Electron microscopy of size and shape of viral DNA in solutions of different ionic strengths

Lang, Dimitrij; Bujard, Hermann; Wolff, B.; Russell, David W.

doi:10.1016/s0022-2836(67)80024-x

Cited by 186 publications

(28 citation statements)

References 58 publications

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“…The lengths of the dsRNA segments in the hybridization mixtures and the lengths of the RNA segments in the unfractionated RNA are summarized in Table 1. After spreading in the presence of formamide at low ionic strength, the lengths of the dsRNA molecules were, as expected (Lang et al, 1967), 19(_+2)~ greater than the lengths measured under standard conditions at an ionic strength of 0.2 M.…”

Section: Electron Microscopy Of Pcv Rna Segmentssupporting

confidence: 79%

Evidence for Sequence Heterogeneity among the Double-stranded RNA Segments of Penicillium chrysogenum Mycovirus

Edmondson¹,

Lang

Gray

1984

Journal of General Virology

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SUMMARYWe 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. INTRODUCTIONMany of the dsRNA mycoviruses (fungal viruses) contain several dsRNA segments of different molecular weights that are encapsidated in separate virus particles (Buck, 1980;Hollings, 1978;Wood, 1973). Evidence is accumulating that some of the individual dsRNA segments contain unique genetic information in these mycoviruses. For example, Romanos et al. (1981) investigated two viruses of the wheat fungus Gaeumannomyces graminis by RNA fingerprinting, solution hybridization, and translation experiments in vitro. They found that the sequences of two dsRNA segments of each virus are unique (< 10 % homology) and that a third segment of one of the viruses is probably a satellite RNA of unique sequence. In the virus-like particles of killer strains ofSaccharomyces cerevisiae and Ustilago maydis, it has been determined by translation studies in vitro, hybridization analysis, sequencing and heteroduplex mapping that genes for capsid and toxin polypeptides reside on different dsRNA segments (Bostian et al., 1980;Field et al., 1983), although resistance to toxin in the U. maydis system can be encoded by either of two dsRNAs of different size (Bostian et al., 1980). There is much less evidence for the uniqueness of genetic information in the dsRNA segments of viruses of Penicillium fungi. Wetmur et al. (1981) concluded from the renaturation kinetics of dsRNA from Penicillium chrysogenum virus (PcV) that there is little homology among the different RNA segments of this virus. However, their study was done on unfractionated RNA from a strain of PcV that was assumed to have only three RNA segments. We have succeeded in isolating each of the four RNA segments of PcV from fungal strain ATCC 9480, and in the present paper we present evidence of conformational and sequence heterogeneity among the segments.

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Section: Electron Microscopy Of Pcv Rna Segmentssupporting

confidence: 79%

Evidence for Sequence Heterogeneity among the Double-stranded RNA Segments of Penicillium chrysogenum Mycovirus

Edmondson¹,

Lang

Gray

1984

Journal of General Virology

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“…In order to do this, the molecule will tend to minimize interactions with the surrounding solution, and as a result the time-averaged shape of a DNA molecule in solution is spherical [82]. dsDNA, however, is not overly flexible, and as a result behaves similarly to a series of semi-flexible rods joined end-to-end [83].…”

Section: Dna In Free Solutionmentioning

confidence: 99%

Interfacing solid‐state nanopores with gel media to slow DNA translocations

et al. 2015

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We demonstrate the ability to slow DNA translocations through solid‐state nanopores by interfacing the trans side of the membrane with gel media. In this work, we focus on two reptation regimes: when the DNA molecule is flexible on the length scale of a gel pore, and when the DNA behaves as persistent segments in tight gel pores. The first regime is investigated using agarose gels, which produce a very wide distribution of translocation times for 5 kbp dsDNA fragments, spanning over three orders of magnitude. The second regime is attained with polyacrylamide gels, which can maintain a tight spread and produce a shift in the distribution of the translocation times by an order of magnitude for 100 bp dsDNA fragments, if intermolecular crowding on the trans side is avoided. While previous approaches have proven successful at slowing DNA passage, they have generally been detrimental to the S/N, capture rate, or experimental simplicity. These results establish that by controlling the regime of DNA movement exiting a nanopore interfaced with a gel medium, it is possible to address the issue of rapid biomolecule translocations through nanopores—presently one of the largest hurdles facing nanopore‐based analysis—without affecting the signal quality or capture efficiency.

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“…The complex was then fixed (10 min, 370) by addition of one-fifth volume of fixation buffer (0.5% glutaraldehyde in the above solution at pH 7.9), as described by Portmann et For experiments using ethidium bromide, DNA was simply diluted to 0.8 Mg/ml in the elution buffer and heated for 15 min at 370 to dissociate the cohesive ends. After addition of ethidium bromide to 15 ,g/ml, the DNA was adsorbed to carbon filmcoated grids, as above.…”

mentioning

confidence: 99%

Discrete length classes of DNA depend on mode of dehydration.

Vollenweider¹,

James²,

Szybalski³

1978

Proc. Natl. Acad. Sci. U.S.A.

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The length of double-stranded coliphage X DNA, as determined by electron microscopy using the benzyldimethylalkyl ammonium chloride technique, depends on the mode of dehydration. The freeze-dried DNA form is the longest (16.5 mr), whereas dehydration in methanol (15.9 Am) or in ethanol (three forms: 15.2 gsm, 13.9 im, and 12.4 gm) results in progressively shorter molecules. These measured lengths of the freeze-dried, methanol-dehydrated, and shortest ethanol-dehydrated forms correspond to the axial rise per nucleotide pair in the B, C, and A forms of DNA, respectively. The remaining forms of ethanol-dehydrated DNA seem to represent novel intermediary conformations of DNA. In agreement with the redicted increment, DNA exposed to ethidium bromide and ried is elongated by 39% (2.9 Am) All size classes show the same relative distribution pattern of bound Escherichia coli RNA polymerase molecules (nucleoside triphosphate:RNA nucleotidytransferase, EC 2.7.7.6), used as intramolecular markers, indicating that the dehydration-caused transitions are uniform.Although x-ray diffraction, x-ray scattering, UV The conformation of double helical DNA (1) has been studied by various methods, including x-rays, circular dichroism (CD), and electron microscopy. X-ray diffraction by oriented DNA fibers reveals three double-helical forms, depending on the relative humidity, the ionic strength, and the nature of the cation. The B form appears at relative humidity 92% for the sodium salts (2); the crystallinity of the fibers is best with lithium salts at 66% relative humidity (3). The A form (4) exists in the presence of sodium, potassium, or rubidium salt when the relative humidity is lower than 75% or after dehydration in 80% ethanol (5). With lithium salts, the A form of DNA could not be obtained but, when the relative humidity is reduced to about 66%, the C form appears (6). Table 1 shows various parameters of the A, B, and C forms of DNA.By using wide-angle x-ray scattering, the structure of DNA in aqueous solution was found to be close to the classical B form (7,8). CD also permits determining various forms of DNA in solution but requires a calibration to assign the CD spectra to the A, B, and C forms. Therefore, Tunis-Schneider and Maestre (13) obtained the CD spectra of the DNA in films under conditions of humidity and ionic strength comparable to those used in x-ray studies. These assignments of standard spectra allowed study of the transition from one form to another in different solvents-e.g., the CD spectra of a number of DNAs were studied as a function of alcohol concentration in aqueous solutions at 0.1-1.0 mM salt concentrations. With increasing concentrations of methanol, the CD changes reflect a B-to-C transition, whereas in 80% ethanol the CD spectrum resembles what one might expect for form A DNA (9, 12) (Table 1). However, it remains unclear whether aggregates of DNA molecules or mixtures of different DNA forms could mimic or mask the true spectrum and thus introduce artifacts (8, 9). 'The costs of publica...

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Electron microscopy of size and shape of viral DNA in solutions of different ionic strengths

Cited by 186 publications

References 58 publications

Evidence for Sequence Heterogeneity among the Double-stranded RNA Segments of Penicillium chrysogenum Mycovirus

Evidence for Sequence Heterogeneity among the Double-stranded RNA Segments of Penicillium chrysogenum Mycovirus

Interfacing solid‐state nanopores with gel media to slow DNA translocations

Discrete length classes of DNA depend on mode of dehydration.

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