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Molecular autoradiography: The β-ray counting from single virus particles and DNA molecules in nuclear emulsions

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... However, this system proved unsatisfactory mainly because of artefacts due to the solubility of the crystals . LEVINTHAL, 1957, devised experiments based on absolute quantitatio n of beta track autoradiography. He suspended virus particles labelled wit h P-32 in thick layers of G-5 emulsion . ...
Article
Results of an investigation into the possibilities for a quantitative ; interpretation of autoradiograms on the basis of grain counting or track counting ; are presented. The relation between track length, number of grains, and initial ; energy of the beta particle is studied, and the blackening produced by a point ; source of a beta emitter, e.g. carbon14, surrounded by nuclear emulsion is ; described in terms of the distribution of grains in space. It is shown how an ; analysis of this type can be carried out for any pure beta emitter. However, the ; present measurements are confined to beta partilces of up to 400 kev. (auth);
... DFP labeled with phosphorus-32 was therefore used here for the latter purpose, and its uptake into single endplates was measured by beta track radioautography . Levinthal and Thomas (1957) have demonstrated the quantitative precision of this technique in an elegant series of experiments on the number of atoms of phosphorus in single virus particles, and its potentialities have been further explored by Levi et al. (1963) . ...
Article
Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-32P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 107 sites per endplate, in the diaphragm, about 3 x 107. Reaction with DFP-32P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 107 and 1.1 x 107per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.
... 4.3. Detecting replication: The Meselsohn–Stahl experiment and autoradiography Levinthal developed a method for detecting the distribution of phosphorus-32 in nucleic acid—by exposing individual labeled phage particles to photographic emulsion to produce tracks on an autoradiograph (Levinthal & Thomas, 1957a). 56 Decay of phos- phorus-32 incorporated into DNA, as point sources, left star-shaped figures in the film; the number of tracks per star gave information about the number of radioactive phosphorus atoms in that source (see Fig. 4). ...
The recent historiography of molecular biology features key technologies, instruments and materials, which offer a different view of the field and its turning points than preceding intellectual and institutional histories. Radioisotopes, in this vein, became essential tools in postwar life science research, including molecular biology, and are here analyzed through their use in experiments on bacteriophage. Isotopes were especially well suited for studying the dynamics of chemical transformation over time, through metabolic pathways or life cycles. Scientists labeled phage with phosphorus-32 in order to trace the transfer of genetic material between parent and progeny in virus reproduction. Initial studies of this type did not resolve the mechanism of generational transfer but unexpectedly gave rise to a new style of molecular radiobiology based on the inactivation of phage by the radioactive decay of incorporated phosphorus-32. These 'suicide experiments', a preoccupation of phage researchers in the mid-1950s, reveal how molecular biologists interacted with the traditions and practices of radiation geneticists as well as those of biochemists as they were seeking to demarcate a new field. The routine use of radiolabels to visualize nucleic acids emerged as an enduring feature of molecular biological experimentation.
Chapter
Prior to speaking of the structure and physical properties of nucleic acids, it would seem desirable to examine their biological role. In this book it has been remarked already several times that DNA is the fundamental genetic substance and nucleic acids are responsible for the synthesis of proteins. It would seem that the time has come to bring out the evidence for these statements.
Article
Full-text available
Studies of the size, composition, and structure of the deoxyribonucleic acid (DNA) of the F and G prototypes of herpes simplex virus (HSV) subtypes 1 and 2 (HSV-1 and HSV-2) showed the following. (i) As previously reported by Good-heart et al. HSV-1 and HSV-2 DNA have a buoyant density of 1.726 and 1.728 g/cm(3), corresponding to 67 and 69 guanine +/- cytosine moles per cent, respectively. The difference in guanine plus cytosine content of the DNA species was confirmed by the finding of a 1 C difference in T(m). (ii) The DNA from purified virus on cocentrifugation with T4 DNA in neutral sucrose density gradients sedimented at 55S, corresponding to 99 +/- 5 million daltons in molecular weight. HSV-1 and HSV-2 DNA could not be differentiated with respect to size. (iii) Cosedimentation of alkali-denatured DNA from purified virus with T4 DNA on alkaline sucrose density gradients consistently yielded several bands of single-stranded HSV DNA ranging from fragments 7 x 10(6) daltons to intact strands 48 x 10(6) daltons in molecular weight.
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The st mutation in the T5 wild-type phage, demonstrated by an increased heat stability of the phage particle, causes changes in the T5 DNA molecule. The five different T5st mutants studied have 2–7% less DNA than the wild-type phage. When the wild-type T5 DNA molecule is first broken by shear it breaks asymmetrically into long and short fragments representing 65 and 35% of the mass of the molecule. When the DNA molecule from the T5st mutants is first broken by shear, it also breaks asymmetrically with a decrease in the average size of the long fragments and an increase in the average size of the short fragments. These changes are specific for each of the st mutants. Other physical parameters of the phage parallel their loss of DNA. Those phage that have lost the greatest amount of DNA are the most heat stable, sediment the slowest, and have the lowest density.
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Molecular weights (M) of the DNA from Escherichia coli bacteriophages T4, T5 and T7 are obtained by averaging the values from several methods. From these values and data for molecular length (l) under specific conditions of preparation for electron microscopy, is derived. The possibility that is not constant for all DNA molecules is discussed. Using sedimentation data an equation relating S20,w0 and M for bulk sedimentation and a second equation for sedimentation in sucrose gradients are presented. The general problems of measurement of M, and S20,w0 and the current state of affairs are discussed.
Article
The molecular weights of T2, T5st+, T5st-0 and T7 DNA's were found by molecular autoradiographic determination of the phosphorus content of the phage DNA and/or the phage particle. The values obtained for the molecular weights of the sodium salts of these DNA's were 132 ± 12 × 106 a.m.u.† for T2, 83 ± 4 × 106 a.m.u. for T5st+, 77 ± 2 × 106 a.m.u. for T5st-0 and 25 ± 2 × 106 a.m.u. for T7. The sedimentation rates of these DNA's relative to T7 DNA (32.6 s) were determined in sucrose gradients. The Ssuc of T2, T5st+ and T5st-0 were 57.0 s, 49.2 s and 48.1 s, respectively. These data and the relative sedimentation rates of T7 “whole” and “half” molecules were used to obtain the relationship: Ssuc = 0.0833M0.350. Emperical relationships for Mversus [η] or S20 · w0 were obtained. These are: log10 ([η] + 5) + 2.774 = 0.653 log10M and . These latter two relationships hold over a molecular weight range of 300,000 to 130 × 106 a.m.u. for duplex DNA. The mass per unit length of DNA molecules measured by the protein film technique was determined to be 19 ± 0.1 × 106 a.m.u. per micron for DNA in films cast on water.
Article
The problem of obtaining meaningful molecular weights for high molecular weight DNA samples by sedimentation equilibrium in a density gradient has been solved by correcting the theory for thermodynamic non-ideality. Several different methods of virial corrections are suggested and examined to determine the best method of obtaining molecular weights at infinite dilution.A pronounced virial effect is experimentally demonstrated for high molecular weight DNA's. The concentration distributions at sedimentation equilibrium are studied as a function of DNA concentration for T7, T5 and T4 coliphage DNA's and the results extrapolated to infinite dilution to obtain molecular weights for these DNA's.The values obtained by this technique are 23.2 × 106 for T7, 65.7 × 106 for T5 and 104 × 106 daltons for T4 DNA. These results are in agreement with recent hydrodynamic predictions but are in disagreement with many literature values. A brief review of the literature is presented to elucidate these discrepancies.
Article
T2 and T4 bacteriophage have been exposed to various treatments which are known to release the encapsulated DNA. The unseparated reaction products have been examined by autoradiography. The results indicate the presence of one large subunit of DNA (molecular weight 45 x 106) for each former phage particle. Some smaller subunits of molecular weight 12 x 106 have been observed. The large subunit is sensitive to very small amounts of DNAase, and is resistant to mixed proteases and cannot be dispersed by banding in cesium chloride density gradients. The sensitivity to fragmentation by P32 decay and the increase in this sensitivity following heat treatment are best explained by assuming that the large subunit is a duplex of polynucleotide strands over most of its length. The presence of hypothetical non-DNA interconnections is considered.
Article
The nucleic acid of the bacteriophage T3 has been studied with the electron microscope in order to determine whether it exists either as a single unit or as several pieces of equal or varying size. The nucleic acid strands, obtained by mild alkali treatment of the virus, were oriented on collodion films and shadowed perpendicular to their length. None of the strands exhibited any branching, being continuous from one end to the other, and their diameters were found to be relatively constant. Measurement of seven such strands revealed a mean length of 14·0 ± 0·6 μ. The relatively good agreement of this value with that arrived at from theoretical calculations can be interpreted to mean that all of the T3 DNA is in one piece.
Article
The DNA molecules were released from T2 and T4 bacteriophage by a variety of procedures and examined by sedimentation equilibrium in CsCl density gradients and by sedimentation velocity and viscosity. The bands that resulted were quite Gaussian and very narrow, corresponding to molecular weights of about 62 × 106. These samples had sedimentation coefficients of about 57 to 63 s, and intrinsic viscosity values as high as 280 (100 cm3/g). When these numbers are interpreted in the light of available theories, or empirical calibrations, they are in agreement with the molecular weight values derived from sedimentation equilibrium. The gradient equilibrium method itself has not yet been adequately calibrated in this molecular weight range, and there are certain important doubts concerning the experimental determination of the sedimentation coefficient and the intrinsic viscosity. If these doubts are set aside, it is possible to conclude that there are two molecules of DNA released from each phage particle, each of approximately the same molecular weight, 62 × 106.
Article
Excerpt INTRODUCTION It is a truism that for most details one's concept of the process of virus infection is based on one's mental image of the objects involved. For example, the creation of a pool of replicating DNA, and the actions and interactions of the objects within this pool, are thought of in terms of physical entities conjured up more or less precisely according to one's needs, knowledge and inclination. Indeed, it is almost impossible to discuss genetic recombination without having recourse to imagery at some stage. And as this imagery becomes of necessity more detailed, so it also becomes more precarious. Many of the subjects of this imagery are theoretically within the reach of autoradiography, so there is no need to justify any review of the application of autoradiography to virus research. However, in a symposium on Animal Virus Multiplication, there is some need to justify taking nearly all one's...
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Amounts of H(3)-thymine-labeled DNA that are transferred from cell to cell in Hfr x F(-) and F(+) x F(-) matings have been measured autoradiographically. The transferred amounts are compared to the DNA content of the bacterial chromosome, which was measured autoradiographically on the basis of a model for the replication of DNA in E. coli (1). Partition of transferred radioactivity among the progeny of F(-) exconjugants was also observed by autoradiography. When F(+) donors were used, it was found that the transferred label often remained intact during growth of the recipients into microcolonies. The class of colonies in which this occurred was shown to contain a uniform amount of radioactivity amounting to 0.5 to 0.9 per cent of the bacterial chromosome. Dispersal of the radioactivity into smaller units was also observed. The fraction of females receiving F from F(+) donors, as measured genetically, was close to the fraction of females receiving DNA-label. The distributions of amounts of radioactivity transferred to females by Hfr males after different periods of mating were shown to fit the predictions of genetic analysis of chromosomal transfer-on the assumption that the total genetic map corresponds to the whole bacterial chromosome. The label transferred by Hfr donors was found to be variably dispersed among the progeny (4 to 5 generations) of the recipient cells.
Article
The chapter discusses the biophysical characterization of bacteriophage nucleic acid. While bacteriophages have molecular weights of millions to hundreds of millions, nevertheless, in a few instances, their chemical composition is known to a considerable extent. In the main, their principal chemical constituents are protein and deoxyribonucleic acid (DNA), with the latter accounting for up to one-half of the particle weight. For the T-even bacteriophages, one generally alludes to three classes of proteins, characterized fortuitously perhaps, by their morphological localizations: (1) a “head” protein, which comprises the membrane surrounding the nucleic acid of the head, and is left behind as a “ghost” when bacteriophages are subjected to osmotic shock and their DNA is released; (2) “tail” proteins, which are involved in the adsorption and penetration phases of infection; and (3) a non-sedimentahle protein, commonly referred to as “internal” protein. The DNA of the T-even bacteriophages is unique since along with the usual constituent bases adenine, thymine, and guanine it contains 5hydroxymethy lcytosine instead of cytosine. Apparently a small amount of 6-methylaminopurine also occurs in T2 DNA, while it has been reported that 5-hydroxymethyl uracil has been isolated from the DNA of the bacteriophage SP8. The physical dimensions and the organization of the nucleic acid of bacteriophage is very much the result of many different areas of biophysical investigation, among which may be mentioned density gradient equilibrium sedimentation, autoradiography, electron microscopy, birefringence studies, and X-ray diffraction. The chapter discusses these techniques in the light of what information each has contributed to the elucidation of the physical state of the nucleic acid within bacteriophage.
Article
The fate of the deoxyribonucleic acid (DNA) molecule during its transfer from the parental to progeny phage was studied, employing phage ϕX-174, which presumably contains one single-stranded DNA molecule. The experimental methods for tracing the parental DNA molecule were based on separation of normal and 5-bromodeoxyuridine (5BU)-labeled viable phage by centrifugation in the CsCl gradient, and on increased sensitivity of 5BU-containing phage to ultraviolet light (UV). When bacteria grown on 5BU-free medium were infected with 5BU-containing (“heavy”) phage, no “heavy” phage was detected by CsCl-gradient centrifugation, even among the early phage progeny released by prematurely opened cells. Similarly, no UV-resistant 5BU-free phage could be found among early phage progeny released by 5BU-labeled bacteria infected with 5BU-free phage. The transfer of DNA from the parental to progeny phage amounts to approximately 50 %. This transfer could also be inferred from the difference between the slopes of the UV-survival curves for early and for late progeny phage. Whichever experimental approach was used, loss of the integrity of the DNA molecule during its transfer from parental to progeny phage was indicated. The results are best explained on the basis of a dispersive mode of DNA replication in phage фX-174. This mechanism would appear to be at variance with the conservative or semiconservative replication of protein-bound, double-stranded DNA observed in other organisms.
Article
Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-(32)P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 10(7) sites per endplate, in the diaphragm, about 3 x 10(7). Reaction with DFP-(32)P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 10(7) and 1.1 x 10(7)per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.
Article
Sedimentation velocity was measured for the following materials: (1) T2 DNA isolated without mechanical breakage and purified by ion-exchange chromatography; (2) the first breakage product produced by stirring T2 DNA; (3) subfractions of (2) separated by chromatography, including putative half molecules; and (4) the quarter-length analogues to (2) and (3) produced by stirring half molecules. Some of these materials were also studied by capillary viscometry and by density gradient centrifugation. Results are summarized in Table 1 and Fig. 6.The results establish a relation between relative molecular weight, sedimenta- tion coefficient, and viscosity over a fourfold range higher than that of DNA preparations studied previously. Quarter length fragments of T2 DNA are similar in physical properties to preparations of bacterial DNA to which a molecular weight of 16 million has been assigned. This and other facts set limits to the molecular weight of T2 DNA, which must lie between 50 and 120 million but cannot be further specified at this time.
Article
The intrinsic viscosity (at zero gradient, zero concentration) of osmotically disrupted T4 bacteriophage has been measured. For a variety of reasons it seems likely that there is very little DNA-protein aggregation in dilute solutions and the measurable viscosity is due to the DNA molecules alone. The magnitude of the intrinsic viscosity is unaltered by changing the growth conditions, or exposing the infected bacteria to moderate doses of X-rays or added 32P β-decay irradiation. This would mean that the DNA molecules are neither stuck together nor broken down by these treatments. Therefore, if these conclusions are correct, the large subunit as seen by autoradiography of the same material is not a consequence of the labelling process.
Chapter
Physiologically active and structurally central substances, such as DNA, RNA, proteins, polysaccharides and lipids are usually after fixation insoluble in a wide range of solvents. The presence, the localization and the quantity of radioactive isotopes incorporated into these compounds, can be demonstrated by autoradiography in organelles, cells or tissues, and often simultaneously in one preparation. Accordingly, autoradiography may be used to solve problems involving the site, initiation, duration and rate of synthesis of substances. Often it is even possible to investigate the type of synthesis, e.g. DNA replication or repair synthesis. The redistribution of substances from parent to daughter cells can also be analysed. Autoradiography is especially useful for demonstrating and measuring small amounts of radioactivity, and is therefore an independent though complementary method to liquid scintillation and gas flow counting techniques. The sensitivity is sufficient to detect a few decays per week.
Chapter
In 1957 Hilde Levi wrote that in almost no analytical method was the term “quantitative” so misused as in autoradiography. If we take the term “quantitative” to mean that absolute amounts of a substance are measured, this would seem to be true. The misuse of the term in this sense, indeed, continued after 1957, and gained ground following the introduction of tritiated thymidine for biological research.
Chapter
Auch der Zustand der nichtwachsenden, aber lebenden und aktiven Zelle ist, wie wir wissen, dynamisch charakterisiert, als Gleichgewichtszustand, in dem der dauernd ablaufende Stoffabbau durch eine Synthese gleichen Umfanges bilanzmäßig kompensiert ist (Rittenberg und Mitarbeiter 1939, Vickery und Mitarbeiter 1939, Hevesy und Mitarbeiter 1940, Schoenheimer 1942, MacVicar und Burris 1948, Mazia und Prescott 1955 u. a.). Überwiegen die Synthesen die abbauenden Vorgänge, so arbeitet die Zelle mit Stoffgewinn, sie wächst. Diese Art des Wachstums, die wir, soweit wir nur die Zunahme der plasmatischen Zellbestandteile ins Auge fassesn, als Plasmawachstum bezeichnen, ist die fundamentale, die gewöhnlich auch den anderen Wachstumsformen, dem Teilungswachstum und dem Streckungswachstum, vorauszugehen hat, diese auch zum Teil noch begleitet.
Chapter
In early isotope studies Cohen (1948b) found that phage particles receive phosphorus assimilated by the bacteria both before and after infection, and Putnam and Kozloff (1950) showed that part of the P32 from labeled parental phage particles is conserved during virus multiplication and transferred to viral progeny.
Chapter
This chapter discusses the life cycle of T2 bacteriophage, which is one of a somewhat an arbitrary family of phages capable of attacking Escherichia coli, strain B. The physical properties, morphology, biochemical make-up, and life cycles of these phages are well known. T2 is the archetype for virulence among viruses because of its ability to commandeer the metabolism of the host cell in the interests of its own reproduction. Hence, it is, in many ways, an ideal model for a study of the processes of cell control. Chemically and metabolically inert by itself, T2 consists externally of a complex of some eight proteins. Its head seems composed of a single protein whose function is to protect and stabilize, both chemically and mechanically, the contained genetic apparatus of the virus. The proteins of T2 in their native state, such as those of viruses in general, are remarkably stable to proteolytic enzymes. When the virus and host cell are mixed in liquid suspension at adequate concentrations, the virus collides with the cell as the result of random Brownian motion. The structural information of the invading phage particle must be transmitted not only heterocatalytically or functionally––that is, into directions for the synthesis of specific proteins––but also autocatalytically––that is, into the replication to form new phage particles.
Article
The project was designed to provide a molecular characterization of the DNA of the T-even bacteriophages, T2 and T4. It also attempted to clarify the problem of a possible bipartite distribution suggested by autoradiographic experiments. The phage were grown on E. coli strain B in synthetic glycerol medium. Highly purified phage stocks were obtained by premature lysis and centrifugation. DNA was released from the phage by osmotic shock and cadmium cyanide-lysine treatment. The DNA solutions were deproteinized (except for whole shockates) by treatment with chloroform-amyl alcohol. Sedimentation studies on purified DNA preparations revealed a single, fairly sharp boundary with a median sedimentation coefficient of 30 s. The intrinsic viscosity was 114 dl./g. The molecular weights determined by light scattering and by density gradient centrifugation were 14 × 106 and 11 × 106 respectively. These values are within the range expected for DNA with the above sedimentation coefficient and intrinsic viscosity. The molecular weight distribution calculated from the sedimentation boundary was found to be much narrower than a Flory (most probable) distribution. An analysis of the sedimentation patterns of whole shockates revealed no evidence for a discrete, large component as suggested by the autoradiograph experiments. A subsequent investigation (Davison, 1959) suggests that the use of a syringe in sedimentation studies may degrade large molecules of DNA. The relationship between the 30 s DNA and that existing within the phage may therefore be uncertain.
Article
Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-32P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 107 sites per endplate, in the diaphragm, about 3 x 107. Reaction with DFP-32P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 107 and 1.1 x 107per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.
Chapter
Prior to speaking of the structure and physical properties of nucleic acids, it would seem desirable to examine their biological role. In this book it has been remarked already several times that DNA is the fundamental genetic substance and nucleic acids are responsible for the synthesis of proteins. It would seem that the time has come to bring out the evidence for these statements.
Article
Of the variety of molecular weight methods applicable to DNA, the sedimentation and/or viscosity method, and the electron microscopic-length method have proved to be the most satisfactory and the most widely accepted. These two techniques are additionally useful because of the information about conformation and topology which they can provide. However, in view of the pitfalls of sedimentation-viscosity measurements and the empirical nature of their relation to molecular weight, and because of the presence of an unknown source of variability in the electron microscopic method,109 absolute molecular weights with less than 10% uncertainty cannot be claimed, and errors as large as 20% are probably not uncommon, as inspection of Table I will suggest. The situation today is nevertheless infinitely improved over what it was a few years ago when errors by factors of two and more were common-place in reported molecular weights of DNA. In addition, there now exist several other independent techniques which provide molecular weight results consistent with those obtained by the two popular methods. Most importantly, “standard” DNA's are now available from the dozen DNA-containing viruses listed in Table I. With further study the molecular weights of the nucleic acids of these and other DNA viruses will be more accurately established. The methodology of DNA molecular weight determinations therefore appears to be capable of meeting the demands put upon it by current experimentation with intact viral chromosomes. Eventually the molecular weights of bacterial chromosomes may also be routinely determined, a possibility suggested by the last three entries in Table I.
Article
Chromatographic analysis by means of a column of basic protein is used to measure the breakage of T2 DNA by stirring. Broken molecules elute from the column at lower salt concentrations than do the original molecules. At a critical low speed of stirring, one can produce single breaks near the centers of the molecules, as shown by the all-or-none character of the initial change in chromatographic behavior, and by the survival of unbroken molecules according to an exponential function of time of stirring. In an analogous way, one can produce fragments resulting from three breaks per molecule, and in general stirring for a long time at a given speed produces a moderately homogeneous collection of fragments whose mean size is smaller, the higher the speed of stirring.The sensitivity of the DNA to breakage by stirring is strongly dependent on the concentration of DNA, being greater the lower the concentration. This self-protective action is greatly reduced when the DNA is broken by stirring.By chromatographic refractionation of the fragments produced by initial breaks, it can be shown that the fragments are not identical in chromatographic properties, presumably because of a continuous distribution of lengths centered about the mean half-length. Unbroken DNA, on the contrary, is homogeneous in chromatographic properties, therefore presumably homogeneous in molecular length. We conclude that DNA can be extracted from T2 by phenol without manipulative breakage, and that it exists in the phage particle in the form of one or more molecules of identical length.
Article
A study of the temperate bacteriophage alpha using a radioautography ; technique is described. (T.R.H.);
Article
A quantitative autoradiographic method is presented for determining amounts of 14C-labeled compounds in individual cells. Reference sources consisting of 14C-metharcrylate are used, which can be removed from the autoradiographs and reused after exposure has been terminated. The thickness of these sources exceeds the maximum β-range of 14C-disintegrations in methacrylate, while length and breadth exceed the maximum β-range many times over. Therefore, conditions of radiation geometry, of backscattering and β-selfabsorption differ in reference source and in individual labeled cells. Autoradiographic evaluations are comparable only if allowance is made for these differing conditions. In addition, the nuclear emulsion covering the cells and the reference source has to be of a constant thickness. Influences of positive or negative chemography are investigated with special attention to different effects on the nuclear emulsion which, on the one hand, covers the cells, and on the other, the reference source. They can be eliminated by appropriate procedures of fixation of the cells, and of the development of the autoradiographs. Using the described method, measurements of the incorporation of 14C-TdR into single cells are made. They are then compared with the results obtained by means of an independent method. This latter method consists basically in the preparation and evaluation of trackautoradiographs of the same cells under conditions of a 100% geometry. The results obtained from the two methods coincide well.
Article
Radiography of small particles, biological materials, and macromolecules ; using beta-sensitive gel emulsions is discussed, and processing facilities and ; methods are described. Beta particles from the small particle studied form stars ; in the emulsion, each prong produced by an electron; thus the number of marked ; atoms in the particle is revealed. The "thorium star" method and the "blob ; counting" method are described, and the processing bath compositions are given. ; (T.R.H.);
Article
Experiments were performed to investigate the distribution of parental 32P-labeled DNA to progeny T2 phages. A radioautographic method was used to render countable the 32P received by individual progeny. A method, taking approximately 1 hr, of phage purification by adsorption onto and elution from a column was used instead of a more time-consuming differential centrifugation process, to attempt to eliminate high radiation damage as a possible source of artifact. The results found, upon observation of the progeny phage emulsions for the presence of large pieces of parental DNA, did not agree with those originally reported (Levinthal, 1956), showing a more heterogeneous population with a greater disposition to pieces smaller than the 20% piece originally found. From control emulsions of the parental phages, this deviation could not be explained by inefficiency of detection by the emulsions. As would be expected from the heterogeneous distribution, the number of progeny phages containing detectable amounts of parental DNA increases with exposure time. From counting the total number of β-ray tracks in the final distributions obtained, it can be seen that approximately 60 to 80% of the transferred radioactivity appears in the progeny in pieces of less than 4 to 5% of the total parental DNA. An experiment to investigate the effect of an increase in radiation damage by a factor of two to the same parental population prior to infection and transfer showed no significant difference in the two progeny populations obtained.
Article
A two-factor cross was performed between bacteriophages labeled with heavy isotopes. Recombinants were found with chromosomes formed entirely or almost entirely of parental DNA. This and other features of the distribution of parental DNA among recombinant phages and among their descendants show that genetic recombination occurs by breakage and joining of double-stranded DNA molecules. Also, there is some indication that a small amount of DNA is removed and resynthesized in the formation of recombinant molecules.
Article
DOI:https://doi.org/10.1103/RevModPhys.24.273
Article
Une technique autoradiographique basée sur l'observation des traces individuelles émises par un élément radioactif au sein d'une cellule a été mise au point en ce qui concerne les cellules de la moelle osseuse. Les résultats obtenus au moyen de glycine-1-C14 ont montré une incorporation particulièrement intense au sein des cellules jeunes; l'incorporation diminue progressivement au cours de la maturation, pour devenir presque nulle dans le cas des hématies. Les leucocytes polynucléaires et les lymphocytes mûrs sont le siège d'une incorporation non négligeable.
Article
The molecular weight and size determined by light scattering and the intrinsic viscosity and the rate of production of dialyzable fragments have been studied as a function of time for the sodium salt of desoxyribose nucleic acid (DNA) prepared from calf thymus. The analysis of the data is based upon the statistics of degradation of single chains and double chains. It is found that following the immediate contraction of the DNA molecule upon lowering the pH to 2.6 by dialysis a gradual degradation of three types of bonding gets underway. The phosphoester bonds break at the rate of about one bond per weight average DNA molecule per 10 hours. The purines are liberated at about 200 times this rate. Substantial evidence indicates that during the early part of the degradation the two original polynucleotide strands making up the native molecule are held together at a few places by what are thought to be residual hydrogen bonds, clusters of hydrogen bonds or regions of extensive chain entanglement. These, however, also decay with the result that the degradation after about 50 hours is simply that of the random scission of single polynucleotide chains which are constantly losing purine groups.
Article
Among the products of osmotic shock of bacteriophage T2 can be identified ghosts, nucleic acid, and a “nonsedimentable” protein amounting to about 3% of the total viral protein. The nonsedimentable protein is an authentic component of the phage and differs from ghost protein both antigenically and by failing to adsorb to bacteria. It is probably not combined with the viral nucleic acid and is not a basic protein, resembling, in fact, the ghost protein in amino acid composition. It is injected into the cell when the virus attaches to bacteria, but it is not an important precursor of the nonsedimentable protein in the viral offspring. Its function is unknown.
Article
A method of high resolution beta track autoradiography applicable to single cells (such as yeast or algae) is described. The cells are suspended in liquid G-5 emulsion of Ilford and low energy beta tracks from C-14 and S-35 are registered quantitatively.
Article
The inactivation of the phages T1, T2, T3, T5, T7, and λ by decay of incorporated P32 has been studied. It was found that these phages fall into two classes of sensitivity to P32 decay: at the same specific activity of P32 in their deoxyribonucleic acid (DNA), T2 and T5 are inactivated three times as rapidly as T1, T3, T7, and λ. Since the strains of the first class were found to contain about three times as much total phosphorus per phage particle as those of the second) it appears that the fraction of all P32 disintegrations which are lethal is very nearly the same in all the strains. This fraction α depends on the temperature at which decay is allowed to proceed, being 0.05 at –196°C., 0.1 at +4°C., and 0.3 at 65°C. Decay of P32 taking place only after the penetration of the DNA of a radioactive phage particle into the interior of the bacterial cell can still prevent the reproduction of the parental phage, albeit inactivation now proceeds at a slightly reduced rate. T2 phages inactivated by decay of P32 can be cross-reactivated; i.e., donate some of their genetic characters to the progeny of a mixed infection with a non-radioactive phage. They do not, however, exhibit any multiplicity reactivation or photoreactivation. The fact that at low temperatures less than one-tenth of the P32 disintegrations are lethal to the phage particle and the dependence of the fraction of lethal disintegrations on temperature can be accounted for by the double stranded structure of the DNA macromolecule.
Article
The bacteriophage T4 containing assimilated radioactive phosphorus is inactivated at a rate proportional to the specific radioactivity of the constituent phosphorus. The beta radiation from the phosphorus makes a negligible contribution to this effect. The inactivation is therefore a direct consequence of the nuclear reaction, which kills the phage with an efficiency of about 1/12. Several phages related to T4 behave similarly. When radioactive phage is grown from a seed of non-radioactive phage, all of the phage progeny are subject to killing by radioactive decay. The phage is killed by beta radiation from P(32) with an efficiency of about 1/100 per ionization within the particle volume. Bacteriophage T4 and its relatives contain about 500,000 atoms of phosphorus per infective particle. Virtually all this phosphorus is adsorbed to bacteria with the specificity characteristic of the infective particles, and none of it can be removed from the particles by the enzyme desoxyribonuclease. The phosphorus content per particle, together with the published data on analytical composition, indicates a particle diameter close to 110 mmicro for the varieties of phage studied.
Article
Following exposure to dilute aqueous solutions of mustard gas, suspensions of E. coli B do not produce DNA although PNA is formed in nearly normal amounts. When the treated cells are infected with virus T(2), DNA is synthesized and RNA is not. The DNA formation continued after the virus titer reached a maximum.
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