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Selective heat inactivation of pneumococcal transforming deoxyribonuclease

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... Weigt et al. (2012) recommended avoiding denatured ethanol if DNA extraction is planned in distant future as the DNA molecule could be degraded and the usefulness of respective samples be compromised. Moreover, acidification triggers DNA depurination (Roger and Hotchkiss 1961;Zimmermann 2008), and it is difficult to amplify high-quality DNA from acidic-based fixatives (O'Leary et al. 1994;Longy et al. 1997;Douglas and Rogers 1998). A low pH in preservation fluids can accelerate the degradation of other molecular components such as lipids (Jones 1976) and subsequent breakdown into other compounds such as glycols, which are leached out of specimens (Carter 2003). ...
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We examined the effects of different types of specimen labels and tags on pH of different concentrations of ethanol typically used for fluid preservation in natural history collections. Labels were immersed in three different concentrations of ethanol, 96% pure undenatured ethanol (EtOH), 96% EtOH denatured with methyl-ethyl ketone (MEK), and 99.8% pure undenatured EtOH, with or without the presence of insect specimens, and the solutions were evaluated after 26 months for changes over time in pH reading. In general, pH readings of all label trials with 96% and 99.8% ethanol increased over time, except for trials of denatured alcohol, which demonstrated lower pH readings in almost all treatments, regardless of label type. Samples that contained labels with ordinary, nonstandardized, not explicitly acid-free printing paper had higher pH readings compared after the trial. Our observations are a good starting point for further experiments to answer research questions related to chemical interactions with labels in ethanol-preserved specimens, including tissue samples for molecular analyses, which can guide collection staff in their daily work.
... The rate of N-glycosylic bond cleavage depends on several factors: pH, temperature, and base and sugar structure. Depurination may occur when oligonucleotides are subjected to stronger acids and/or thermal stress or when conducting detritylation for an extended period of time (Greer & Zamenhof, 1962;Kochetkov & Budovskii, 1972;Lindahl & Karlstrom, 1973;Lindahl & Nyberg, 1972;Roger & Hotchkiss, 1961;Zamenhof & Arikawa, 1966). Depurination may be accelerated in the presence of chemical agents (especially methylating and ethylating alkylating agents) or chemical mutagens (alfatoxins, nitrogen mustards, etc.) but not polycyclic aromatic hydrocarbons and aromatic amines (Loeb & Preston, 1986). ...
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Phosphorothioate (PS) oligonucleotides are a rapidly rising class of drugs with significant therapeutic applications. However, due to their complex structure and multi-step synthesis and purification processes, generation of low level impurities and degradation products are common. Therefore, they require significant investment in quality control and impurity identification. This requires the development of advanced methods for analysis, characterization and quantitation. In addition, the presence of the PS linkage leads to the formation of chiral centers which can affect their biological properties and therapeutic efficiency.
... Or must other structural conditions prevail for these processes to occur? There are now physicochemical means for attacking such genetic questions (Rolfe and Ephrussi-Taylor, 1961; Roger and Hotchkiss, 1961; Guild, 1963). The finding that the mutation of one species links with, and in some cases replaces, a mutation of another species as a result of interspecific transformation is important for still another reason. ...
Article
Ravin, Arnold W. (University of Rochester, Rochester, N.Y.), and Joscelyn D. H. De Sa. Genetic linkage of mutational sites affecting similar characters in pneumococcus and streptococcus. J. Bacteriol. 87:86-96. 1964.-By interspecific transformation, deoxyribonucleic acid (DNA) determinants conferring resistance to high levels of streptomycin in pneumococcus were found to be allelic with DNA determinants conferring low levels of streptomycin resistance in the Challis and NBSI strains of streptococcus. The reciprocal transformation (low resistance pneumococcus x high resistance streptococcus) led to the same conclusion. In addition, determinants controlling resistance to erythromycin in pneumococcus and the Challis strain of streptococcus were found to become closely linked after interspecific transformation. Modifier genes influencing the phenotype conferred by mutations at the streptomycin-resistance locus differentiate species to a certain extent. The results demonstrate that transformations between pneumococcus and streptococcus are not due to episomes, but involve recombinational events in which genetic material of the host species is replaced by homologous material that performed a similar function in the donor species.
... This paper shows that, to the contrary, B. subtilis DNA molecules carrying the same marker can vary widely in sedimentation velocity and melting temperature and, to a lesser extent, in CsCl buoyant density. The latter two characteristics have frequently been used to distinguish DNA fragments carrying one genetic marker from those carrying another marker (11,14,29). ' This understanding of the heterogeneity of B. subtilis DNA can be useful in interpreting the results of various transformation experiments. ...
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In a Bacillus subtilis deoxyribonucleic acid (DNA) preparation, extracted and purified by the Marmur procedure, the DNA molecules carrying a particular marker are heterogeneous with respect to molecular weight, buoyant density, and thermal stability. This finding constitutes evidence against unique points of breakage during DNA isolation. The variation in buoyant density suggests a local compositional heterogeneity in the chromosomal region of certain markers. The variation in molecular weight provides an explanation for the results of certain transformation experiments that are otherwise poorly understood. An example of such a result is the observation that acridine orange increases the efficiency of differential thermal inactivation of markers. An explanation of this phenomenon is suggested by the demonstration that acridine orange can decrease the natural intramarker heterogeneity in melting behavior.
Article
After partial denaturation at temperatures which inactivate only some genetic markers, pneumococcal DNA is shown to consist of a mixture of intact and denatured molecules. Two boundaries are obtained in sedimentation velocity measurements, and two bands are resolved after centrifugation in a cesium chloride density-gradient. These correspond to untreated native DNA and fully denatured DNA.When DNA, partially denatured at 90·0°C, is eluted from a methylated albumin column, with salt solutions of increasing concentration, a native fraction and two denatured fractions are separated. The following properties are observed for the native fraction, as compared with untreated native DNA: the transforming activity of the preserved genetic markers is increased 1·5-fold; the intrinsic viscosity is high and unchanged (85 dl./g); the sedimentation coefficient is slightly lowered (S20,w=23s). It is concluded that the resolved native fraction is similar in molecular weight and configuration to intact native DNA, and is enriched in those molecules which are stable at 90·0°C.The two fractions of denatured DNA are similar in physical properties to fully denatured DNA, yet they are shown by rechromatography to be distinctly different fractions. They also differ in capacity to regain transforming activity under optimal conditions for renaturation; the early column fraction shows no increase in activity, and the later fraction can be reactivated as efficiently as the unfractionated denatured DNA.The combined results indicate that “all-or-none” denaturation occurs. There is no evidence for the formation of partially denatured molecules, and all of the molecules which bear a particular genetic marker either remain intact or are fully denatured. On this basis, as well as the molecular homogeneity indicated by the sharp sedimenting boundaries, it is proposed that chromosomal breakage during isolation of pneumococcal DNA occurs at predetermined, regularly spaced intervals.
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When transforming Haemophilus influenzae DNA is denatured, a small percentage of its biological activity survives denaturation. It can be shown, by using hydroxyapatite chromatography, that the residual transforming activity is carried by about 10% of DNA molecules, which exhibit the chromatographic properties of native DNA and, therefore, can be separated from the bulk of inactive, denatured molecules.The “native-like” fraction of DNA was investigated for its melting behavior, reversibility, renaturability, concentration-response curves and competitive ability against native DNA. In all these properties, this fraction is similar, yet not identical, with native DNA. It has been shown that the different properties of native-like DNA, compared to native DNA, cannot be solely explained by the fact that native-like DNA is contaminated by denatured material. The native-like molecules are, therefore, intrinsically different from native molecules; they appear to have disordered regions and/or single-stranded ends in otherwise double-stranded structures; furthermore, they are heterogeneous in their secondary structures. The native-like fraction seems to be formed by molecules the strands of which never came apart during the melting process, perhaps because of the existence of inter-strand cross-links of unknown nature.
Article
Depurination by heat of DNA and DNA constituents, in solution and in the dry state, has been further investigated. The extent of depurination in solution varies inversely with the ionic strength and pH. Depurination at elevated temperatures in solution appears to be mainly an acid-catalysed hydrolysis. Studies of depurination in the dry state, in the same conditions of heating used to induce mutations in dry cells and spores, indicated that approximately 30 molecules of purine are liberated per molecule of DNA, probably by the pyrolytic breakage of the N-glycosidic bond and/or destruction of the sugar. In solution and in the dry state, the nature and the extent of depurination of DNA by heat differs from that of the constituent deoxymononucleotides. Depurination, which appears to occur throughout the entire molecule of DNA, results in most conditions in a slightly greater liberation of guanine compared with adenine. The energies of activation of depurination of DNA in solution and in the dry state were calculated. The extent of depurination was unaffected by incorporation of 5-bromouracil into the DNA. Correlations between depurination and changes in the viscosity and transforming activity of DNA and the nature and extent of heat-induced mutational sites are discussed.
Article
The experiments described in this paper distinguish two processes which lead to a reversal of DNA denaturation and to the re-formation of long-stacked arrays of nucleotide pairs. Type I reversibility is a very rapid intramolecular process which occurs in DNA preparations from viral, bacterial and animal sources, in solvents varying greatly in ionic strength. Type II reversibility (“renaturation”, Marmur & Lane, 1960; Doty, Marmur, Eigner & Schildkraut, 1960; Marmur & Doty, 1961) has the properties of an intermolecular process, is much slower, and occurs only in relatively homogeneous DNA preparations at moderately high ionic strength. Data on the dependence of type I reversibility upon DNA size and composition are consistent with the proposal that type I reversibility is controlled by GC-rich nucleotide pair sequences which act as “nuclei” for the re-establishment of long-range order. Under the conditions of these experiments, such nuclei control the re-stacking of extremely long sections of polynucleotide chain. Consequently, type I reversibility is strongly affected by molecular weight changes in the range 0·5 to 10 × 106.GC-rich “nuclei” may, however, be dissociated by heating. The loss of type I reversibility is accordingly associated with the final stages of the dissociation of a double helix. However, if complementary polynucleotide chains of DNA are linked covalently, type I reversibility is permanent. (Such material is called “reversible” DNA.)The interpretation of experiments on the stability of DNA secondary structure, especially those involving bacterial transformation assays, is discussed in the light of these results.
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Micrococcin P1, 1, is an antibiotic that exhibits noteworthy antibacterial, antiprotozoal, antimalarial,cytotoxic, and gene-modulating activities. This notwithstanding, its precise structure remained undefined until mid-2009, when our group carried out the total synthesis of a substance that proved to be spectroscopically and polarimetrically identical to the natural product. This work lifted all structural and stereochemical ambiguities that have surrounded micrococcin since its discovery. Of course,knowledge of the precise structure of the molecule is essential in understanding intimate details of its mode of action and in charting possible medicinal chemistry activity. This review summarizes present knowledge about the sources, properties, and chemical synthesis of micrococcin P1.
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The kinetics and mechanism of the deprotection (detritylation) of 5'-O-(4,4'-dimethoxytrityl)-2'-deoxythymidine nucleoside catalysed by dichloroacetic acid to give a 4,4'-dimethoxytrityl carbocation have been studied in toluene, dichloromethane and acetonitrile. There is little or no effect of solvent polarity on the equilibrium and rate constants. Entropies of activation are highly negative approximately -105 J K(-1) mol(-1) and similarly show little variation with solvent. Addition of small amounts of water to the reaction medium reduces the detritylation rate, presumably through its effect on the solution acidity. All observations are compatible with detritylation occurring through a concerted general acid-catalysed mechanism rather than a stepwise A1 process.
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It has previously been demonstrated that the DNA molecules containing a genetic marker from one region of a bacterial genome undergo complete strand separation at temperatures usually different from the molecules containing a genetic marker from another region of the genome. The experiments also showed that if a group of molecules undergo complete strand separation over a narrow temperature range, of the order of one to three degrees, it is highly likely that they all come from one region of the bacterial genome. The purpose of the work reported here was to establish appropriate procedures for doing a similar analysis of the DNA molecules containing the integrated SV4O DNA in transformed mouse cells. One result of interest is that in the 11A8 cell line about 40 per cent of the integrated SV40 DNA detectable by an RNA-DNA hybridization assay can be accounted for by a group of molecules which undergo complete strand separation within a 1.0 °C interval.
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Fragments of bihelical bacteriophage Φ80 DNA have been generated by enzymatic cleavage at denaturation-sensitive sites. The DNA is exposed to subcritical denaturation conditions which are known to result in the generation of small single-stranded loops (Inman, R. B. (1967) J. Mol. Biol. 28, 103–116), presumably at loci enriched for A · T base pairs. The loops are “fixed” by reaction with formaldehyde. In rapid succession the unreacted formaldehyde is removed and the DNA is digested with the single strand-specific endonuclease from Neurospora crassa. Polyacrylamide agarose gel electrophoresis is used to monitor the reaction or isolate the DNA fragments. This procedure provides one approach to studying the chromosomal distribution of regions that are strongly biased toward A · T or G · C base pairs and also afford a flexible complement to the site-specific endonucleases in isolating and studying specific regions of the chromosome.
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The in vivo stability of 3-methyladenine in rat liver DNA has been assessed in animals treated with low doses of 14C-labelled methyl methanesulphonate. The half life for the loss of this base is approximately 3 h. The biological implications of this rapid in vivo loss of 3-methyladenine and the possibility of enzyme involvement are considered in relation to the results of in vitro measurements.
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Using the stepwise elution technique, native doublestranded DNA is eluted by 0.20 M and 0.25 M potassium phosphate buffer, pH 6.8: occasionally minor fractions are eluted by 0.30 M and 0.50 M phosphate. Rechromatography experiments showed that the multipeak pattern obtained is an artifact. Indeed, when using the gradient elution technique all native DNA preparations investigated in the present work were eluted as single peaks.A different chromatographic behaviour was shown by singlestranded DNA from Φ X174 phage, yeast mitochondrial DNA, and glucosylated DNA.No significant changes in the physical, chemical or biological properties of native DNA are caused by the adsorption-elution process. The native DNA's studied here were not fractionated by hydroxyapatite columns as far as their base composition and biological properties are concerned. A very limited extent of fractionation on the basis of molecular weight was observed. It was shown that elution can be performed at constant ionic strength and in the presence of a variety of organic molecules.
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Transforming deoxyribonucleate, isolated from bacteria grown in medium containing 32P at high specific activity, loses biological activity as a consequence of 32P disintegration. The three single markers examined are inactivated at the same rate. Linkage groups are inactivated at the rate to be expected for the independent inactivation of each of the single markers present in the group.From the rate of inactivation of single markers it has been possible to set a lower limit of 900 nucleotide pairs on the size of the entity that must remain intact for the retention of biological activity.An examination of the rates of inactivation of linkage groups shows that a lethal event in one member of a linkage group has little or no effect on the activity of the other member or members of the linkage group. In addition, it appears that 32P disintegrations between markers in a linkage group do not destroy linkage.
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Assays of the transforming activity of fractions obtained from a density-gradient of thermally denatured Bacillus subtilis DNA show that the residual activity is located away from the bulk of the DNA (denatured) and at densities corresponding to native or nearly native DNA. By using biologically formed hybrid DNA in which one strand has an enhanced density, it is shown that the two strands of the molecules bearing the residual activity do not separate under denaturing conditions. Thus the residual transforming activity of this DNA (1 to 4%) is due to a denaturation-resistant fraction. The inactive, denatured DNA is not taken up by competent cells, unless it is converted to the bihelical form by renaturation, in which case about 40% of the transforming activity of native DNA is restored.
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DNA preparations of Bacillus subtilis, were assayed for transforming activity for three unlinked genetic markers (adenine, leucine, methionine) in different stages of de- and renaturation induced by the gradual withdrawal of electrolytes by dialysis and their subsequent restitution. Experiments on denaturation by heat or alkali and thermal renaturation of transforming DNA were also performed. The biological assays were correlated with temperature-absorbance measurements and determinations of the buoyant densities of the preparations.
Bacillus subtilis DNA was heated at constant temperatures within the helix-coil transition interval. Slight increases in the temperature caused the denaturation of greater fractions of DNA. The native molecules were separated from the heat-sensitive ones by means of nitrocellulose chromatography. The fractions thus obtained were characterized for their base composition and ability to hybridize with ribosomal RNA (rRNA). As expected, heat resistance is inversely correlated with thymidine frequency and directly correlated with the content in sequences complementary to rRNA (rDNA).
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Some regions of a bacterial genome can be partially purified on the basis of their relatively high resistance to heat-induced complete separation of their strands. A method is described here for calculating the order of purification that should be achievable using this purification procedure. The method is used with previously published data on Bacillus subtilis DNA of mol. wt. 20·106. For this DNA it is estimated that about 10% of the genome should be purifiable by a factor of three or more. About 3% of the genome should be purifiable by a factor of ten or more.
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The steps involved in bacterial transformation can be divided into two main categories, those leading to the juxtapositioning (synapsis) of the donor and recipient genes and those involved in the subsequent recombinational processes. The elucidation of the mechanism by which the latter step is accomplished is of general importance to the field of genetics since similar mechanisms could quite possibly be responsible for genetic exchange reactions in all living systems. On the other hand, one might consider studies concerned with the juxaposition reactions of a more limited value in view of the uniqueness of bacterial transformation as regards the ability of purified DNA to transfer information and thus to induce hertiable genetic alterations. But, to the contrary, the understanding of the basic mechanisms involved in the process of DNA transport in bacterial transformation might be of great importance since it may provide insights into the manner in which the surfaces of more complex cells can interact with and respond to informational macromolecules in the environment.
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Conditions for the “reversible” and “irreversible” uptake of denatured DNA of Hemophilus influenzae by receptor cells have been described. At a pH of 4·8, cells bound “irreversibly” twice as much denatured DNA as they could bind native DNA under optimal conditions. Spheroplasts, produced by the penicillin technique, and cell wall material, prepared according to the procedure of Hancock & Park (1958), also bound denatured DNA “irreversibly” at low pH. The denatured DNA, bound “irreversibly” to cells, did not penetrate the cells at low pH; however, these cells transformed with relatively low efficiency when returned to neutral pH. The efficiency of transformation of cells that had taken up DNA at pH 4·8 was greatly increased by subsequent treatment with EDTA at pH 7. Fractions from equilibrium density centrifugation provided direct physical proof in support of the idea that the transformations were produced by fully denatured DNA.
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Molecules of DNA isolated from bacteriophage λdg can be broken in half when exposed to hydrodynamic shear. There are two types of half-molecules as demonstrated by their biological activity: those that contain the gal+ and m6+ genes (gal+-halves), and those that contain the iλ and mi+ genes (iλ-halves). This is the result expected if the linkage map for vegetative λdg phage were also cleaved at its center to form two linkage groups.Although the two types of half-molecules co-sediment in a sucrose gradient, they can be separated from each other by chromatography on methylated serum albumin columns, 50% of the total DNA being associated with each activity. The buoyant densities of the isolated half-molecules differ by 0·006 g/cm3, suggesting that the guanine-cytosine content of the gal+-half is 0·52 while that of the iλ-half is 0·46. The thermal denaturation curves of each half-molecule as well as the whole molecule are compatible with this suggestion.
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A recording thermospectrophotometer was assembled mainly from commercially available components, including a UV monochromator, an automatic cuvette positioner, a programmed thermostatically controlled circulating bath, an optical density converter, and a thermistortype thermometer, with the outputs of the latter two automatically plotted by an X-Y recorder. The instrument permits the measurement of the thermal stability of nucleic acids at the ambient temperature (and also of irreversible denaturation) by automatic recording of the O.D.260 versus temperature curve for up to four samples per run. The construction of the instrument, the preparation of the DNA samples, and a few applications are described.
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Sensitized photoinactivation of transforming Bacillus subtilis markers, involving 313 nm irradiation in the presence of acetophenone, was studied with both eight-fold auxotrophic uvr+ and uvr− recipients. The results allow the following conclusions: (i) Two types of inactivating lesions, dimers and non-repairable DNA breaks (pre- dominantly of a single-stranded nature), are induced upon sensitized photoinactivation. The ratio of single-stranded breaks to dimers is approximately 1:90. The breaks account for approximately 20% of the total marker sensitivities (assayed on uvr− recipients and expressed as the slopes of the inactivation curves relating the square roots of the reciprocal transforming activities to dose of irradiation). The breaks do not appear to involve cleavage of phosphodiester bonds; (ii) dimers are predominantly responsible for differential marker inactivation in transforming DNA irradiated in the acetophenone as well as in the 254 nm UV system of irradiation, and differential repair of markers is mainly due to differential repair of dimers in the marked segments of DNA; (iii) As judged from the photoreactivable sector, excellent correlation exists between the frequency of dimers in the marked segments of DNA and the marker sensitivity to irradiation on the uvr− recipient; (iv) For 6 out of the 8 markers studied good correlation exists between the sensitivities to irradiation (assayed on the uvr− recipient) and the sensitivities to heat denaturation, indicating that the A-T content of the DNA segments carrying the markers is a factor in their sensitivity to irradiation.These results lend further support to the hypothesis that differential marker inactivation by short wavelength irradiation is mainly due to differences in the frequency and distribution of potential photochemical lesions in the transforming segments of DNA.
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A sample of triply marked pneumococcal DNA has been thermally denatured at pH 7 and ionic strengths of 0·1 M and 3 × 10−4M. The inactivation temperatures for the markers have been compared to the hyperchromicity midpoint and the marker densities reported by Rolfe & Ephrussi-Taylor (1961). We find that: (1) at low ionic strength, the transition breadth for inactivation is much greater than at high μ; (2) the inactivation midpoint temperatures at low μ are much greater than the ambient hyperchromicity Tm, while at high μ, this difference is small; (c) the differences between inactivation midpoints for different markers are increased at low μ. We suggest that the inactivation of each marker involves the denaturation of CG-rich critical regions. The critical regions for low ionic strength denaturation are shorter than the critical regions for high ionic strength denaturation. They are richer in GC content and more heterogeneous in base composition. We interpret the increased transition breadth at low ionic strength as evidence for a series of partially denatured states for each marker molecule.
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Nucleobases serve as ideal targets where drugs bind and exert their anticancer activities. Cisplatin (cisPt) preferentially coordinates to 2′-deoxyguanosine (dGuo) residues within DNA. The dGuo adducts that are formed alter the DNA structure, contributing to inhibition of function and ultimately cancer cell death. Despite its success as an anticancer drug, cisPt has a number of drawbacks that reduce its efficacy, including repair of adducts and drug resistance. Some approaches to overcome this problem involve development of compounds that coordinate to other purine nucleobases, including those found in RNA. In this work, amino acid-linked platinum(II) (AAPt) compounds of alanine and ornithine (AlaPt and OrnPt, respectively) were studied. Their reactivity preferences for DNA and RNA purine nucleosides (i.e., 2′-deoxyadenosine (dAdo), adenosine (Ado), dGuo, and guanosine (Guo)) were determined. The chosen compounds form predominantly monofunctional adducts by reacting at the N1, N3, or N7 positions of purine nucleobases. In addition, features of AAPt compounds that impact the glycosidic bond stability of Ado residues were explored. The glycosidic bond cleavage is activated differentially for AlaPt-Ado and OrnPt-Ado isomers. Formation of unique adducts at non-canonical residues and subsequent destabilization of the glycosidic bonds are important features that could circumvent platinum-based drug resistance. Graphic abstract Open image in new window
Chapter
The direct evidence that nucleic acids are the genetic material is based mainly on the results of experiments with micro-organisms.
Chapter
The bacterium Diplococcus pneumoniae was first described less than a century ago. Following its discovery in 1881 by both Pasteur and Sternberg and the demonstration by Fraenkel and Weichselbaum five years later that the Pneumococcus was the causative agent of lobar pneumonia in man, interest in this organism had centered on its pathogenic properties and on the defense of the host against them (Breed et al., 1957). Griffith’s work on pneumococcal transformations (Griffith, 1928) led to the discovery that the chemical nature of the genetic material for all cellular forms of life is deoxyribonucleate. The classic study of Avery, MacLeod, and McCarty on the nature of the substance inducing transformation of pneumococcal types was published in 1944, and it marked the beginning of modern genetics and of molecular biology.
Article
IRMPD action spectroscopy studies of protonated 2'-deoxycytidine and cytidine, [dCyd+H](+) and [Cyd+H](+), have established that both N3 and O2 protonated conformers coexist in the gas phase. Threshold collision-induced dissociation (CID) of [dCyd+H](+) and [Cyd+H](+) is investigated here using guided ion beam tandem mass spectrometry techniques to elucidate the mechanisms and energetics for N-glycosidic bond cleavage. N-glycosidic bond cleavage is observed as the major dissociation pathways resulting in competitive elimination of either protonated or neutral cytosine for both protonated cytosine nucleosides. Electronic structure calculations are performed to map the potential energy surfaces (PESs) for both N-glycosidic bond cleavage pathways observed. The molecular parameters derived from theoretical calculations are employed for thermochemical analysis of the energy-dependent CID data to determine the minimum energies required to cleave the N-glycosidic bond along each pathway. B3LYP and MP2(full) computed activation energies for N-glycosidic bond cleavage associated with elimination of protonated and neutral cytosine, respectively, are compared to measured values to evaluate the efficacy of these theoretical methods in describing the dissociation mechanisms and PESs for N-glycosidic bond cleavage. The 2'-hydroxyl of [Cyd+H](+) is found to enhance the stability of the N-glycosidic bond vs that of [Cyd+H](+). O2 protonation is found to control the threshold energies for N-glycosidic bond cleavage as loss of neutral cytosine from the O2 protonated conformers is found to require ~25 kJ/mol less energy than the N3 protonated analogues, and the activation energies computed using B3LYP exhibit excellent agreement with the measured thresholds for the O2 protonated conformers.
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The effect of heating and quick cooling on the infectivity of partially purified FX 174 replicative form was examined. A sharp increase, up to 300 fold, in infectivity was observed over a range of 4–8° C. The biologicalTM (defined as half-maximal infectivity) shows a linear dependence on the logarithm of ionic strength. The G-C content of the replicative form, as calculated from a comparison of the biologicalTM values with opticalTM values obtained byMarmur andDoty onE. coli DNA, is 45% (theoretical value=43%). The results provide a simple biological assay for the replicative form; they also indicate a high specificity of our competent bacteria for the heated replicative form.
Article
The difference of the DNA base ratios for different organisms and their homogeneity for the same organism are explained by evolution from a common organism, using the following assumptions: (1) Most base pairs in DNA can undergo changes that have no or only an insignificant selective effect such that organisms experiencing many of these changes can survive as well as others experiencing only few changes. (2) For each DNA species one kind of base pair, e.g. G-C, has been altered more frequently than the other one, resulting in a shift of the base ratio. It can then be shown: (1) the number of base pair changes, required to explain the present disparity in the base ratio of different organisms, is about one or more per base pair. (2) The disparity of the base ratio may have been caused either by differences in the relative rates with which A-T versus G-C pairs have been attacked, or by differences in the number of base pair changes which the different DNA species have undergone. (3) The influence of the initial base frequency distribution on the present distribution must have decreased exponentially with increasing number of base pair changes, but it may still affect the present distribution. Eventually, aftermany base pair changes, the variance of the base frequency distribution about the mean base frequency (or base ratio) would become much smaller than the observed variance, if the constraint of biological information on DNA changes were negligible; this final variance depends only on the present mean base frequency even if that continues to change. Thus it is easy to explain the homogeneity of the present base ratios within one organism. (4) If the larger observed variance would still contain a contribution by the initial base distribution one could determine which base pair changes have been preferred during evolution. In the second part it is shown how accurately determined dinucleotide frequencies may reveal which organisms are evolutionarily more related than others; this cannot be concluded from the base ratio alone. This relationship may disclose which detailed types of base pair changes occurred predominantly during DNA evolution; in addition, it may allow one to decide whether DNA evolution moves towards more or toward less randomness of the base sequences.
Article
Sensibility to X rays and ultraviolet rays of a transforming deoxyribonucleic acid in single- and double-stranded formsTransforming DNA from Pneumococcus in its natural state (double stranded) and in a denatured state (single stranded) has been irradiated with X-rays and ultraviolet light. The survival of the genetic marker, “resistance to streptomycin”, has been determined as a function of irradiation dose in three different experimental procedures: (a) denaturation, irradiation, condensation; (b) denaturation, condensation, irradiation; (c) irradiation, denaturation, condensation.The action of X-rays has been studied under conditions of indirect effect (dilute aqueous solution) and semi-direct effect (sample frozen in 1 % yeast extract).The results obtained in each case are compared and discussed. In particular, the focus rests on their significance in regard to the biologically active structure of the selected marker and the respective mode of action of X-rays and ultraviolet light on this structure.RésuméLe DNA transformant du pneumocoque à l'état natif (bifilaire) et à l'état dénaturé (unifilaire) a été irradié avec des rayons X et ultraviolets. La survie du marqueur “résistance à la streptomycine” a été déterminé en fonctionde la dose rayonnement á chacune des trois étapes des trois schémas suivants: (a) dénaturation, irradiation, radiation, condensation; (b) dénaturation, condensation, irradiation; (c) irradiation, dénaturation, condensation.L'action des rayons X a été étudiée dans les conditions d'effet indirect (solution aqueuse diluée) et d'effet semi-direct (solution à 1 % d'extrait de levure congelée).Les résultats obtenus dans chaque cas sont comparés et discutés. On considère en particulier leur signification à l'égard de: (a) la structure biologiquement active du marqueur; (b) les modes d'action des rayons X et des rayons ultraviolets sur cette structure.
Article
Ravin, Arnold W. (University of Rochester, Rochester, N.Y.), and Joscelyn D. H. De Sa. Genetic linkage of mutational sites affecting similar characters in pneumococcus and streptococcus. J. Bacteriol. 87 86–96. 1964.—By interspecific transformation, deoxyribonucleic acid (DNA) determinants conferring resistance to high levels of streptomycin in pneumococcus were found to be allelic with DNA determinants conferring low levels of streptomycin resistance in the Challis and NBSI strains of streptococcus. The reciprocal transformation (low resistance pneumococcus × high resistance streptococcus) led to the same conclusion. In addition, determinants controlling resistance to erythromycin in pneumococcus and the Challis strain of streptococcus were found to become closely linked after interspecific transformation. Modifier genes influencing the phenotype conferred by mutations at the streptomycin-resistance locus differentiate species to a certain extent. The results demonstrate that transformations between pneumococcus and streptococcus are not due to episomes, but involve recombinational events in which genetic material of the host species is replaced by homologous material that performed a similar function in the donor species.
Article
A kinetic study is reported for the acid-catalyzed hydrolysis of oxygen (O)-linked biaryl ether 8-2'-deoxyguanosine (dG) adducts produced by phenolic toxins following metabolism into phenoxyl radical intermediates. Strikingly, the reaction rate of hydrolysis at pH 1 decreases as electron-withdrawing chlorine (Cl) substituents are added to the phenoxyl ring. The Hammett plot for hydrolysis at pH 1 shows a linear negative slope with ρX = -0.65, implying that increased Cl-substitution diminishes the rate of hydrolysis by lowering N(7) basicity. Spectrophotometric titration provided an N(7)H(+) pKa value of 1.1 for the unsubstituted adduct 8-phenoxy-dG (Ph-O-dG). Model pyridine compounds suggest N(7)H(+) pKa values of 0.92 and 0.37 for 4-Cl-Ph-O-dG and 2,6-dichloro-Ph-O-dG (DCP-O-dG), respectively. Density functional theory (DFT) calculations also highlight the ability of the 8-phenoxy substituent to lower N(7) basicity and predict a preference for N(3)-protonation for highly chlorinated O-linked 8-dG adducts in water. The calculations also provide a rationale for the hydrolytic reactivity of O-linked 8-dG adducts in the gas-phase, as determined using electrospray mass spectrometry (ESI-MS). The inclusion of our data now establishes that the order of hydrolytic reactivity at neutral pH for bulky 8-dG adducts is N-linked > C-linked > O-linked, which correlates with their relative ease of N(7)-protonation.
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An oligoribonucleotide has been synthesized in solution, using an ionic-liquid-based soluble tag at a scale several hundred times that of a standard solid-phase synthesis approach. Ogilvie's 2′-TBDMS strategy was adopted, and because of the resultant increase in lipophilicity, it allowed an easier purification of the growing oligomer compared with the previously observed for DNA, which does not require 2′ protection. The procedure is illustrated by the synthesis of the pentaribonucleotide sequence AGAUC, corresponding to a segment of the tRNAfMet from E. coli.Key words: solution-phase RNA synthesis, ionic-liquid tag.
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Bacterial transformation in relation to DNA transport and competence in Streptococcus pneumoniae (also called Diplococcus pneumoniae) is discussed. This species will serve as a model with which to compare transformation in other bacterial species, particularly Bacillus subtilis and Haemophilus influenzae, with emphasis on the many similarities as well as differences.
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1. Pneumococcal DNA having transforming activities for resistance to streptomycin, aminopterin, canavanine, micrococcin and sulfonamides was chromatographed on columns of a polycarboxylate resin (IRC-50) and columns of methylated albumin-kieselguhr. Material determining the last three properties was eluted at constant proportion with DNA, whereas activities for the first two predominated in the earlier fractions. Transforming activity for the genetically linked pair, streptomycin-sulfonamide resistances, was distributed about a position intermediate between the peak positions of the single markers.
Chapter
IntroductionThe Nature of Thermal ActionExperimental FindingsTheoretical DiscussionTheoretical Partition Functions for Proteins and DNAEnergy of the Hydrogen BondLoss of Infectivity of VirusesConclusion Acknowledgements
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Maize plants were cultivated in temperatures in the area 15 to 25° C. The pollen grains were screened for waxy mutants. An increase of the spontaneous mutation rate with temperature was indicated.
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The lethal and cytoplasmic mutagenic effects of 52C incubation during the cell cycle of a haploid strain of Saccharomyces cerevisiae were examined. Both effects varied periodically in a rather parallel pattern: the maximum thermosensitivity was seen at budding time, corresponding to the S period (Williamson, 1965). The 52C induction of a nuclear forward mutation was also examined: canavanine-resistant mutants were induced by this treatment. Exponentially growing cells were much more sensitive than resting cells to the different effects of heating which were studied. On the other hand, on comparing asynchronous cultures of 6 different radiosensitive mutants only one (xrs5) showed a greater thermosensitivity than the corresponding wild type.
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Genetic analysis has been carried out on a number of arginine mutants of Bacillus subtilis 168 which could be grouped into three separate classes by nutritional data. Reciprocal transformation experiments revealed linkage among mutants classified within a particular nutritional group. The genetic units responsible for the synthesis of three distinct biochemical reactions involved in arginine formation were found not to be linked.Physical-chemical studies indicated that one of the genetic markers studied could be distinguished from the other two by its resistance to heat denaturtion at 94°. The parallel thermal inactivation at 93.8° of an arginine locus loosely linked to an erythromycin resistance marker, and the absence of linkage of two other arginine loci to the same erythromycin marker, suggests the existence of separate transforming DNA molecules responsible for separate biochemical steps in arginine biosyntheis in B. subtilis.Studies of ornithine transcarbamylase revealed that the enzyme is repressed by exogenous concentrations of arginine in excess of 20 μg/ml in this organism. The levels of the enzyme as well as the degree of repressibility were found to vary in some of the mutants examined. Transformation experiments to arginine independence with an ornithine transcarbamylase-deficient strain as recipient and DNA extracted from donor cells producing varying levels of ornithine transcarbamylase, and differing in degree of repressiblity, resulted only in the transfer of the structural gene.
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The objective of this study is to determine whether differential denaturation of various genes can be observed. By treatments with heat or by titration with hydroxyl ions at subcritical levels, genetic markers from Haemophilus influenzae have been inactivated in the order of streptomycin resistance > erythromycin resistance > novobiocin resistance. The kinetics and extent of inactivation depends on whether a thermal or alkaline method is used. Results obtained with an assay system which allows the expression of single-stranded DNA are complementary to those testing the transforming activity of double-stranded DNA.
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The lipids from the electric organ of the ray, Torpedo marmorata, have been isolated and characterized. The major lipids were cholesterol, choline phospholipids, ethanolamine phospholipids, and sphingomyelins. The major fatty acids of ethanolamine phospholipids were 18:1, 18:0, 22:6, and 20:4. More than 50% of the acids in choline phospholipids were 16:0. The sphingomyelins consisted of five major ceramide species, all with sphingosine and the fatty acids 14:0, 15:0, 16:0, 22:1, and 24:1. The fatty acid 15:0 was mostly branched (n-2), a fatty acid earlier identified in sphingomyelins of the rectal gland of spiny dogfish. All long-chain bases were dihydroxy bases with a small percentage of branched chains. Sulfatides (cerebroside sulfate) made up the largest glycolipid fraction. The polar moiety wase galactose-3-sulfate. The fatty acids were normal and 2-hydroxy; the homologue 24:1 was the most abundant in both types of fatty acids. Most fatty acids were higher homologues of mono-unsaturated acids, but normal 18:0 fatty acid was also found. The long-chain bases were both dihydroxy and trihydroxy, with very small amounts of branched chains. The two major ceramide species of sulfatides were sphingosine combined with normal and hydroxy 24:1 fatty acids, respectively. Smaller amounts of trihydroxy base (18:0) were found linked to hydroxy 24:1 fatty acid, but not to its normal homologue. The cerebrosides contained the two major species mentioned above but lacked the trihydroxy base-hydroxy fatty acid species. The ratio of the activity of Na+-K+-dependent ATPase (EC 3.6.1.3) and the concentration of sulfatides was similar to ratios found for other tissues with normal and increased Na+ and K+ transporting capacity. The significance of this finding is discussed.
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The transforming principles of Hemophilus influenzae have been purified by a new method including fractional extraction. The active molecule behaves in these extractions like the bulk of the DNA preparation. The minimal amount of DNA necessary for transformation appeared to be of the same order of magnitude as the amount of DNA in a single cell. Quantitative study has been made of the resistance of transforming activity to various agents. When subjected to heat, the temperature at which the activity starts to decrease corresponds rather closely to the temperature at which the viscosity of the bulk of the DNA preparations starts to decrease. Similar correspondence was found when the transforming principle was subjected to pH changes. This is further evidence that the behavior of the active molecules is similar to the behavior of the average DNA molecule of the preparation. The activity is reduced by exposure to low ionic strength and by dehydration. Desoxyribonuclease in concentrations less than 10–4 γ/cc. is able to destroy the activity; a lag period during which the activity but not the viscosity decreases has been observed. NaNO2 at pH 5.3, HCHO and 10–5 M Fe++ reduce or destroy the activity; the importance of intact amino groups in the DNA molecule for the activity is discussed. Several protein-denaturing, sterilizing, and mutagenic agents have been found to have no effect on the transforming activity.
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Japanese quail cells transformed by the envelope-defective Bryan high-titer strain of Rous sarcoma virus [R(-)Q] were used as a source of the Rous sarcoma virus genome in three kinds of assays. (i) The simplest and most sensitive assay for infectious, endogenous viruses of the chicken belonging to subgroup E involved infection of a mixture of R(-)Q cells and turkey cells with the sample and assay of supernatants of these cells for focus formation on subgroup E susceptible cells. (ii) Inactivated Sendai virus-induced fusion of R(-)Q cells with live test cells was found to be a specific method for detection of chick helper factor. Focus formation by supernatant of the fused cells on subgroup E susceptible cells was correlated with the presence of subgroup E envelope glycoprotein on the plasma membranes of test cells. Whole blood cells as well as fibroblasts could be used in this assay. (iii) A method of assay for exogenous lymphoid leukosis viruses in which mixed cultures of R(-)Q cells and C/E cells and assay of supernatants for focus formation on C/E cells was as sensitive as assays presently used for exogenous lymphoid leukosis virus. Because no infectious Rous sarcoma virus was used as part of the procedure, the assays for infectious virus described here yielded pure pseudotypes of the input virus, an advantage for determining purity and subgroup of the input virus.
Article
The transforming principles of Hemophilus influenzae have been purified by a new method including fractional extraction. The active molecule behaves in these extractions like the bulk of the DNA preparation. The minimal amount of DNA necessary for transformation appeared to be of the same order of magnitude as the amount of DNA in a single cell. Quantitative study has been made of the resistance of transforming activity to various agents. When subjected to heat, the temperature at which the activity starts to decrease corresponds rather closely to the temperature at which the viscosity of the bulk of the DNA preparations starts to decrease. Similar correspondence was found when the transforming principle was subjected to pH changes. This is further evidence that the behavior of the active molecules is similar to the behavior of the average DNA molecule of the preparation. The activity is reduced by exposure to low ionic strength and by dehydration. Desoxyribonuclease in concentrations less than 10(-4) gamma/cc. is able to destroy the activity; a lag period during which the activity but not the viscosity decreases has been observed. NaNO(2) at pH 5.3, HCHO and 10(-5)M Fe(++) reduce or destroy the activity; the importance of intact amino groups in the DNA molecule for the activity is discussed. Several protein-denaturing, sterilizing, and mutagenic agents have been found to have no effect on the transforming activity.
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Excerpt This article will describe the transformation of pneumococci in the direction of high sulfonamide resistance, a process during which the group of closely linked genetic determinants is disseminated in fragments of varying size among the transformed cells. The transformation of bacteria by deoxyribonucleate (DNA) from related but different strains has been shown to have the features of a genetic transfer. Development of quantitative systems for transfer of drug resistance markers in pneumococcus allowed the demonstration that the entities transferred contained single mutated units rather than all the markers of the donor (Hotchkiss, 1951). When prepared as soluble, dispersed DNA, the genetic material of pneumococcus seemed to be subject to independent reassortment of the genes governing capsule synthesis and several levels of penicillin and streptomycin resistance. These and similar examples of reassortment described since, have been taken as evidence that individual DNA molecules bear a relation to individual functional genes. It...
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The loss of capacity of pneumococcal DNA to effect transformation as the result of exposure to several degradative agents has been studied, together with the concomitant decline in cellular incorporation of the treated DNA. The results indicate a one-hit process for the loss of genetic activity, and are in accord with the hypothesis that cellular incorporation reflects the number average molecular weight of the DNA. On this basis it has been possible to define the length of a critical DNA segment essential to genetic activity. Its value for the traits studied is about 8% of the intact molecule.Treatment by heat or ultraviolet yields multicomponent inactivation curves. The former appears to be the result of two easily distinguishable processes. Of all the agents tested, only ultraviolet discriminated between the optochin and streptomycin resistance markers. The quantum yield for ultraviolet inactivation has been calculated.
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An enzyme of Pneumococcus responsible for the conversion of maltose to glucose and oligosaccharides was investigated. The genetic analysis of a group of mutants lacking this enzyme was accomplished by studying DNA-mediated transformations of the different strains.Transformation of the different mutants to maltose utilization by wild-type DNA occurred with different frequencies. Recombination of genetic material of the maltose-negative strains also gave rise to positive cells. Analysis of the recombinations indicated that the regions corresponding to the different markers are linked and arranged in a linear fashion. Some larger regions overlapped several smaller ones indicating that single mutations altered regions in the genetic material of different size.Heating the DNA used in transforming the mutant strains destroyed most rapidly the ability to transform the factors corresponding to the larger genetic regions. Factors corresponding to smaller regions were inactivated less rapidly. This is taken to indicate that a DNA molecule partly denatured by heat still retains part of its genetic activity.It is concluded, furthermore, that genetic properties of markers, such as size, linkage, and discreteness, are reflections of the actual distribution of determinants within DNA molecules.
Article
Excerpt The process known in bacteriology as transformation has been demonstrated chiefly by the induction of heritable modifications in certain species-specific or type-specific antigens (Avery, MacLeod, and McCarty, 1944; Taylor, 1949; Austrian and MacLeod, 1949; Alexander and Leidy, 1951; Boivin, 1947). In each case, desoxyribonucleate-containing extracts from a donor strain of bacteria conferred one of the heritable properties of this strain upon a receptor when the latter was grown in the presence of the sterile extract. While such systems offer the possibility of studying the genetic relationships of the various antigens of Pneumococcus, for example, they seem still more uniquely suited to elucidate the broader biochemical principles which implicate nucleic acids in the genetic mechanisms of perhaps all forms of life. In particular, if desoxyribonucleate extracts were available which could induce several different transformations, it should be possible to study the inheritance of these different biological effects simultaneously. In work directed
Article
Density-gradient centrifugation has revealed that the population of DNA molecules from E. coli is relatively homogeneous with respect to buoyant density in a solution of cesium chloride.(1) Because of the notably small apparent atomic volume of nitrogen in aqueous solutions of organic compounds(2) and because the guanine-cytosine base pair is more rich in nitrogen than is the adenine-thymine pair, it was considered that the density homogeneity among E. coli DNA molecules might reflect a high degree of homogeneity with respect to base composition. To investigate this possibility, an examination has been made of the relationship between buoyant density in cesium chloride solution and base composition of DNA from various sources.
Article
It is clear that the correlation between the structure of deoxyribonucleic acid (DNA) and its function as a genetic determinant could be greatly increased if a means could be found of separating and reforming the two complementary strands. In this and the succeeding paper' some success along these lines is reported. This paper will deal with the evidence provided by employing the transforming activity of DNA from Diplococcus pneumoniae while the succeeding paper' will summarize physical chemical evidence for strand separation and reunion. Bacterial transformation offers a unique approach to this problem since the activity of DNA isolated from genetically marked. strains can be assayed after being subjected to various treatments. We have concentrated on thermal treatment to accomplish our goal for several reasons. First, the accumulated experience in this Laboratory2-5 has shown that exposure of DNA to carefully controlled temperatures for certain periods of time can denature the DNA molecules with minimal damage to their chemical structure; this is a prerequisite to strand separation and furthermore the ease of precise control of the temperature offers an obvious means of providing nearly reversible conditions which would optimize the chances of reuniting the DNA strands. Moreover, it has been shown in one case that thermal treatment did lead to strand separation" and there is considerable evidence that transforming activity falls sharply in the region of thermal denaturation." 7, 8 By utilizing these observations and by giving particular attention to the rate of cooling from the elevated temperature we have been able to demonstrate a pattern of inactivation and restoration of biological activity consistent with strand separation and specific reunion. It appears that this reunion can take place between complementary strands or between strands of two closely related molecules from mutant strains of D. pneumoniae. Experimental Details.-The strains of D. pneumoniae and the transformation techniques employed have been described previously.9-"1 The isolation of DNA from D. pneumoniae and other bacterial species was carried out by a procedure described elsewhere.12 The samples of pneumococcal DNA had sedimentation constants, S20, measured in 0.15 M NaCl plus 0.015 M sodium citrate in the range of 24 to 26 S (corresponding to molecular weights of 8-10 million). In most experiments pneumococcal DNA was dissolved in 0.15 M NaCl plus 0.015 M sodium citrate (hereafter referred to as standard saline-citrate) and heated in small tubes or flasks by immersion in boiling water. When fast cooling was employed, samples were transferred to tubes precooled in ice water. In cases where slow cooling was desired, samples were placed in an insulated water bath (six liters) at the elevated temperature, and allowed to cool with the heaters turned off. In a typical experiment the drop in temperature took the following time course: 89?0 (minutes), 80?-15, 70?-40, 60?-75 and 50?-130. The relationship between concentration and transforming activity of DNA which has been heated at 100? 453
We are particularly indebted to Dr. Ginoza and to Dr. Bruno H. Zimm, who, knowing of our early involvement in these studies, kindly arranged simultaneous publication in these PROCEEDINGS
  • Paul Doty
  • Julius Marmur
  • William With Dr
  • Ginoza
Paul Doty and Julius Marmur and some months ago with Dr. William Ginoza. We are particularly indebted to Dr. Ginoza and to Dr. Bruno H. Zimm, who, knowing of our early involvement in these studies, kindly arranged simultaneous publication in these PROCEEDINGS (See pp. 633-652).
  • R D P Hotchkiss
Hotchkiss, R. D., Methods in Enzymology, ed. S. P. Colowick and N. 0. Kaplan (New York: Academic Press, Inc., 1957), vol. 3, pp. 692 and 712. 10 Doty, P., J. Marmur, and N. Sueoka, Brookhaven Symposia in Biol., 12, 1 (1959).
  • S A Rice
  • P Doty
Rice, S. A., and P. Doty, J. Am. Chem. Soc., 79 3937 (1957).