here the use of EM has provided a direct visualization of the form and architecture of coaggregates revealing a dense interweaving of presynaptic filaments and dsDNA.

Slow interaction of 5'-adenylyl-beta,gamma-imidodiphosphate with Escherichia coli DNA gyrase. Evidence for cooperativity in nucleotide binding.

We have examined the kinetics of interaction between Escherichia coli DNA gyrase and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP) in the presence and absence of ATP. In the absence of ATP, [alpha-32P]ADPNP binds extremely slowly to gyrase, with an apparent second-order rate constant (k1) of 120 M-1 min-1. Similarly, the limited negative supercoiling of closed-circular DNA caused by ADPNP binding is slow, requiring at least 2 h to reach completion in the presence of 100 microM ADPNP. A very slow but detectable rate of dissociation of ADPNP from gyrase was measured, with a rate constant of 3.5 x 10(-4) min-1. The calculated dissociation constant for ADPNP is thus 2.9 microM. ADPNP is a potent competitive inhibitor of ATP-dependent DNA supercoiling. Inhibition is established much more rapidly than can be accounted for by the slow rate of ADPNP binding in the absence of ATP. We have found that ATP can accelerate the rate of [32P]ADPNP binding by more than 15-fold (k1 = 1,850 M-1 min-1). The ATP-promoted rate enhancement requires the presence of DNA; in the absence of DNA, ATP has no effect on the rate of binding. Relaxed closed-circular, nicked-circular, and linear pBR322 DNA are all equally effective cofactors for ATP-stimulated binding of ADPNP. After a short lag, the presence of ATP also greatly speeds up ADPNP dissociation from gyrase bound initially to closed-circular DNA, with the restoration of DNA supercoiling activity. This effect is not observed in the presence of nicked-circular or linear DNA, suggesting that ADPNP dissociates more rapidly from gyrase bound to supercoiled DNA. The results of ADPNP binding provide evidence for cooperative interactions between the nucleotide binding sites. To account for these data, a model is proposed for the interaction of nucleotides at the two ATP binding sites on DNA gyrase.

Effect of ethidium on the torsion constants of linear and supercoiled DNAs

The torsion elastic constants (α) of linear pBR322 (4363 bp) and pUC8 (2717 bp) DNAs and supercoiled pBR322 and pJMSII (4375 bp) DNAs are measured in 0.1 M NaCl as a function of added ethidium/base-pair (EB/BP) ratio by studying the fluorescence polarization anisotropy (FPA) of the intercalated ethidium. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single photon counting detection. Previously developed theory for the emission anisotropy is generalized to incorporate rotations of the transition dipole due to excitation transfer. The excitation transfers are simulated by a Monte Carlo procedure (Genest et al., Biophys. Chem. 1 (1974) 266–278) and the consequent rotations of the transition dipole are superposed on the Brownian rotations. After accounting for excitation transfer, the torsion constants of the linear DNAs are found to be essentially independent of intercalated ethidium up to a binding ratio r = 0.10 dye/bp. Dynamic light scattering measurements on linear pUC8 DNA confirm that the torsion constant is independent of binding ratio up to r = 0.20 dye/bp. If α d denotes the torsion constant between ethidium and a base-pair, and α 0 that between two base-pairs, then our data imply that α d/α 0 lies in the range 0.65 to 1.64 with a most probable value of 1.0. The torsion constants of supercoiled DNAs decrease substantially with increasing binding ratio even after accounting for excitation transfer. At the binding ratio r* = 0.064, where the superhelix density vanishes and superhelical strain is completely relaxed, the torsion constant of the supercoiled pBR322 DNA/dye complex lies below that of the corresponding linear DNA/dye complex by about 30%. This contradicts the conventional view according to which linear, nicked circular, and supercoiled DNA/dye complexes with r = r* should coexist with the same concentration of free dye, display the same distribution of bound dye, and exhibit identical secondary structures, twisting and bending rigidities, and FPA dynamics. These and other observations imply the existence of metastable secondary structure in freshly relaxed supercoiled DNAs. A tentative explanation is presented for these and other unexpected observations on supercoiled DNAS.

In vitro rescue of an integrated hybrid adeno-associated virus/simian virus 40 genome

In an in vitro simian virus 40 (SV40) DNA replication assay, we have observed excision of a hybrid adeno-associated virus (AAV)/SV40 insert from a plasmid construct. The excision was dependent on the presence of the palindromic AAV terminal repeat and greatly enhanced by the addition of the SV40 T antigen to the reaction. Analysis of the excision product supports a model in which the palindromic terminal sequences of AAV form a cruciform structure (equivalent to a Holliday recombination intermediate), which is cleaved and resealed so that the excision products are linear duplex pBR322 and linear duplex AAV/SV40 insert. Both the excised linear duplex pBR322 and the excised linear duplex AAV/SV40 insert have each terminus covalently crosslinked by one copy of the palindromic region of the AAV terminal repeat region folded on itself. The excision process may be a model system for cellular homologous recombination. The process as observed was either concomitant with or subsequent to DNA replication.

Effects of chloroquine on the torsional dynamics and rigidities of linear and supercoiled DNAs at low ionic strength.

The magnitude and uniformity of the torsion elastic constant (alpha) of linear and supercoiled pBR322 DNAs are measured in 3 mM Tris as a function of added chloroquine/basepair ratio (chl/bp) by studying the fluorescence polarization anisotropy of intercalated ethidium dye. The time-resolved FPA is measured using a picosecond dye-laser for excitation and time-correlated single-photon counting detection. For both linear and supercoiled DNAs, alpha remains uniform except at the very highest chl/bp ratio examined. For the linear DNA, alpha decreases from 5.0 x 10(-12) dyne-cm at chl/bp = 0 to about 3.5 x 10(-12) dyne-cm at chl/bp = 0.5, and remains at that value up to chl/bp = 5, whereupon it increases back up to its original value. For the supercoiled DNA, alpha remains constant at about 5.2 x 10(-12) dyne-cm from chl/bp = 0 up to chl/bp = 5, whereupon it increases in parallel with the linear DNA. The effect of chloroquine on the secondary structure, torsion constant, and torsional dynamics evidently differs substantially between linear and supercoiled DNAs, even under conditions where the supercoiled DNA is completely relaxed and both DNAs bind the same amount of dye. This strongly contradicts any notion that the local structures of linear and relaxed supercoiled DNA/dye complexes with the same binding ratio are identical. The increase in apparent alpha at chl/bp = 5 for both DNAs may be due to stacking of the chloroquine in the major groove and consequent stiffening of the filament.

Interaction of chloroquine with linear and supercoiled DNAs. Effect on the torsional dynamics, rigidity, and twist energy parameter.

The magnitude and uniformity of the torsion elastic constant (alpha) of linear pBR322 DNA and supercoiled pBR322 DNAs with high-twist (sigma = -0.083) and normal-twist (sigma = -0.48) are measured in 0.1 M NaCl as a function of added chloroquine/base-pair ratio (chl/bp) by studying the fluorescence polarization anisotrophy (FPA) of intercalated ethidium dye. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single-photon counting detection. A general theory is developed for the binding of ligands that unwind superhelical DNAs, and the simultaneous binding of two different intercalators is treated in detail. The equilibrium constant (K) for binding chloroquine to linear pBR322 DNA and the number (r) of bound chloroquines per base pair are determined from the relative amplitude ratio of the slow (normally intercalated) and fast (free) components in the decay of the (probe) ethidium fluorescence intensity as a function of chl/bp. For chloroquine binding to supercoiled pBR322 DNAs, the intrinsic binding constant is assumed to be the same as for the linear DNA, but the twist energy parameter ET (N times the free energy to change the linking number from 0 to 1 in units of kBT) is regarded as adjustable. Using the best-fit ET, the binding ratios r are calculated for each chl/bp ratio. Twist energy parameters are also determined for ethidium binding to these supercoiled DNAs by competitive dialysis. For chloroquine binding, we obtain ET = 360 and 460 respectively for the normal-twist and high-twist supercoiled DNAs. For ethidium binding the corresponding values are ET = 280 +/- 70 and 347 +/- 50. Like other dye-binding values, these are substantially lower than those obtained by ligation methods. In the absence of chloroquine, the torsion constants of all three DNAs are virtually identical, alpha = (5.0 +/- 0.4) x 10(-12) dyn.cm. For linear pBR322 DNA, the magnitude and uniformity of alpha remain unaltered by intercalated chloroquine up to r = 0.19. This finding argues that the FPA is not significantly relaxed by diffusion of any kinks or solitons. If alpha d denotes the torsion constant between a dye and a base pair and alpha 0 that between two base pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65-1.64, with a most probable value of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS)

Tris(tetramethylphenanthroline)ruthenium(II): a chiral probe that cleaves A-DNA conformations.

lambda-Tris(3,4,7,8-tetramethyl-1,10-phenanthroline)ruthenium(II) [lambda-Ru(TMP)2+3] was found to be a distinctive molecular tool to examine the local variations in conformation along the strand. The metal complex binds cooperatively to A-form helices of various base sequences under conditions where little or no binding was found to analogous B-form DNAs. Photoactivated DNA cleavage may be coupled to this conformation-specific binding by taking advantage of the photophysical properties of ruthenium(II) complexes. lambda-Ru(TMP)2+3 cleaves preferentially 3H-labeled A-form polynucleotides upon irradiation with visible light. The photoinduced DNA strand scission is likely to be mediated by singlet oxygen, which leads to a preferential cleavage of guanine residues. Comparative mapping of cleavage sites on a linear pBR322 fragment for tris(phenanthroline)ruthenium(II), which binds to B-DNA and cleaves also by sensitization of singlet oxygen, and for Ru(TMP)2+3 shows the selective binding of lambda-Ru(TMP)2+3 to conformationally distinct sites along the fragment. These sites correspond to 5- to 13-base-pair homopyrimidine stretches.

Purification and analysis of DNA‐binding proteins from Streptomyces hygroscopicus

AbstractPurification by DNA‐cellulose chromatography of acid‐soluble DNA‐binding proteins (ASB) from Streptomyces hygroscopicus is described. Two main fractions, ASBII and ASBIII, containing different major protein components were isolated. The dominating protein of ASBIII (19 kd) could be enriched on Streptomyces DNA‐cellulose.DNA‐binding properties of ASBIII were studied by using a filter retention assay, thermal melting, and by following the DNA template activity in the RNA polymerase system of E. coli. Binding to supercoiled and linear pBR322 DNA occurred effectively at 0.05 M NaCl while increasing salt concentrations up to 0.15 M and 0.2 M NaCl caused dissociation of the complexes. Single‐stranded DNA showed a slightly lower affinity for the ASBIII protein. ASBIII blocked the DNA template activity of the RNA polymerase reaction in vitro. The binding properties and role of ASB‐proteins are discussed with respect to their possible influence on the chromosomal state of the DNA.

PBR322 DNA inhibits simian virus 40 gene expression in Xenopus laevis oocytes.

SV40 DNA form I is expressed efficiently after its injection into the nuclei of Xenopus laevis oocytes, resulting in the synthesis of RNA and protein products of both viral late and early transcription units. However it was observed that injection of SV40 genes cloned into pBR322 or related plasmids yielded vastly reduced quantities of viral DNA and proteins. If SV40 DNA was cleaved from the plasmid, and then recircularized prior to microinjection, viral expression was regained. The inhibition by plasmid DNA was not confined to an effect in cis because coinjection of circular pBR322 DNA along with SV40 DNA, as separate entities, also blocked viral RNA and protein synthesis. As circular but not linear pBR322 DNA was actively transcribed by polymerase II in oocytes, even in the presence of SV40 DNA, it is likely that pBR322 competes for transcription factors required for viral gene expression. Injection of pBR322 as early as two hours after injection of SV40 DNA into the oocyte nucleus did not inhibit SV40 RNA synthesis, indicating that once initiated, SV40 transcription is stable and insensitive to the competition by plasmid DNA. A plasmid vector was developed that allows expression of SV40 DNA in Xenopus laevis oocytes.

Differences in the kinetic properties of BamHI endonuclease and methylase with linear DNA substrates.

BamHI endonuclease and methylase were found to exhibit a kinetic preference for linear pBR322 DNA substrates containing the recognition site in a central location. The Km values for substrates having the recognition site in a terminal location were approximately 3-fold greater than those with a centrally located site. This phenomenon may be partially due to facilitated transfer of the enzymes to the recognition site over nonspecific flanking sequences. The exploitation of facilitated transfer by these enzymes has been inferred from studies demonstrating kinetic preferences for longer DNA substrates. The reaction rates of the endonuclease were 9-fold greater with full-length pBR322 DNA than with a 74-base pair derivative. The methylase exhibits a kinetic preference for longer substrates but only under conditions of comparatively higher DNA concentrations. In addition, the methylase has the property of increasing long chain preference with increasing salt concentrations up to 120 mM. Increasing salt concentrations decreased the endonuclease's preference for longer substrates. Nonspecific inhibition studies revealed qualitative and quantitative differences between the two enzymes under catalytic conditions. These studies suggest that BamHI endonuclease and methylase interact with nonspecific DNA in different ways.

Mechanism of intramolecular recyclization and deletion formation following transformation of Escherichia coli with linearized plasmid DNA.

The deletion end-points of a number of type I (less than monomeric) plasmid deletants obtained by transforming recA+ or recA- E. coli with linear pBR322 DNA were determined by DNA sequencing. In both monodirectional and bidirectional deletions the recyclization point was normally characterized by recombination between directly repeated sequences of between 4 and 10 bp present on each arm of the linearized pBR322 molecule. Frequently, short tracts of uninterrupted homology involved in recombinational recircularization were embedded in regions of relative non-homology. A model predicting the probability of matching sequences in either end of a linear plasmid molecule is presented. It is proposed that exonucleolytic processing of the exposed termini of linear plasmid molecules generates substrates for subsequent recombinational recyclization and deletion. The activity of host recombination and repair functions in recircularizing linear DNA molecules explains the generation of many of the aberrant recombinant DNA constructs obtained during gene cloning procedures.

Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli.

Monomeric pBR322 DNA that had been linearized at its unique SalI site transformed wild-type Escherichia coli with 10(2) to 10(3) times less efficiency than CCC plasmid DNA. Dose-response experiments indicated that a single linear plasmid 'molecule' was sufficient to produce a transformant. Transformation with linearized pBR322 DNA was reduced 10 to 40 fold in recA1 , recBC- or recF- backgrounds. In contrast, transformation with CCC DNA was unaffected by the rec status of the host. Transformation with linear pBR322 DNA was increased 3-fold in a DNA ligase-overproducing ( lop11 ) mutant and decreased to a similar degree by transient inactivation of ligase in a ligts7 mutant. A proportion (ranging from about 9% in the wild-type to 42% in a recBC, lop11 mutant) of the transformants obtained with SalI-linearized pBR322 monomeric DNA contained deleted plasmids. Deletion rates were generally higher in rec- strains. Dephosphorylation of the termini on linear DNA or the creation of blunt-ended pBR322 molecules (by end-filling the SalI 5' protrusions or by cleavage with PvuII) decreased the transformation frequency whilst increasing the deletion rate. Linear pBR322 dimeric DNA gave transformation frequencies in recA+ and recA- strains that were reduced only 3 to 7 fold respectively relative to frequencies obtained with dimeric CCC DNA. Furthermore, in contrast to transformation with linear monomeric DNA, deletions were not observed. We propose that the majority of transformants arise, not by simple intracellular reannealing and ligation of the two cohesive SalI-termini of a linear molecule, but by intramolecular recombination.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and properties of nuclease gamma from Ustilago maydis.

We have purified an acid-soluble DNA endonuclease, termed nuclease gamma, from Ustilago cell extracts. The enzyme is nearly homogeneous, purified 1700-fold. The protein appears to be globular with a molecular weight in the range 17,000 to 21,000. It requires a divalent cation and is optimally active at slightly alkaline pH. The enzyme prefers duplex DNA as substrate but will slowly cleave single-stranded DNA. Cleavage of covalently closed duplex DNA is unaltered by changes in superhelix density. Divalent cations direct the mode by which the enzyme cleaves duplex DNA. When Mg2+ or Ca2+ is added, the enzyme nicks one strand of the duplex. When Mn2+, Co2+, or Zn2+ is added, the enzyme can introduce double strand breaks. Oligonucleotides terminated with 5'-phosphoryl and 3'-hydroxyl groups are the products of hydrolysis. DNA fragments generated can be religated to linear pBR322 DNA with completely base-paired ends.