Linear Duplex Molecules Research Articles

Replication process of the parvovirus H-1. II. Isolation and characterization of H-1 replicative form DNA.

The Parvovirus H-1 replicates autonomously in hamster embryo cells. A DNA synthetic event, called HA-DNA synthesis, upon which subsequent viral RNA and viral hemagglutinin synthesis is dependent, is initiated in late S phase of the infected cell (18). It was postulated that HA-DNA represents parental viral replicative form DNA (RF DNA). This study describes the isolation and characterization of H-1 RF DNA as part of the continuing study of the mechanisms and control of DNA replication in the eukaryotic cell. The H-1 RF DNA is a linear duplex molecule containing the viral strand and its complement. The complementary strands of the RF DNA have been separated by equilibrium density gradient centrifugation. The RF DNA has a buoyant density of 1.705 in neutral CsCl and an estimated guanine plus cytosine (GC) content of 45.9%. It has a sedimentation coefficient of 17S. The calculated molecular weight of 3.7 x 10(6) is twice that of the single-stranded virion DNA. H-1 virions contain DNA that is homogeneous and free of complementary strands.

Cleavage of circular, superhelical simian virus 40 DNA to a linear duplex by S1 nuclease.

S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.

Studies on Bacillus subtilis bacteriophage φ15

The growth characteristics and physicochemical properties of Bacillus subtilis bacteriophage φ15 have been investigated and compared with φ29. Differences in growth characteristics of these phages were found in various stages of spore germination and outgrowth which may account for the absence of plaque formation by φ15 on wild-type cells. Both φ15 and φ29 genomes were incorporated into spores when cells were infected at a certain critical time during host cell development. φ15 is morphologically indistinguishable from φ29. The DNA of φ15 is a linear duplex molecule having a mean length of 6.1 μm. The S value of this DNA is 23 ± 0.4 S. These values correspond to a molecular weight of approximately 12 × 10 6 daltons. At least seven structural proteins of φ15 were recognized in SDS-acrylamide gel electrophoresis. The sum of the molecular weights of φ15 proteins is approximately 354,000 daltons. This corresponds to about 59% of the coding capacity of double-stranded DNA of φ15. φ15 DNA was found to hybridize with φ29 DNA.

Endonuclease activity associated with the DNA protein complex of minicircular DNA of Escherichia coli 15.

The DNA · protein complex (form I*) of minicircular DNA of Escherichia coli has associated with it an endonucleolytic activity which converts the supercoiled form I into an open circular form II by introducing one single‐stranded break per molecule. This nick can occur with similar frequency in either strand. Prolonged incubation results in the formation of linear duplex molecules (form III). The endonuclease requires a divalent cation (Mg2+ or Mn2+) and is inhibited by high salt concentration and transfer RNA. It might provide a swivel mechanism during the replication of minicircular DNA.

Cleavage of Simian virus 40 DNA at a unique site by a bacterial restriction enzyme.

The R(1) restriction endonuclease of Escherichia coli converts covalently-closed circular Simian Virus 40 (SV40) DNA to unit-length linear duplex molecules. Cleavage occurs at a unique site, since denaturation and renaturation of these linear molecules yield linear but no circular molecules. The distance from the cleavage site to the SV40 DNA sequence contained in the adenovirus-SV40 hybrid, Ad2(+)ND(1), is 0.11 of the length of SV40 DNA. T4 gene 32 protein binds to SV40 DNA in a region 0.45 of the length of SV40 DNA from the R(1) cleavage site. E. coli B restriction endonuclease can cleave SV40 DNA at several sites.

Properties of sex factor and related episomes isolated from purified Escherichia coli zygote cells

Whole cells of 13C, 15N-labeled (heavy) Escherichia coli and non-labeled (light) E. coli were separated as discrete bands by centrifugation in a preformed sucrose-CsCl density gradient. By this technique, primary zygote cells were freed from donor cells in a mating mixture in which [ 3H]thymidine labeled heavy donor cells carrying sex factor (F +) or a related episome had been crossed with light recipient cells. Radioactive DNA transferred from the donor cells to recipient cells during mating, was extracted from the purified zygotes and analyzed by equilibrium centrifugation and zone sedimentation. Most of the F + DNA isolated by this technique, behaves as linear duplex molecules with a weight of 1.0 × 10 8 daltons. The major components in F′8, F′ lac and F' try DNA preparations isolated similarly have molecular weights of 1.0 × 10 8, 1.6 × 10 8 and 2.1 × 10 8, respectively. The transferred E. coli sex factor (F +) replicates semiconservatively in the zygotes, probably once in a generation time of the recipient cells.

Physical and biological properties of phage phi 29 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.

Terminal repetition in non-permuted T3 and T7 bacteriophage DNA molecules

Evidence in the preceding paper indicates that the DNA molecules from bacteriophage T2 contain terminally repetitious nucleotide sequences. In the first part of this paper, we describe similar, but more extensive, experiments performed on T3 and T7 DNA molecules. Partial degradation of either phage DNA molecule by exonuclease III allows efficient conversion of the linear molecules into circular ones. Fragments of these molecules, which are not expected to be terminally repetitious, can be partially degraded under identical conditions, but they do not anneal to form circles. These facts indicate that T3 and T7, like T2 DNA, have terminally repetitious sequences. The second part of the paper deals with the non-permuted character of T3 and T7 DNA molecules. The complementary strands of each species of molecule were completely separated with alkali and then annealed. In both cases, this treatment produced predominantly linear duplex molecules of a length equal to that of the undenatured molecules. From this we conclude that the collection of molecules is non-permuted (i.e., they are a unique collection of sequences). When this experiment is taken together with earlier equivalent experiments on T2 DNA molecules, which are readily cyclized by this treatment, it appears that alkali denaturation and annealing is a rigorous test for the presence of multiple permutations. In sum, T3 and T7 DNA molecules are terminally repetitious but non-permuted; T2 DNA is terminally repetitious and permuted.

Changes in the structure and activity of λ DNA in a superinfected immune bacterium

Labeled intracellular λ DNA isolated from superinfected lysogenic Escherichia coli K12 exhibits a very low level of infectivity. Sedimentation studies revealed that the residual infectivity was due to a few fully infectious molecules (species III), but that a majority of the labeled phage DNA was present as two new and non-infectious species (I and II). At neutral pH, intracellular species I sedimented 1·9 times faster and species II 1·14 times faster than λ DNA isolated from mature phage. In alkali, species II co-sedimented with denatured mature DNA molecules while species I moved 3·8 times faster. During storage, tritiated species I is converted slowly into II and also loses its distinctive capacity to renature readily after alkali treatment. It is suggested that intracellular species I is a condensed form of λ DNA, species II is a circular molecule and species III is a normal linear duplex molecule similar to those obtained from mature phage.