DNA synthesis in nucleotide-permeable Escherichia coli cells

Hess, Ulrich; Dürwald, Hildegard; Hoffmann‐Berling, Hartmut

doi:10.1016/0022-2836(73)90090-9

Cited by 19 publications

(5 citation statements)

References 27 publications

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“…DNA through alkaline sucrose (5-20%, pH 12.5), two major products were observed (Figure 4). By comparison with unmodified DNA in a parallel gradient (not shown), the first peak representing 20% of the radioactive label was found to sediment with approximately 45 S. This is the value reported for covalently closed supercoiled -RFI (Hess et al, 1973).…”

Section: Resultssupporting

confidence: 64%

“…In addition, we observed that the width of the melting transition, , was slightly broader (1 -2 °C) for phosphorothioate fd-RF DNA was synthesized in 5 X 9 H512 ether-treated cells in the presence of [a-32P]dTTP with either dATP, dCTP and dGTP (20 µ each), or 50 µ dATPaS instead of dATP. DNA products were isolated as described (Hess et al, 1973). Separation of RFI and RFII was achieved in a neutral sucrose gradient (5 to 20%, SW27 rotor, 22 000 rpm for 12 h at 20 °C).…”

Section: Resultsmentioning

confidence: 99%

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Incorporation of phosphorothioate groups into fd and φX174 DNA

Vosberg¹,

Eckstein²

1977

Biochemistry

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We have synthesized fd and phi X174DNA in the presence of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP alpha S) and the corresponding phosphorothioate derivatives of dCTP and dTTP using ether-permeabilized E. coli cells or crude cell extracts of E. coli DNA polymerase I. Reaction rates of enzymes involved in the formation or breakdown of DNA are decreased in the presence of phosphorothioates. The amount of label incorporated with [35S]dATP alpha S suggests that the dAMP has been completely substituted by 2'-deoxyadenosine 5'-0-phosphorothioate (dAMPS). The substituted DNAs have the same sedimentation coefficients, similar buoyant density, infectivity, and thermal stability as the unsubstituted DNAs. The procedure therefore allows specific modification at the 5' position of dA, dC, or dT in the DNA. In view of the recent demonstration of specific binding of Pt2+ complexes to the phosphorothioate analogue of poly[r(A-U)] (Strothkamp, K.G., and Lippard, S.J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 2536), the synthesis of phosphorothioate containing DNA may be of use for DNA sequencing by electron microscopy.

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Section: Resultssupporting

confidence: 64%

Section: Resultsmentioning

confidence: 99%

Incorporation of phosphorothioate groups into fd and φX174 DNA

Vosberg¹,

Eckstein²

1977

Biochemistry

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“…of a single bead (200 to 250 base pairs) have been found in studies of DNA replication in eukaryotic cells (17,18), in certain phages (19), and more recently in E. coli (20). Also the reported properties of DNA in the E. coli folded chromosome (nucleoid) are consistent with this study.…”

Section: Discussionmentioning

confidence: 99%

Visualization of prokaryotic DNA in a regularly condensed chromatin-like fiber.

Griffith¹

1976

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

137

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Electron microscopy of disrupted Escherichia coli cells under certain conditions revealed loops of a fiber 120 A in diameter which were attached to the cell envelope and showed a 130 A repeating beaded substructure. These fibers were detected only when the cells were lysed in 0.15 M NaCl solutions directly on the electron microscope supporting films and if the dehydration steps began within 2 min of lysis.Under these conditions examination of cells lysogenic for phage X after superinfection with X wild type or deletion mutants disclosed short loops of a 120 A diameter fiber free of the cell envelope. Because the contour length of these loops was proportionate to the DNA content of the superinfecting X phage, it was concluded that the fibers contained DNA condensed 6.5-fold in blocks of about 250 base pairs. Packaging DNA is a problem common to all cells. In higher cell nuclei the histones stabilize DNA in a condensed structure termed chromatin. This nucleoprotein complex can be isolated and visualized as a network of fibers with a 120 A diameter and a regularly repeating bundled substructure (2-4). Comparison of the size of a bacterial cell and the total length of DNA it contains makes some condensation of its DNA likely. But prokaryotic cells do not possess histones, and consequently it is of interest to ask how their DNA might be kept in a condensed state in the cell; no such condensation has yet been observed. Prokaryotic cells do contain large amounts of Mg++ and the small basic polyamines (5), and these alone or together with proteins could complex DNA. Assuming that such a condensed or complexed state exists within the cell but is labile upon lysis, a technique for lysing bacteria in physiologic salt solutions directly on the electron microscope supporting film and for preparing samples for electron microscopy has been developed to approach a visualization of the structure of the putative condensed DNA. Observations presented here with Escherichia coli and phage X indicate that immediately after lysis, prokaryotic DNA can be found in fibers having physical parameters remarkably similar to those found for the eukaryotic unit fiber (3, 4, 6). The significance of this finding to studies of higher cell chromosome structure and the relation of these bacterial fibers to the state of DNA in vvo are discussed. MATERIALS AND METHODSGrowth of Bacteria (10) were immersed in the wells for 4 min, rinsed with buffer B, and placed in wells filled with buffer B containing 1% Triton X-100. The micro titer plate with the supports was then warmed from 00 to 370 for 1-5 min. The supports were removed, dehydrated, and shadowed with tungsten (10), the washes beginning with 50% ethanol. Electron micrographs were taken with a Philips EM 300 and dimensions measured directly on the original micrographs by optically projecting them onto an electronic digitizer tablet. RESULTSVisualization of E. coli cells disrupted on the electron microscope supporting film Several images of E. coli cells treated as described above are i...

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“…However, in these experiments, there is probably some residual ligase acti vity or, in the case of phage, the bacterial ligase may be doing the joining (Newman & Hanawalt, 1968;Sugimoto et a1., 1968;Pauling & Hamm, 1969;Olivera & Lundquist, 1971). There is a report that small sized fragments seen 20 at low concentrations of deoxynucleotides (see below) do not require the NAD (nicotinamide-adenine dinucleotide)-dependent ligase to join into larger pieces (Hess et al, 1973), but there is probably some other ligase that joins these pieces.…”

Section: Enzymes Needed For Joiningmentioning

confidence: 99%

“…Whether the other strand is also made discontinuously is still very much an open question. On the basis of the assumption that more than 50% of acid-precipitable radioactivity in small pieces after a short pulse-label indicates that both strands are being synthesized discontinuously, many groups have claimed to observe totally discontinuous synthesis (Okazaki et al , 1968a(Okazaki et al , , h, 1973Sadowski et al, 1968;Yudelevich et al, 1968;Fareed et al, 1973;Goldstein & Rutman, 1973;Gottesman et al, 1973a;Hess et al, 1973;Kohn et al J 1974;Gautschi & Clarkson, 1975;Mendelsohn et a1. , 1975;Tseng & Goulian, 1975a).…”

Section: Are Both Strands Made Discontinuously?mentioning

confidence: 99%