Phased adenine tracts in double-stranded RNA do not induce sequence-directed bending

Tracts of four to six adenines phased with the DNA helix produce a sequence-directed bending of the helix axis. Here, using gel electrophoresis and electron microscopy (EM), we have asked whether a similar motif will induce bending in a duplex RNA helix. Single-stranded RNAs were transcribed either from short synthetic DNA templates or from Crithidia fasciculata kinetoplast bent DNA, and the complementary single-stranded RNAs were annealed to produce duplex RNA molecules containing blocks of four to six adenines. Electrophoresis on polyacrylamide gels revealed no retardation of the RNAs containing phased blocks of adenines relative to duplex RNAs lacking such blocks. Examination by EM showed most of the molecules to be straight or only slightly bent. Thus, in contrast to DNA duplexes, phased adenine tracts do not induce sequence-directed bending in double-stranded RNA. Analysis of the distribution of molecule shapes for the highly bent C. fasciculata DNA showed that the adenine blocks do not act cooperatively to induce DNA bending and that the molecules must equilibrate between a spectrum of bent shapes.     

Physical heterogeneity of mouse thymus lymphocytes.

Mouse thymocytes have been separated by velocity sedimentation in a density gradient. The resulting fractions have been analyzed using electrophoretic light scattering. The electrophoretic distributions of the individual sedimentation fractions reveal the presence of physically distinct subpopulations. Comparison of the mean mobilities of each fraction indicates that the faster-sedimenting cells tend to have a higher electrophoretic mobility.     

Cytoplasmic DNA synthesis in rhinovirus type 14-inoculated KB cells.

Rhinovirus type 14 (RV14) incuced a transient statistically significant stimulation in synthesis of DNA which appeared between 0 and 3 h post-inoculation in the cytoplasm of high density monolayer cultures of KB cells. Newly synthesized DNA was measured by incorporation of [3H] thymidine into acid-insoluble DNAase-sensitive material and the cytoplasmic location established by cell fractionation and electron microscope radioautographic methods. A minimum of 10 plaque-forming units per cell of RV14 was required to stimulate DNA synthesis which did not occur above 34.5 degrees C, a temperature optimal for virus replication. Cytoplasmic DNA taken from RV14-infected or control cells could be differentiated from the bulk of cell (nuclear) DNA by several criteria, including: (1) RV14 induction of synthesis; (2) lower buoyant density and greater heterogeneity in CsCl and ethidium bromide/CsCl gradients; and (3) a different kinetic complexity upon reannealing. The Cot 1/2 value of cytoplasmic DNA, calclated as 50--100 from reassociation profiles, was about 10-fold less complex than the Cot 1/2 value of nuclear DNA (800-1000). These data rule out the possibility that cytoplasmic DNA arises by random breakage of nuclear DNA during cell disruption and extraction and are compatible with the hypothesis that inoculation of KB cells with RV14 results in stimulation of synthesis of a specific class of cell DNA which is detected in the cytoplasm.     

Curved helix segments can uniquely orient the topology of supertwisted DNA

To show that DNA containing sharp sequence-directed curvature can preferentially establish ends of supertwisted domains, a highly curved DNA from Crithidia fasciculata was cloned into two sites separated by 28% in pBR325. When this construct (pJGC2) was examined by electron microscopy, 63% of the supercoiled molecules were branched with three or more arms, and the remaining molecules appeared as linear interwound rods. The distance between the tips of two of the arms for the branched molecules measured within 2% of 28% of the DNA contour for 32% of the pJGC2 molecules, as contrasted with only 6.6% for the poorly branched pBR325 DNA. When one of the curved segments in pJGC2 was replaced by a highly curved fragment from SV40, similar results were obtained.