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Effects of mechanical shear on genetic activity of Bacillus subtilis DNA

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... ments within a particular preparation vary widely in molecular weight, with the transforming activity tending to associate with the higher molecular weight DNA (3,25). This is not necessarily inconsistent with unique break points, since such break points need not occur at equal distances. ...
... [The term "average molecular weight" is intended only to imply the molecular weight calculated from the position of the peak of the activity under analysis. It would not be precisely accurate to call it either a number average or a weight average since, although the transforming activity of a DNA molecule increases with molecular weight (25), there is no evidence for any simple mathematical relationship between the two parameters.] With this as a molecular weight standard, the average molecular weights of the long and short molecules were calculated to be 34 X 106 and 13 X 106, respectively. ...
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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.
... This is because many forms of DNA damage, including single and double strand breaks, loss or modification of bases and oxidation or chemical modifications of bonds, can directly or indirectly lead to a reduction of average molecular weight. [4,15,26,27] Moreover, HMW DNA is desirable or required for use in many research applications. [2,28,29] Indeed, the 'percent above threshold' approach used here has been proposed as a standard metric for reporting DNA quality. ...
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DESS is a formulation widely used to preserve DNA in biological tissue samples. Although it contains three ingredients, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA) and sodium chloride (NaCl), it is frequently referred to as a DMSO-based preservative. The effectiveness of DESS has been confirmed for a variety of taxa and tissues, however, to our knowledge, the contributions of each component of DESS to DNA preservation have not been evaluated. To address this question, we stored tissues of three aquatic taxa, Mytilus edulis (blue mussel), Faxonius virilis (virile crayfish) and Alitta virens (clam worm) in DESS, each component of DESS individually and solutions containing all combinations of two components of DESS. After storage at room temperature for intervals ranging from one day to six months, we extracted DNA from each tissue and measured the percentage of high molecular weight (HMW) DNA recovered (%R) and normalized HMW DNA yield (nY). Here, HMW DNA is defined as fragments >10 kb. For comparison, we also measured the %R and nY of HMW DNA from extracts of fresh tissues and those stored in 95% EtOH over the same time intervals. We found that in cases where DESS performed most effectively (yielding ≥ 20%R of HMW DNA), all solutions containing EDTA were as or more effective than DESS. Conversely, in cases where DESS performed more poorly, none of the six DESS-variant storage solutions provided better protection of HMW DNA than DESS. Moreover, for all taxa and storage intervals longer than one day, tissues stored in solutions containing DMSO alone, NaCl alone or DMSO and NaCl in combination resulted in %R and nY of HMW DNA significantly lower than those of fresh tissues. These results indicate that for the taxa, solutions and time intervals examined, only EDTA contributed directly to preservation of high molecular weight DNA.
... MAK-frac-tionated complementary strands exhibit size differences (21,24) and show a variation in the self-annealing activity of the H strands (20,23). It is known that in B. subtilis the highest transforming activity tends to associate with the higher molecular weight DNA fragments (1,17). We strongly believe that the observed differences in transforming efficiencies reported here and by others are due in part to hybrids containing DNA strands of unequal length. ...
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The annealing properties as measured by the restoration of transforming activity and hypochromicity of methylated albumin-kieselguhr (MAK)-fractionated complementary strands of Bacillus subtilis deoxyribonucleic acid (DNA) are presented. Temperature-absorbance measurements performed on annealed mixtures of various L and H strand fractions indicated the existence of a complementarity gradient between the two MAK peaks. The markers purA16, leu-8, metB(5), thr-5, and the linked marker hisB(2)-try-2 exhibited different bimodal distributions on MAK columns. The transforming efficiency of heteroduplex mixtures, prepared by cross-annealing resolved complementary strands of wild-type and recipient DNA, was compared. The transforming efficiency of the wild-type L and H strands was equal in one preparation and unequal in a second preparation. It was found that in the second strand preparation the heteroduplex DNA containing the H strand from wild type was more efficient for all of the markers tested. The variations in transforming efficiencies of the complementary strands in heteroduplex molecules reported here and by others are due in part to strands of unequal length and probably to the self-annealing property of the H strands. At present, no conclusion could be made regarding the existence of strand selection bias during integration of donor DNA in competent B. subtilis cells.
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Bacillus subtilis and T2 phage have been grown on a medium containing the following isotopically labeled compounds: 15N2H4Cl, thymidine-3H, deuterated sugars, and deuterated amino acids. The growth rates, titers, and thymidine-3H uptake are reported. 15N2H3H-DNA has been isolated from these two organisms. Some physical properties of these DNA samples are reported. Studies on genetic activity of the B. subtilis DNA do not reveal any change produced by the substitution of 15N2H3H for 14N1H.
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The activity of chorismate mutase plays a strategic role in mediating the flow of chorismate between the synthesis of tyrosine and phenylalanine on the one hand, and of tryptophan and a number of aromatic vitamins on the other hand. A gene specifying chorismate mutase in strain 23 of Bacillus subtilis was introduced into the genetic background of strain 168 by deoxyribonucleate transformation. The newly introduced chorismate mutase has a specific activity which is 10 times greater than that of strain 168. This hybrid strain was used to study the effect of the altered activity of chorismate mutase upon the routing of common pathway precursors into the divergent terminal branches of the aromatic amino acid pathway of biosynthesis. 1. Compared to strain 168, the hybrid strain (carrying chorismate mutase-H) was more resistant to growth inhibition by low concentrations of d-tyrosine and less resistant to growth inhibition by low concentrations of 5-methyltryptophan. The hybrid strain was resistant to a weak inhibition of growth which may occur in derivatives of strain 168 in the presence of the combination of tryptophan and tyrosine. The hybrid strain also possesses an increased capacity for incorporation of exogenous shikimate. 2. Chorismate mutase was product-inhibited by prephenate, a finding which we suggest is physiologically significant—a part of the pattern of sequential feedback inhibition which regulates the aromatic pathway in B. subtilis. 3. The considerable ability of the hybrid strain to adjust to the unbalancing influence of chorismate mutase-H is attributed to the compensatory capabilities of allosteric and repressive controls existing in the pathway. Accordingly, when these regulatory controls in the terminal branches of the pathway were lost by mutation, the full potential of an alteration in level of chorismate mutase to unbalance the flow of common precursors to the terminal branches of the pathway was unmasked. The dependence of the aromatic pathway in B. subtilis upon the regulatory enzymes in its terminal branches for the appropriate function of chorismate mutase leads us to describe this chorismate mutase as an example of a subordinate enzyme. 4. Hybrid strains of B. subtilis are widely distributed, often inadvertently. The properties of hybrid strains explain a number of findings in the literature.
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1.1. The loss of capacity of Bacillus subtilis DNA to effect transformation as the result of exposure to carzinophillin has been studied, together with some properties of transforming DNA inactivated by mitomycin C.2.2. When purified DNA of B. subtilis was treated with carzinophillin in vitro, the frequency of transformation of 4 markers (tryptophan, histidine, tyrosine and shikimic acid) decreased at the same rate and the sensitivity of the marker complex was about equal to that of the single markers alone. Such a complete overlapping effect was also observed with mitomycin.3.3. Unlike normal DNA, transforming DNA inactivated by carzinophillin was relatively resistant to further inactivation by heating at 100° for 5 min and completely resistant to further inactivation by heating to 50° for 30 min.4.4. Carzinophillin-inactivated transforming DNA was reactivated to some extent by conditions under which carzinophillin-induced cross-linking in the DNA molecule was removed.5.5. These findings are compatible with the previous assumption that carzinophillin, like mitomycin, induces covalent interstrand cross-linking in DNA analogous to nitrogen mustard, without, however, inducing single-strand breaks.
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Takahashi, I. (McMaster University, Hamilton, Ontario, Canada). Joint transfer of genetic markers in Bacillus subtilis. J. Bacteriol. 91 101–105. 1966.—To compare the processes of genetic incorporation in transduction and transformation in Bacillus subtilis, several groups of linked markers were selected and the degree of linkage was determined by the two means of genetic exchange. Bacteriophage PBS 1 was used in transduction experiments. In all cases, frequencies of joint transfer, as expressed by the cotransfer index or by percentage of joint transfer, were higher in transduction than in transformation. With a pair of closely linked markers, the frequency of joint transduction was only slightly higher than that of joint transformation. On the other hand, a considerably higher degree of linkage was obtained by transduction when loosely linked markers were examined. It appears that the size of donor chromosome transferred by transducing phage particles is much larger than that incorporated by recipient cells in transformation. It is suggested that transduction in B. subtilis may be a useful tool in extending further the linkage groups established by the transformation technique.
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Biological, physical, and chromatographic properties of methylated albuminkieselguhr (MAK)-fractionated complementary strands, designated as light (L) and heavy (H), of Bacillus subtilis deoxyribonucleic acid (DNA) are presented. The pattern of transforming activity along the MAK elution profile of alkilidenatured DNA shows that the residually active molecules selectively fractionated ahead of the L strand fraction, whereas the most active self-annealed molecules fractionated preferentially at the trailing end of the H strand fraction. The restoration rate of transforming activity in the late-eluting H molecules was rapid and independent of concentration during the annealing reaction. The data suggest that the self-annealing activity in the H strand is due in part to the formation of intrastrand secondary structures. Hydroxyapatite chromatography of self-annealed L and H strands yielded a major fraction (I) of highly purified strand preparations devoid of transforming activity and hypochromicity, and a minor "nativelike" fraction (II). Sedimentation velocity measurements show that, in addition to the mutual complementary nature of the L and H fractions, they differ in molecular size and possibly configuration.
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In phage P1 lysates, two kinds of particles of different sizes have been observed. The small particles are 0·05 g cm−3 lighter than the normal particles, which are predominant. The small particles consist of the defective phage particles containing incomplete phage genomes and of the transducing particles carrying bacterial DNA. The size of the DNA molecule of the small particle carrying either phage DNA or bacterial DNA is 40% of that of the normal P1 DNA. Reflecting the reduced amount of DNA in a particle, the small phage particles are defective but can produce infective particles by multiple infection. Some of the bacterial markers, such as thr and leu, which can be co-transduced by a normal transducing particle, cannot be transduced simultaneously by a small transducing particle. The frequency of co-transduction of closely linked markers by the small particles is less than that by the normal particles. From their densities and the DNA content, the protein content of a small particle is calculated to be 67% of that of a normal particle. Mechanisms of determination of length of phage DNA molecules are discussed.
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Strauss, Norman (Yale University, New Haven, Conn.). Configuration of transforming deoxyribonucleic acid during entry into Bacillus subtilis. J. Bacteriol. 89 288–293. 1965.—A correlation was obtained between map distance and the length of the lag period preceding the appearance of pairs of genetic traits after the addition of deoxyribonucleic acid (DNA) to a competent culture of Bacillus subtilis. The results are taken to indicate that DNA enters competent cells in lengthwise fashion. The smallest length of transforming DNA which can participate in a recombination event, and the number of nucleotide pairs which enter the cell per unit time, have been estimated. The evidence indicates that only part of the lag period is devoted to the transport of DNA into the cell. The significance of these results with respect to the mechanism of entry of DNA into the cell is discussed.
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A method has been described for the isolation of DNA from micro-organisms which yields stable, biologically active, highly polymerized preparations relatively free from protein and RNA. Alternative methods of cell disruption and DNA isolation have been described and compared. DNA capable of transforming homologous strains has been used to test various steps in the procedure and preparations have been obtained possessing high specific activities. Representative samples have been characterized for their thermal stability and sedimentation behaviour.
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A readily prepared fractionating column is described that separates several ribonucleic and deoxyribonucleic acids from each other, including the closely similar nucleic acids of T2 and T4 bacteriophages. The column also distinguishes DNA that has been denatured by heat and DNA that has been broken by hydrodynamic shear from the starting material.Some notes are recorded also concerning the preparation of nucleic acids from bacteria and phage by the phenol method.
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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
Molecules of DNA isolated from bacteriophage λ break in half, but no further, when subjected to the appropriate amount of hydrodynamic shear. The half-molecules can be separated from whole molecules by zone sedimentation in a sucrose gradient.The phage particles produced by bacteria exposed to λ DNA and active “helper” phage reveal the genes carried into the bacterium by the DNA. Whole molecules of λ DNA appear to be the entire phage chromosome since they carry genes from all parts of the recombination map: m6, m5, h, s, i and mi.One of the two half-molecules produced by shear breakage of λ DNA is capable of transferring s, i and mi, but not m6, m5 or h. Since this incomplete complement is a continuous segment of the linkage map of λ, it is argued that the sequence of genes on the recombination map is the same as the sequence of the nucleotides or blocks of nucleotides which correspond to each of the genes in a molecule of λ DNA.
Article
32P-labeled T2 phage DNA has been subjected to controlled shear forces and the tensile force on the molecule causing scission of the double helix has been determined. Calculation shows that this rupturing force is compatible with the bond strengths. The significance of the molecular weight of DNA normally isolated from organisms is discussed.
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13 These values for S are only provisional since a single distance at a single time was measured. Corrections have not been made for temperature and the sucrose gradient. 14
13 These values for S are only provisional since a single distance at a single time was measured. Corrections have not been made for temperature and the sucrose gradient. 14 Mandell, J., and A. D. Hershey, Anal. Biochem., 1, 66 (1960).