Propidium Diiodide Research Articles

Bacteriophage φx174 DNA synthesis in a replication-deficient host: Determination of the origin of φX DNA replication

We have reinvestigated bacteriophage φX DNA synthesis in rep 3 − cells. In these cells adsorption, transcription and translation of φX occurs normally, but φX replicative form DNA replication is blocked. After infection, φX DNA was isolated and separated by propidium diiodide/CsCl gradients into RFI † † Abbreviations used: RFI, replicative form DNA with both strands closed; RFII, replicative form DNA with one or more discontinuities in either strand; CAM, chloramphenicol; m.o.i., multiplicity of infection; PrI 2, propidium diiodide. HaeIII and HindII, restriction endonucleases from Haemophilus aegyptius and Haemophilus influenzae Rd, respectively. Z and R, DNA fragments produced by HaeIII and HindII, respectively. and RFII, followed by chromatography on BND-cellulose. In addition to the results of Francke & Ray (1971, 1972), we found an increasing amount of viral strand DNA synthesis in each fraction as time of infection proceeds. The occurrence of viral strand DNA synthesis is dependent on the presence of a functional cistron A product. In the presence of 150 μg chloramphenicol/ml or in infections with am18 (gene A) no viral strand DNA synthesis could be detected. After completion of the parental RF molecule, [ 3H]thymidine enters almost exclusively into viral strand DNA. The greater part of this viral strand DNA is found in genome-length φX. DNA in both RFI and RFII. Alkaline sucrose sedimentation of the RFII indicates that post-infection viral DNA label is only in linear φX DNA. These results strongly suggest that an extensive nick translation of the viral strand of RFII, followed occasionally by a conversion to RFI, takes place after synthesis of a functional cistron A product. This conclusion could be confirmed by restriction enzyme analysis of φX RF DNA, isolated from rep 3 − cells infected with 32P-labeled phage in the presence of [ 3H]thymidine. With the restriction enzyme HaeIII a gradient in the 3H 32P ratio of the fragments was obtained extrapolating into the fragment Z 6B. From this gradient the origin of the φX DNA nick translation could be located in the left half of Z 6B at approximately 65% (185 base-pairs) from the Z 6A-Z 6B junction.

The structure of kinetoplast DNA: I. Properties of the intact multi-circular complex from Crithidia luciliae

1. We have developed a modified isolation procedure that yields kinetoplast DNA networks containing more than 90% closed circular DNA, as judged by two criteria: 1. (a) In 0.15 M NaCl 0.015 M sodium citrate (pH 7.0), less than 10% of the intact kinetoplast DNA melts in the temperature region of sonicated kinetoplast DNA. In 7.2 M NaClO 4 the kinetoplast DNA melts with a T m 26° C higher than sonicated kinetoplast DNA. Even after complete melting in 7.2 M NaClO 4 at 90° C, the network remains intact, as judged by regain of hypochromicity on cooling and analysis in CsCl containing propidium diiodide. 2. (b) In alkaline sucrose gradients more than 90% of the kinetoplast DNA sediments in a single peak. 2. In CsCl gradients containing ethidium bromide or propidium diiodide intact kinetoplast DNA gives a single uni-modal band showing an extremely restricted dye uptake. From the position of the band relative to the bands of PM2 DNA, the superhelix density of these networks is calculated to be +3.9 twists per 1000 base pairs. The superhelix density of closed mini-circles, efficiently liberated from the networks by shear in a French press, is −0.5 twists per 1000 base pairs. We attribute the high superhelix density (the highest yet observed in any DNA) of intact networks to their compact, highly catenated structure, leading to an additional constraint on dye uptake, superimposed on the restriction due to closed circularity.

Restricted uptake of ethidium bromide and propidium diiodide by denatured closed circular DNA in buoyant cesium chloride

Alkali-denatured closed circular DNA forms, on neutralization, a relatively stable species first described by Pouwels et al. (1968). In contrast to single-stranded DNA, this denatured two-stranded closed circular DNA species bands densely and co-bands approximately with closed circular duplex DNA in ethidium bromide-CsCl equilibrium density gradients. In CsCl gradients containing propidium diiodide, denDNA I is denser than DNA I, nicked circular DNA and single-stranded φX174 viral DNA. The magnitude of the separations between the above DNAs allows preparative isolation of each when all four are present in the same gradient. The denDNA I has a novel open circular appearance in the electron microscope when cast on standard aqueous hypophases. This species becomes tightly twisted when cast on either aqueous or formamide hypophases containing ethidium bromide. We have concluded from these observations that the high buoyant density of denDNA I in dye-CsCl gradients, relative to single-stranded DNA, is the result of a restricted uptake of dye.

An electron microscopic method for studying nucleic acid-protein complexes. Visualization of RNA polymerase bound to the DNA of bacteriophages T7 and T3.

AbstractDouble‐stranded DNA can be readily adsorbed on mica or directly on carbon coated grids from the surface of solutions containing ethidium bromide, actinomine, or propidium diiodide. The DNA molecules are unfolded, well separated, and show a length distribution similar to molecules prepared by protein monolayer techniques.Since the intercalating dyes tested do not lead to an increased apparent diameter of the nucleic acid the method is useful for the study of nucleic acid–protein complexes. As a model, the binding of E. coli RNA polymerase to phage T7 and T3 DNA was examined under different conditions. The enzyme can easily be identified and its position along the DNA molecule can be mapped.

In vivo effects of intercalating drugs on the superhelix density of mitochondrial DNA isolated from human and mouse cells in culture

The degree of supercoiling (superhelix density) of closed circular mitochondrial DNA was found to increase three- to fivefold when the intercalating drug ethidium bromide was added to exponentially growing cultures of HeLa cells and to an SV40 virus-transformed line of mouse cells (SV3T3). This structural change was complete within ten hours in cells growing at a normal rate in the presence of 0.1 μg of the drug/ml., and within a shorter time at 1 μg/ml. Mitochondrial DNA synthesis, as indicated by incorporation of [ 32P]orthophosphate and [ 3H]thymidine, is essentially completely inhibited at the higher drug level and is greatly reduced at the lower drug level. The above results demonstrate that mitochondrial DNA is subject to nicking-closing cycles in the presence of the drug. Four acridine derivatives, known to unwind DNA upon binding, have similar in vivo effects on the superhelix density of mitochondrial DNA. The ethidium analogue, propidium di-iodide, has no effect, but appears on the basis of fluorescence microscopy to be excluded from the cells. Actinomycin D also has no effect. Ethidium bromide had no detectable effect on the base composition of mitochondrial DNA as indicated by buoyant density studies in neutral and alkaline cesium chloride. The drug had no effect on the distribution of complex mitochondrial DNA (circular dimers and catenated oligomers) measured with the electron microscope.