Dimitrij Lang

Darstellung und längenmessungen des gesamten desoxyribonucleinsäure-inhaltes von T2-bakteriophagen

Abstract Electron micrographs of DNA macromolecules are prepared by spreading a suitable protein-salt solution with T2 phages to a mixed monolayer on a water-air interface. During the development of the film an osmotic shock occurs which confines the whole DNA content as a single thread near the phage ghost. The surface film is transferred to the support (carbonized formvar) by adsorption and removal of the water. The contrast arises by platinum deposition during the rotation of the specimens in a shadow-casting chamber. With a frequency of about 30%, unequivocal measurements of the DNA-thread system are possible. The first data of an average T2-DNA length of 49±4 μ are discussed in relation to the possible sources of error.

Molecular weights of coliphages and coliphage DNA. 3. Contour length and molecular weight of DNA from bacteriophages T4, T5 and T7, and from bovine papilloma virus.

Abstract DNA has been prepared for electron microscopy at ionic strength 0.20. The lengths of DNA molecules from the following viruses were found to be: bovine papilloma virus, (2.49 ± 0.05)μ (circular DNA); T7, (12.15 ± 0.25)μ; T5, (36.2 ± 0.8)μ; and T4, (52.0 ± 1.0)μ. The limits given are estimated systematic errors of unknown sign plus sample standard deviations. In units of T7 DNA, the relative lengths and their maximum errors are: bovine papilloma virus, 0.205 ± 0.003; T5, 2.98 ± 0.06; and T4, 4.28 ± 0.06; these values should equal relative molecular weights except for T4 DNA (4.71). Based on the mean value of 25.1 × 10 6 daltons, obtained from three recent and independent determinations of T7 DNA molecular weight, the molar linear density of duplex DNA as prepared by standardized electron microscopy was found to be 2.07 × 10 10 for DNA with common bases, 2.28 × 10 10 for T4 DNA, and 2.23 × 10 10 daltons/cm for T2 DNA. Consequently, molecular weights of the following viral DNAs are: bovine papilloma virus, 5.15 × 10 6 ; T5, 74.9 × 10 6 ; T4, 119 × 10 6 ; and T2, 116 × 10 6 daltons.

Regular superstructures of purified DNA in ethanolic solutions

Abstract Aqueous solutions of purified DNA from bacteriophage T7 were subjected to various concentrations of ethanol and visualized by electron microscopy. Compact, linear, unbranched particles with uniform diameters were found which have three distinct lengths, 3.04±0.04 μm, 0.69±0.01 μm and 0.159±0.014μm, with increasing diameters. An analysis of the observations revealed supercoils of first, second and third order for the above lengths. DNA supercoiling may be the consequence of dehydration by ethanol and drying and, in native chromatin, of dehydration by histone.

Dehydrated circular DNA: electron microscopy of ethanol-condensed molecules.

Abstract The ethanol-induced condensation of linear DNA from phage φ29, and circular DNA from phage PM2 and plasmid R6K, on air-dried electron microscope specimen grids has been analyzed by measuring size and shape of the resulting particles. Upon such dehydration, single DNA molecules are condensed into tight particles of three different classes of superstructure, appearing shorter and thicker with increasing ethanol concentration, as described earlier with coliphage T7 DNA (Lang, 1973), except that circularity of DNA impairs the formation of condensates of second and third order to a degree that depends on the molecular weight. It is suggested that tight supercoils are formed by induction of numerous kinks in the double helix between short, straight DNA segments. This would be consistent with DNA kinks proposed by Crick & Klug (1975).

CONFIGURATION AND LONGITUDINAL DISTRIBUTION OF DNA MOLECULES IN SOLUTION

Abstract A diffusion method is described for visualization of DNA molecules which is equivalent to the spreading of a mixed DNA-protein solution. Macromolecules diffusing in a solution of about 5×10−8 g DNA per ml are adsorbed by a cytochrome c-film at the air-water interface. Subsequently they are transferred to electron microscopic supports. In the enlarged two-dimensional image the end-to-end distances and the length distribution are measured for different DNA preparations (from cod fish, trout, holothuria and T2 phage). Following recalculation in the three-dimensional state a relation is given for the mean square of the end-to-end distances, h 2 , and the length, L, as ( h 2 ) 1 2 = 1.3 × 10 −1 L(1 + √ 1 + 2.3 × 10 −3 L at ionic strengths from 0.09 to 0.35, according to a theory by Katchalsky and Lifson for diluted linear polyelectrolytes. The molecular shape is not a random coil or a rod but intermediate on account of electrostatic interaction. An application of the results on the theory of Kuhn, Kuhn and Buchner yields equations for the diffusion coefficient or other characteristics of DNA as a function of length or molecular weight. The length distribution of DNA, after disintegration by preparation and storage, usually has the form of a descending exponential function. Within the accuracy of the length distribution it is allowed to deduce random scissions along the native DNA molecule.

A thin quartz cell suitable for vacuum ultraviolet absorption and circular dichroism measurements.

The design of a thin quartz cell suitable for absorption and circular dichroism measurements in the vacuum ultraviolet is described. Important features of the cell are (1) that it can be disassembled for cleaning and reproducibly reassembled with path lengths up to 0.3 mm, and (2) that strain in the windows from the compressed sample can be relieved by a sample overflow port. The latter feature allows the cell to be used for circular dichroism as well as absorption measurements.

In vitro assembly of the outer shell of bacteriophage ϕ6 nucleocapsid

Following dissociation of bacteriophage phi 6 nucleocapsid (NC) by EDTA, a particle composed of protein P8 and corresponding to the outer shell of the NC was assembled in vitro in the presence of Ca2+ and Mg2+. Assembly was obtained from soluble protein constituents above 100 micrograms/ml and was optimal within a temperature range of 22-30 degrees. Assembly did not require the presence of genomic RNA. Crosslinking results of intact NCs and in vitro-assembled outer shells suggested that protein P8 dimers are the structural subunits of the shell. Analysis of the assembly kinetics by electron microscopy suggested that ring-like particles of uniform size, packed in flat hexagonal arrays, are intermediates in outer shell assembly.

Evidence for sequence heterogeneity among the double-stranded RNA segments of Penicillium chrysogenum mycovirus.

We have examined the dsRNA segments of the multipartite virus from Penicillium chrysogenum (ATCC 9480). A fourth RNA segment has been detected recently and, in the present work, we separated the four RNA segments and looked for possible homologies among them by thermal denaturation and electron microscopical heteroduplex studies. Differences were found between the differential melting profiles of the separated RNA segments, and no extensive regions of homology were detected among the various RNA segments by heteroduplex analyses. The results suggest that each RNA segment contains unique sequences. A comparison of the lengths of the RNA segments determined by electron microscopy with those determined by gel electrophoresis suggests that one of the segments has a distinct tertiary structure.

Dependence of the U.V. Survival of transforming DNA on the amount of DNA uptake per cell

SummaryUV irradiation of transforming DNA from Haemophilus influenzae, carrying a streptomycin resistance marker (Sr), results in decreased transforming activity. At high DNA concentration the “marker survival” is lower than it is at low concentration. The transition from high to low survival occurs at concentrations ranging from 2.5×10-3 to 2.5×10-2 μg/ml; in this range the probability that transformed cells take up DNA fragments in addition to the marked one increases rapidly. A similar effect of DNA concentration on the percentage of transformants is observed for a mixture of unirradiated and irradiated DNA, where virtually all of the transformants originate from the unirradiated component. This eliminates the possible explanation that the concentration dependence of UV survival of a marker reflects increasing competition for a cellular repair system.It is concluded that the lower marker survival obtained at high DNA concentration involves lethality due to UV lesions present in the additional irradiated DNA taken up by the cell. Thus the steeper marker survival curve is due to the increasing UV dose which the additional DNa necessarily receives when a marker survival curve is being established. Intergration of UV lesions rendering a chromosomal DNA strand inviable is suggested by a slight delay in cell multiplication after uptake of irradiated and — to a lesser extent — unirradiated DNA. Acriflavine at a concentration of 0.5μg/ml enhances the effect of DNA concentration on marker survival. Similarly the number of transformants obtained with unirradiated DNA in the presence of acriflavine is more strongly decreased at high than at low DNA concentration. It is suggested that each event of DNA integration involves a small change for lethality, which is enhanced if the DNA carries UV lesions or if acriflavine is present.