Defective Bacteriophage PBSH in Bacillus subtilis: I. Induction, Purification, and Physical Properties of the Bacteriophage and Its Deoxyribonucleic Acid

PBSH, a defective phage of Bacillus subtilis strain 168, is described. Conditions are given for optimal induction of the prophage with mitomycin C. After a latent period of 90 min, cells were lysed and phage-like particles were released with a burst size of approximately 100 to 400 phage per bacterium. Since no known host supports phage replication after infection, burst size was determined by electron microscope count. Purification procedures and criteria for purity are described. The molecular weight of deoxyribonucleic acid (DNA) extracted from PBSH was estimated by length measurement and sedimentation. No circular DNA molecules were found by either technique. PBSH DNA molecules are linear, double-stranded, and of homogeneous molecular weight, about 12 x 10(6) daltons. There is no evidence for single-strand breaks. The majority of PBSH DNA molecules show a sedimentation behavior dependent on ionic strength. It is inferred that most of the DNA molecules are less hydrodynamically rigid than native DNA having a similar average base composition and molecular weight. Possible reasons for the sedimentation behavior are discussed.     

DNA of a Human Hepatitis B Virus Candidate

Particles containing DNA polymerase (Dane particles) were purified from the plasma of chronic carriers of hepatitis B antigen. After a DNA polymerase reaction with purified Dane particle preparations treated with Nonidet P-40 detergent, Dane particle core structures containing radioactive DNA product were isolated by sedimentation in a sucrose density gradient. The radioactive DNA was extracted with sodium dodecyl sulfate and isolated by band sedimentation in a preformed CsCl gradient. Examination of the radioactive DNA band by electron microscopy revealed exclusively circular double-stranded DNA molecules approximately 0.78 mum in length. Identical circular molecules were observed when DNA was isolated by a similar procedure from particles that had not undergone a DNA polymerase reaction. The molecules were completely degraded by DNase 1. When Dane particle core structures were treated with DNase 1 before DNA extraction, only 0.78-mum circular DNA molecules were detected. Without DNase treatment of core structures, linear molecules with lengths between 0.5 and 12 mum, in addition to the 0.78-mum circles were found. These results suggest that the 0.78-mum circular molecules were in a protected position within Dane particle cores and the linear molecules were not within core structures. Length measurements on 225 circular molecules revealed a mean length of 0.78 +/- 0.09 mum which would correspond to a molecular weight of around 1.6 x 10(6). The circular molecules probably serve as primer-template for the DNA polymerase reaction carried out by Dane particle cores. Thermal denaturation and buoyant density measurements on the Dane particle DNA polymerase reaction product revealed a guanosine plus cytosine content of 48 to 49%.     

Sedimentation velocity properties of catenated DNA molecules from HeLa cell mitochondria

Catenated molecules of closed circular DNA have been isolated from the mitochondrial DNA of HeLs cells. The sedimentation coefficients of several purified species have been investigated. The catenated dimer, made up of two interlocked duplex circles, sediments at 51 S in its superhelical (closed) form. Treatment with pancreatic DNase to relax the duplex circles converts the 51 S doubly closed dimer to a 42 S singly open species, then to a 36 S doubly open catenated dimer. The triply closed trimer sediments at 63 S and is converted to a 45 S triply open form by DNase. Electron microscopy of the DNA samples before and after DNase treatment shows that under the conditions used DNase does not change the catenated nature of the DNA. The measured sedimentation coefficients, have been compared with those estimated from previously proposed correlations of sedimentation coefficient and molecular weight, and with the sedimentation coefficients for catenated DNA presented by Wang. When all the interlocked circles in a catenane are relaxed, the DNA sediments about 5–10% faster than a relaxed multiple-length circular molecule of the same molecular weight. The sedimentation coefficient, 36 S, of the fully relaxed catenated dimer is 1.4 times that of the relaxed monomer.     

Circular Epstein-Barr virus genomes of reduced size in a human lymphoid cell line of infectious mononucleosis origin.

Circular Epstein-Barr virus (EBV) DNA molecules have been purified and characterized from a human lymphoid cell line derived from a case of heterophile antibody-positive, blood transfusion-induced infectious mononucleosis, 883L. The circular EBV DNA in three cell lines obtained by transformation of human umbilical cord blood leukocytes with a strain of EBV originally derived from 883L was also studied. As estimated from sedimentation velocity data and electron microscopy, the circular EBV DNA molecules are 10 to 15% smaller than either the circular EBV DNA previously found intracellularly in several other types of EBV-transformed cells or the linear EBV DNA present extracellularly in virus particles. In addition, the EBV-transformed cord blood cell lines studied here differed from other EBV-transformed cells in that integrated virus DNA sequences could not be detected.     

Physical and Biological Properties of Phage φ29 Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) molecules having a mean length of 5.8 mum were released from purified Bacillus subtilis bacteriophage phi29 with 2 m sodium perchlorate. Small 0.1 to 0.2-mum molecules were also detected in these DNA preparations. Since intact single chains annealed to form linear duplex molecules, phage phi29 DNA was found to be nonpermuted. The molecular weights of single chains of phi29 DNA were approximately half that of native DNA, as determined by analytical band sedimentation in CsCl, indicating that phi29 DNA is composed of two continuous polynucleotide chains. The molecular weight values of native and annealed phi29 DNA from sedimentation agreed with the molecular weight values obtained from electron microscopy. The infectivity of phi29 DNA was reduced to a low level by alkaline denaturation and was partially restored by annealing.     

Isolation of coliphage lambda ghosts able to adsorb onto bacterial cells

We have examined three methods of γ ghost production, starting with the [³H]eucine-labelled phage, purified by CsCl density gradient sedimentation. Ghosts obtained by the osmotic shock or by incubation in 5 M LiCl do not adsorb on bacteria. Ghosts obtained by the treatment with the chelating agent EDTA and purified by CsCl density gradient sedimentation posses well preserved adsorption properties and are virtually free of DNA and infectious phage particles.     

Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule.

The structure of DNA from the temperate Bacillus subtilis phage phi105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis. The DNA contains six EcoRI cleavage sites. Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA. In neutral gradients, the majority of whole phi105 DNA sedimented as nicked circles and the remainder as oligomers. No unit-length linear structures were detected. The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease. On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, phi105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA.     

Location of DNA ends in P2, 186, P4 and lambda bacteriophage heads

When mature phage particles were suspended in a solution containing formaldehyde (0.07 m-Na⁺, pH 9.0, 10% HCHO for 10 min at 23 °C) and the mixture then spread for electron microscopy in the presence of 50% formamide and cytochrome c, the phage lysed and a high proportion of the DNA molecules were seen to be attached to phage tails. The phage tails were found to be attached at only one end of each DNA molecule and denaturation mapping showed that this end was unique for each of the phages P2, 186, P4 and λ. It is argued that in these mature phage particles one specific end of the DNA molecule is present at the head-tail attachment site.     

PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends

PY54 is a temperate phage isolated from Yersinia enterocolitica. Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram-negative bacteria, the phage genome has 3'-protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN-like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage.     

Bacteriophage phiNS11: a lipid-containing phage of acidophilic thermophilic bacteria. IV. Sedimentation coefficient, diffusion coefficient, partial specific volume, and particle weight of the phage.

The particle weight (molecular weight) of phiNS11 was determined from the sedimentation coefficient, diffusion coefficient, and partial specific volume of the phage. The sedimentation coefficient of the phage (S(0)20, W) is 416 +/- 2.7S. The diffusion coefficient D(0)20, W), which was determined by quasielastic light scattering measurement, is (0.57 +/- 0.03) x 10(-7) cm2/s. The partial specific volume was determined by the mechanical oscillation technique to be 0.747 +/- 0.007 cm3/g. Based on these values, the particle weight of the phage was calculated to be (70.3 +/- 4.3) x 10(6) daltons, which agrees well with the particle weight (69--72 x 10(6) daltons) estimated from the molecular weight of phage DNA and the content of DNA. The Stokes radius of the phage particle was calculated to be 37.7 +/- 2 nm and hydration of the phage was estimated to be 1.18 cm3/g of dry phage. From the particle weight and the chemical composition of the phage, we estimated that one phage particle contains one double-stranded DNA molecule, 16,000 residues of fatty acid, 72 protein I molecules, 920 protein II, 42 protein III, 48 protein IV, 290 protein V molecules, and 3,700 molecules of polyamines.     

Effects of genome size on bacteriophage phi X174 DNA packaging in vitro

Effects of the size of template DNA on the DNA packaging reaction of bacteriophage phi X174 were studied using plasmids of various sizes which contain the phi X174 origin of DNA replication and the in vitro phage synthesizing system (Aoyama, A., Hamatake, R. K., and Hayashi, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4195-4199). DNA between 78.5% and 101% of the length of phi X174 DNA produced infectious particles efficiently. Packaging of DNA smaller or larger than this range produced uninfectious defective particles. Although these particles contained circular single-stranded DNA, they suffered structural changes which altered the sedimentation properties or the ability to adsorb to the cells. Mutant phage were found from the packaging reaction of DNA larger than 101% of phi X174 DNA. These mutants deleted the termination region of DNA, suggesting that they were produced by early termination of the phage synthesizing reaction.     

Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA

Intracellular events following infection of competent Haemophilus influenzae by HPlcl phage, or transfection by DNA from the phage, were examined. Physical separation of a large fraction of the intracellular phage DNA from the bulk of the host DNA was achieved by lysis of infected or transfected cells with digitonin, followed by low-speed centrifugation. The small amount of bacterial DNA remaining with the phage DNA in the supernatants could be distinguished from phage DNA by its ability to yield transformants. After infection by whole phage, three forms of intracellular phage DNA were observable by sedimentation velocity analysis: form III, the slowest-sedimenting one; form II, which sedimented 1.1 times faster than III, and form I, which sedimented 1.6 times faster than III. It was shown by electron microscopy, velocity sedimentation in alkali, and equilibrium sedimentation with ethidium bromide, that forms I, II and III are twisted circles, open circles, and linear duplexes, respectively.After the entry of phage DNA into wild-type cells in transfection, the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. Some of the fast-sedimenting molecules are presumably concatemers and are generated by recombination. In strain rec₁^? the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in wild-type cells. In strain rec₂^? there is little degradation of phage DNA, and the proportion of fast-sedimenting molecules is much smaller than in wild-type cells. Since rec₁^? and rec₂^? are transfected with much lower efficiency than wild type, our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for more efficient transfection.     

Bacteriophage N3 of Haemophilus influenzae. IV. Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA

Intracellular events following infection of competent Haemophilus influenzae cells by N3 phage or transfection by DNA from phage were examined. After infection by whole phage three forms of intracellular phage DNA were observed by sedimentation velocity analysis. These forms are probably twisted circles, open circles and linear duplexes. In transfection only about 15% of the phage DNA is efficiently taken up by the competent cells. After entry of phage DNA into wild-type cells in transfection the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. These presumably concatamer forms are generated by recombination. In strain rec-1 the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in the wild-type cells. Since rec-1 is transfected with much lower efficiency than the wild-type our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for efficient transfection. These results also show that although phage N3 codes for its own recombination system it cannot operate in the early stages of transfection and succesful transfection is entirely dependent upon the host recombination system.     

Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins.

The proteins encoded by the adeno-associated virus type 2 (AAV-2) rep and cap genes obtained during a productive infection of HeLa cells with AAV-2 and adenovirus type 2 were fractionated according to solubility, cellular localization, and sedimentation properties. The majority of Rep and Cap proteins accumulated in the nucleus, where they distributed into a soluble and an insoluble fraction. Analysis of the soluble nuclear fraction of capsid proteins by sucrose density gradients showed that they formed at least three steady-state pools: a monomer pool sedimenting at about 6S, a pool of oligomeric intermediates sedimenting between 10 and 15S, and a broad pool of assembly products with a peak between 60 and 110S, the known sedimentation positions of empty and full capsids. While the soluble nuclear monomer and oligomer pool contained predominantly only two capsid proteins, the 30 to 180S assembly products contained VP1, VP2, and VP3 in a stoichiometry similar to that of purified virions. They probably represent different intermediates in capsid assembly, DNA encapsidation, and capsid maturation. In contrast, the cytoplasmic fraction of capsid proteins showed a pattern of oligomers continuously increasing in size without a defined peak, suggesting that assembly of 60S particles occurs in the nucleus. Soluble nuclear Rep proteins were distributed over the whole sedimentation range, probably as a result of association with AAV DNA. Subfractions of the Rep proteins with defined sedimentation values were obtained in the soluble nuclear and cytoplasmic fractions. We were able to coimmunoprecipitate capsid proteins sedimenting between 60 and 110S with antibodies against Rep proteins, suggesting that they exist in common complexes possibly involved in AAV DNA packaging. Antibodies against the capsid proteins, however, precipitated Rep78 and Rep68 predominantly with a peak around 30S representing a second complex containing Rep and Cap proteins.     

The presence of RNA in a double helix inhibits its interaction with histone protein

The binding of core histones (H2A, H2B, H3, H4) to a circular plasmid DNA and to a circular DNA-RNA hybrid molecule of similar size has been compared. Circular hybrid molecules were formed from single stranded fd DNA by synthesis of the complimentary strand with ribonucleotides using wheat germ RNA polymerase II. Upon reconstitution of plasmid DNA circles with histone, the sedimentation profiles of the DNA remained sharp by increased several fold in rate. Material from the peak fractions of these sedimentations appeared to be condensed circular loops of nucleosomes when examined by electron microscopy (EM), and the mass ratio of DNA to histone (at the histone concentrations which produced the fastest sedimentations) was typical of native chromatin. In contrast, the sedimentation behavior of DNA-RNA hybrid circles after addition of histone remained unchanged except for a minor fraction which exhibited a broad and faster sedimentation rate. Examination by EM revealed that most of the molecules appeared identical to protein free hybrid circles while the minor, faster sedimenting fraction appeared to be two or more circles bound together by protein aggregates. Finally, a linear molecule consisting of about 3000 base pairs of duplex DNA covalently joined on both ends to 1500 base pairs of RNA-DNA hybrid helix was constructed. Reconstitution of this molecule with core histone showed nucleosome formation only on the central DNA duplex region. Isopycnic banding of fixed hybrid-histone mixtures showed that little or no histone had bound to the bulk of the full hybrid molecules. We suggest that the presence of RNA in a nucleic acid duplex inhibits the condensation of the duplex into a nucleosomal structure by histone.     

Induction of prophage SPO2 in Bacillus subtilis: isolation of excised prophage DNA as a covently closed circle.

Bacillus subtilis tryC2, thyA, thyB, lysogenic for the phage DNA polymerase negative mutant SPO2 susL244, was induced under conditions preventing phage and bacterial DNA synthesis. The biological activity of DNA from induced cells and from uninduced controls was assayed by transformation and transfection, respectively. About 50% of the phage DNA biological activity in DNA extracted from induced cells was resistant to exposure to pH 11.8 TO 11.9. This DNA was operationally defined as alkali-resistant phage DNA. Transforming bacterial DNA from uninduced or induced cells and transfecting DNA from uninduced cells were more than 95% inactivated after exposure to high pH. The alkali-resistant phage DNA was characterized by sucrose gradient centrifugation, by centrifugation in cesium chloride-propidium iodide, and by electron microscopy. It was found to consist of a majority of covalently closed circular DNA molecules. Length measurements of a few relaxed circular molecules indicate a molecular weight of these similar to that previously found for mature SPO2DNA. Attempts to isolate similar covalently closed circular phage DNA from induced bacteria lysogenic for SPO2 phage with a functional DNA polymerase gene were unsuccessful. The gene order in mature and prophage SPO2 was determined by rescue of single and double markers from the respective type of DNA. The data obtained show that prophage DNA is (genetically) permuted relative to mature DNA. The phage attachment site is suggested to be located between genes I and J.     

Multiple origins and circular structures in replicating T5 bacteriophage DNA.

Replicating T5 phage DNA was gently isolated using NaI density gradient centrifugation and examined by electron microscopy. At the beginning of phage DNA synthesis, linear unit-length T5 DNA molecules containing from one to four replicating "eye-loops" were consistently observed. Replication in these molecules was found to proceed bidirectionally from multiple, internal origins. A primary origin of replication is located near the center of the T5 genome, which does not coincide with the location of any of the nicks (single-strand breaks) found in mature T5 DNA. The initiation of replication at the various origins within an individual molecule does not appear to follow any definite temporal sequence. At later times in the infection, we have observed a significant number of circular T5 DNA molecules-both replicating and nonreplicating-whose average circumference is approximately the length of mature T5 DNA minus the terminal redundancy. The replicating circular molecules appear to be either in a theta configuration, a sigma configuration with the tails all being less than the length of the circle, or a combination of theta and sigma forms.     

Properties of Thermoactinomyces vulgaris phage Ta1 and its extracted DNA

The virulent phage Ta1 was obtained in good yields from infected cultures of Thermoactinomyces vulgaris 1227. The purified phage was found to sediment with a single band, the sedimentation constant being (519 +/- 14)S, and to exhibit a typical nucleoprotein behaviour in UV-spectrophotometric and CD experiments. The Ta1 phage consists of a hexagonal head about 0.056 micrometers in diameter and a very short tail. It is morphologically similar to the temperate Salmonella phage P22. The nucleic acid extracted from the phage was found to be a double-stranded linear DNA with a G+C content of 42 mole-% as deduced both from its melting temperature and buoyant density in CsCl. Analytical sedimentation revealed a high degree of molecular homogeneity of Ta1 Dna. the sedimentation constant of this DNA amounts to (35.9 +/- 0.3)S, corresponding to a DNA molecular weight of about 29 millions daltons. The biological activity of Ta1 DNA was indicated by its ability to infect the mycelium of the components T. vulgaris strain 1227 and to give rise to mature phages.     

Atomic force and electron microscopy of high molecular weight circular DNA complexes with synthetic oligopeptide trivaline

Intramolecular compact structures formed by high molecular weight circular superhelical DNA molecules due to interaction with synthetic oligopeptide trivaline (1) were studied by atomic force and electron microscopy. Three DNA preparations were used: plasmids pTbol, pRX10 and cosmid 27,877, with sizes 6,120 bp, 10,500 bp and 44,890 bp respectively. Plasmid pTbo1 and pRX10 preparations along with monomers contained significant amount of dimers and trimers. Main structures in all preparations observed were compact particles, which coincide in their appearance and compaction coefficient (3,5-3,7) with triple rings described earlier. The size and structure characteristics of triple rings and other compact particles on atomic force images in general coincide with those obtained by EM (2). AFM (3) images allow to get additional information about the ultrastructural organization and arrangement of DNA fibers within the compact structures. Along with triple rings in pTbol and pRX10-TVP complexes significant amount of compact structures were observed having the shape of two or three compact rings attached to each other by a region of compact fibre. Basing on the data of contour length measurements and the shape of the particles it was concluded that these structures were formed due to compaction of dimeric and trimeric circular DNA molecules. Structures consisting of several attached to each other triple rings were not found for pTbol, pRX10 monomers or cosmid preparations--TVP complexes where only single triple rings were observed. The conclusion is made that initiation of compact fibre formation within the circular molecules depends on the primary structure and for dimeric or trimeric circular molecules two or three compaction initiation points are present, located in each monomer unit within one circular DNA molecule. The nucleotide sequence dependent compaction mechanism providing independent compaction of portions of one circular molecule can be of interest for understanding of DNA compaction processes in vivo.     

Direct transfer of coliphage lambda DNA from Escherichia coli to cellular slime mold Dictyostelium discoideum

Dictyostelium discoideum myxamoebae were cultured with Escherichia coli cells infected with lambda phage in the presence of chloramphenicol. After eliminating the uningested bacteria by repeated centrifugation in a Percoll gradient, we examined the myxamoeba cytoplasm (not the food vacuole) for the presence of phage DNA. A significant amount of DNA extracted from the myxamoebae was hybridizable with purified phage lambda DNA, and capable of forming phage particles when packaged in vitro with phage lambda proteins. The EcoRI restriction maps of the phages recovered from the plaques were identical to that of the infecting phage. These results strongly suggest that phage DNA molecules were taken up by the cellular slime mold cells and that at least some fraction existed in intact form.