Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA.

In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.     

Cleavage of Epstein-Barr virus DNA by restriction endonucleases EcoRI, HindIII and BamI.

The cleavage of the DNAs of the B95-8 and P3HR-1 virus strains of Epstein-Barr virus by the restriction endonucleases EcoRI, HindIII and BamI was investigated using a new technique for quantitative evaluation of the fluorescence of ethidium stained DNA fragments separated on agarose gels. The results obtained with B95-8 DNA showed that in addition to the limited repetitions of nucleotide sequences observed in the EcoRI and HindIII cleavage patterns, the molecule contained a BamI fragment with a molecular mass of 2.0 megadaltons which was present in a total of about 11 copies and localized to a limited part of the DNA molecule. The same sequences were also present in the P3HR-1 DNA albeit in a lower molar ratio. P3HR-1 DNA yielded restriction enzyme cleavage patterns suggesting DNA sequence heterogeneity of P3HR-1 virus. No fragment was present in more than about 4 copies per molecule of P3HR-1 DNA. Comparison of the restriction enzyme cleavage patterns of P3HR-1 and B95-8 DNA revealed a high degree of structural homology emphasized by nucleic acid hybridization experiments with EBV complementary RNA synthesized in vitro.     

A map of the sites on bacteriophage PM2 DNA for the restriction endonucleases HindIII and HpaII

The map of the seven sites for the restriction endonuclease HindIII³ and the single site for endo R.HpaII on PM2 DNA was determined. This map was oriented with respect to the denaturation map of this DNA (Brack et al., 1975) by partial denaturation mapping of the fragments. A new method for localizing restriction fragments by DNA-DNA hybridization and electron microscopy is described.     

Molecular-biological study of vaccinia virus genome. I. Cloning of vaccinia virus DNA fragments in bacterial vectors

The HindIII DNA fragments of vaccinia virus strain L-IVP were cloned in pBR322 bacterial plasmid. A hybrid plasmids collection of pVHn series contains all fragments of virus genome except terminal HindIII-B and HindIII-G, and also a large HindIII-A. The latter was cloned in cosmid pHC79. The obtained collection of hybrid DNA molecules allows to carry out a wide range of molecular biological experiments on the vaccinia virus genome.     

Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I

Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently. Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA. These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria. Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature. Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation. Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission. Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies. The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein. Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions. This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme. Noncovalent binding is therefore independent of competence in transesterification. It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes.     

Simian adenovirus 20 genome. I. DNA mapping using restriction enconucleases BamHI, EcoRi, XbaI, HindIII

The effect of specific restriction endonuclease on the simian adenovirus SV20 DNA was studied. It was shown that endonucleases SalI, XbaI, EcoRI, BamHI, HindIII cleaved the viral DNA into 3, 4, 5, 5, 8 specific fragments respectively. The sequence of fragments (physical map) was determined and found to be B-C-A for enzyme SalI, C-D-B-A--for enzyme Xbal, E-A-C-D-B--for enzyme EcoRI, B-E-C-A-D--for enzyme BamHI and B-E-A-C-(GH)-D-F--for enzyme HindIII. The G-C content of specific fragments was studied. The "right"-"left" orientation of the physical map of the simian adenovirus 20 DNA based on the G-C content was made in respect with the nomenclature of human adenoviruses.     

Site-specific DNA cleavage by vaccinia virus DNA topoisomerase I. Role of nucleotide sequence and DNA secondary structure.

Cleavage of linear duplex DNA by purified vaccinia virus DNA topoisomerase I occurs at a conserved sequence element (5'-C/T)CCTT decreases) in the incised DNA strand. Oligonucleotides spanning the high affinity cleavage site CCCTT at nucleotide 2457 in pUC19 DNA are cleaved efficiently in vitro, but only when hybridized to a complementary DNA molecule. As few as 6 nucleotides proximal to the cleavage site and 6 nucleotides downstream of the site are sufficient to support exclusive cleavage at the high affinity site (position +1). Single nucleotide substitutions within the consensus pentamer have deleterious effects on the equilibria of the topoisomerase binding and DNA cleavage reactions. The effects of base mismatch within the pentamer are more dramatic than are the effects of mutations that preserve base complementarity. Competition experiments indicate that topoisomerase binds preferentially to DNA sites containing the wild-type pentamer element. Single-stranded DNA containing the sequence CCCTT in the cleaved stand is a more effective competitor than is single-stranded DNA containing the complementary sequence in the noncleaved strand.     

Comparative restriction analysis of HindIII-F-fragments of DNA from 4 strains of vaccinia virus

The localization of KpnI, SacI, XhoI, AvaI, PstI, BglI, BamHI, EcoRI, PmiI, SalI, BglII, restriction endonuclease cleavage sites in HindIII-F-fragments of DNA from vaccinia strains WR, Copenhagen, LIVP and neurovaccine has been detected. The fragments have been shown to differ in the number of AvaI, EcoRI and BamHI sites. The fragments also differ from the analogue of Tian Tan vaccinia strain in the pattern of restriction by AvaI, XhoI, PstI, EcoRI and BamHI endonucleases.     

DNA binding and aggregation properties of the vaccinia virus I3L gene product.

The vaccinia virus I3L gene encodes a single-stranded DNA-binding protein which may play a role in viral replication and genetic recombination. We have purified native and recombinant forms of gpI3L and characterized both the DNA-binding reaction and the structural properties of DNA-protein complexes. The purified proteins displayed anomalous electrophoretic properties in the presence of sodium dodecyl sulfate, behaving as if they were 4-kDa larger than the true mass. Agarose gel shift analysis was used to monitor the formation of complexes composed of single-stranded DNA plus gpI3L protein. These experiments detected two different DNA binding modes whose formation was dependent upon the protein density. The transition between the two binding modes occurred at a nucleotide to protein ratio of about 31 nucleotides per gpI3L monomer. S1 nuclease protection assay revealed that at saturating protein densities, each gpI3L monomer occludes 9.5 +/- 2.5 nucleotides. In the presence of magnesium, gpI3L promoted the formation of large DNA aggregates from which double-stranded DNA was excluded. Electron microscopy showed that, in the absence of magnesium and at low protein densities, gpI3L forms beaded structures on DNA. At high protein density the complexes display a smoother and less compacted morphology. In the presence of magnesium the complexes contained long fibrous and tangled arrays. These results suggest that gpI3L can form octameric complexes on DNA much like those formed by Escherichia coli single-stranded DNA protein. Moreover, the capacity to aggregate DNA may provide an environment in which hybrid DNA formation could occur during DNA replication.     

Restriction enzyme mapping of vaccinia virus DNA.

The cleavage sites for the restriction enzymes Bg/I, HindIII, KpnI, SalI, SmaI, and XhoI were located, from primary data, on the DNA isolated from the WR strain of vaccinia virus. Bg/I and SmaI divide the DNA into five segments which can be isolated by sucrose gradient centrifugation. These large segments provide a convenient means to group segments produced by other enzymes. The construction of physical maps was initiated by identifying the segments at each end of the DNA and then finding segments which were adjacent to these terminal sections. This was accomplished by isolating large shear fragments which contained the covalently linked termini of the DNA. Most of the data needed to derive the maps were obtained by isolating segments produce by one enzyme and then cleaving these individual segments with a second enzyme.     

High-frequency homologous recombination in vaccinia virus DNA.

A recombinant vaccinia virus genome was constructed in which the viral thymidine kinase (tk) gene was placed between direct repeats of a 1.5-kilobase-pair DNA sequence of heterologous origin. When forced to replicate in tk- cells in the presence of methotrexate (i.e., under tk+-selective conditions), the recombinant maintained its tk+ phenotype. Under nonselective conditions, however, the tk gene was frequently excised by both inter- and intramolecular recombination events because the repeated sequences provided substantial targets for homologous DNA recombination. Unique DNA products of intramolecular recombination were detected in the cytoplasm of infected cells soon after the onset of viral DNA replication, and their appearance was blocked by inhibitors of DNA synthesis. During repeated passage of the virus under nonselective conditions, the tk+ fraction decreased with first-order kinetics at a rate that reflected the frequency of recombination per cycle of virus replication. Eventually, a residual population of stable tk+ viruses remained, and analyses of the genome structures of individual members of this population showed that some of them appeared to be the products of nonhomologous DNA recombination.     

Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.     

Electron microscopic visualization of restriction sites on DNA molecules.

DNA molecules were adsorbed to a polylysine-treated carbon film and digested directly on the film by restriction enzymes. After washing the film with 1 M NaCl, 0.4% Kodak Photo-Flo and 9% formamide, each cleavage site introduced was visualized as a gap under the electron microscope. By measuring the gapped positions on linear DNA molecules induced by other enzymes, a single EcoRI site on a lambda dv1 molecule and three HinHI sites on an fd1RF molecule were mapped at the positions expected from the cleavage maps, respectively. This electron-microscopic procedure may be useful for the construction of a cleavage map.     

HindII, HindIII, and HpaI restriction fragment maps of bacteriophage lambda DNA

The site-specific restriction endonucleases isolated from Hemophilus influenzae strains Rc (HincII) and Rd (HindII + III), and Hemophilus parainfluenzae (HpaI) were used to digest bacteriophage lambda DNA into 34, 40, and 15 specific fragments, respectively. The sites cleaved by each of these enzymes were localized on the lambda physical map and the fragments resulting from these cleavages were electrophoretically identified on gels by (1) analysis of the digestion profiles of deletion and transducing derivatives of lambda; and (2) digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. This paper presents the HindII, HindIII, and HpaI restriction fragment maps for the entire lambda genome, and the data used to derive these maps for the region of the lambda genome between the attachment site (at 57.3% lambda) and the right vegetative end (100% lambda). The data for mapping the left arm of lambda may be found in the accompanying paper (Robinson and Landy, 1977).     

Resolution of poxvirus telomeres: processing of vaccinia virus concatemer junctions by conservative strand exchange.

The replication of vaccinia virus proceeds through concatemeric intermediates which are resolved into unit-length DNA. In vaccinia virus-infected cells, plasmids containing the vaccinia virus DNA junction fragment that connects concatemers are resolved into linear minichromosomes of vector DNA flanked by hairpin loops. Resolution requires two copies of a specific nucleotide sequence conserved among poxviruses and found proximal to the hairpin loop. This study demonstrates that orientation of each sequence with respect to the other as well as to the axis of symmetry is critical for resolution, the processing of plasmids containing heterologous pairs of resolution sites is influenced by mismatched nucleotides between the sites, and the vaccinia virus hairpin in the linear minichromosome is a heteroduplex composed of DNA from each strand of the concatemer junction. A model incorporating site-specific recombination and orientated branch migration is proposed to account for resolution of the vaccinia virus concatemer junction.     

Conditional growth of Escherichia coli caused by expression of vaccinia virus DNA topoisomerase I.

Active vaccinia virus topoisomerase I is expressed in Escherichia coli containing plasmid p1940 (S. Shuman, M. Golder, and B. Moss, J. Biol. Chem. 263:16401-16407, 1988). Growth curves showed a decline of 2 to 3 logs in the number of viable cells at 42 degrees C after shift from 30 degrees C because of increased vaccinia virus topoisomerase I level. Mutations in the gyrA and gyrB genes allowed cells to grow equally well at 42 and 30 degrees C. The presence of gyrase inhibitor also improved growth at 42 degrees C.