Branch capture reactions: effect of recipient structure.

Branch capture reactions (BCR) contain two DNA species: (i) a recipient restriction fragment terminating in an overhang and (ii) a displacer-linker duplex terminating in a displacer tail complementary to the overhang as well as contiguous nucleotides within the recipient duplex. Branched complexes containing both species are captured by ligation of the linker to the recipient overhang. Specificity depends upon branch migration and is increased by substitution of bromodeoxycytidine for deoxycytidine in the displacer. BCR rates and specificities were determined for recipient overhangs that were (i) 5' and 3', (ii) 3 and 4 nucleotides long, and (iii) 0-100% G+C. Model systems permitted independent determination of G+C and branching effects on ligation rates and verification of rapid equilibrium between the branched complex and its component species. With all 4-base overhangs, recipient duplexes permitting extensive branch migration became saturated with displacer-linker duplexes. With increasing G+C, increasing ligation at competing sites led to decreased BCR specificity. BCR may be used to label a DNA fragment prior to electrophoresis, mark a fragment for affinity chromatography, or introduce a new overhang sequence compatible with a restriction endonuclease site in a cloning vector. A protocol was confirmed for mapping restriction sites in cloned DNA.     

DNA protection with the DNA methylase M . BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII

BamHI fragments of the Bacillus brevis var. GB plasmid pAD1 have been cloned in Escherichia coli HB101 using pBR322 plasmid as a vector. The analysis of the recombinant plasmids showed that additional PstI sites had appeared in cloned fragments of pAD1. Methylation of the recombinant plasmids in vitro by enzymes from B. brevis GB cells blocks cleavage at these additional PstI sites of cloned pAD1 fragments and at the PstI site of pBR322. Among DNA methylases of B. brevis GB, the cytosine DNA methylase M . BbvI is the most likely agent modifying the recognition sequences of PstI. The methylase can modify cytosine residues in PstI or PvuII sites if these recognition sequences are linked to G at 5'- or to C at 3'-termini. In particular, in vitro methylation of the SV40 DNA by B. brevis GB methylases protects one of the two PstI sites and two of the three PvuII sites. The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.     

Recovery of DNA fragments inserted by the "tailing" method: regeneration of PstI restriction sites

A general method has been developed for the recovery of any DNA fragment inserted into a cloning vehicle containing a single endonuclease PstI site. Endonuclease PstI sites are regenerated by the addition of one or more deoxyguanosine residues to the 3' termini of the PstI-cleaved vehicle by terminal deoxynucleotidyl transferase. Chain elongation by terminal deoxynucleotidyl transferase is then continued with dITP, dATP or dGTP. A plasmid vehicle, pAO1, containing a single PstI site has been constructed. Insertional (foreign) DNA fragments that were "tailed" with dCTP have been annealed to PstI-cleaved pAO1 that was "tailed" with dGTP. When the annealed fragments were used to transform competent Escherichia coli cells, the single-stranded DNA gaps in the recombinant plasmids were repaired. Plasmids recovered from transformed bacteria could be cleaved by PstI into the insertional DNA with dG:dC tracts and linear pAO1 molecules.     

Plasmid vector for cloning, determination of nucleotide sequence and directed assembly of DNA fragments

A plasmid vector pNIMB has been constructed (starting) from the pUR222 plasmid as a result of substitution of the polylinker containing restriction sites: PstI, SalGI, AccI, HindII, BamHI EcoRI and by other synthetic linkers with additional sites for HindIII and HgaI. Plasmid pNIMB does not differ from the parent one phenotypically. Compared to pUR222 the vector contains an additional site for cloning HindIII fragments of DNA and allows to clone SalGI/BamHI- and PstI/SalGI-fragments. Cloning of DNA fragments in all seven unique sites of pNiMB gives the possibility for sequencing the fragments avoiding their isolation from the gel. Moreover, this vector may be useful for cloning and directed assembly of chemically synthesised DNA fragments when the endonuclease HgaI sites are used.     

Branch capture reactions: displacers derived from asymmetric PCR.

Branch capture reactions (BCR) contain three DNA species: (i) a recipient restriction fragment terminating in an overhang, (ii) a displacer strand containing two adjacent sequences, with one complementary to the overhang and to contiguous nucleotides within the recipient duplex and (iii) a linker which is complementary to the second displacer sequence. Branched complexes containing all three species may be captured by ligation of the linker to the recipient overhang. The use of 5-MedC in the displacer facilitates BCR. High temperature ligation with a thermostable enzyme increased specificity for ligation to the correct recipient in a complex mixture of restriction fragments. Displacer synthesis by PCR permitted separate reactions of formation of stable displacement complexes and of high-temperature ligation. Ethylene glycol-containing buffer permitted PCR with 5-MedCTP or high G + C products using thermostable polymerases. BCR may be used to modify the ends of one recipient DNA duplex in a population of duplex DNA fragments. Modification of the recipient could be used to facilitate detection, affinity chromatography or cloning. By using PCR to obtain a BCR displacer, the sequence non-homologous to the recipient duplex may be expanded to include the sequence of a selectable marker, thus facilitating chromosome walking.     

Clone banks of the mung bean, pea and spinach chloroplast genomes

All but one of the PstI restriction fragments from mung bean, pea, and spinach chloroplast DNAs have been stably cloned into pBR322. Large fragments (15-54 kb) were cloned at low efficiencies which decreased with increasing fragment length. However, plasmids containing fragments above 25-30 kb were too unstable to be useful. In particular, pBR322 derivatives containing the largest mung bean and spinach fragments (34 kb and 54 kb, respectively) are extremely unstable and rapidly delete parts of the plasmid sequence. The PstI fragments of mung bean chloroplast DNA which cover the 34-kb PstI fragment have been cloned into pACYC177. After a search of several thousand recombinants we were unable to recover a clone containing a 12.2-kb pea chloroplast PstI fragment and suggest that some property of its sequence may be inimical to the cloning process. The identity of the cloned fragments to native chloroplast DNA restriction fragments is demonstrated by restriction analysis and the ability to construct detailed restriction maps of the mung bean and pea chloroplast genomes.     

Incorporation of 5-bromodeoxycytidine in the adenovirus 2 replication origin interferes with nuclear factor 1 binding.

We have studied the binding of nuclear factor 1 (NFI), a human sequence-specific DNA-binding protein, to a DNA fragment substituted in vitro with 5-bromodeoxycytidine (5-BrdC). Even at low substitution grades binding of NFI to its recognition sequence was considerably lower than with the unsubstituted control fragment. We developed a procedure to cleave substituted DNA specifically at a BrdC residue and searched for contacts between NFI and 5-BrdC residues by an interference assay. Surprisingly, no specific contacts were found in or near the recognition sequence. It appeared instead that interference was inversely related to the distance of a 5-BrdC residue from the NFI binding site. Models to explain these results, including a possible sliding mechanism, are discussed.     

The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development

In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule. One of the deletions (delta C3) was obtained in a previously described phi C31c+::vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA. After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c+ phage but not from its delta C3 derivative. Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions. PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the delta C3 derivative with retention of the vph selective marker. With the insertion of a 6.03-kb PstI fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9 kb. In the course of this work, several ClaI sites in phi C31::pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam+ Escherichia coli strain. This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb.     

Isolation and characterization of the Streptomyces cattleya temperate phage TG1

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.     

Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease.

The EcoRV mutant D90A which carries an amino acid substitution in its active center does not cleave DNA. Therefore, it is possible to perform DNA binding experiments with the EcoRV-D90A mutant both in the absence and in the presence of Mg2+. Like wild-type EcoRV [Taylor et al. (1991) Biochemistry 30, 8743-8753], it does not show a pronounced specificity for binding to its recognition site in the absence of Mg2+ as judged by the appearance of multiple shifted bands in an electrophoretic mobility shift assay with a 377-bp DNA fragment carrying a single EcoRV recognition sequence. In the presence of Mg2+, however, only one band corresponding to a 1:1 complex appears even with a high excess of protein over DNA. This complex most likely is the specific one, because its formation is suppressed much more effectively by a 13-bp oligodeoxynucleotide with an EcoRV site than by a corresponding oligodeoxynucleotide without an EcoRV site. The preferential interaction of the EcoRV-D90A mutant with specific DNA in the presence of Mg2+ was also demonstrated directly: a 20-bp oligodeoxynucleotide with an EcoRV site is bound with KAss = 4 x 10(8) M-1, while a corresponding oligodeoxynucleotide without an EcoRV site is bound with KAss less than or equal to 1 x 10(5) M-1. From these data it appears that Mg2+ confers DNA binding specificity to this mutant by lowering the affinity to nonspecific sites and raising the affinity to specific sites as compared to binding in the absence of Mg2+. It is concluded that this is also true for wild-type EcoRV.